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Novel stereochemical probes for DNA polymerases: nucleoside triphosphate beta,gamma-CXY analogues

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Novel stereochemical probes for DNA polymerases: nucleoside triphosphate beta,gamma-CXY analogues

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Content NOVEL STEREOCHEMICAL PROBES FOR DNA POLYMERASES: NUCLEOSIDE TRIPHOSPHATE BETA,GAMMA-CXY ANALOGUES by YUEWU A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Copyright 2012 Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) DECEMBER 2013 YueWu DEDICATION To my parents, for their unconditional love and incredible support. 1 ACKNOWLEDGEMENTS First of all I would like to thank Professor Charles E. McKenna for giv ing me the opportunity to study chemistry and do research with the McKen na group. I first met Professor McKenna when I visited USC at the Chemis try Department Open House looking forward to studying in drug discovery. Over the years, Professor McKenna has been a great mentor to me: teaching me about chemistry and research, giving great advice to prepare me for screening and qualifYing exams, and spending a lot of time with me writing scientific articles. I am especially grateful for his kindness, support, and understanding that helped me going through the most difficult time during my Ph.D. study. Next, I would like to thank Dr. Boris A. Kashemirov for his daily guid ance on my graduate research, inspiring ideas, and invaluable training that prepared me for being an organic chemist. The work presented here could not be accomplished without his help. I also really appreciate his sugges tions on career and life. My graduate research at USC is part of a multidisciplinary project and has benefited from extensive collaboration with our knowledgeable and passionate collaborators. I would like to particularly thank Professor Myron F. Goodman and Dr. Samuel H. Wilson for having organized the NIH-funded 11 program project of which this research was a part (NIH grant U19CA105010). The work presented here contains contributions from scientists in the Wilson lab, as well as the Goodman lab, and some members of the McKenna lab. In Chapter 1, Keriann Oertell performed the direct competition kinetic experiments of pol j3 incorporating }3, y-CHX dGTP (X = F, Cl, Br) diastere omers from their -1: 1 synthetic mixtures and prepared the samples for NMR analysis. Keriann also performed the entire pre-steady state kinetic analyses with pol j3 using the individual diastereomers that I synthesized. Part of Chapter 2 was published in Journal of American Chemical Society (2012, 134 (4), 8734). Dr. Valeria M. Zakharova from McKenna lab synthesized the (R)- and (S)-}3, y-CHF dGTP diastereomers based on the same method and Dr. Vinod Batra from Wilson lab crystallized the ternary complexes of the indi vidual diastereomers of }3, y-CHF dGTP and }3, y-CHCl dGTP with pol j3 and DNA, resolved their structures and determined the absolute configurations at the }3, y-bridging carbon. Thank you to Professor Barry C. Thompson, Professor Myron F. Goodman and Professor Peter Z. Qin for service on my dissertation commit tee, as well as Professor G. K. Surya Prakash and Professor Julio A. Camarero for membership in my qualifying committee, and Professor Clay C. Wang for membership in my screening committee. I am also thankful to Professors Nicos A. Petasis, Richard W. Roberts, Kyung W. Jung, Peter Z. Qin, G. K. Surya Prakash and Myron F. Goodman, whose classes I attended during the first two years of the program and learned a lot from. Ill Thank you to Allan Kershaw for faithfully maintaining the USC NMR facility and constantly providing valuable insights and support in the design of NMR experiments. The chemistry department administrative team always kept things running smoothly: Marie de la Torre, Michele Dea, Katie McKissick, Heather Connor, and Inah Kang, thank you to all. Thank you to the entire McKenna lab, former and current members. In particular, thank you to Dr. Michaela Serpi, my roommate in OCW 207 for 3 years, for coaching me hand by hand on experimental skills. Thank you to Dr. Thomas G. Upton for starting such a fruitful project and those compounds I inherited from him in the beginning of my research. Thanks are due to Dr. BrianT. Chamberlain, Dr. Ivan S. Krylov, and Dr. Valeria M. Zakharova for chemistry discussions, great friendship, lending and sharing chemicals and devices. Above all, I would like to express my full gratitude to my parents, Jianxin Wu and Qingmei Zhou, for their tremendous support, encourage ment and unconditional love. lV TABLE OF CONTENTS DEDICATION .................................................................................... i ACKNOWLEDGEMENTS ................................................................... 11 LIST 0~ TAIILES ........................................................................... \Till LIST OF FIGURES ........................................................................... ix LIST OF SCHEMES .......................................................................... X AIJSTFtACT ..................................................................................... ){i Chapter 1 : (R/ S)-./3, y-CXY dGTP analogues: The effect of bridging carbon stereochemistry on the kinetics of DNA pol./3 .................... 14 1.1 Introduction ................................................................................... 14 1.2 ReSlllts and discussion ................................................................... 19 1.2.1 Synthesis ofthe mixedfi,y-CXY dGTP diastereomer pairs ................ 19 1.2.2 Resolve the s1p NMR of(R/SJ-fi,y-CXY dGTP diastereomer mixtures.23 1.2.3 Direct competition experiments of pol fi incorporating~ 1:1 diastereomer pairs conducted by 19F and s1p NMR .................................... 29 1.2.4 Separate pre-steady-state kinetic analyses ofwild-type polfi and R 183A mutant of pol fi with the individual diastereomers ......................... 37 1.3 Conclusion ..................................................................................... 45 1.4 Experimental .................................................................................. 45 1.4.1 Materials and methods ..................................................................... 45 1.4.2 Competition kinetic experiment studied by 19F and s1p NMR ........... .47 1.4.3 Synthesis of2'-deoxyguanosine 5'-triphosphate fi, y-CXY analogues .50 1.5 References ...................................................................................... 60 v Chapter 2 : (R)- or (S)-}3, y-CXY dNTP diastereomers: Synthesis, discrete 19F and a1p NMR signatures and absolute configurations of new stereochemical probes for DNA polymerases ........................... 66 2.1 Introduction ................................................................................... 66 2.2 Results and discussion ................................................................... 68 2.2.1 Synthesis of the individual diastereomers of 14a- d ........................ 68 2.2.2 Absolute configurations of the stereoisomers and correlation with discrete 19F and s1p NMR signatures ......................................................... 72 2.2.3 Attempts to use (Rj-(+)-phenylethanol as the chiral auxiliary ............ 76 2.2.4 Attempts to synthesize the (Rj- and (S)-jl, y-CHBr dGTP diastereomers ................................................................................................................. 78 2.3. Conclusion .................................................................................... 80 2.4 Experimental .................................................................................. 81 2.4.1 Materials and methods ..................................................................... 81 2.4.2 Synthetic procedures ....................................................................... 83 2.5 References .................................................................................... 109 Chapter 3 : fi, y-C(sp2) nucleotide analogues ................................... 113 3.1 Introduction ................................................................................. 113 3.2 Results and discussion ................................................................. 115 3.2.1 Synthesis of tetraethyl ethene-1, 1-diylbis(phosphonate), 3 ............. 115 3.2.2 Attempts to synthesize thejl,y-(C=O) dGTP analogue, 13 ............... 117 3.3 Conclusion ................................................................................... 118 3.4 Experimental ................................................................................ 119 3.4.1 Materials and methods ................................................................... 119 3. 4. 2 Synthetic procedures ..................................................................... 120 3.5 References .................................................................................... 125 Vl BIBLIOGRAPHY ........................................................................... 128 Appendix A. Chapter 1 supporting data ....................................... 140 Appendix B. Chapter 2 supporting data ....................................... 163 Appendix C. Chapter 3 supporting data ....................................... 234 Vll LIST OF TABLES Table 1.1 The 19F, and 'H-decoupled s1p NMR spectral data 6 (ppm) and J (Hz) for the tetraisopropyl esters of a-halogenated methylenebis(phosphonate)s, 2 - 8 (CDCls) ......................................................................................................... 21 Table 1.2 19F and s1p NMR spectral data 6 (ppm) and J (Hz) for j3, y-CXY dGTP analogues 15a/b- 18a/b (D20, pH 9.8- 10.0) ............................................... 28 Table 1.3 Relative rate of pol.f3-catalyzed incorporation of (R)- and (S)-.f3, y-CHX dGTP diastereomers ........................................................................................ 36 Table 1.4 Presteady state parameters for correct and incorrect incorporation of (R)- and (S)-CHF or (R)- and (S)-CHCl diastereomers by wt pol.f3 ...................... 42 Table 1.5 Presteady state parameters for correct incorporation of (R)- and (S)- CHF or (R)- and (S)-CHCl diastereomers by R183A pol.f3 ................................. 43 Table 2.1 Relative s1p and 19F NMR chemical shifts and absolute configurations of j3, y-CXY dGTP diastereomer pairs in D20a ................................................... 74 Vlll LIST OF FIGURES Figure 1. 1 Diastereomer mixtures of a,j3-CXY and .f3, y-CXY (X * Y) analogues of dNTP obtained from direct coupling with the prochiral bisphosphonates ......... 15 Figure 1.2 Representative analytical SAX HPLC trace of morpholidate coupling reaction ........................................................................................................... 23 Figure 1. 3 31 P NMR spectra of 17a/b at pH 7.0 (pink) and pH 10.0 (blue) ........ 25 Figure 1.4 31p NMR spectra of 16a/b before (blue) and after (pink) treatment with Chelex-100, both at pH 10.0 .................................................................... 25 Figure 1. 5 31 P NMR spectra of the Pil resonances of j3, y-CHBr dGTP analogue (17a/b), at pH 9.9 after treatment with Chelex-100, acquired at different operating frequencies: a) at 162 MHz, b) at 202 MHz ....................................... 27 Figure 1.6 Reaction scheme and 19F NMR spectra of the reaction mixture of 15a/b .............................................................................................................. 33 Figure 1.7 Reaction scheme and 31p NMR spectra of the reaction mixture of 16a/b .............................................................................................................. 35 Figure 1.8 Pre-steady-state incorporation into single-gap DNA substrate reactions with (S)-CHF (15a) and (Rj-CHF (15b) diastereomers ........................ 39 Figure 1.9 Pre-steady-state incorporation of (S)-CHCl (16a) and (Rj-CHCl (16b) diastereomers into single-gap DNA ................................................................. .4 1 Figure 1.10 Pre-steady-state incorporation by R183A pol.f3 into single-gap DNA substrate reactions with separate diastereomers ............................................. 44 Figure 2.1 31p NMR spectra (202 MHz, D 2 0) of (a) a diastereomeric mixture of 7a-lf7a-2 at pH 9.8, (b) diastereomer 7a-l at pH 9.8, and (c) diastereomer 7a-2 ~pH10.0 ....................................................................................................... 70 Figure 2.2 Detailed view ofthe incoming nucleotide (Rj-j3,y-CHF dGTP (12b-2) in the active site of the X-ray crystal structure of its temary complex with pol.f3 and DNA (PDB entry 4D09) ............................................................................. 73 Figure 2.3 31p NMR spectra (202 MHz, D20) ofPa in 14a: (a) Artificial mixture of 14a-lf 14a-2 at pH 10.2 .................................................................................. 75 Figure 3.1 The common bisphosphonates with sp2 bridging carbon .............. 114 IX LIST OF SCHEMES Scheme 1.1 Synthesis of the mixed j3, y-CXY dGTP (X * Y) diastereomer pairs . ....................................................................................................................... 20 Scheme 1.2 Sketch depicting the direct competition reaction conducted for the NMR analysis .................................................................................................. 30 Scheme 2.1 Synthesis of the chiral bisphosphonate synthons, 7 ... .................. 68 Scheme 2.2 Synthesis of the target diastereomeric nucleotides, 14 . ................ 72 Scheme 2.3 Attempts to use (Rj-(+)-1-phenylethanol as the chiral auxiliary ..... 76 Scheme 2.4 Retrosynthetic analysis of j3, y-CHBr-dGTP diastereomers, 24a/b. 78 Scheme 2.5 Attempt to synthesize the chiral synthon of (bromomethylene)bis- (phosphonic acid) ............................................................................................ 79 Scheme 3. 1 Synthesis of j3, y- ( C=CH2) dTTP analogue, 8 ................................. 116 Scheme 3.2 Synthesis oftetrasodium carbonylbis(phosphonate), 11 ............. 118 X ABSTRACT 2'-Deoxynucleoside 5'-triphosphate analogues with jl,y-bridging oxygen replaced by a CXY group are useful chemical probes to investigate DNA poly merase catalytic and base-selection mechanisms. A long-standing limitation of such probes has been that conventional synthetic methods generate the ana logues as mixtures of two diastereomer components in equal amounts when the bridging carbon substitution is nonequivalent (X ;< Y). X-ray structural studies of DNA polymerase jl (pol .fJJ carried out with -1:1 mixture of jl, y-CXY dGTP diastereomers revealed stereospecific binding exclusively with the fluorine containing diastereomer analogues. These fmdings provided strong impetus in investigating the effect of stereochemical interactions of the jl, y-diastereomers on pol jl kinetics, the stereochemical effect that could not be addressed in the previous linear free energy relationship (LFER) correlation plotting log( kpol) of the -1: 1 mixed diastereomers vs. the pKa4 of the prochiral bisphosphonate leaving group. A general solution to the long-standing synthetic challenge was devel oped with eight examples ofjl, y-CXY dNTP diastereomers: (S)- and (Rj-CHCl, (S) and (Rj-CHF, and (S)- and (Rj-CFCl dGTP diastereomers, as well as (S)- and (Rj CHCl dATP diastereomers. Central to their preparation was the synthesis of the HPLC separable diastereomeric bisphosphonate synthons, the P,C dimorpholidate derivatives of the (Rj-mandelic acid monoesters of the parent prochiral bisphosphonic acids. Selective acidic hydrolysis of the P-N bond afforded "portaf' diastereomers, which were readily coupled to morpholine- Xl activated dGMP or dAMP. Removal of the chiral auxiliary by hydrogenolysis gave the individual diastereomeric nucleotides, of which the diastereomeric purities were confirmed by s1p andfor 19F NMR spectroscopy. After treatment with Chelex-100 to remove traces of paramagnetic ions, at pH -10 the diastere omer pairs exhibit discrete Pa and Pil s1p resonances, and discrete 19F resonanc es for fluorine-containing diastereomer pairs. The more upfield P a and more downfleld P.il resonances, as well as the more upfield 19F NMR resonances for the j3, y-CHF dGTP diastereomer, were assigned to the R configuration at the P Jl CHX-Pv carbons, based on the absolute conflgnrations of the individual dia stereomers as determined from the X-ray crystallographic structures of their temary complexes with DNA and polymerase .f3. An NMR method that was developed to idenwy the individual diastere omers in their mixture solutions was successfully applied to analyze kinetic assays in which the (R)- and (S)-j3,y-CHX dGTP (X = F, Cl, Br) diastereomers were competing for pol .f3 incorporation. Selective incorporation by pol .f3 was observed for all three diastereomer pairs, with stereospecificities of 3 (jl,y-CHF), 7 (jl, y-CHCl) and 2-3 (jl, y-CHBr). The (R)-stereoisomer was preferentially insert ed for both the j3, y-CHF and .f3, y-CHCl analogues. In the case of j3, y-CHBr analogue, although the absolute conflgnration of the preferred stereoisomer has not been identified yet, the preferred isomer also showed more upfield P a and more downfleld P.il resonances, similar to those of the (R)-CHF and (R)-CHCl stereoisomers. Pre-steady-state kinetics of pol.f3 were performed with the fully charac terized individual diastereomers of (R)- and (S)-.f3, y-CHF dGTP and (R)- and (S)- Xll jl,y-CHCl dGTP. For both diastereomer pairs, the (Rj-stereoisomer was favored over the (~-stereoisomer for G·C correct incorporation, with stereospecificities [(/\pol/ Kd)R/(kpol/ Kd)s] of 3.8 and 6.3, respectively, and also for G·T misincorpora tion with stereospecificities of 11 and 7.8, respectively. The pre-steady state kinetics and direct competition assays showed similar stereospecificities with jl, y-CHCl and jl, y-CHF diastereomer pairs for G·C correct incorporation. Xlll Chapter 1 : (R/ S)-}3, y-CXY dGTP analogues: The effect of bridging carbon stereochemistry on the kinetics of DNA pol }3* 1.1 Introduction Nucleotide bisphosphonate analogues in which a pyrophosphate bridging oxygen is replaced by a methylene carbon were first described by Myers. 1-3 Subsequently, it was suggested that the bisphosphonate moiety more closely mimics pyrophosphate when the bridging carbon is fluorinated. 4-7 P a-CXY- P!l substitution will block nucleotidyl transfer catalyzed by polymerases, whereas P!l-CXY-Pv substitution (Figure 1.1) produces a dNTP substrate analogue with leaving group properties that can be tuned by the substituents. Conventional synthesis of a,j3- or j3, y-CXY nucleotide analogues involves direct coupling of nucleosides or nucleoside monophosphates with bisphosphonates, respective- ly.s-1o When the substituents on the bridging carbon are not equivalent, the a,./3- CXY and ./3, y-CXY (X ;< Y) triphosphate analogues are obtained as mixtures of two diastereomers due to the generation of a new chiral center by coupling with the prochiral bisphosphonates (Figure 1.1). Several j3,y-CXY nucleotide ana- logues have been investigated as enzymatic probes for DNA, viral RNA or RNA- directed DNA polymerases,ll-19 but the potential for a stereospecific interaction Sections 1.2.3, 1.2.4 and related experimental procedures are reprinted with permis sion from Oertell, K., Wu, Y., Zakharova, V. M., Kashemirov, B. A., Shock, D. D., Beard W. A., Wilson, S. H., McKenna, C. E., Goodman, M. F., Biochemistry. 2012, 51(43), 8491-850 1. Copyright 2012 American Chemical Society. 14 of these diastereomeric analogues with the enzyme active site has received little attention until recently.2o-21 ""~ X Y Jase 'd 0 0 -:. .J O 011 HO,p,.O'p-4-. p..-0 I I I 0 OH OH OH ox vo HO II \/II OH 'p-"'p' I I OH OH HO.~.O ' OH J OH OH a,,B-analogues p,y-analogues p,y-analogues Figure 1.1 Diastereomer mixtures of a,frCXY and j3,y-CXY (X¢. Y) analogues of dNTP obtained from direct coupling with the prochiral bisphosphonates. A series of a,j3- and }3, y-CXY dNTP analogues has been reported as struc- tural, functional and fidelity probes in a study of the thus far obscure transition state of DNA polymerase j3 (pol ftJ .1o, 22- 26 DNA pol j3 is a key polymerase in short-patch base excision repair (BER) . 27 - 29 This is the predominant BER path- way in humans and is crucial for maintaining genome integrity. Pol j3 has been an extensively studied model for understanding polymerase fidelity. 29-so The use of a systematically varied }3, y-CXY group to probe for a leaving group effect in the catalytic process revealed a base match-dependent chemical transition step, supported by a negative correlation between log( /\pol) vs pKa4 of the bisphospho- nate leaving group. 22 - 23 The result is consistent with the findings of the theoreti- cal analyses by Lin et al. 31 - 32 and the experimental studies by Tsai et al. using 15 stopped-flow fluorescence assays and steady-state fluorescence spectroscopy.33- 34 Unexpected results were found in mispairing experiments when dGTP ana logues were mispaired with dC in the DNA template; the linear free energy relationship (LFER) data for pol .f3 incorporating analogues of dihalogenated bisphosphonates correlated to a different trend line than the other analogues, indicating a leaving group effect on fidelity.22-23 In these LFER studies however, the chemically different (R)- and (SJ-.f3, y-CXY dGTP (X ;< Y) diastereomers were used only as diastereomeric mixtures and analyzed as single compounds.22-23 Although the pKa4 value of the leaving group is presumably the same for both the (R)- and (SJ-stereoisomers, the halogen on the same leaving group has different orientations for the (R)- and (S)-stereoisomers, due to preorganization upon substrate binding and, therefore, involves in interactions with different residues at the enzyme active site and the negative charge generated as the pyrophosphate bond breaking. It is therefore essential to analyze the kinetics of pol.f3 incorporating the individual diastereomers separately. Understanding the effect of j3, y-CHX stereochemical interaction on the pol.f3 transition state would help to further understand the interactions at the transition state that deter mine the fidelity of DNA pol.f3 and explain the separation of LEFR lines between the dihalogenated and monohalogenated triphosphate analogues. Interestingly, X-ray crystallographic studies of temary complexes formed from diastereomeric j3,y-CXY dGTP analogue mixtures incubated with binary DNA-pol.f3 complex crystals have revealed the presence of only one diastereomer in the active site for monofluorinated analogues (X = H, Cl, Me; Y = F) associat ed with an interaction between the F atom and the guanidinium moiety of 16 Arg183.20-21, 35-36 Other jl, y-monohalogenated (X = H; Y = Cl, Br), monomethylat ed (X= H, Y =Me), and heterodihalogenated (X= Cl, Y = Br) analogues populat ed the active site evenly in crystal complexes.2o-21 The stereospecific binding within the DNA poljl active site exclusively with the monofluorinated analogues is associated with an electrostatic interaction between the F atom and the guanidinium moiety of Arg183,20-21 which becomes an additional experimental example in the long-standing vigorous debate about the existence of hydrogen bonds involving C-F groups and H donors such as NH or OH.37-47 Examples of "arginine fluorophilicity'' were identified by a systematic search of a protein structure database, providing support of the interaction between F and arginine guanidinium found in DNA pol jl.4s Chamberlain et al. recently reported the similar stereospecific binding of pol jl with a mono fluorinated a,jl-analogue in a crystal structure, associated with a non-covalent weak interaction between the fluorine atom of the ligand and the active-site structural water proximal to Asp276.25 These observations with X-ray crystallography also provided a strong im petus to study the effect of the jl, y-bridging carbon stereomchemistry on the kinetics of DNA pol jl that was previously neglected in the LFER studies. The challenges to address this question are: 1) the method to unambiguously idenwy the individual diastereomers in their mixture solutions so that direct competition analysis with DNA pol jl can be performed; and 2) the preparation of the individual diastereomers (Chapter 2) for transient-state kinetic analysis, which provides the kpo1 and Kd of pol jl incorporating each individual diastere- omer. 17 The resolved 19F NMR spectra of the mixed diastereomers of j3, y-CXY dGTP analogues (X = H, Cl, CHs; Y = F) and the NMR simulations based on the experimental spectra were reported previously, confirming the existence of two diastereomers in nearly equal amounts.2o As s1p NMR is more general for char acterization of the triphosphate analogues, especially the analogues without fluorine substituents, it would be more useful to resolve the diastereomer resonances in the s1p NMR spectra. In this chapter, the method that unambigu ously identifies the individual diastereomers by 19F and s1p NMR and the appli cation of the NMR method in the analysis of DNA pol j3 incorporating the dia stereomer pairs ( 15a/b - 17a/b) under direct competition condition are de scribed. The detailed synthetic procedures of the mixed diastereomers of monohalogenated (X = H, Y = F, Cl, Br) and unequally dihalogenated (X = F, Y = Cl) dGTP analogues (15a/b- 18a/b) are also provided. Independent transient-state kinetic analyses with the available individual diastereomers of the j3, y-CHX dGTP (X = F, Cl) analogues ( 15a/b, 16a/b) (syn thesis described in Chapter 2) were performed to explore the stereospecificity for correct incorporation opposite template C and misincorporation opposite T. The kinetics of the wild-type (wt) pol j3 incorporating the individual diastere omers was compared with those of a mutant pol j3 in which Arg183 was re placed with alanine (R183A) in order to examine whether a specific electrostatic interaction between Arg183 and the fluorine atom is implicated in any observed stereospecificity of the (Rj-isomer, which is relevant to assessing the potential of Arg183 as an element in the future design of selective pol j3 inhibitors. 18 1.2 Results and discussion 1.2 .1 Synthesis of the mixed j3, y-CXY dGTP diastereomer pairs Conventional methods of synthesizing j3, y-CXY dNTP analogues generally involve activation of the nucleoside monophosphate via either a morpholidate or imidazolidate intermediate. The preferred morpholidate method49-so employs a two-step process which involves the conversion of the nucleoside monophos phate to the corresponding morpholidate by DCC coupling and then the conju gation with the appropriate bisphosphonate. The imidazolidate method"· 17 activates the nucleoside monophosphate using 1,1'-carbonyldiimdazole followed by addition of the corresponding bisphosphonate in one-step as a one-pot synthesis, or altematively, as a two-step synthesis similar to the morpholidate method. The morpholidate method, as compared to the imidazolidate method, requires relatively long reaction times (up to 48 hrs) and produces modest yields, but it avoids the generation of side-products that are difficult to remove and circumvents the protectionfdeprotection tactics for certain nucleosides. 9 In order to achieve the highest purity for reliable kinetic studies of polymerases, the j3, y-CXY dNTP (G or T) analogues synthesized by DCC-mediated morphol idate coupling were purified by dual-pass HPLC (SAX and RP C,s), which pro vided the product in high purity, free of detectable contaminating nucleotides. 9. 22-23, 26 19 Scheme 1.1 Synthesis of the mixedj3,y-CXY dGTP (X'¢ Y) diastereomer pairs. 0 0 0 0 0 0 11 11 II II II II iPr-0-,P'-"'Il_-0-iPr I IPr-0-,P)(P,-0-IPr II HO-_P)(P,-OH iPr-0 0-iPr - IPr-0 X y 0-IPr - HO X y OH 1 2-8 9-12 2 X,Y = 11' 3, 9,15&/b: X,Y = B,F 4 X,Y = Cl S,10,16a/b: X,Y = B,Cl 6 X,Y- Br 7,11,17&/b: X,Y = B,Br 8,12,18&/b: X,Y = F,Cl lv fNlNH oXYo o N N).._NH HO 11)l._11 O 11 O-d 2 N£0 f I NH 0 N NANH 11 -d 2 HO-p-0 111 1 0 - OH OH 13 1\ 0 . 0 N-~-0-1- \_/ I OH 14 ... ~ ~- -~- 0 ---- OH OH OH OH 15a/b-18a/b i) 2, 3: NaH, Salectfluor, DMF (anh), 0 •c; 4: NaOCI, 0 •c; 5: (from 4) Na,SO., H 2 0: EtOH, 0 •c; 6: Br 2 , NaOH, H 2 0, 0 OC; 7: (from 6) SnCI 2 , H 2 0: EtOH, 0 °Cj 8: (from 3) NaOCI, 0 OC. II) for 9-11: HCI, reflux, 4 hrs; for 12: HBr, reflux, 4 hrs. Ill) morphollne, DCC, t-BuOH: H 2 0, reflux, 4 hrs. lv) Bu 3 N, DMSO (anh). The bisphosphonate precursors 2 - 8 were prepared by halogenation of tetraisopropyl methylenebis(phosphonate), 1, (Scheme 1.1).7. 51-52 Elec- trophilic fluorination of the carbanion of 1, prepared using 1 equiv. of NaH, with Selectfluor™ provided the monofluorinated ester 3 in major amount and difluorinated ester 2 as the by-product. 7, 52 Esters 4 - 8 were synthe- sized following known procedures. The dichloro- and (dibromometh- ylene)bis(phosphonate) esters 4 and 6 were obtained in quantitative yield by oxidation of 1 with the corresponding hypohalite reagent.51 Reduction of esters 4 and 6 provided the corresponding monohalogenated bisophspho- nates 5 and 7 in high yields.51 The [chloro(fluoro)methylene]bis(phosphonate) ester 8 was prepared by oxidiation of 3 with sodium hypochlorite.51 20 Table 1.1 The 19 F, and 1 H-decoupled 31 P NMR spectral data 15 (ppm) and J (Hz) for the tetraisopropyl esters of a-halogenated methylenebis(phosphonate)s, 2- 8 (CDCh). Literature values are provided for comparison7, 9, 26,51 ln references 7 and 51, the chemical shifts were reported as neat compounds and the 31 P NMR was 1 H-coupled. ln references 9 and 26, the chemical shifts were reported in CDCh and the 31 P NMR was 1 H-decoupled. Compound 31 P NMR 19 F NMR Ref 7 Ref 51 Ref 9 Ref 26 (1981) (1988) (2008) (2012) 2 (CF2) 2.65 (t) -124.02 (t) 31P: 2.8 (tt) 31P:3.5(t) 31P: 2.7 (t) 2 Jpp 87.4 Hz 2 Jpp 87.2 Hz 2 Jpp 84Hz '•F: -123.3 (t) 2 Jpp 87.3 Hz 3 JPH 3Hz '•F: -124. 1 (t) 19 F: -121 (t) 2 Jpp 87.2 Hz 2 Jpp 85Hz 3 (CHF) 10.07 (d) -228.48 (dt) 31P: 10.7 (ddt) 31P: 10.8 (t) 31P: 10.1 (d) 2 JPF 64.9 Hz 2 JFP 64.9 Hz 2 JPF 63Hz 19 F: -226. 1 (t) 2 JPF 65Hz 2 JFH 45.4 Hz 2 JPH 12 Hz 19 F: -228.5 (t) 3 JPH 3Hz 2 Jpp 65Hz 19 F: -221 (t) 2 JFH 45Hz 2 Jpp 63Hz 2 JFH 44Hz 4(CCb) 7.29 (s) 7.3 (t) 7.9 (s) 3 JPH 6Hz 5 (CHCl) 12.16 (s) 12.2 (s) 12.6 (s) 2 JPH 18Hz 3 JPH 6Hz 6 (CBr2) 7.73 (s) 7.5 (s) 8.1 (s) 3 JPH 6Hz 7 (CHBr) 12.15 (s) 12.3 (s) 12.1 (s) 2 JPH 17Hz 3 JPH 6Hz 8 (CFCl) 4.92 (d) -147.01 (t) 31P: 4.9 (ddt) 31 P: 5 (t) 2 Jpp 77.7 Hz 2 Jpp 77.0 Hz 2 Jpp 77Hz '•F: -145.8 (t) 3 JPH 6Hz '•F: -150 (t) 2 Jpp 77Hz 21 Acid hydrolysis of esters 3, 5 and 8 in refluxing HCl and 7 in reflux mg HBr afforded the free bisphosphonic acids, 9 - 12, in quantitative yields. 7, s1 The mono tri-n-butylammonium salts of the bisphosphonic acids, 9- 12, were prepared using 1.5 equiv. oftri-n-butylamine for the following step, conjugation reaction with the morpholidate of nucleoside monophos phate. 2'-Deoxyguanidine 5'-phosphomorpholidate 14 was prepared from the commercially available dGMP mono sodium salt (13) by DCC coupling with better than 90% yield as monitored by 31 P NMR. 49-so Conjugation of the morpholidate intermediate 14 with the tri-n-butylammonium salts of the corresponding bisphosphonic acids (9 - 12) in anhydrous DMSO at room temperature provided the }3, y-CXY dGTP analogues, 15a/b - 18a/b. 9, 49-so The progress of the reaction was monitored by analytical SAX HPLC using a gradient of 0-50% 0.5 M LiCl (pH = 8) with the starting material 14, dGMP side product 13, and the desired product 15a/b - 18a/b well separated in the chromatogram (Figure 1.2.1). Typically after 48 hrs, 60% of the mor pholidate intermediate (14) was converted to the triphosphate analogue with -10% of 14 hydrolyzed back to dGMP, 13. The desired products were puri fied by dual-pass HPLC with a preparative SAX colunm using a gradient of 0-100% 0.5 M TEAB (pH = 7.5), followed by preparative RP-C,s column purification under isocratic condition using 0.1 M TEAB 3.5% acetonitrile buffer (pH = 7.8).9. 22-23,26 The final products 15a/b - 18a/b were obtained on milligram scale as triethylammonium salts and dissolved in 50 mM 22 Tris·HCl buffer (pH = 8) before being delivered for kinetic analysis. The concentrations of the analogues were determined by UV absorbance at A252 (£= 13700).53 0 . 0 min s . t{NH 0 Cl 0 0 N NANH HO-~-f-~-0-~-0 0 11 ' 11 II -d 2 HO F OH OH Chrom . 1 OH 18a/b 30 . 0 mins . Figure 1.2 Representative analytical SAX HPLC trace of morpholidate coupling reac tion. Shown: reaction of dGMP-morpholidate (14) with [chloro(fluoro)]bis(phosphonic acid) after 48 hrs. Peak 1 = 14, peak 2 = dGMP (13), peak 3 = 18a/b. Conditions: Pump A: H20; pump B: 0.5 M (0-50% linear) LiCl gradient over 30 min; flow rate: 4 mL/min. 1.2.2 Resolve the 31p NMR of (R/ S)-j3,y-CXY dGTP diastereomer mixtures The existence of diastereomer pairs of j3,y-CXY NTP analouges has long been realized but the spectroscopic evidence and the ratio between the two diastereomers in each synthetic mixture have not been reported. 35 Recently, the first resolved 19 F NMRs of the mixed }3, y-CFX-dGTP (X = H, Cl, CHs) diastere- omer pairs were obtained at pH 10 showing a roughly 1:1 ratio between the two diastereomers, and the chemical shifts of the individual diastereomers were calculated based on simulation using NUTS (Acom, Inc) .2o-21 The literature s1p NMR data, however, have so far been reported that the diastereomer resonances of }3, y-CXY NTP analogues coincide.2o- 21. ss, 54 A similar P-CHX-PP triphosphate system with one adenosine on each terminal phosphate is the only example 23 showing discrete s1p resonances for different diastereomers, but the conditions of the NMR are not reported clearly. 55 It is known that the nature of the s1p NMR chemical shifts and coupling constants of nucleoside triphosphates is very sensitive to conditions of the media, especially the pH and the counterions.56-57 The polyprotic triphosphates have the transition from the trianions to the tetranionic state NTP4- at pH close to 7.0.58 The s1p NMRs of triphosphate analogues at neutral or slightly basic pH have broad signals due to equilibrium between the trianion and the tetranion, and electron delocalization of the trianion (Figure 1.3, pink). A much better resolution can be obtained with sharper s1p siguals when the pH is adjusted to -10.0 using Na2COs (Figure 1.3, blue). Bisphosphonates are known to chelate with divalent metal ions to form complexes. Therefore, the presence of divalent metal ions is a common cause for significant broadening in line width although the pH has already been adjusted to -10.0 (Figure 1.4, blue). The s1p NMR resolution can be siguificantly improved after the chelating metal 1ons are removed by treating the same sample with Chelex-100 (Figure 1.4, pink). 24 N:Co (( I NH o Bro o N NANH2 HO, II)__ "~0, "~00 ~ ~ ~ 0 OH OH OH 1711/b (1:1) OH J~ pH 7 7_ 7 7 . 1!'1 Hi 7.4 7. 3 PP,. \ ,, \ ,' \ , \ ,' \ , \ ,' ) N pH 7 \ , \ _,/ pH 10 ~ ~ • J . ~~~~ v~~~~~ I I I I 1 1 pH 10 10 8 6 4 -10 PPM Figure 1.3 31 P NMR spectra of 17a/b at pH 7.0 (pink) and pH 10.0 (blue). NJ[o (( I NH o 9 1 o o N NANH H0,11A11~0,11~0~ 2 ~ ~ ~ 0 OH OH OH 11alb (1:1) OH With Chelex With Chelex I I I I 10 9 8 7 -9.5 -10.0 -10.5 Figure 1.4 3 1 P NMR spectra of 16a/b before (blue) and after (pink) treatment with Chelex-100, both at pH 10.0. 25 The s1p NMRs ofjl,y-CXY dGTP analogues (15a/b- 18a/b) acquired near pH 10 without the influence of divalent metal ions have shown discrete dia stereomer resonances for Pa and Pil. For example, two doublets for Pa and two doublets of doublets for P.il were observed for jl, y-CHBr dGTP (Figure 1.3, blue) and jl,y-CHCl dGTP (Figure 1.4, pink). The diastereomer resonances of P.il for the jl, y fluorinated compounds are also resolved but appeared to be much more complicated due to additional splitting by fluorine (Appendix A, Figure A29). In order to assign the correct 19F and s1p diastereomer resonances, it is necessary to differentiate the coupling constants (J) and the chemical shift differences (l'.6) between the diastereomeric resonances. The coupling constants (J) are inde pendent of the operating frequency, whereas the chemical shift differences (l'.O) are frequency dependent. 59 Therefore, at different operating frequencies, the Jvalues remain the same but the l'.6 values change proportionally to the change of frequency as it is shown for the Pll diastereomer resonances of 17a/b (Figure 1.5). The 6 and J assignments of compounds 15a/b - 18a/b were unambiguously confirmed using the same technique for both 19F and 31p NMRs (Appendix A, Figures A37- 40), but the correlation between the relative resonances and the absolute configurations at the jl, y-bridging carbon remained unaddressed here. The 19F, 31p NMR and the J values of compounds (15a/b- 18a/b) are included in Table 1.2. 26 a aS (7.6 Hz) 7.4 162 MHz b as (9.4 Hz) 202 MHz ~z) Jpp (5.5 Hz) ~ 7.2 7.4 7.2 dG-CHBr (17a/b) Pp Figure 1.5 3lp NMR spectra of the Pfi resonances of j3,y-CHBr dGTP analogue (17a/b), at pH 9. 9 after treatment with Chelex-1 00, acquired at different operating frequencies: a) at 162 MHz, b) at 202 MHz. 27 Table 1.2 19 F and 31 P NMR spectral data 15 (ppm) and J (Hz) for }3,y-CXY dGTP analogues 15a/b - 18a/b (D20, pH 9.8 - 10.0). The NMR spectra of compounds 15a/b- 17a/b were acquired using Varian 400-MR, and compounds 18a/b was studied using VNMRS 500. Literature values are incomplete and the diastereomer resonances are only resolved for 19 F NMR 20 Compound F F' Py, Py' p~ p~· Pa Pa' 15a/b -218.42 (ddd) -218.48 (ddd) 7.92 (dd) 5.81 (ddd) 5.78 (ddd) -9.97 (d) -9.99 (d) (CHF) JFP 65.1 JFP 65.1 Jpp 55.2 Jpp 64.9 Jpp 64.9 Jpp 28.0 Jpp 28.0 JFP 55.1 JFP 55.1 Jpp 14.8 Jpp 28.0 Jpp 28.0 JFH 45.6 JFH 45.6 Jpp 15.2 Jpp 15.2 16a/b 9.45 (d) 7.75 (dd) 7.71 (dd) -10.04 (d) -10.06 (d) (CHCl) Jpp 6.7 Jpp 26.9 Jpp 26.9 Jpp 27.1 Jpp 27.1 Jpp 6.7 Jpp 6.7 17a/b 8.83 (d) 7.34 (dd) 7.30 (dd) -10.15(d) -10.20 (d) (CHBr) Jpp 5.1 Jpp 26.4 Jpp 26.4 Jpp 26.4 Jpp 26.4 Jpp 5.5 Jpp 5.5 18a/b -139.37 (dd) -139.33 (dd) 6.69 (dd) 0.42 (ddd) 0.40 (ddd) -9.97 (d) -10.05 (d) (CFCl) JFP 78.5 JFP 78.5 Jpp 64.4 Jpp 78.3 Jpp 78.3 Jpp 31.1 Jpp 31.1 JFP 64.6 JFP 64.6 Jpp 32.9 Jpp 32.9 Jpp 32.9 Jpp 31.2 Jpp 31.2 28 1.2.3 Direct competition experiments of pol j3 incorporating ~1:1 diastereomer pairs conducted by 19F and 31p NMR The method that resolves the diastereomer resonances of (Rf Sj-./3, y-CXY dGTP (15a/b- 18a/b) in 19F and s1p NMRs spectra provides the opportunity to conduct the analysis of two diastereomers in the same solution competing directly for incorporation by pol ./3, which is the condition incapable to be stud ied by conventional methods used for transient-state kinetic study even with the separated diastereomers available. The relative concentrations of the unre acted j3,y-CXY dGTP diastereomers, as determined by 19F andfor s1p NMR analysis on the reaction mixtures after some amount of pol .f3-catalyzed incorpo ration has occurred, would provide direct evidences for the preference of pol j3 in regards of a particular diastereomer pair. In order to apply the NMR technique in the study of enzyme kinetics with DNA pol ./3, it is critical to match the scales of the two experiments as NMR is usually on a millimolar scale whereas an enzyme kinetic study is usually on a nanomolar to micromolar scale. The 19F resonances of ./3, y-CHF dGTP analogue (15a/b) is split into doublets of doublets of doublets by one proton and two phosphorus, in which case higher concentration is required than for resonances with less splitting. The s1p decoupled 19F NMR of j3,y-CHF dGTP analogue (15a/b) would supposedly help to detect at lower concentration by reducing number of splittings, but adverse effects due to line broadening caused by decoupling were found such as low S/N ratio and sigrtificant overlapping with the diastereomeric resonances which ultimately limited the application of this technique in our quantitative kinetic studies with DNA pol j3. Using the default 29 settings from Varian 400MR for 19F NMR, the lowest net concentration was found to be 500 pM for compound 15a/b in order to obtain a well resolved 19 F spectrum with good S/N ratio after 26000 scans. The same concentration also gave the 31 P spectrum with good quality, using the default settings from Varian 400MR except the digital resolution was increased to 0.3 Hzjpt due to the small f'...{j between the diastereomer resonances. Scheme 1.2 Sketch depicting the direct competition reaction conducted for the NMR analysis. N _J ~ Jl. .~NH 0 X 0 0 N NA NH 2 H0-~_1_~-0-~-0-d I I I 0 OH OH OH 158/b-1811/b OH -CGC I polp --GCGCCCCCCAG-- 3, 158/b: X= F 5, 1611/b: Cl 7, 1711/b: Br --CGCGGGGGG + --GCGCCCCCCAG-- 0 0 II II HO-P P.-OH Hd Y 'oH X 3,5, 7 The experiment was designed to start with 1 mM total nucleotide ana- logue in the reaction mixture and after a significant amount ( -30%~ of the analogue was incorporated by pol j3 the reaction was quenched. DNA pol j3 is known to fill short gaps of up to 6 nucleotides by a progressive mechanism, 60 - 61 and therefore a continuous 6-nucleotide gap of C was introduced to the DNA template so that the required DNA template concentration was reduced to be 50 pM (Scheme 1.2). The downstream primer was not used to avoid further dilut- ing the DNA template concentration. After the reaction mixture was incubated for several hours to reach approximately 30°/o incorporation, the DNA product 30 together with the dGMP that was incorporated into the DNA were then removed from the reaction mixture by filtration. The pH of the reaction mixture was then adjusted using Na 2 C0 3 to pH 10 and the 19F and s1p NMRs were acquired (Figures 1.6 and 1.7). Figure 1.6 shows the 19F NMR spectra of a reaction mixture consisting of DNA, wt pol.f3, and the synthetic diastereomer pair of j3, y-CHF dGTP ( 15a/b). The 19F NMR were recorded before and after 5 hrs reaction with pol .f3 as an offset overlay, with time zero (pink) above the spectrum after the reaction (blue). Figure 1.6B shows the entire region of interest in the 19F NMR spectrum, allows calculation of the reaction progress based on the integral of (fluorometh ylene) bis(phosphonic acid) (9), the product from j3, y- CHF d GTP analogue ( 15a/b) after incorporation, relative to the integral of the unreacted 15a/b remaining in solution. As revealed by integration, the ratio between 3 and unreacted 15a/b in the reaction mixture was 31:69, indicating that the amount of j3,y-CHF dGTP analogue ( 15a/b) reacted corresponds to 31% utilization of the analogue. The amount of incorporation was adjusted to 30% based on the s1p NMR of the same reaction mixture (Appendix A, Figure A41) which shows a trace amount of dGMP (1 o/<j, a product formed from hydrolysis of the triphosphate as opposed to being incorporated into the DNA by pol.f3. Focusing on resonances from the unreacted triphosphate analogue (Figure 1.6C), it is apparent that after the reaction the two distal peaks are no longer equal in integration. Calculation based on the integral ratio between the two distal peaks after the reaction indicated that both diastereomers must had been incorporated and that the relative consumption of isomers during the reaction had occurred in a 3: 1 ratio. 31 Analysis of the s1p NMR spectra (Appendix A, Figure A41) of the same reaction mixture provides the data that are consistent with the 19F NMR analysis. Later in Chapter 2 as the individual diastereomers and their crystal structures be came available, we are able to conclude that pol.f3 has a preference for inserting the (R)-j3, y-CHF isomer from the -1:1 diastereomer pair. On the other hand, this experiment shows unequivocally that the (Sj-j3, y-CHF dGTP diasteromer is also a substrate of pol.f3 under the mixed diastereomer condition, despite not being detected in the X-ray crystal structure of a temary complex formed by soaking binary DNA-pol.f3 crystals in the solution of the mixed (R/SJ-CHF diastereomers. 32 A B c 0 N:C ~ I NH 0 ~ 0 0 N NANH 2 poljl O F O DNAt,..ll + HO,~A~,OH HO, II ;A.,. II ,0, II ,O'd D~nl subatnrte I I p p p 0 OH OH OH OH OM RIS OH 15alb 19 F NMR t = 0 pH 10.1 I ~J- _Fj-~ 14.0 -214.5 19 F NMR t = 0 pH 10.1 15.6 -215.8 -215.0 -216.0 -216.2 -215.5 -216.4 I tsa/b I 9 0 0 _.,.,......-~ 1 1sa1b 1 -216.0 -216.5 -217.0 Ni:o (( I NH 0 F 0 0 N NANH2 HO,II A II,O,Io,O'd ~ ~R) ~ ~ 0 OH OH OH OH 15b PPM -216.6 -216.8 -217.0 -217.2 -217.4 PPM Figure 1.6 Reaction scheme and 19 F NMR spectra of the reaction mixture of 15a/b. (A) Scheme depicting the direct competition reaction carried out for the NMR analysis. (B) The ~1:1 mixture of (R/S)-)3,y-CHF-dGTP analogue (15a/b) was incubated with DNA substrate with wt pol )3. The 19 F NMR spectra were taken at t = 0 (pink) and t = 5 hrs (blue) at 162 MHz and pH 9.8 with D20 capillary as external reference. (C) Zoom in of the resonances corresponding to the (Rj S)-)3,y-CHF-dGTP analogue (15a/b) at t = 0 (pink) and t = 5 hrs (blue). 33 The s1p NMR spectra of the reaction mixture of pol jl incorporating (Rf Sj jl,y-CHCl dGTP (16a/b) is shown in Figure 1.7. The top (pink) spectrum was obtained at t = 0, and the bottom (blue) spectrum was at t = 8 hrs. Based on the integral ratio between the (chloromethylene)bis(phosphonic acid) (10) formed and the remaining jl, y-CHCl dGTP analogue ( 16a/b), the amount of consump tion of 16a/b was calculated to be 27o/o. Since 3% of 16a/b was hydrolyzed as indicated by the existence of small amount of dGMP, the actual amount of 16a/b that was incorporated into DNA was therefore 24% (Figure 1.7B). From the observation of P a resonances (Figure 1. 7C), the relative consumption of the (Rj- and (Sj-diastereomers can be calculated; the leftmost peak is attributed solely to the (Sj-diastereomer and the rightmost one solely to the (R) diastereomer. The diastereomers were consumed during the reaction in a 7:1 (R:Sj ratio. The same experiment was performed with pol jl incorporating jl,y CHBr dGTP analogue (17a/b), of which the two diastereomsers were consumed in a 2-3: 1 (fast : slow) ratio. The results of the three experiments of pol jl incor porating the diastereomers of jl, y-CHX dGTP (X = F, Cl, Br) analogues ( 15a/b - 17a/b) under direct competition conditions are included in Table 1.3. 34 A 0 N=CNH ~I \ I )l HO.~,A~..-0-~..-0'd N NHz 1fl. • ' ,T 0 OH OH OH R/S OH !\. 18a/b B Pv Pill 31 P NMR t = 0 pH 10.1 " '" C) co '<!" ... N C") ..t tO N It') ,.... ,.... t = 8 hr [!!] pH 10 ~ 15 10 c 0 N£ ~ I NH 0 ~ 1 o 0 N NANH HO,~-tsJ-~~0-~~0'd 2 I I l et 0 OH OH OH OH 1811 -9.6 -9.8 5 polfl DNAcrt+CI + 10 ..... p a '" " '"" ;;!; dGMP { from hydrolysis of 16a/b J .1. 0 -10.0 -5 -10 PPM N=Co ~ I NH o c 1 o o N NANH2 HO,II.Jv-11~0,11~0'-d ~ ( ~ ~u 0 OH OH OH -10.2 OH 18b -10.4 PPM Figure 1. 7 Reaction scheme and 3 1 P NMR spectra of the reaction mixture of 16a/b. (A) Scheme depicting the direct competition reaction carried out for the NMR analysis. (B) The ~1: 1 mixture of (Rj S)-j3,y-CHC1 dGTP analogue (16a/b) was incubated with DNA substrate with wt pol j3. The 31 P NMR spectra were taken at t = 0 (pink) and t = 8 hrs (blue) at 162 MHz and pH 9.8 with D20 capillary as external reference. (C) Zoom in of the Pa resonances of (Rj S)-j3,y-CHC1 dGTP analogue (16a/b) at t = 0 (pink) and t = 8 hrs (blue). 35 Table 1.3 Relative rate of pol )3-catalyzed incorporation of (R)- and (S)-)3,y-CHX dGTP diastereomers Compound jl,y-CHF dGTP (15a/b) jl,y-CHCl dGTP (16a/b) jl,y-CHBr dGTP (17a/b) Reaction Time (hrs) 5 8 12 Rate Ratio Percent (Fast: Slow) Completion 3: 1 30% (Rj : (S) 7: 1 24% (Rj : (S) 2-3: 1 48% The interesting results found in the kinetic studies by NMR have shown that DNA pol jl selectively incorporates all three monohalogenated dGTP ana- logues (CHF, CHCl and CHBr), although in crystal structures stereospecific binding was only found with the monofluorinated dGTP analogues. Both dia- stereomers were incorporated with one diastereomer faster than the other. The ratio between the fast and slow diastereomers shows that the selectivity for the monofluoro and mono bromo dGTP analogues is similar (3: 1) while it is bigger for the monochloro dGTP analogue (7: 1). The trend, however, does not simply follow either the trend of a steric effect or an electrostatic effect of the substitu- ents, which implies that multiple factors are involved in stabilizing the transi- tion state. It was found later when the individual diastereomers of the jl, y-CHF and jl, y-CHCl dGTP analogues became available (Chapter 2) that the diastere- omers with faster reaction rates have R configuration at the jl, y-bridging carbon (both CHF and CHCl compounds). 36 1.2.4 Separate pre-steady-state kinetic analyses of wild-type pol j3 and R183A mutant of pol.f3 with the individual diastereomers The DNA incorporation reactions of the individual (R)- and (Sj-j3, y-CHF dGTP diastereomers catalyzed by pol j3 were analyzed using quench-flow transient-state kinetic assays. For each diastereomer, exponential time courses with different analogue concentrations were determined to measure the correct incorporation opposite dC (Figure 1.8) and misincorporation opposite dT (Figure 1.8B). The percentage of primer extended is plotted versus time, and the datum for each concentration is fit to the first-order exponential y = a(l - e-kt), where a is the maximal percent of primer extension and k is the observed rate constant. The observed rate constant (kob,) is plotted versus the corresponding analogue concentration, and the data fit to the rectangular hyperbola kob, = kpo11Jl, y-CHF dGTP]/(Kd + IJl, y-CHF dGTP]) to give the kpol and Kd parameters (Figure 1.8 and Table 1.4). The stereospecificity (S) is given by the /\pol/ Kd ratios for the stereoisomers; S = (/\pol/ Kd)R/ (kpol/ Kd)s (Table 1.4). Both dGTP diastereomers were incorporated opposite dC in the single-nucleotide gapped DNA substrate, visualized as the addition of a single deoxynucleotide to the 3'-end of the primer strand (Figure 1.8A, P+ 1 PAGE band). The (R) diastereomer was incorporated with a 2-fold larger kpol and a 1.5-fold smaller apparent Kd than the (SJ-diastereomer, resulting in an S of 3.8 (Table 1.4), which agrees with the S of 3 obtained by the NMR method, within the estimated error. The agreement between the NMR and kinetic methods indicates that DNA polymerase substrate specificity measured with two dNTPs competing directly for incorporation into DNA is equivalent to using kinetics to determine specifici- 37 ty by measuring lcpo1/ Kd values for dNTP substrates in separate reactions 52 and is in accord with a model by Fersht.63 Elevated concentrations of (R)- and (S)-jl, y-CHF dGTP substrates (125-2000 vM) were used to measure misincorporation opposite template T (Figure l.SB, P + 1 and P + 2 PAGE bands). The formation of mispairs opposite T shows a much larger disparity between the catalytic rates for the (R)- and (S) diastereomers (kpol,R/ kpo1,s - 14.4) versus that observed for incorporation oppo site C (kpol,R/ kpo1,s - 2.0), whereas the overall apparent binding constants were similar (i.e., within a factor of 2), for incorporations opposite either C or T (Figure 1.8 and Table 1.4). The P + 2 gel band (Figure l.SB, left-hand gel) represents a correct incorporation opposite the downstream C occurring via strand displacement at the high substrate concentrations required for the detection of misincorporation. 38 A ~ dGMP·p-CHF·p 3lp ___ gc gt___ pol~ --- cgCca--- 3lp ___ gcG gt:_ __ --- cgC c· + p·CHF· p (S)·i3,y-CHF-dGTP 20 ~=if;;/' >·;:.---- .2 I . . ~ ., B 1.0 t;, ~ ' ~~ I J 10 LS 1. 2 .S 3 hi P+l --~~- p-- ::. r.-. .:t 1'i :1. 1 r>.~ n.; <; t o; .s I'J ~ 20 ' t' ~ ' ;t 0 s (') ----- - - - 11:: dGMP·p· CHF· p B n p ___ gc gt_ pol 0 ---cgTca-- 32p ---gcG gt - ___ cg Tca-- + (RH.y-CHF-dGTP ;;; lli) ,;;/~,- . ·~ •,</ .:: 60 · '" ~ 1/ 4: 4-o:. ; .li ' ~201 E ·c c.. · 0 ;.o 1 J > 20 2:.o 30 (s) P +2 P+l - --- - p ~-;--~ 3 ~ ~~· (S)- 13, y-CHF-dGTP P+l p --------------- :! ]'i 1':\!i 1 .. 1'i .~0 ~~l 0 10 20 30 (dGT P·CHF] (11M) p-CHF· p 2.----------. 1.5 1 500 1000 lSO O 2000 [dGTP·CHF) (JlM) Figure 1.8 Pre-steady-state incorporation into single-gap DNA substrate reactions with (S)-CHF (15a) and (R)-CHF (15b) diastereomers. (A) Correct incorporation opposite dC of the (R)-diastereomer (left) and (S)-diastereomer (right). The plot of percentage primer extension vs time is shown for all six analogue concentrations [1 (•), 2 (o), 4 (l'), 8 (.6.), 16 (•), and 32 pM (o)[, below which is a representative gel showing a time course with 1 pM analogue. The corresponding reaction time is shown below each lane. P represents the unextended primer and P + 1 the extension by a single dGMP. The observed rate constant is plotted opposite the corresponding analogue concentration for both the (R) and (S)-diastereomers on the same plot for the purpose of comparison. (B) Misincorpo ration opposite dT of the (R)-diastereomer (left) and (S)-diastereomer (right). The plot of percentage primer extension vs time is shown for all six analogue concentrations [ 125 (•), 250 (o), 500 (l'), 1000 (.6.), 1500 (•), and 2000 pM (o)[, below which is a representa tive gel showing a time course with 125 pM analogue. P represents the unextended primer, P + 1 the extension by a single dGMP, and P + 2 a second incorporation by displacement of the downstream oligonucleotide, which is observed for only the R diastereomer. The corresponding reaction time is shown below each lane. The observed rate constant is plotted opposite the corresponding analogue concentration for both the (R)- and (S)-diastereomers on the same plot for the purpose of comparison. The reac tions were conducted in triplicate and have a standard error of the mean of ±15%; the figure shows a representative data set. 39 The individual (R)- and (S)-jl,y-CHCl dGTP diastereomers were used as substrates in pre-steady-state assays with pol jl (Figure 1.9). As with the fluorine analogues, time courses were determined at six concentrations of each substrate for correct incorporation opposite dC (Figure 1.9A) and misincorpora tion opposite dT (Figure 1.9B). The data were plotted and analyzed in the same way as with the fluorine analogues. The values of kpo1 and Kd are listed in Table 1.4. As observed in the NMR experiment, both diastereomers are incorporated by pol jl; however, the (R)-diastereomer with the chlorine atom oriented toward the R183 residue is incorporated with a slight catalytic advantage (7.6 s _, vs 4.8 s- 1 ) and an apparent binding constant -3-fold lower (3.2 llMJ than that of the (S)-diastereomer (11 llMJ (Table 1.2). The stereospecificity ratio [(/\pol/ Kd)R/(kpol/ Kd)s] of 6.3 is in agreement with the value of 7 obtained using the NMR method. Therefore, the observed relative incorporation of (R)-CHCl over (S)-CHCl is slightly larger than the approximate detection limit for the distribution of each diastereomer in the tertiary crystal structure, which showed equal populations of(R)-CHCl and (S)-CHCl. 40 A ~ dGMP-p-CHCI-p 3 2 p ___ gc gt___ poll! --- cgCac:aa--- l2p --- gcG gt __ _ ___ cgC c + p-CHCI-p (R)-~,y-CHCI-dGTP P+t ---~ p ~- ....... --- CI.M 0.1 O.lt 1 " tn (!I fr dGMP-p-CHCI-p (5)-~, y-CHCI-dGTP P+l p ~-=--~---;;.:) 8 6 .e ~- -"' 4 · B"P ___ gcq.gt_ polll " P--- gcG gt __ --- cgTca-- - ---CgT ca-- -+ p-CHCI-p (R)-jl,y-CHCI-dGTP _::.---- -- ; [dGTP -CHCI] (ItM) (R)-~,y-CHC I-dGTP (SH,y-CHCI-dGTP o.2o_~ . ----------; ~ ao ... ..c.:;::.:::-=-.=:~~ ·~ :: /'~: ... ---~:.-~----- ! 20 ~~;:~~~~~--~-=-.::--= ~ o · 10 40 flO go iOO 1 20 ($) P< ·l p ~----- :'1:1 .J 6:: · ¥1:' 100 120 (:) ;:;-- (R)-jl,y-CHCI-dGTP A ..... <······' ____ _ _ __ ,. 0.15.· ~ 0.10 .:./.' ,_... .. -·· / ~/-- , ... 0.05· / (5)-~,y-<:HCI-dGTP ... (~;--·-- __ _...__ - - · o 100 200 3 oo 400 5oo 600 [dGTP-CHCI] (~o~M) Figure 1.9 Pre-steady-state incorporation of (S)-CHCl (16a) and (R)-CHCl (16b) dia stereomers into single-gap DNA. (A) Correct incorporation opposite dC of (R)-CHCl (left) and (S)-CHCl (right). The cartoon shows the generalized scheme for the reaction. A plot of the percentage of primer extension vs. time for six concentrations of the CHCl ana logue is shown for one of three repeats of each reaction [1 (•), 2 (o), 4 ('"), 8 (.t..), 16 (•), and 32 pM (o)[. Below each plot is a time course shown on a representative gel for the first concentration (1 pM), where P represents the unextended primer and P + 1 the extension of the primer by a single incorporation. The corresponding reaction time is shown below each lane. Next to these gels is a plot of the observed rate constant vs. the corresponding analogue concentration for both diastereomers. (B) Misincorporation opposite dT of (R)-CHCl (left) and (S)-CHCl (right). As for the correct pairing, the per centage of primer extension vs. time for each concentration of analogue is shown for both diastereomers [25 (•), 50 (o), 100 ("), 200 (.t..), 400 (•), and 600 pM (o)[ with a representative gel for each set of reactions; the corresponding reaction time is shown below each lane. The observed rate constant is plotted vs. the corresponding analogue concentration on the same plot for each diastereomer. The reactions were conducted in triplicate and have a standard error of the mean of ±15%; the figure shows a repre sentative data set. 41 Table 1.4 Presteady state parameters for correct and incorrect incorporation of (R)- and (S)-CHF or (R)- and (S)-CHCl diastereomers by wt pol }3. Kpo!, Kd and stereospecificity values are reported as the mean ± standard error of three replicates. Stereospecificity is defined as ( k,ol/ Kd) R/ ( Kpo1/ Kd) s. Pairing Diastereomer k,o1 (s- 1 ) Kd (llMJ Stereospecificity (S)-CHF (15a) 9.2 ± 2.3 10 ± 1 (R)-CHF (15b) 18 ± 3 6.2 ± 2.2 3.8 ± 0.4 Opposite C (S)-CHCl (16a) 4.8 ± 0.4 11 ± 1 (R)-CHCl (16b) 7.6 ± 1.2 3.2 ± 0.8 6.3 ± 0.8 (S)-CHF (15a) 0.16 ± 0.01 590 ± 110 (R)-CHF (15b) 2.3 ± 0.4 740 ± 140 11 ± 1 Opposite T (S)-CHCl (16a) 0.051 ± 0.003 540 ± 30 (R)-CHCl (16b) 0.26 ± 0.01 290 ± 20 7.8 ± 0.4 To examine further the hypothesis of a specific interaction between the fluorine atom of the (Rj-diastereomer and the guanidinium group of Arg183, we repeated the kinetic analysis with the separate (Rj- and (S)-CHF diastereomers using a poljl mutant in which Arg183 was replaced with alanine (Figure 1.10). No measurable difference was found between the catalytic insertion rate con- stants for the separate diastereomers (k,o!,R = 0.27 s-1; Kpo!,s = 0.29 s- 1 ), while there is a significant relative reduction in the apparent binding constant of the (S)-diastereomer (Kd,R = 6.8 vM; Kd,s = 39 vM) (Table 1.5). The overall stereo- specificity favoring the incorporation of the (Rj-diastereomer has been increased by -1.5-fold for the mutant polymerase, stemming principally from a sizable 42 (-6-fold) destabilization in the binding of the (S)-isomer relative to that of the (R)-isomer, which may suggest a local repulsive effect. For the Cl diastereomers, a specificity difference between the (R)-CHCl and (S)-CHCl diastereomers re- mains when R 183 is replaced with Ala (Figure l.lOB and Table 1.3). The difference between the (R)- and (S)-diastereomers in both the catalytic rate constant (-2-fold) and Kd (-3-fold) remains the same for both the wt and R183A mutant poljl (Table 1.3). Table 1.5 Pre-steady-state parameters for correct incorporation of (R)- and (S)-CHF or (R)- and (S)-CHCl diastereomers by R 183A pol }3. kpol and Kd values are reported as the mean ± standard error of three replicates. Diastereomer /\pol (s- 1 ) Kd (llMJ (S)-CHF (15a) 0.29 ± 0.06 39 ± 8 (R)-CHF (15b) 0.27 ± 0.02 6.8 ± 1.0 (S)-CHCl (16a) 0.88 ± 0.13 300 ± 96 (R)-CHCl (16b) 1.5±0.1 110 ± 3 43 P+1 p- " " "' P+l p - --- IS . VI <15 f.!l If:: dGMP-p-CHCI· p B ll p ___ gc gt ___ R'l83Apo l~ 32p ___ gcGgt __ _ --- cgCca --- --- cgC ,._ __ (R)-~.y-CHCI-dGTP (5)-~,y-CHCI-dGTP P--~--- Ql!i O.!i J J~ ~Q t!) + p-CHF· p o 20 jr----{;::~,-,)~~;:- .:v-_-:=.~-=-~:- -~:= - ~=r_P=- _- _-_- ; - . ~ 0.15 i! 0.10 C• -"' {5)-~,y-CHF-dGTP ---~ ............ ---------- -- 0.05 ! !• ..... > ........ :~:.-···· :;.··· ti 10 20 (<) [dG TP-CHF] (UM) + p.CHCI·p 1.0 0.8 ----------, (R)·~.v·CHCI·dGTP .... -······ .... --·· .... -·· ~ 0.6 / ... ----~ J 0.4 / ' / (5\-a,y-CHCI- dGTP .. _ .. • 0.2 ;::, _ __ .; .--------<---------· o 30 Go 90 120 iso [dGTP-CHCI] (UM) Figure 1.10 Pre-steady-state incorporation by R183A pol j3 into single-gap DNA sub strate reactions with separate diastereomers. The plot of percentage primer extension vs. time is shown for all six analogue concentrations [ 1 ( • ), 2 ( o), 4 ( T), 8 ( .t..), 16 (•), and 32 pM (o)), below which is a representative gel showing a time course with 1 pM ana logue. P represents the unextended primer and P + 1 the extension by a single dGMP. The corresponding reaction time is shown below each lane. The observed rate constant is plotted opposite the corresponding analogue concentration for both the (R)- and (S) diastereomers on the same plot for the purpose of comparison. (B) Correct incorporation opposite dC of (R)-./3,y-CHC1 dG1P (left) and (S)-./3,y-CHC1 dG1P (right) by R183A pol ./3. The plot of percentage primer extension vs. time is shown for each analogue concentra tion [5 (•), 10 (o), 20 (T), 40 (.t..), 80 (•), and 160 pM (o)) with a representative gel picture underneath; the corresponding reaction time is shown below each lane. The observed rate constant is plotted vs. the corresponding analogue concentration. The reactions were conducted in triplicate and have a standard error of the mean of ±15%; the figure shows a representative data set. 44 1.3 Conclusion In conclusion, a series of j3, y-CXY dGTP analogues (X;< Y) 15a/b- 18a/b have been synthesized according to available methods. A method to resolve the diastereomer Pa and Pil resonances by s1p NMR was developed and the /'1.6 and J values were unambiguously identified at different operating frequencies. The effect of .f3,y-bridging carbon stereochemistry on the kinetics of DNA pol j3 was examined in the direct competition experiments with the mixed diastereomer pairs of the monohalogenated analogues (15a/b - 17a/b) analyzed by s1p andfor 19F NMR, showing a preferential insertion of one isomer over the other for each pair of diastereomers that were examined. The pre-steady state exper iment were performed examining pol j3 incorporating the individual diastere omers into the single gap DNA substrate, which similarly showed preferential insertion of the (Rj-CHF (15a) and (Rj-CHCl (16a) diastereomers. 1.4 Experimental 1.4.1 Materials and methods Tetraisopropyl methylenebis(phosphonate) (1) was kindly provided by Al bright and Wilson Americas, Inc. and 1-chloromethyl-4-fluoro-1,4-diazonia bicyclo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor™) was purchased from Alfa Aesar, Inc. All other reagents were purchased from Sigma Aldrich, Inc. and 45 used as obtailled, unless specified otherwise. Wilmad® coaxial insert (stem L 50 mm) was purchased from Sigma Aldrich, Inc. Preparative HPLC was performed using a Varian ProStar equipped with a Shimadzu SPD-10A UV detector (0.5 mm path length) with detection at 256 nm. Preparative reverse phase (RP) HPLC was conducted using a Varian 21.4 mm x 250 mm Microsorb 100-5 C,s column eluted isocratically with 0.1 M tri ethylammonium bicarbonate (TEAB) pH 7.5 buffer contailling 3.5% acetonitrile pumped at 8 mLfmin. The preparative strong anion exchange (SAX) HPLC was performed using a Macherey-Nagel 21.4 mm x 250 mm SP15/25 Nucleogel column eluted with buffer A (H20) and buffer B (0.5 M TEAB, pH 7.5) using a gradient that was increased from 0-60% over the first 10 min, maintained at 40% from 10-16 min, and then increased to 100% from 16 - 25 min with 8 mLfmin flow rate. The analytical HPLC analysis was conducted on a Varian PureGel SAX 10 mm x 100 mm 7 vL column eluted with buffer A (H20) and buffer B (0.5 M LiCl) using a linear gradient that was increased from 0 - 50% over 30 min at a 4 mLfmin flow rate. The fmal products after dual preparative HPLC purifica tion, obtained as the triethylammonium salts, were quantitated by UV absorb ance using the extinction coefficient (Am= = 253 nm, £ = 13700) of the natural dGTP compound at pH 7.53 'H, 19F and s1p NMR spectra were obtained on Varian 400-MR, VNMRS- 500 and Bruker AMX-500 2-Channel and VNMRS-600 3-Channel NMR spec trometers. The NMR operating frequencies are listed with their respective assignments. Multiplicities are quoted as singlet (s), doublet (d), triplet (t), unresolved multiplet (m), doublet of doublets (dd), doublet of doublet of dou- 46 blets (ddd), doublet of triplets (dt) or broad signal (br). All chemical shifts ( 6) are in parts per million (ppm) relative to residual CHsOH in CDsOD (6 3.34, 1 H NMR), CHCh in CDCh (6 7.26, 'H NMR), HDO in D 2 0 (6 4.79, 'H NMR),64 exter nal85% H 3 P0 4 (6 0.00, s1p NMR), or extemal hexafluorobenzene, C 6 F 6 (6 -164.9, 19F NMR).65 s1p NMR spectra were proton-decoupled unless otherwise specified, and 'H, 19F, and s1p coupling constants (Jvalues) are given in Hz. The concen tration of the NMR samples was in the range of2- 5 mgfmL. The pH values of the NMR samples were measured in 99.9% D20 and are reported without deuterium isotope correction. 'H NMR spectra (2- 11, 15a/b- 18a/b), 19F NMR spectra (2, 3, 8, 9, 12, 15a/b, 18a/b), s1p NMR spectra (2 - 12, 15a/b - 18a/b), 19F NMR spectra under different operating frequencies (15a/b- 18a/b), s1p NMR spectra under different operating frequencies (16a/b - 18a/b), and s1p NMR spectra of the reaction mixtures after incorporation by DNA pol.f3 (15a/b - 17a/b) are pre sented in Appendix A. 1.4.2 Competition kinetic experiment studied by 19F and 31p NMR DNA synthesis, purification, and annealing The primer (5'-TAT TAC CGC GCT GAT GCG C) and the template (5' GCG TTG TIC CGA CCC CCC GCG CAT CAG CGC GGT AAT A) were synthe sized on a solid phase DNA synthesizer and purified by 16% denaturing poly acrylamide gel electrophoresis, followed by desalting using oligonucleotide purification cartridges. Primer, 1 mol equiv, was mixed with 1.2 mol equiv 47 template, heated to 95 oc and cooled slowly to room temperature to anneal the strands. Protein and reaction buffer preparation. Wild-type poljl was purified as previously reported. 56 The reaction buffer for all experiments consisted of 50 mM Tris-Cl, 20 mM KCl, 20 mM NaCl, 10 mM MgCb, 1 mM DTT, and 6% glycerol at pH 8.0, 37 °C. Assay sample preparation Nine individual reaction tubes (100 11L fmal volume in each) containing 50 11M unlabeled and annealed primer/template were incubated with 100 11M wt pol jl (fmal concentrations) in reaction buffer for 3 minutes at 37 oc, followed by addition of (Rf S)-jl, y-CHF dGTP (15a/b) to a fmal concentration of 1 mM (500 11M of each diastereomer). The reactions were allowed to proceed for 5 hrs, followed by heating to 95 oc for ten minutes to melt the DNA. The reaction mixtures were pooled and centrifuged using a Microcon YM-3 column to ex clude any reactants or products >3 kDa. The pH of all NMR samples was ad justed to 9.8 - 10.0 with Na2COs and trace divalent metal ions were removed with Chelex®-100. The resulting reaction mixture, 500 11-L with a net concentra tion of the two diastereomers of -0.7 mM was then loaded into an NMR tube following insertion of a coaxial insert containing D20 as an extemal reference. NMR spectra of compounds 15a/b before incorporation by pol jl were obtained in D20 with a net concentration of 10 mM. In an identical fashion, the reaction of (Rf S)-jl, y-CHCl dGTP ( 16a/b) was carried out except the reaction time was 48 extended to 8 hrs, and the reaction of (Rf S)-jl, y-CHCl dGTP (17a/b) was done in 12 hrs. Settings of NMR acquisition The chemical shifts are relative to external85% H 3 P0 4 (6 0.00, s1p NMR), or external hexafluorobenzene, C 6 F 6 (6 -164.9, 19F NMR).65 19F NMR ofjl,y-CHF dGTP analogue(15a/b) before incorporation by pol jl was acquired using Varian 400-MR default setting with 75188 Hz sweep width and -67773 Hz offset. s1p NMRs of jl,y-CHF dGTP (15a/b), jl,y-CHCl dGTP (16a/b) and jl,y-CHBr dGTP ( 17a/b) analogues before incorporation by pol jl were acquired using Varian 400-MR default setting except 4882 Hz sweep width, 109 Hz offset and 0.30 Hzfpoint digital resolution. 19F NMR of the assay sample was acquired using 4.600 vsec pulse width, 1.0 sec relaxation delay, 1.59 Hzfpoint digital resolu tion, 52038 Hz sweep width, -81878 Hz offset, 0.63 sec acquisition time and 26000 number of acquisition. s1p NMR of the assay samples were acquired using 4.650 vsec pulse width, 1.0 sec relaxation delay, 0.30 Hzfpoint digital resolution, 4882 Hz sweep width, 109 Hz offset, 3.36 sec acquisition time and 12000 number of acquisition. Relative Consumption Calculation The relative consumption was defmed as the ratio of the amounts of (R) jl,y-CHF dGTP (15a) vs. (S)-jl,y-CHF dGTP (15b) incorporated after 5 hrs of reaction. The starting (Rf S)-CHF was set to be 100 integral, 50 for each dia stereomer (Figure 1.6, pink). At t = 5 hrs, (fluoromethylene)bis(phosphonic acid) (3), the incorporation and hydrolysis product of 15a/b, was 31 integral; and the 49 unreacted (RI S)-CHF was 69 (Figure 1.6B, blue). s1p NMR (Appendix A., Figure A41) showed 1 integral of dGMP at t = 5 hrs, which was the product of 15a/b through hydrolysis assuming 0.5 from each diastereomer. Focusing on the unreacted region of 15a/b (Figure 1.6C), the two distal peaks each represents a different diastereomer. The ratio between the unreacted (Rj-CHF (15a) and the unreacted (S)-CHF (15b) was calculated by the ratio between the integrals of the two distal peaks, which was 26.8 I 42 = 0.638. Therefore, the amount of (Rj CHF (15a) left in the reaction was calculated as 69 x [0.638 I (1 + 0.638)] = 26.9, and the (S)-CHF (15b) remained was 69 x [1 I (1 + 0.638)] = 42.1. The amount of (Rj-CHF (15a) incorporated was then calculated as 50 - 0.5 - 26.9 = 22.6, and that of(S)-CHF (15b) was 7.4. The relative consumption was therefore calculated as R:S = 22.6 I 7.4 = 3.05 "' 3. The relative consumption for (RI S) jl,y-CHCl dGTP (16a/b) and (RIS)-jl,y-CHBr dGTP (17a/b) analogues was calculated similarly from the s1p NMR spectra. The amount of remaining tri phosphate analogue was calculated by averaging the integrals of P a, P!l and P v· The amount of the bis(phosphonic acid) formed in the reaction mixture was calculated by dividing the integral of the bis(phosphonic acid) by 2 due to the two equivalent P atoms in the molecule. 1.4. 3 Synthesis of 2 '-deoxyguanosine 5 '-triphosphate j3, y-CXY analogues Tetraisopropyl esters of a-halogenated methylenebis(phosphonate)s, 2- 8 The a-halogenated tetraisopropyl methylenebis(phosphonate)s (2 - 8) were prepared according to the literature procedures reported by McKenna et 50 a/.51-52 with modifications partly reported by Upton9.t and Chamberlain.26 The characterization data ('H, s1p, 19F NMR) are in agreement with the reported values (Table 1.1). 7. 9. 26. s1 Tetraisopropyl (difluoromethylene)bis(phosphonate), 2 and tetraisopropyl (fluoromethylene)bis(phosphonate), 3 NaH (1.16 gas a 60% oil immersion, 29.0 mmol) was added into a 250 mL 3-neck flask. Under N2, the flask was cooled to 0 'C and 140 mL of anhy- drous DMF was added. With vigorous stirring at 0 'C, tetraisopropyl meth- ylenebis(phosphonate) (TipMBP, 1, 10.10 g, 29.0 mmol) was added dropwise using a gas tight syringe and allowed to react for 20 min at 0 'C. The first portion of Selectfluor™ (6.68 g, 18.4 mmol) was added into the reaction mixture. After 10 min, the second portion of Selectfluor™ (6.68, 18.4 mmol) was added. The reaction mixture was first stirred under N2 at 0 'C for 30 min and then 1 hr at rt. Completion of the reaction was monitored by s1p NMR showing 10% of TipDFBP (2), 52% ofTipMFBP (3), and 38% ofunreacted 1. For a better yield of 3 after column chromatography, it is important to minimize the amount of 2, which is eluted slightly faster than 3. The reaction was quenched with 200 mL of saturated NH4Cl solution and then extracted with ice cold dichloromethane (DCM) (50mL x 4). The solvent of the combined organic extracts was removed under vacuum. The crude was again dissolved in 50 mL of DCM and washed with H20 (25 mL x 3) to remove the excess ofDMF. After removal ofDCM under t Typographical error reported by Upton in the synthesis of tetraisopropyl [chlo ro(fluoro)methylene[bis(phosphonate) is corrected. 51 vacuum, 10.20 g of the crude product containing 1, 2 and 3 was obtained. Purification of 4.00 g crude by silica gel column chromatography (10 - 50% EtOAcfDCM) afforded 1.91 g of 3 (40o/<j, 0.393 g of 2 (8o/<j and 0.643g of 3 with a trace amount of 2. TipDFBP (2): colorless oil; 1 H NMR (600 MHz, CDCls): 6 4.95-4.90 (m, 4H), 1.41- 1.38 (m, 24H); 19F NMR (564 MHz, CDCb): 6 -124.02 (t, JFP = 87.2 Hz); s1p NMR (243 MHz, CDCls): 6 2.65 (d, JPF = 87.4 Hz). Lit?: 'H: 6 4.93 (m, 40CH), 1.40 (d, J= 6Hz, 8CH 3 ); 19F (neat): 6-121 (t, JFP =85Hz); s1p NMR (neat): 6 2.8 (tt, JPF = 84 Hz, JPH = 3 Hz). TipMFBP (3): colorless oil; 1 H NMR (500 MHz, CDCb): 6 5.34 - 5.22 (m, 4H), 5.27 (dt, JHF = 45.8 Hz, JHP = 13.7 Hz, 1H), 1.80- 1.78 (m, 24H); 19F NMR (470 MHz, CDCb): 6 -228.48 (dt, JFP = 64.7 Hz, JFH = 45.4 Hz); 31 P NMR (202 MHz, CDCls): 6 10.07 (d, JPF = 64.9 Hz). Lit?: 1 H NMR (CDCb): 6 4.82 (dt, JHP =14Hz, JHF =44Hz, CFH), 4.77 (m, 40CH), 1.26 (d, J =6Hz, 8CHs); 19F NMR (neat): 6 -221 (dt, JFP =63Hz, JFH = 44Hz); s1p NMR (neat): 6 10.7 (ddt, JPF =63Hz, JPH = 12Hz, JPH =3Hz). Tetraisopropyl (dichloromethylene)bis(phosphonate), 4 TipMBP (1, 10.3 g, 30 mmol) was added dropwise to an ice chilled solu tion of 5.25% NaOCl (Clorox®, 332 g, 234 mmol) with vigorous stirring. The reaction mixture was then stirred at rt for 30 min. After completion, a white precipitate formed and the solution was extracted with hexane (50mL x 5). The combined organic extracts were dried over MgS04 and the solvent was removed under vacuum to yield 12.4 g of 4 (quant.). Colorless oil; 1 H (CDCb, 500 MHz): 6 4.95 (m, 4H), 1.406 (d, J= 6.2 Hz, 24H); s1p (CDCb, 202 MHz): 6 7.29 (s). Lit51: 52 'H NMR (270 MHz, CDCh): 6 1.38 (d, 3JHH = 6 Hz, 8CHs), 4.95 (m, 40CH); s1p NMR (109 MHz, CDCh): 6 7.3 (s). Tetraisopropyl (chloromethylene)bis(phosphonate), 5 An ice chilled 320 mL H20 solution of Na2SOs (14.02 g, 111.25 mmol) was added dropwise into the ice chilled solution of 4 (12.5 g, 30.3 mmol) in 80 mL EtOH. The reaction was stirred at 0 'C for 15 min and then 1 hr at rt. The reaction mixture was extracted with DCM (50 mL x 5). The combined organic extracts were dried with MgS04 and the solvent was removed under vacuum to yield 10.8 g of 5 (95o/<j. Colorless oil; 1 H NMR (CDCh, 500 MHz): 6 4.90 - 4.82 (m, 4H), 3.88 (t, JHP =17.6 Hz, 1H), 1.38- 1.36 (m, 24H); s1p NMR (CDCh, 202 MHz): 6 12.16 (s). Lit51: 'H NMR (270 MHz, CDCh): 6 1.35 (d, 3JHH = 6.5 Hz, 8CH 3 ), 3.85 (t, 2JHP = 17.5 Hz, CHCl); s1p (109 MHz, CDCh): 6 12.2 (s). Tetraisopropyl (dibromomethylene)bis(phosphonate), 6 To an ice chilled 200 mL H20 solution ofNaOH (10.8 g, 272 mmol), 21.7 g ofBr2 (13.6 mmol) was added slowly with vigorous stirring. TipMBP, 1, (10.0 g, 29.2 mmol) was then added dropwise. The reaction mixture was stirred at 0 'C for 10 min and then 5 min at rt. The milky yellow solution was extracted with DCM (50 mL x 4). The combined organic extracts were dried over MgS0 4 and the solvent was removed under vacuum to yield 14.8 g of 6 (quant.). Colorless oil; 'H NMR (500 MHz, CDCh): 6 5.00-4.94 (m, 4H), 1.43- 1.41 (m, 24H); s1p NMR (202 MHz, CDCh): 6 7.73 (s). Lit 51 : 1 H NMR (270 MHz, CDCh): 6 4.95 (m, 40CH), 1.39 (d, J =6Hz, 8CH 3 ); s1p NMR (109 MHz, CDCh): 6 7.5 (t, JPH =6Hz). 53 Tetraisopropyl (bromomethylene)bis(phosphonate), 7 A 40 mL H20 solution of 6.67 g SnCb·H20 (29.8 mmol) prepared and added dropwise into the ice chilled solution of 6 (14.80 g, 29.6 mmol) in 20 mL EtOH with vigorous stirring. The reaction mixture was stirred at 0 'C for 5 min and then 5 min at rt. The reaction mixture was extracted with DCM (25 mL x 4). The combined organic extracts were dried over MgS04 and the solvent was removed under vacuum to yield 12.31 g of 7 (98o/~. Colorless oil; 1 H NMR (500 MHz, CDCb): 6 4.88-4.77 (m, 4H), 3.74 (t, JHP = 17.2 Hz, lH), 1.37- 1.34 (m, 24H); s1p NMR (202 MHz, CDCb): 6 12.15 (s). Lit51: 'H NMR (270 MHz, CDCb): 6 4.85 (m, 40CH), 3.72 (t, JHP =17Hz, CHBr), 1.35 (d, J= 6.5 Hz, 8CH 3 ); s1p (109 MHz, CDCb): 6 12.3 (dt, JPH =17Hz, JPH =6Hz). Tetraisopropyl [chloro(fluoro)methylene]bis(phosphonate), 8 Compound 3 (1.91 g, 1.61 mmol) was added dropwise into an ice-chilled 80 mL 5.25% NaOCl solution (Clorox®, 72 g, 15.5 mmol) with vigorous stirring. The reaction was then stirred at rt for 90 min. After completion, a white precipi tate formed and the solution was extracted with DCM (50 mL x 4). The com bined organic extracts were dried over MgS04 and the solvent was removed under vacuum. Purification by silica gel column chromatography (10 - 30% EtOAcfDCM) afforded 1.90 g of 8 (91o/~. Colorless oil; 1 H NMR (500 MHz, CDCb): 6 4.99-4.91 (m, 4H), 1.42- 1.39 (m, 24H); s1p NMR (202 MHz, CDCh): 6 4.92 (d, JPF = 77.7 Hz); 19F NMR (470 MHz, CDCb): 6-147.01 (t, JFP = 77.0 Hz). Lit51: 'H (270 MHz, CDCh): 6 4.82 (m, 40CH), 1.38 (d, J = 6.5 Hz, 8CH 3 ); s1p 54 NMR (109 MHz, CDCb): 6 4.9 (ddt, JPF 77 Hz, JPH 6 Hz); 19F (254 MHz, CDCb): 6-150 (dt, JFP =77Hz). General procedure for syntheses of bisphosphonic acids, 9 - 12 The bisphosphonic acids (9 - 12) were prepared according to litera ture procedures. 7, 9, s1 Approximately 5 g of corresponding ester 3, 5 or 8 was dissolved in -40 mL 12 M HCl and refluxed for 4 hrs. In preparation of the acid 11 from ester 7, 40 mL 49% HBr was used instead. Completion of the reaction was monitored by 31p NMR. The solvent was then removed under vacuum and the remaining residue was dissolved in MeOH and dried under vacuum for several times to completely remove HCl or HBr. The yield was quantitative. For self-consistency, the NMR data of compounds 9 - 12 are reported for the free acids. (Fluoromethylene)bis(phosphonic acid), 9 Colorless oil; 1 H NMR (500 MHz, CDsOD): 6 3.98 (dt, JHF = 45.7 Hz, JHP = 13.3 Hz); 19 F NMR (470 MHz, CDsOD): 6 229.41 (dt, JFP = 65.6 Hz, JFH = 45.8 Hz); s1p NMR (202 MHz, CD 3 0D): 6 11.56 (d, JPF = 65.5 Hz). Lit?: 19F NMR: 6-225 (dt, JFH =46Hz, JFP =63Hz); s1p NMR: 6 10.5 (dt, JPF =64Hz). (Chloromethylene)bis(phosphonic acid), 10 Colorless crystals; 'H NMR (500 MHz, D20): 6 3.98 (t, JHP = 16.5 Hz); s1p NMR (202 MHz, D 2 0): 6 12.72 (s). Lit9: 'H NMR (D 2 0): 6 3.8 (dd); s1p NMR (D 2 0): 6 11.8(s). 55 (Bromomethylene)bis(phosphonic acid), 11 Colorless film; 'H NMR (500 MHz, D 2 0): 6 3.80 (t, JHP = 15.4 Hz); s1p NMR (202 MHz, D 2 0): 6 12.33 (s). Lit9: 'H NMR (D 2 0): 6 3.5 (dd); s1p NMR (D 2 0): 6 11.4 (s). [Chloro(fluoro)methylene]bis(phosphonic acid), 12 Colorless f:tlm; 19F NMR (564 MHz, D20): 6-144.83 (t, JFP = 75.0 Hz); s1p NMR (243 MHz, D 2 0): 6 6.52 (d, JPF = 75.1 Hz). Lit9: 19F (D 2 0): 6-145.4 (t); s1p (D20): 4.4 (d). 2'-Deoxy-5'-0.[hydroxy(morpholin-4-yl)phosphoryl]guanosine, 14 The procedure adapted from the original synthesis of nucleoside 5' phosphoromorpholidate.49-so 2'-deoxyguanosine 5'-monophos-phate mono Na• salt (13), 123 mg (0.303 mmol) was dissolved in 10 mL of t-BuOH:H20 (1:1) solution. The pH of the solution was adjusted to 2 using 1 M HCl. Distilled morpholine (80 vL, 81 mg, 0.930 mmol) was added to the solution and then the reaction mixture was heated till a steady reflux was reached. DCC (198 mg, 0.930 mmol) was dissolved in 2 mL of t-BuOH and 1/8 of this solution was added dropwise through the condenser to the reaction mixture every 15 min for 2 hrs. Following addition of DCC, the reaction was refluxed for another 2 hrs. Completion of the reaction was monitored by s1p NMR, which showed a single peak at -7 ppm. The solvent was then removed and the resulting crude mixture was resuspended in water. White precipitates were removed by filtration and water was removed under vacuum to give 14 as an amorphous yellow solid 56 which was used in coupling without further purification. Yield was 95% deter mined by s1p NMR. s1p NMR (202 MHz, D 2 0, pH -7): 6 7.40 (s). General procedure for syntheses of 2'-deoxyguanosine 5'-triphosphate }3, v CXY analogues, 15a/b - 18a/b The compounds 15a/b - 18a/b were synthesized according to literature procedures.9. 2o-2s Approximately 240 mg (4 equiv.) of the corresponding bisphosphonic acid 9- 12 was dissolved in 10 mL of EtOH:H20 (1: 1) and then 450 vL of BusN (1.5 equiv.) was added into the solution. After reacting for 15 min, the solvent was removed under vacuum and the remaining oil was dried by co-evaporation with anhydrous DMF for at least 3 times. Compound 14 (1 equiv., 125 mg, 0.303 mmol) was also dried by co-evaporation with anhydrous DMF for at least 3 times and then dissolved in 3 mL of anhydrous DMSO. The solution of compound 14 was then added into the flask containing the dried tri n-butyl ammonium salt of the corresponding bisphosphonic acid (9 - 12). After the reaction was stirred for 48 hrs, DMSO was removed on rotovap by co evaporation with EtOH and the reaction mixture was dissolved in 2 mL of 0.1 M TEAB buffer, pH 7.5. The crudes were then purified by dual preparative HPLC purification (SAX then reverse phase C,s) to yield the final compounds 15a/b - 18a/b (-30 o/<j. Analytical SAX HPLC analysis demonstrated that purity was;, 99%. For self-consistency, the chemical shifts are reported at adjusted pH ( -10). The diastereomer resonances of the F (15a/b and 18a/b), Pa and Pil nuclei are resolved for the individual isomers, and therefore the diastereomer resonances are assigned asP and P', or F and F'. The literature values ('Hand s1p NMR) are 57 incomplete and reported without specifYing the pH and the diastereomer reso nances for P a and Pil nuclei have not been reported. 9, 2o-21 2'-Deoxy-5'-0-[({[fluoro(phosphono)methyl](hydroxy)phosphoryl}oxy)(hydro xy)phosphoryl]guanosine, J3, y-CHF dGTP analogue, 15a/b Colorless film; 1 H NMR (400 MHz, D20, pH 9.8): 6 8.08 (s, 1H), 6.28 (t, J = 6.8 Hz, 1H), 4.97-4.91 (dt, JHF = 45.6 Hz, JHP = 12.4 Hz, 1H), 4.24-4.16 (m, 3H), 2.83- 2.76 (m, 1H), 2.53- 2.47 (m, lH); 19F NMR (376 MHz, D 2 0, pH 9.8): 6-218.42 (ddd, JFP = 65.1 Hz, JFP = 55.1 Hz, JFH = 45.6 Hz, F), -218.48 (ddd, JFP = 65.1 Hz, JFP = 55.1 Hz, JFH = 45.6 Hz, F); s1p NMR (162 MHz, D 2 0, pH 9.8): 6 7.92 (dd, JPF = 55.2 Hz, Jpp = 14.8 Hz, Pvand Py), 5.81 (ddd, JPF = 64.9 Hz, Jpp = 28.0 Hz, Jpp = 15.2 Hz, PJl), 5.78 (dd, JPF = 64.9 Hz, Jpp = 28.0 Hz, Jpp = 15.2 Hz, PJl), -9.97 (d, Jpp = 28.0 Hz, P;j, -9.99 (d, Jpp = 28.0 Hz, Pal Lit2o: 19F NMR (D 2 0, pH 10): 6-218.61 and -218.67 (ddd, JFP = 66.9 Hz, JFP = 55.9 Hz, JFH = 44.7 Hz). 5'-0-[({[Chloro(phosphono)methyl](hydroxy)phosphoryl}oxy)(hydroxy)phos phoryl]-2'-guanosine, }3, y-CHCl-dGTP analogue, 16a/b Colorless f:tlm; 1 H NMR (400 MHz, D20, pH 9.8): 6 8.05 (s, 1H), 6.31 (t, J = 7.2 Hz, 1H), 4.24-4.13 (m, 3H), 3.90 (t, J= 14.8 Hz, 1H), 2.77-2.70 (m, 1H), 2.53- 2.47 (m, 1H); s1p (162 MHz, D 2 0, pH 9.8): 6 9.45 (d, Jpp = 6.7 Hz, Pv and Py), 7.75 (dd, Jpp = 26.9 Hz, Jpp = 6.7 Hz, PJl), 7.71 (dd, Jpp = 26.9 Hz, Jpp = 6.7 Hz, PJl), -10.04 (d, Jpp = 27.1 Hz, P;j, -10.06 (d, Jpp = 27.1 Hz, Pal 58 5'-0-[({[Bromo(phosphono)methyl](hydroxy)phosphoryl}oxy)(hydroxy)phos phoryl]-2'-guanosine, }3, y-CHBr dGTP analogue, 17a/b Colorless f:tlm; 1 H NMR (400 MHz, D20, pH 9.9): 6 7.90 (s, 1H), 6.15 (dd, J = 7.8, 6.3 Hz, 1H), 4.10- 3.97 (m, 3H), 3.68 (dd, JHP = 16.5 Hz, JHP = 14.6 Hz, 1H), 2.67-2.60 (m, 1H), 2.37- 2.31 (m, 1H); s1p NMR (162 MHz, D20, pH 9.9): 6 8.83 (d, Jpp = 4.9 Hz, Pv and Py), 7.34 (dd, Jpp = 26.4 Hz, Jpp = 5.5 Hz, P!l), 7.30 (dd, Jpp = 26.4 Hz, Jpp = 5.5 Hz, PJl), -10.15 (d, Jpp = 26.4 Hz, Pa), -10.20 (d, Jpp = 26.4 Hz, Pal 5'-0-[({[Chloro(fluoro)phosphonomethyl](hydroxy)phosphoryl}oxy)(hydroxy) phosphoryl]-2'-guanosine, J3, y-CFCl dGTP analogue, 18a/b Colorless f:tlm; 1 H NMR (500 MHz, D20, pH 10.0): 6 8.06 (s, 1H), 6.31 (t, J = 6.8 Hz, 1H), 4.24-4.14 (m, 3H), 2.79- 2.74 (m, 1H), 2.52- 2.47 (m, 1H); 19F NMR (470 MHz, D20, pH 10.0): 6 -139.33 (dd, JFP = 78.5 Hz, JFP = 64.6 Hz, F), -139.37 (dd, JFP = 78.5 Hz, JFP = 64.6 Hz, F); s1p NMR (202 MHz, D20, pH 10.0): 6 6.69 (dd, JPF = 64.4 Hz, Jpp = 32.9 Hz, Py and Py), 0.42 (ddd, JPF = 78.3 Hz, Jpp = 32.9 Hz, Jpp = 31.2 Hz, PJl), 0.40 (dd, JPF = 78.3 Hz, Jpp = 32.9 Hz, Jpp = 31.2 Hz, PJl), -9.97 (d, Jpp = 31.1 Hz, Pa), -10.05 (d, Jpp = 31.1 Hz, Pal Lit2o: 19F NMR (D20, pH 10): 6-136.49 and -136.51. 59 1.5 References 1. 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H., Studies of gapped DNA substrate binding by mammalian DNA polymerase fJ. Dependence on 5'-phosphate group. J Bioi. Chem 1994, 269 (27), 18096-18101. 61. Singhal, R. K.; Wilson, S. H., Short gap-filling synthesis by DNA polymerase /]is processive. J Bioi. Chem 1993, 268 (21), 15906-15911. 62. Bertram, J. G.; Oertell, K.; Petruska, J.; Goodman, M. F., DNA polymerase fidelity: comparing direct competition of right and wrong dNTP substrates with steady state and pre-steady state kinetics. Biochemistry 2010, 49 (1), 20-28. 63. Fersht, A. R., Enzyme structure and mechanism. 2nd ed.; W.H. Freeman & Co.: New York, 1985. 64. Gottlieb, H. E.; Kotlyar, V.; Nudelman, A., NMR chemical shifts of common laboratory solvents as trace impurities. J Org. Chem 1997, 62 (21), 7512-7515. 65. Ravikumar, I.; Saha, S.; Ghosh, P., Dual-host approach for liquid-liquid extraction of potassium fluoride/chloride via formation of an integrated 1-D polymeric complex. Chem Commun. 2011, 47(16), 4721-4723. 66. Beard, W. A.; Wilson, S. H., Purification and domain-mapping of mammalian DNA polymerase fJ. DNA Replication 1995, 262, 98-107. 65 Chapter 2 : (R)- or (S)-}3, y-CXY dNTP diastereomers: Synthesis, discrete 19 F and 31 P NMR signatures and absolute configura- tions of new stereochemical probes for DNA polymerases* 2.1 Introduction Nucleotide analogue stereoisomers resulting from replacing a nonbridg- ing Pa or Pil oxygen with B, S, or Se have been isolated by HPLC, or, in the case of ATPaS and ATPJlS, by selective enzymatic depletion.l-5 In contrast, separation of diastereomers where a bridging oxygen is replaced by a CXY group has remained a long-standing challenge. The individual diastereomers of a,j3- CMe(Ns) dATP were recently prepared via the corresponding dADP isomers, which could be separated by preparative reversed-phase C 18 HPLC.6 However, efforts to separate the a,j3-CH(N 3 ) stereo isomers were unsuccessful. 6 (R/ Sj-j3, y- CH(Ns) dGTP and (R/ Sj-j3, y-CMe(Ns) dGTP analogues also proved refractory to this method,6 suggesting that both the size of substituents and the distance of the CXY group from the chiral deoxyribose affect the separation by chromatog- raphy. It seemed apparent that preparation of the elusive individual .f3, y-CXY + Sections 2.1, 2.2.1, 2.2.2 and related experimental procedures are reproduced or adapted from Wu, Y., Zakharova, V. M., Kashemirov, B. A., Goodman, M. F., Batra, V. K., Wilson, S. H., and McKenna, C. E. Journal of AmBrican Chemical Society. 2012, 134 (21), 8734-8737. Copyright 2012 American Chemical Society. The X-ray crystallograph ic results described in this chapter were obtained in the laboratory of Dr. Sammuel Wilson at NlEHS (Research Triangle, NC) by Dr. Vinod Batra, whose permission to include their data is gratefully acknowledged. The monofluoro compounds ( lb - 14b l/b-2) were prepared by Dr. V. M. Zakharova in our laboratory. 66 stereoisomers, particularly the highly desirable monohalo derivatives, 7-lo re quired a new approach, ideally one achieving stereochemical separation at the bisphosphonate level. The main idea of the synthesis is to fix the chirality at the bridging car bon of the prochiral a-halo bisphosphonate prior to conjugation using a novel chiral auxiliary strategy. After separation, the intermediate is conjugated con ventionally with the targeted dNMP. As the incorporated nucleoside suffices to maintain the bisphosphonate stereochemistry, the chiral auxiliary can then be removed reductively under mild, nonracemizing conditions. Here the detailed syntheses of jl,y-CXY dNTP analogues (X = H, Cl; Y = F, Cl; N = G, A) (14a- 1/14a-2- 14d-l/14d-2) using (R)-(-)-methyl mandelate as the chiral auxiliary are described. The absolute configuration assignments of all six monohalo diastereomers, jl, y-CHX dGTP (X = F, Cl) (14a-l, 14-2, 14b-l, 14b-2) and jl, y CHCl dATP (14d-l, 14d-2), and the correlation of the absolute configurations with the analytically discrete 19F and s1p NMR signatures are provided. An initial attempt to use (R)-(+)-phenylethanol as the chiral auxiliary to develop the strategy of chiral synthon is discussed. An attempt to synthesize individual jl, y CHBr dGTP diastereomers which contain a bromine substituent that is sensi tive to reductive conditions is also discussed. 67 2.2 Results and discussion 2.2.1 Synthesis of the individual diastereomers of 14a- d Synthesis of the chiral bisphosphonate synthons 7a-lf7a-2, 7b-1/7b-2 and 7c-1/7c-2 is outlined in Scheme 2.1. The previously synthesized (Chapter 1) a-halo bisphosphonic acid la,ll lb1o, 1 2-13 or lcll was heated at reflux with trimethylorthoformate to afford the corresponding tetramethyl ester 2a, 14 - 15 2b or 2c.16 Monodemethylation using 1 equiv of Nai in acetone 17-18 afforded the racemic trimethyl esters 3a - c, which were converted to their acid forms on Dowex (H+) and then esterified with (R)-(-)-methyl mandelate by Mitsunobu coupling, giving esters 4a - c with inversion at the chiral center of the auxilia- ry. 19 These mixtures of diastereomers were subjected to selective silyldemeth- ylation with BTMS 20 - 21 in anhydrous CH3CN followed by methanolysis to afford the (S)-mandelyl bisphosphonates Sa-c as diastereomer pairs. Scheme 2.1 Synthesis of the chiral bisphosphonate synthons, 7. 0 0 II Y II Meo-,P...J....'\-oMe MaO [ OMa X II - o o £ II Y II ~ MaO-,P--.j...-F!,-o OMa MaO i OMJ O 2a-e la - 7a-l/7a-2: X= Cl, Y = B lb - 7b-1/7b-2: X= F , Y = B lc - 7c-l/7c-2: X= F , Y = Cl o o £ II Y II I\ Ho-,P--.j...-P,- 0 N 0 N :· · OH O \._/ ( 0 ) X 7a-1/7a-2 7b-1/7b-2 7c-1/7c-2 X= F, Cl; Y = H, Cl Separation by HPLC vi - o o £ II Y II Ho-P--.j...-P,-o OH HO i OH 0 Ba-1/&a-2 Bb-1/&b-2 Bc-1/Bc-2 v - X= Cl, Y = H Separation by HPLC X= F, Y = H No separation by HPLC 4a-e ! w o o £ II Y II Ho-,P-- 1-P,-o OMe HO I OH 0 5a-1/5a-2 5b-1/5b-2 5c-1/5c-2 X=F,CI;Y= H No Separation by HPLC I) CH(OCH3) 3 , reflux, 1-3 hnJ.II) 2a, 2b: Nal, acetone, rt; 2c: Nal, acetone, -20 °C. Ill) 1). DOWEX H+; 2). DIAD, PPh:~o dioxane (anh), N 2 , (R)-methylmandelata. lv) 1). BTMS, CH 3 CN (anh); 2). MeOH. v) N~C0 3 , H 2 0, pH 8. vi) DCC, morphollne, t-BuOH: HzO (1:1). 68 Attempts to separate the stereoisomers of 5 by RP-C,s HPLC were not successful. However, after facile (pH 8) hydrolysis of the carboxylate methyl ester (which proceeded without loss of stereochemistry at the benzyl carbon), the resulting mandelic acid bisphosphonate esters 6a-l/6a-2 could be separat ed by preparative RP-C,s HPLC under isocratic conditions. Unfortunately, the a fluoro bisphosphonate diastereomers 6b-l/6b-2 could not be resolved by this method, even on analytical scale. Separation of 6c-l/6c-2 was not attempted. To explore the effect of masking both the carboxylic and phosphonic acid groups on improving the chromatographic separability, the mixtures of dia stereomers of 6 were converted to the corresponding P,C-dimorpholinamides 7, which grawyingly made possible the facile preparative isolation of all six dia stereomers 7a-l, 7a-2, 7b-l, 7b-2, 7c-l and 7c-2 by preparative HPLC. Dimor pholidation of compounds 6a-l/a-2 was performed successfully with over 90% conversion under the conditions developed by Moffatt commonly used for morpholidation of nucleoside monophosphates, which used t-BuOH:H20 (1:1) as solvent.22-23 Using the same conditions, dimorpholidation of the fluorinated compounds 6b-l/b-2 or 6c-l/c-2 generated mixtures of dimorpholidates (7b- 1/b-2 or 7c-l/c-2) in no more than 50% yield and monomorpholidates (12b- 1/b-2 or 12c-l/c-2) which were masked only on the carboxylic group. The reaction could not be driven to completion either by addition of more reagents or by continued heating. The diastereomer mixtures of monomorpholidates however were not separable by HPLC chromatography. Using anhydrous t-BuOH instead of aqueous t-BuOH dramatically improved the yield of the target dimorpholidates and shortened the reaction time. The s1p NMR spectra of 69 7a before and after separation are shown in Figure 2.1. The relatively more downfield chemical shift for each diastereomer corresponds to the benzyl ester phosphonate P nucleus, based on 1 H- 31 P gradient heteronuclear multiple bond correlation (Appendix B, Figure B34 and B38) between the downfield phospho- rus peak and the benzyl proton. '(') ""': to I N "' §' I .... I .. I R< 0 r\ II (SJ 0 N-P P.-0 N) \___/ I 'v" I \__ HO ~: OH Cl 0 a 7a-1/a-2 'I.J 0 N <=! 0 .... I ..: a p ~ 0 r\ ~II a. ( J b 0 N-P~~-0 N) \___/ HO ~ OH \__ 7a-1 Cl o \......! 0 .... 0 0 .... .... a p ~ 0 r\ jill a. ( 1 0 N-r~~-0 N) c '---! HO OH \__ 7a-2 Cl o 12.5 12.0 11.5 PPM Figure 2.1 31 P NMR spectra (202 MHz, D2 0) of (a) a diastereomeric mixture of 7a-1/7a- 2 at pH 9.8, (b) diastereomer 7a-1 at pH 9.8, and (c) diastereomer 7a-2 at pH 10.0. The absolute configuration assignments are based on X-ray crystallographic analysis of the downstream nucleotide products in a ternary complex with DNA pol f3 and DNA (see Figure 2.2 and Appendix B, Figures B 125 - 127). 70 Formation of the target nucleotides was carried out by conjugation with a 5'-activated dNMP (Scheme 2.2). The isolated 7a - c stereoisomers were first exchanged on a Dowex H+ column, and the pH of the eluate was adjusted to 1 using 1 M HCl to complete the hydrolysis of the P-N bond, giving the "portar' monoesters 12a- c. Acid P-N hydrolysis completed in 30 min for compouds 7a and 7b, but 3 days for compounds 7c. Each of these diastereomers was coupled with dGMP-morpholidate (10)7- 10 or dAMP-morpholidate (11) [prepared by N,N dicyclohexylcarbodiimide coupling of dGMP (8) or dAMP (9) with morpholine]22- 23 by stirring in anhydrous DMSO for 3 days to afford the nucleoside triphos phate analogues 13a - c. These compounds were purified by strong anion exchange HPLC and obtained as the triethylammonium salts. Removal of the chiral morpholinamide auxiliary by hydrogenolysis over 10 wt% Pd/C in 0.1 M triethylammonium bicarbonatejMeOH (1: 1, pH 8) gave the deprotected individ ual diastereomers of jl, y-CHCl dGTP (14a-l/14a-2), jl, y-CHF dGTP ( 14b-l/14b- 2), jl, y-CFCl dGTP (14c-l/14c-2) and jl, y-CHCl dATP (14d-l/14d-2), which were then purified by RP-C,s HPLC and obtained as triethylammonium salts. 71 Scheme 2.2 Synthesis of the target diastereomeric nucleotides, 14. ar(SJ ~ «(SJ ~";;::, 0 0 0 0 b II Y II 1\ I II Y II 1\ HO-,P...J,..P,-o N 0 _____.. HO-,P...J....FI\-o N 0 N : OH \_/ HO : OH (SJ I \_/ ( ) x 0 x 0 7a-1 or7a-2 12a-1 or12a-2 ar(R) J o 7b-1 or7b-2 12b-1 or12b-2 6 0 o~lo 0 7c-1 or 7c-2 12c-1 or 12c-2 /' (SJ 0-~A~-0-~-0 I N I !') I I 0 l____lll_____ 0~ OH OH OH r--------- 1 -;;::, 13a-1 or 13a-2 OH J & 13b-1or13b-2 J 13c-1 or 13c-2 0 1\ 0 13d-1 or 13d-2 H0-~-0 ii 0 N-~-0 ! I 0 _____.,. \._/ I ~ ~ ~ OH 8, 10: B = G OH ar(R) J 8-9 9, 11: B • A 10-11 0~ Yo 0 Ho II '-./ II 0 II Q -p-"-p- -p- I (S} I I Q OH OH OH 7a-1, 7a-2, 12a-1, 12a-2 - 14a-1, 14a-2: X= r, Y = B, B = G 7b-1, 7b-2, 12b-1, 12b-2 - 14b-1, 14b-2: X= C1, Y = H, B = G 7c-1, 7c-2, 12c-1, 12c-2 - 14c-1, 14c-2: X= r, Y = Cl, B = G 13d-1, 13d-2 - 14d-1, 14d-2: X = Cl, Y = B, B = A OH 14a-1 or 14a-2 14b-1 or 14b-2 14c-1 or 14c-2 14d-1 or 14d-3 I} Morphollna, DCC, t-8uOH: H~ (1 :1 }, reflux, 4 hrs. II} 1 ). DOWEX W; 2). 1 M HCI. Ill) Bu 3 N, DMSO (an h). lv} 10% Pd/C, H 2 , 0.1 M TEAS: MeOH (1:1}, pH 8. 2.2.2 Absolute configurations of the stereoisomers and correlation with discrete 19F and 31p NMR signatures The 19 F NMR spectra of the mixed 14b-1/14b-2 and 14c-1/14c-2 dia- stereomer pairs as obtained by conventional synthesis were previously reported to display nonoverlapping peaks for the two diastereomers. 7, 1o The chemical shifts and correct coupling constants of the two diastereomers were derived by simulation7 but could not be assigned to a specific configuration at the }3, y- bridging carbon. The absolute configuration at the chiral CHF carbon of 14b-2 was found to be R by X-ray crystallographic analysis of its temary complex with DNA and pol j3 (Figure 2.2; PDB entry 4D09), allowing the assignment of the more upfield 19 F resonance to this diastereomer (Table 2.1). It was possible to 72 obtain the absolute configurations of the other three diastereomers ( 14b-1, 14a- 1, and 14a-2) similarly (Appendix B, Figures B125- B127; PDB entries 4 DOA, 4DOC, and 4008, respectively). The absolute configurations at the P-CXY-P' carbon of compounds 7a/b, 12a/b a nd 13a/b were derived from the absolu te configurations of compounds 14a/b. The absolute configurations at the ]3, y- bridging carbon of 13d-1/d-2 and 14d-1/d-2 were derived from the corre- sponding chiral synthons 7a-1 and 7a-2. The X-ray crystallographic analysis of the ternary complex of 14c-1 or 14c-2 with DNA and pol j3 has not yet per- formed, and thus the absolute configurations at the ]3, y-CFCl bridging carbon remain undefined. Figure 2.2 Detailed view of the incoming nucleotide (R) -.,.B,y-CHF dGTP (12b-2) in the active site of the X-ray crystal structure of its ternary complex with pol .,.B and DNA (PDB entry 4 D09). The Arg183 , Asp 190, and Asp 192 side chains in the enzyme active site are shown, along with the nucleotide-binding magnesium and a water molecule. The interatomic distance between the F atom and N r(2 of Arg183 is 3. 11 A. 73 The ability to detect discrete s1p resonances for non-F containing ana- logues such as 14a-l/14a-2 would render them more generally useful as stereoprobes. At pH -10 and after removal of traces of paramagnetic metal ions by passage through Chelex-100, the diastereomeric Pa and Pil resonances of 14a-l/14a-2, 14b-l/14b-2, 14c-l/14c-2 and 14d-l/14d-2 proved to be observable at different operating frequencies (0.30 Hzfpoint digital resolution). As shown in Figure 2.3, a 2:1 mixture of 14a-l/14a-2 exhibits a s1p /'1.6 of 5.4 Hz for P a at 202 MHz, leading to the assignment of the more downfield signal to the (S)-isomer, 14a-l. The P.il resonances (Appendix B, Figure B93), which are separated by 8.5 Hz under the same conditions, show the reverse relationship (Table 2.1). Table 2.1 Relative 31 P and 19 F NMR chemical shifts and absolute configurations of }3,y CXY dGTP diastereomer pairs in D20a Compounds s1p NMR 31PNMR 19FNMR A.C.a Pa pjl CHX 14a-l (dG-CHCl) Db Uc N/A s 14a-2 (dG-CHCl) u D N/A R 14b-l (dG-CHF) D u D s 14b-2 (dG-CHF) u D u R 14d-l (dA-CHCl) D u N/A s 14d-2 (dA-CHCl) u D N/A R "A. C. =absolute configuration; bD = downfield; cU = upfield. 14a-1/14a-2: Pa 1'16 = 5.4 Hz, P.ll 1'16 = 8.5 Hz (202 MHz, pH 10.2); 14b-l/14b-2: Pa 1'16 = 2.4 Hz, P.ll 1'16 = 5.3 Hz (162 MHz, pH 10.5); 19 F 1'16 = 22.6 Hz (376 MHz, pH 10.5). 1'16 values are measured from the NMRs of the artificial mixtures. 74 N:Co ~ I NH o Clo o N NANH2 HO, 11.).._ 11 ,.o, 11 ,.o'd ~ ~ ~ 0 OH OH OH 14a-1/a-2 OH (87% of 14a-1 and 33% of 14a-2) -10.0 -10.5 -11.0 PPM Figure 2.3 31 P NMR spectra (202 MHz, D 2 0) of Pain 14a: (a) Artificial mixture of 14a- 1/14a-2 at pH 10.2. 14a-1 was added in excess, demonstrating that the 14a-2 signal is more upfield (U in Table 2.1) by 1'16 = 5.4 Hz (0.027 ppm). (b) Diastereomer 14a-1 at pH 10.6. (c) Diastereomer 14a-2 at pH 10.3. 75 2.2.3 Attempts to use (R)-(+)-phenylethanol as the chiral auxiliary Scheme 2.3 Attempts to use (R)-(+)-1-phenylethanol as the chiral auxiliary. 0 0 0 - II II = MeO~,Py ~-o.-:'::-- MeO , OMrfSJ Cl 15 ~iii o~ 0 0 ~ II II = M.o~,Py~-o.-:=::-. 0 ,. 0 (If) + - (' - + Na Cl Na 21 0 0 0 - ii -----· II II = Ho~,P.........-~-o.-:=::-. HO ~ OH (SJ Cl 18alb No HPLC Separation 0 0 II II Meo-,PyP,-oH MaO , OMa Cl 17 0 0 II II Mao-,PyP,-oH MaO ; OH Cl 19 0 0 II II Ho-,P"v"' P,-oH MeO I OMa Cl 18 0 0 II II Ho-,P.........-P,-oH MaO ~ OH Cl 20 J :0 0 ~{rNH f o a o 0 ~{rNH, ! ~o-~-L~-0-~-01 n I z ~o-~-L~-0-~-01 n I ' : 1 >10 v : 1110 : c OH OM: OH OH -----• i c OH OH :H OH ' ---------------------------------------------- I) 1) DOWEX W; 2) DIAD, PPh 31 dioxane (anh), N 2 , (R}-(+)-1-phanylethanol.ll) 1) BTMS, CH 3 CN; 2) MaOH. iii) 2 aq. Nal, acetone. iv) Bu 3 N, DMSO (anh), dGMP-rnorpholidate. v) Nal, to reaction mixture of lv. (R)-(+)-1-phenylethanol was the first chiral auxiliary studied to estabilish the idea of building chiral bisphosphonate synthons that are separable by chromatography (Scheme 2.3). Mitsunobu coupling of the trimethyl ester 3a with (R)-(+)-1-phenylethanol afforded the mixed ester 15. Reaction with BTMS failed to afford the desired compound 16. Instead, the a-methylbenzyl ester was removed exclusively followed by partial demethylation to give a mixture of three 76 different partial methyl esters ( 17 - 20) (Appendix B, Figure B 122). The reac tion of silylation with BTMS completes in two steps, a nucleophilic substitution on the silicon of the trimethylsilyl group with the phosphoryl oxygen replacing bromide followed by nucleophilic attack on the alkoxy carbon by the bromide generated in the first step.24 Competitive experiments with BTMS indicated that the relative reactivities of dimethyl, diethyl and diisopropyl phosphonates are ca. 1/0.25/0.04,21 supporting an SN2 mechanism during the step of nucleophilic attack by bromide. Vepsiiliiinen et al. reported the syntheses of a series of bisphosphonate partial esters utilizing different dealkylation methods.25-27 One effective approach is the regioselective silylation of bisphosphonate mixed tetraesters based on the relative reactivity of the esters: Me > 1 '-alkyl > 2' -alkyl > Ph,21, 2s which is typical for an SN2 mechanism. Only a few and contradictory examples were found in the literature comparing the relative reactivity of methyl and benzyl esters in hydrolysis.2B-29 The a-methylbenzyl ester used in our approach was much more reactive during silylation with BTMS presumably due to the formation of a stable carbocation which is significantly stabilized by resonance structures, the electron-donating methyl substituent on the carbo cation and hyperconjugation, and therefore undergoes SN 1 instead of SN2 reaction. By replacing the electron-donating methyl group with an electron withdrawing carboxyl ester group [(Rj-(-)-methyl mandelate], SN1 reaction is disfavored due to destablization of the carbocation, and hence selective demeth ylation by BTMS is successfully achieved (Scheme 2.1). 77 Altematively, reaction of compound 15 with Nal selectively removed one methyl group from each phosphonate to afford compound 21 as a mixture of two diastereomers, which unfortunately could not be separated by RP-C1s HPLC. Conjugation of compound 21 with dGMP morpholidate (10) afforded compound 22 (confirmed by mass spectrometry) with a-methylbenzyl ester on Py and methyl ester on P~ theoretically. Demethylation by adding Nai directly to the reaction mixture of22 did not provide the desired product 23. 2.2.4 Attempts to synthesize the (R)- and (S)-)3,y-CHBr dGTP diastereomers Scheme 2.4 Retrosynthetic analysis of }3,y-CHBr-dGTP diastereomers, 24a/b. N:Co ;.' I NH o Br o o ~N ).._ Ho II I II O 11 O'd N NH2 -p-'--p- -p- OH OH OH 0 N:Co '/ I NH o Br o o ~N ).._ H ll i ll II 'd N NH2 Chiral Auxiliary-N-p-'--p-0-p-0 OH OH OH 0 2411/b OH 258/b OH 0 0 0 0 ~ II II Ho-,Py f\-oH HO OH Br II II HO-P P.-OH ' Y ' HO HN-Chiral Auxiliary Br 27 2811/b The j3,y-CHBr dGTP analogue (24a/b) contains a reactive bromo func- tional group that can be easily reduced by hydrogenolysis. A chiral auxiliary attached via an acid labile phosphoramide linkage was therefore studied as the altemative method for the synthesis of (R)- and (S)-)3, y-CHBr dGTP diastere- omers (Scheme 2.4). McKenna et al. previously reported a P-N conjugate with 78 C-ethyl protected amino acids and cleavage of the phosphoramide bond under mild acidic conditions.so Scheme 2.5 Attempt to synthesize the chiral synthon of (bromomethylene)bis(phos phonic acid). 27 ! ;; - -----------~~------------- _, ~ .. \~ Q~ ~------------------ , ' ' 0 0 : 0 0 : II II Ill : II II _ +' MeO-,P--...,.....ft..-oMe ------• :Mea-,P-....,.....P,-o Na: MaO I OMe ' MaO ~ OMa : Br : Br : lv ------· ' 29 ' 30 ~------------------- 0 0 0 0 II II Meo-,P........_,.. P,-oMa MaO OMe II II + Mao-,P........_,..Ft._-6 Na MaO OMa 31 32 ' 0 0 0 (S) \ II II CH, MeO-,P-....,.....P.,-NH 0 MaO $ OMa Br 33 i) 1) DCC, t-BuOH:H 2 0 (1:1), (L)·phanylalanina ethyl aster hydrochloride, reflux; or 2) EDC, (L)· phenylalanine ethyle ester hydrochloride, H 2 0. ii) trimethylorthoformate, reflux. iii) 1) Nal, acetone; or 2) ElaN, acetonitrile; or 3) OlEA, acetonitrile; or 4) Bu 3 N, acetonitrile; or 5) NaOH, MeOH. iv) PPh 3 , (L)-phenylalanine ethyl ester hydrochloride, DIAD, dioxane (anh). There are at least two ways the phosphoramide conjugate of (bromo- methylene)bis(phosphonic acid) (27) with C-ethyl-protected (L)-phenylalanine (28) could be synthesized (Scheme 2.5). Direct coupling of 27 with (L)- phenylalanine ethyl ester hydrochloride in 1:1 ratio was attempted using aque- ous DCC coupling and aqueous EDC couplingso but no desired product (28) was obtained. The altemative approach was to prepare the (bromometh- ylene)bis(phosphonate) partial ester (30) via monodemethylation of tetramethyl 79 (bromomethylene)bis(phosphonate) (29). The partial ester (30) was supposed to react with oxalyl chloride to afford the phosphoryl chloride which then reacted with (L)-phenylalanine ethyl ester hydrochloride to afford compound 33. Re moval of the methyl esters by BTMS could afford the desired phosphoramide compound (28).31 The tetramethyl ester (29) was obtained by methylation of (bromomethylene)bis(phosphonic acid) (27) using trimethylorthoformate under reflux. Monodemethylation of 29 was attempted using different reagents, including Nal, NaOH, triethylamine, tri-n-butylamine, and N,N- diisopropylethylamine. With all reagents that were used, debromination was the major reaction that occurred. Reaction with triethylamine provided the best result which, however, only afforded a 20% yield of compound 30 (Appendix B, Figure B124). More than 50% of the starting material was debrominated to tetramethyl methylenebis(phosphonate) (31) of which demethylation was con tinued to afford compound 32. Due to the low yield and difficulty in product purification, this approach was not pursued further. 2.3. Conclusion In conclusion, the first examples of individual .f3, y-CXY dNTP diastere omers 14a - d have been successfully prepared using an approach based on constructing a "portar' chiral bisphosphonate synthon. The absolute configura tions of diastereomers 14a-l/a-2, 14b-l/b-2 and 14d-l/d-2 have been corre lated with discrete features of their s1p and 19F NMR spectra. The synthetic strategy developed should be adaptable to the synthesis of some cognate nude- 80 otide bisphosphonate diastereomers. The availability of the individual diastere omers of the dGTP analogues 14a - c now made it possible to perform inde pendent transient-state kinetic analyses to explore stereospecificity for correct incorporation opposite C and misincorporation opposite T.32 2.4 Experimental 2.4.1 Materials and methods 2'-Deoxyguanosine 5'-monophosphate monosodium salt monohydrate (8), 2'-deoxyadenosine 5'-monophosphate (9), (Rj-(-)-methyl mandelate (ee: 97o/<j and (Rj-(+)-phenylethanol (ee: 97o/<j were purchased from Sigma Aldrich, Inc. Synthesis of the a-halogenated methylenebis(phosphonic acid)s (1a - c, and 27)11-13 and 2'-deoxyguanosine 5'-monophosphate morpholidate (dGMP morpholidate) (10)22-23 were described in Chapter 1. Compounds 2- 7, 11- 14, 15, and 20 - 21 were synthesized as described below. All other reagents were purchased from commercial sources and used as obtained, unless specified otherwise. 'H, 19F and 31p NMR spectra were obtained on Varian 400-MR, VNMRS-500 and Bruker AMX-500 2-Channel and VNMRS-600 3-Channel NMR spectrometers. Multiplicities are quoted as singlet (s), doublet (d), triplet (t), unresolved multiplet (m), doublet of doublets (dd), doublet of doublet of dou blets (ddd), doublet of triplets (dt) or broad signal (br). All chemical shifts (6) are in parts per million (ppm) relative to residual CH30H in CD30D (6 3.34, 1 H NMR), CHCh in CDCh (6 7.26, 1 H NMR), HDO in D20 (6 4.79, 1 H NMR), 33 inter nal PPhsO (6 28, 31p NMR),34 external85%H 3 P0 4 (6 0.00, 31p NMR) or external 81 CFCh (6 0.00, 19F NMR). s1p NMR spectra were proton-decoupled, and 'H, 19F, and s1p coupling constants (J values) are given in Hz. The concentration of the NMR samples was in the range of 2 - 5 mgfmL. Preparative HPLC was per formed using a Varian ProStar equipped with a Shimadzu SPD-lOA UV detector (0.5 mm path length) with detection at the wavelength specified in Table B1. Mass spectrometry was performed on a Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI source in the negative ion mode. Compound IUPAC names were assigned using ACD/Labs, Release 12.00, Product Version 12.0 1. LC-MS was performed on Finnigan LCQ Deca XP Max mass spectrometer in negative mode with a Finnigan Survey or FDA 158 Plus detector (1 em path length) and MS Pump Plus, all controlled using Xcalibur software, version 2.0.7. Images of 'H NMR spectra (2 - 7, 12 - 14), 19F NMR (2b,c - 7b,c, 12b,c- 14b,c), 31p NMR spectra (2- 7, 11- 14, 15, 20-21, and the BTMS reaction mixture of 15), 'H-31P NMR gHMBC spectra (7a-1, 7a-2), HPLC traces (7, 13 - 14), LR mass spectra (7a-1, 7a-2) and LC-MS (7b) are pre sented in Appendix B. Crystallization of the pol J3 substrate complexes Binary complex crystals of human pol.f3 with dideoxy-terminated primer m a !-nucleotide gapped DNA were grown as previously described.1o The se quence of the template strand (16-mer) was 5'- CCG ACC GCG CAT CAG C- 3'. The primer strand (9-mer) sequence was 5'- GCT GAT GCG -3'. The downstream oligonucleotide (5-mer) was phosphorylated, and the sequence was 5'- GTC GG - 3'. The soaking of binary complex crystals with artificial mother liquor (50 mM 82 imidazole, pH 7.5, 20% PEG3350, 90 mM sodium acetate, 100 mM MgCb with 2.5 mM of 14a-l, 14a-2, 14b-l or 14b-2, and 12% ethylene glycol) resulted in temary complex crystals. Diffraction quality data were then collected for the temary complex crystal as described below. Data collection and structure determination Data were collected at 100 K on a CCD detector system mounted on a MiraMax®-007HF (Rigaku Corporation) rotating anode generator. Data were integrated and reduced with HKL2000 software.35 The temary complex struc ture was solved by molecular replacement using 2PXl'o as a reference model. The structure was refmed using PHENlX and manual model building using 0. The crystallographic statistics are reported in Appendix B, Table B2. Figure 2.2 in the Section 2.2.2 and Figures B125- B128 in Appendix B was prepared using Chimera. 36 2.4.2 Synthetic procedures Tetramethyl (chloromethanediyl)bis(phosphonate), 2a (Chloromethanediyl)bis(phosphonic acid) la, 1.00 g (4.75 mmol) was dis solved in 10 mL of trirnethyl orthoformate (91 mmol, 9.7 g). The reaction mix ture was set to reflux for 1 h. Warm water (65'C) was circulated in the conden ser to allow evaporation of the byproducts, MeOH and trimethylformate, in order to drive the reaction to completion. Excess trirnethyl orthoformate was 83 removed under vacuum to yield 1.048 g (83o/~ of compound 2a, which was obtained as a colorless oil. The compound was used for the next step without further purification. 1 H NMR (500 MHz; CDCh): 6 4.08 (t, JHP = 17.8 Hz, 1H), 3.91 (m, 12H); s1p NMR (202 MHz; CDCh): 6 16.24 (s). Lit'4-ls; 'H (CDCh): 4.0 (1H, t, J= 18Hz), 3.7 (12 H, m); s1p (CDCh): 15.5. Tetramethyl (fluoromethanediyl)bis(phosphonate), 2b Following the procedure for synthesis of 2a, 0.418 g (2.16 mmol) of (fluo romethanediyl)bis(phosphonic acid) lb was methylated to yield 0.535 g (>99o/~ of compound 2b. After removal of solvents a colorless oil was obtained. 1 H NMR (500 MHz; CDsOD): 6 5.67 (dt, JHF = 44.8 Hz, JHP = 14.3 Hz, 1H), 3.89 (br, 12H); 19F NMR (470 MHz; CD 3 0D): 6 -231.97 (dt, JFH = 44.1 Hz, JFP = 63.1 Hz); s1p NMR (202 MHz; CDsOD): 6 13.11 (d, JPF = 62.3 Hz). Tetramethyl [chloro(fluoro)methanediyl]bis(phosphonate), 2c Following the procedure for synthesis of 2a, 1.45 g (6.4 mmol) of chloro fluorobis(phosphonic acid) lc was methylated after reflux for 3 hrs to yield 1.73 g (95o/~ of compound 2c. After removal of solvents a brown oil was obtained. 1 H NMR (500 MHz; CDCh): 6 4.02- 3.98 (m, 12H); 19F NMR (470 MHz, CDCh): 6 -146.22 (t, JFP = 75.5 Hz); s1p NMR (202 MHz, CDCh): 6 8.423 (d, JPF = 75.9 Hz). Sodium methyl [chloro(dimethoxyphosphoryl)methyl]phosphonate, 3a Compound 2a, 2.36 g (8.85 mmol) was dissolved in 5 mL of acetone, fol lowed by addition of Nal (8.85 mmol, 1.33 g). After the Nal was completely dissolved, the reaction mixture was allowed to stand at rt ovemight. White crystals, the di-demethylated compound, slowly precipitated out of the solution 84 and were removed by filtration. The orgaruc phase was concentrated under vacuum. The residue was redissolved in 10 mL of H20 and unreacted tetrame thyl ester was extracted with CHCh (20 mL x 3). The aqueous phase was dried under vacuum to yield 1.176 g (49o/~ of compound 3a, which was obtained as a colorless, viscous oil. 1 H NMR (500 MHz; D20; pH 7.0): 6 4.40 (t, JHP = 16.6 Hz, 1H), 3.91- 3.88 (m, 6H), 3.69 (d, JHP = 10.8 Hz, 3H). s1p NMR (202 MHz; D 2 0; pH 7.0): 6 22.59 (d, Jpp = 3.9 Hz, 1P), 9.62 (d, Jpp = 3.9 Hz, 1P). Sodium methyl [(dimethoxyphosphoryl)(fluoro)methyl]phosphonate, 3b Following the procedure for synthesis of 3a, 0.53 g (2.11 mmol) of com pound 2b was monodemethylated to yield 0.277 g (51o/~ of compound 3b. After removal of solvent a colorless film was obtained. s1p NMR (202 MHz; CD 3 0D): 6 19.37 (dd, Jpp = 15.2 Hz, JPF = 66.1, 1P), 6.93 (dd, Jpp = 15.2 Hz, JPF = 54.7, 1P). Sodium methyl [chloro(dimethoxyphosphoryl)fluoromethyl]phosphonate, 3b Compound 2c, 1.33 g (4.67 mmol) was dissolved in 150 mL of acetone and chilled on ice. Following the addition of 0.9 equiv. of Nal (4.20 mmol, 0.23 g) that was dissolved in acetone, the reaction mixture was mixed well and allowed to react at 0 'C over 2 days. The white crystals were collected by filtra tion and washed with acetone. The crystals were resuspended in EtOH and the insoluble di-demethylated product was removed by filtration. The solvent was dried under vacuum to yield 1.12 g (82o/~ of compound 3c, which was obtained as a white powder. 1 H NMR (500 MHz, CDsOD): 6 3.99- 3.961 (m, 6H) 3.78 (d, JHP = 10.4 Hz, 3H); 19F NMR (470 MHz, CD 3 0D): 6 -143.84 (dd, JFP = 65.1 Hz, 85 JFP = 81.3 Hz); s1p NMR (202 MHz, CDsOD): 6 12.51 (dd, Jpp = 34.8 Hz, JPF = 81.4 Hz, 1P), 4.17 (dd, Jpp = 34.7 Hz, JPF = 65.2 Hz, 1P). Methyl (7S)-7-benzyl-4-chloro-3,5-dimethoxy-2,6-dioxa-3,5-diphosphaocta n-8-oate-3,5-dioxide, 4a Monosodium salt 3a, 365 mg (1.33 mmol) was dissolved in 1 mL of MeOH, loaded onto a column of strong cation exchange DOWEX resin in acidic form (5 mL) and then eluted from the column using MeOH. The eluate was concentrated under vacuum and dried by repeated co-evaporation with anhy drous dioxane until the total weight of the flask remained constant. The prod uct was then redissolved in 2 mL of anhydrous dioxane, followed by sequential addition ofPPhs (1.99 mmol, 523 mg) and (Rj-(-)-methyl mandelate (1.99 mmol, 332 mg). Distilled anhydrous diisopropylazodicarboxylate (DIAD) (1.99 mmol, 404 mg, 395 vL) was dissolved in 1 mL of anhydrous dioxane and added to the reaction mixture dropwise under N2. The reaction mixture was stirred under N2 at rt overnight. After reaction was complete (monitored by s1p NMR), volatiles were removed under vacuum. The crude product was purified by column chro matography on silica gel (10% MeOHfether) to yield 346 mg (65o/<j of 4a as a mixture of four diastereomers (31 P NMR). After removal of solvent a colorless oil was obtained. Altematively, impurities were removed by crystallization (15% hexanefdiethyl ether) and the organic phase was concentrated under vacuum to afford 4a. Mixture 4a purified by the second method contained some PPhsO impurity, but was used for the next step without further purification. s1p NMR (202 MHz; CDCh): 6 16- 15 (m). 86 Methyl (7 S)-7-benzyl-4-fluoro-3,5-dimethoxy-2,6-dioxa-3,5-diphosphaocta n-8-oate-3,5-dioxide, 4b Following the procedure for synthesis of4a, 270 mg (1.05 mmol) ofmon osodium salt 3b was alkylated by (Rj-(-)-methyl mandelate. Preparative TLC (50% hexane/ethyl acetate) purification gave 298 mg (74o/<j of the product 4b as a mixture of four diastereomers (31P NMR). After removal of solvent a colorless oil was obtained. s1p NMR (202 MHz; CD 3 0D): 6 14- 12 (m). Methyl (7 S)-4-chloro-4-fluoro-3,5-dimethoxy-7 -phenyl-2,6-dioxa-3,5-dipho sphaoctan-8-oate-3,5-dioxide, 4c Following the procedure for synthesis of4a, 977 mg (3.34 mmol) ofmon osodium salt 3c was alkylated by (Rj-(-)-methyl mandelate. The reaction was 50% complete by s1p NMR and stopped proceeding after PPhs was consumed. The solvent was removed under vacuum and the crude was re-dissolved in DCM. The unreacted mono acid of 3c was extracted with water at pH 8 adjust ed by Na2COs. The organic phase was dried under vacuum. The oily crude was first crystalized using 15% hexane/ether to remove some of the major by products. The organic phase was again dried under vacuum and the resulting crude was purified by silica gel column chromatography (2.5-15% ace tonefDCM) to afford 354 mg (25o/<j of product 4c as a mixture of four diastere omers (31P NMR). Colorless oil; 19F NMR (470 MHz, CDCh): 6-146.31- -147.26; s1p NMR (202 MHz, CDCh): 6 8.61- 6.72. (Chloro{hydroxy[( 1 S)-2-methoxy-2-oxo-1-phenylethoxy]phosphoryl}methyl) phosphonic acid, 5a-1/5a-2 87 Compound 4a (32 mg, 82 vmol) was dissolved in 5 mL of anhydrous ace tonitrile, followed by addition of 6 eq of freshly redistilled bromotrimethylsilane (BTMS) (494 vmol, 75 mg). The reaction mixture was stirred at rt for 1 h. Completion of the reaction was confirmed by mass spectrometry by monitoring the peak at 357 mfz. Excess BTMS was removed under vacuum. The residue was dissolved in 10 mL of MeOH and stirred at rt for 15 min. Volatiles were removed under vacuum producing a crude mixture of compounds Sa-1/Sa-2, which was obtained as a colorless film. The compound was not further charac terized and used in the next step without purification. (Flu oro{ hydroxy[( 1 S)-2-methoxy-2-oxo-1-phenylethoxy]phosphoryl}methyl) phosphonic acid, Sb-1/Sb-2 Following the procedure for synthesis of Sa-1/Sa-2 and monitored by s1p NMR, silyldemethylation of 100 mg (0.26 mmol) of 4b gave the mixture Sb- 1/Sb-2. After removal of solvent a colorless film was obtained. The compound was not further characterized and used in the next step without purification. [Chloro(fluoro){hydroxy[( 1 S)-2-methoxy-2-oxo-1-phenylethoxy]phosphoryl} methyl]phosphonic acid, Sc-1/Sc-2 Following the procedure for synthesis of Sa-1/Sa-2 and monitored by s1p NMR (2 hrs), silyldemethylation of 170 mg (0.41 mmol) of 4c gave the mixture Sc-1/Sc-2. After removal of solvent a colorless film was obtained. The com pound was not further characterized and used in the next step without purifica tion. 88 (2S)-({[(S)-chloro(phosphono)methyl](hydroxy)phosphoryl}oxy)(phenyl)etha noic acid, 6a-l The crude mixture of Sa-1/a-2 was dissolved in 10 mL of water and the pH was adjusted to 8 using Na2COs. The solution was washed with CHCh (30 mL x 3) to remove contaminating PPhsO. The aqueous layer was stirred over night, and then concentrated under vacuum to provide sodium salts 6a-l/6a-2 in 80% yield (by s1p NMR overall from 4a). Purification and separation of dia stereomers 6a-l/6a-2 was performed on HPLC using a Varian Microsorb C,s HPLC column (5 vm, 250 mm x 21.4 mm) with 3.5% CHsCN in 0.1 N tri ethylammonium bicarbonate (TEAB) buffer pH 7.2 at a flow rate of 15.0 mLfmin. The UV detector was operated at 256 nm. Diastereomer 6a-l eluted at 10.5 min and was obtained as a triethylammonium salt. After removal of sol vent a colorless film was obtained. 1 H NMR (500 MHz; D20; pH 10.3): 6 7.58 - 7.35 (m, 5H), 5.54 (d, JHP = 8.8 Hz, 1H), 3.68 (t, JHP = 15.8 Hz, 1H). s1p NMR (202 MHz; D20; pH 10.3): 6 15.54 (d, Jpp = 4.6 Hz, 1P), 9.76 (d, Jpp = 4.9 Hz, 1P). (2S)-({[(R)-chloro(phosphono)methyl](hydroxy)phosphoryl}oxy)(phenyl)etha- noic acid, 6a-2 Following the procedure for synthesis and isolation of 6a-l, HPLC sepa ration provided individual diastereomer 6a-2, which eluted at 11.5 min and was obtained as a triethyammonium salt. After removal of solvent a colorless film was obtained. 1 H NMR (500 MHz; D20; pH 9.8): 6 7.55- 7.36 (m, 5H), 5.56 (d, JHP = 9.0 Hz, 1H), 3.66 (t, JHP = 15.8 Hz, 1H). 31 P NMR (202 MHz; D20; pH 9.8): 614.03 (d, Jpp = 5.2 Hz, 1P), 9.71 (d, Jpp = 5.6 Hz, 1P). 89 (2S)-({[fluoro(phosphono)methyl](hydroxy)phosphoryl}oxy)(phenyl)ethanoic acid, 6b-1/6b-2 Following the procedure for synthesis of 6a-1, the methylrnandelate moi ety of the crude mixture Sb-1/Sb-2 was hydrolyzed to give diastereomers 6b- 1/6b-2 in 83% yield (by s1p NMR, overall from 4b). After removal of solvent a colorless film was obtained. HPLC separation of 6b-1/6b-2 was not successful using similar or modified conditions of separation for 6a-1/6a-2. s1p NMR (202 MHz; CDsOD): 6 13.48- 12.73 (m, 1P), 7.87 -7.43 (m, 1P). (2S)-({[chloro(fluoro)phosphonomethyl](hydroxy)phosphoryl}oxy)(phenyl)et hanoic acid, 6c-1/6c-2 Following the procedure for synthesis of 6a-1, the methylrnandelate moi ety of the crude mixture Sc-1/Sc-2 was hydrolyzed to give diastereomers 6c- 1/6c-2 in 90% yield (by s1p NMR, overall from 4c). After removal of solvent a colorless f:tlm was obtained. HPLC separation of 6c-1/6c-2 was not performed. 19F NMR (202 MHz, D 2 0, pH 10.2): 6 -139.27- -139.62; s1p NMR (202 MHz, D20, pH 10.2): 6 9.01 -7.12. [(R)-Chloro{hydroxy[( 1 S)-2-(morpholin-4-yl)-2-oxo-1-phenylethoxy]phospho ryl}methyl]morpholin-4-ylphosphinic acid, 7a-1 The mixture of diastereomers 6a-1/6a-2 (30.4 mg, 88.3 vmol) was dis solved in 10 mL of t-BuOH:H20 (1: 1), followed by addition of morpholine (706.4 vmol, 61.5 mg, 61 vL). The reaction mixture was f:trst stirred at rt for 15 min and then set to reflux. Dicyclohexylcarbodiimide (DCC) (1.79 mmol, 379 mg) was dissolved in 3 mL of t-BuOH and divided into 12 aliquots. Every 15 min, 90 one aliquot was added dropwise to the reaction mixture under reflux. After 3 h, DCC addition was complete, and reflux was continued for another 2 h. The reaction completion was confirmed by mass spectrometry by monitoring the peak at 481 mfz. After reaction was complete, the mixture was cooled tort and solvent was removed under vacuum. The residue was resuspended in 2 mL of water. Solids were removed by filtration and the aqueous layer was concentrat ed under vacuum to yield 33.5 mg (78o/~ by HPLC) of 7a-l/a-2 as a mixture of diastereomers. Purification and separation of 7a-l/7a-2 was performed on preparative HPLC using a Varian Microsorb C,s HPLC column (5 vm, 250 mm x 21.4 mm) with 15% CHsCN in 0.1 N triethylammonium bicarbonate (TEAB) buffer pH 7.4 at a flow rate of 8.0 mLfmin. The UV detector was operated at 256 nm. Diastereomer 7a-l eluted at 14.2 min and was obtained as a tri ethylammonium salt. After removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz; D20; pH 9.8): 6 7.55-7.46 (m, 5H), 6.20 (d, JHP = 8.7 Hz, 1H), 3.82- 3.76 (m, 5H), 3.63 (m, 8H), 3.16- 3.14 (m, 4H). s1p NMR (202 MHz; D 2 0; pH 9.8): 6 11.79- 11.63 (JPP = 5.3 Hz, 2P). MS [M-H]-: calcd for C17H24ClN20sP2-, 481. 1, found 481. 1. [(S)-Chloro{hydroxy[(lS)-2-(morpholin-4-yl)-2-oxo-1-phenylethoxy]phospho ryl}methyl]morpholin-4-ylphosphinic acid, 7a-2 Following the procedure for synthesis and separation of 7a-l, HPLC sep aration provided diastereomer 7a-2, which eluted at 15.2 min and was obtained as triethylammonium salt. After removal of solvent a colorless film was obtained. 1 H NMR (500 MHz; D20; pH 10.0): 6 7.56-7.45 (m, 5H), 6.16 (d, JHP = 8.6 Hz, 91 1H), 3.84- 3.78 (m, 5H), 3.65 - 3.63 (m, 8H), 3.16-3.13 (m, 4H). s1p NMR (202 MHz; D20; pH 10.0): 6 12.46 (d, Jpp = 4.5 Hz, 1P), 11.67 (d, Jpp = 4.6 Hz, 1P). MS [M-H]-: calcd for C17H24ClN20sP2-, 481.1, found 481.1. [(R)-Fluoro{hydroxyl[( 1 S)-2-(morpholin-4-yl)-2-oxo-1-phenylethoxyl]phosph oryl}methyl]morpholin-4-ylphosphinic acid, 7b-1 Following the procedure for synthesis and separation of 7a-1, 72.2 mg (0.22 mmol) of the diastereomer mixture 6b-1/6b-2 was reacted with morpho line to yield 89.2 mg (87o/~ of 7b-1/7b-2 as a diastereomer mixture. HPLC separation provided individual diastereomer 7b-1, which eluted at 14.3 min and was obtained as a triethylammonium salt. After removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz; D20; pH 10.3): 6 7.47 - 7.40 (m, 5H), 6.09 (d, JHP = 8.3 Hz, 1H), 4.64 (dt, JHP = 12.2 Hz, JHF =45.4 Hz, 1H), 3.73 (m, 4H), 3.60 (m, 8H), 3.02 (m, 4H). s1p NMR (202 MHz; D 2 0; pH 10.0): 6 9.77 (dd, Jpp = 12.7 Hz, JPF = 62.0 Hz, 1P), 9.62 (dd, Jpp = 12.7 Hz, JPF = 58.8 Hz 1P). 19F (470 MHz; CDsOD): 6 -218.46 (dt, JFH = 45.3 Hz, JFP = 59 Hz). LC-MS [M-H]-: calcd for C17H24FN20sP2-, 465.10, found 465.05. [(S)-Fluoro{hydroxyl[(1S)-2-(morpholin-4-yl)-2-oxo-1-phenylethoxyl]phosph oryl}methyl]morpholin-4-ylphosphinic acid, 7b-2 Following the procedure for synthesis and separation of 7a-1, HPLC sep aration provided individual diastereomer 7b-2, which eluted at 15.5 min and was obtained as a triethylammonium salt. After removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz; D20; pH 10.3): 6 7.46 - 7.39 (m, 5H), 6.08 (d, JHP = 8.8 Hz, 1H), 4.66 (dt, JHP = 12.2 Hz, JHF =42.6 Hz, 1H), 3.73 (m, 92 4H), 3.58 (m, 8H), 3.05 (m, 4H). s1p NMR (202 MHz; D20; pH 10.0): 6 10.22 (dd, Jpp = 12.7 Hz, JPF = 63.6 Hz, 1P), 9.70 (dd, Jpp = 12.7 Hz, JPF = 60.4 Hz 1P). 19F (470 MHz; CDsOD): 6 -218.26 (dt, JFH = 45.3 Hz, JFP = 61 Hz). LC-MS [M-H]-: calcd for C17H24FN20sP2-, 465.10, found 465.09. [Chloro(fluoro){hydroxy[( 1 S)-2-(morpholin-4-yl)-2-oxo-1-phenylethoxy]phos phoryl}methyl]morpholin-4-ylphosphonic acid, 7c-1 (HPLC P1) Following the procedure for synthesis of 7a-1, diastereomers 7c-1/7c-2 were synthesized from 6c. The reaction stopped at 50% completion of morphol idation on the phosphate group, and amidation on the carboxyilic group was completed. Yield could be improved to 100% by using only t-BuOH as the solvent. HPLC separation using Phenomenex C1s column eluted 15% CHsCN with in 0.1 N triethylammonium bicarbonate (TEAB) buffer pH 7.4 at a flow rate of 8.0 mLfmin provided the individual diastereomer 7c-1, which eluted at 20.6 min and was obtained as a triethylammonium salt. After removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz, D20, pH 10.0): 6 7.55-7.43 (m, 5H), 6.2 (d, JHP = 8.0 Hz, 1H), 3.64- 3.54 (m, 8 H). 19F NMR (470 MHz, D20, pH 10.0): 6 -139.10 (dd, JFP = 74.1 Hz, JFP = 68.3 Hz). s1p NMR (202 MHz, D20, pH 10.0): 6 7.32 (Jpp = 32.7 Hz, JFP = 68.2 Hz), 6.22 (Jpp = 32.7 Hz, JFP = 74.2 Hz). [Chloro(fluoro){hydroxy[( 1 S)-2-(morpholin-4-yl)-2-oxo-1-phenylethoxy]phos phoryl}methyl]morpholin-4-ylphosphonic acid, 7c-2 (HPLC P2) Diastereomer 7c-2 was synthesized in one pot with 7c-1. HPLC separa tion under the same system for 7c-1 provided individual diastereomer 7c-2, which eluted at 22.0 min and was obtained as a triethylammonium salt. After 93 removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz, D20, pH 10.0): 6 7.56 - 7.45 (m, 5H), 6.27 (d, JHP = 8.2 Hz, 1H), 3.67 - 3.60 (m, 8 H), 3.22- 3.19 (m, 4H). 19F NMR (470 MHz, D 2 0, pH 10.0): 6 -138.25 (dd, JFP = 72.9 Hz, JFP = 68.7 Hz). s1p NMR (202 MHz, D 2 0, pH 10.0): 6 7.36 (Jpp = 32.3 Hz, JFP = 67.9 Hz), 6.15 (JPP = 32.5 Hz, JFP = 74.4 Hz). [(R)-Chloro{hydroxy[( 1 S)-2-(morpholin-4-yl)-2-oxo-1-phenyl-ethoxy]phospho ryl}methyl]phosphonic acid, 12a-1 The triethylammonium salt of the individual diastereomer 7a-1 24 mg (50 vmol) was dissolved in 15 mL of water, followed by addition of strong cation exchange Dowex resin in acidic form (5 mL), and the mixture was stirred at rt for 30 min. The Dowex resin was removed by filtration. A couple of drops of 1 M HCl were added to the filtrate, and stirring continued for another 30 min to complete the hydrolysis of the phosphoramide. Completion of hydrolysis was confirmed by mass spectrometry by monitoring the peak at 412 mfz. The solvent was removed under vacuum to yield 20.6 mg (>99o/<j of compound 12a- 1. After removal of solvent a colorless film was obtained. The compound was not further characterized and used in the next step without purification. [(S)-Chloro{hydroxy[(1S)-2-(morpholin-4-yl)-2-oxo-1-phenyl-ethoxy]phospho ryl}methyl]phosphonic acid, 12a-2 Following the procedure for synthesis of 12a-1, 30.1 mg (62.5 vmol) of 7a-2 was hydrolyzed to yield 25.8 mg (>99o/<j of compound 12a-2. After removal of solvent a colorless f:tlm was obtained. The compound was not further charac terized and used in the next step without purification. 94 [(R)-Fluoro{hydroxy[( 1 S)-2-(morpholin-4-yl)-2-oxo-1-phenyl-ethoxy]phospho ryl}methyl]phosphonic acid, 12b-1 Following the procedure for synthesis of 12a-1, 30 mg (64.3vmol) of7b- 1 was hydrolyzed to yield 25.4 mg (>99o/~ of compound 12b-1. After removal of solvent a colorless f:tlm was obtained. The compound was not further character ized and used in the next step without purification. [( S)-Fluoro{hydroxy[( 1 S)-2-(morpholin-4-yl)-2-oxo-1-phenyl-ethoxy]phospho ryl}methyl]phosphonic acid, 12b-2 Following the procedure for synthesis of 12a-1, 35 mg (75.1 vmol) of 7b- 2 was hydrolyzed to yield 30 mg (>99o/~ of compound 12b-2. After removal of solvent a colorless f:tlm was obtained. The compound was not further character ized and used in the next step without purification. [Chloro(fluoro){hydroxy[( 1 S)-2-(morpholin-4-yl)-2-oxo-1-phenylethoxy ]phos phoryl}methyl]phosphonic acid, 12c-1 (HPLC P1) Following the same procedure for synthesis of 12a-1, 22 mg (44vmol) of 7c-1 was hydrolyzed to yield 17 mg (90o/~ of compound 12c-1. The P-N bond was completely hydrolyzed after 3 days at pH 2, and 10% of the product was further hydrolyzed on the amide moiety. After removal of solvent a colorless f:tlm was obtained. The compound was used in the next step without purification. 19F NMR (470 MHz, D20): 6 -142.78 (dd, JFP = 73.5 Hz). 95 [Chloro(fluoro){hydroxy[( 1 S)-2-(morpholin-4-yl)-2-oxo-1-phenylethoxy]phos phoryl}methyl]phosphonic acid, 12c-1 (HPLC P2) Following the same procedure for synthesis of 12a-1, 17 mg (34.5 vmol) of 7c-2 was hydrolyzed to yield 13.4 mg (90o/~ of compound 12c-2. The P-N bond was completely hydrolyzed after 3 days at pH 2, and 10% of the product was further hydrolyzed on the amide moiety. After removal of solvent, a color less film was obtained. The compound was used in the next step without purifi cation. 19F NMR (470 MHz, D 2 0): 6-142.94 (dd, JFP = 74.3 Hz). 5'-0.[ ({[( S)-Chloro{hydroxy[( 1 S)-2-(morpho lin -4-y 1)-2-oxo-1-pheny lethoxy ]ph osphoryl}methyl](hydroxy)phosphoryl}oxy)(hydroxy)phosphoryl]-2'-deoxy- guanosine, 13a-1 The individual diastereomer 12a-1 (25 mg, 60.5 vmol) was dissolved in 5 mL of EtOH. Tributylamine in EtOH (1: 10) was slowly added to the mixture to reach pH 4.5. After mixing for 30 min at rt, the solvent was removed under vacuum and dried by co-evaporation with anhydrous DMF (3 mL x 3). The compound was then mixed with 2 mL solution of 1.0 eq of dGMP-morpholidate (90.8 vmol, 37.7 mg) in anhydrous DMSO. Additional 0.5 eq of dGMP morpholidate (45.4vmol, 18.8 mg) was added to the reaction mixture after 24 h. The reaction mixture was stirred under rt for another 48 h. Completion of reaction was confirmed by mass spectrometry by monitoring the peak at 7 41 mfz and by s1p NMR. Purification of 13a-1 was performed on a Macherey-Nagel Nucleogel SAX 1000-10 25 mm x 15 em preparative column, using a gradient (0-10 min, 55% 10-16 min, 55% 16-25 min, 100o/~ of0.5 N triethylammonium 96 bicarbonate (TEAB) buffer pH 7.4 at a flow rate of 9 mLfmin. Compound 13a-1 was eluted at 18.5 min to give 15.77 mg (35o/~ and obtained as a triethylammo nium salt with 2 - 5% impurity of diguanosine diphosphate (dGppdG). After removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz; D20; pH 9.8): 6 8.03 (s, 1H), 7.53- 7.41 (m, 5H), 6.30 (dd, J = 8.1 Hz, 6.3 Hz, 1H), 6.22 (d, JHP = 8.7 Hz, 1H), 4.74 (m, 1H), 4.23-4.04 (m, 4H), 3.73-3.56 (m, 8H), 2.75 -2.69 (m, 1H), 2.50-2.45 (m, 1H). s1p NMR (202 MHz; D 2 0; pH 9.8): 6 10.96 (d, Jpp = 8.0 Hz, PJ, 2.68 (dd, Jpp = 26.6 Hz, Jpp = 8.4 Hz, P!l), -10.39 (d, Jpp = 26.8 Hz, Pa). 5'-0-[ ({[(R)-Chloro{hydroxy[ ( 1 S)-2-(morp holin -4-y 1)-2-oxo-1-pheny lethoxy]ph osphoryl}methyl](hydroxy)phosphoryl}oxy)(hydroxy)phosphoryl]-2'-deoxy guanosine, 13a-2 Following the procedure of synthesis and purification for 13a-1, 20 mg (48.4 vmol) of compound 12a-2 was conjugated with dGMP-morpholidate (10) to yield 18.5 mg (50o/~ of compound 13a-2 obtained as a triethylammonium salt after HPLC purification, eluted at 18.8 min with 2 - 5% diguanosine diphos phate (dGppdG). After removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz; D20; pH 10.0): 6 8.05 (s, 1H), 7.54-7.41 (m, 5H), 6.31 (dd, J= 7.7 Hz, 6.8 Hz, 1H), 6.21 (d, JHP = 8.3 Hz 1H), 4.75 (m, 1H), 4.24- 4.06 (m, 4H), 3.64- 3.54 (m, 8H), 2.79 - 2.74 (m, lH), 2.51 - 2.46 (m, lH). s1p NMR (202 MHz; D20; pH 10.0): 6 11.38 (d, Jpp = 8.1 Hz, PJ, 2.76 (dd, Jpp = 26.7 Hz, Jpp = 8.5 Hz, PJl), -10.43 (d, Jpp = 27.2 Hz, Pa). 97 2'-Deoxy -5'-0-[( {[ ( S)-fluoro{hydroxy[( 1 S)-2-(morpholin -4-y 1)-2-oxo-1-p heny 1- ethoxy]phosphory1}methy1](hydroxy)phosphory1}oxy)(hydroxy)phosphory1] guanosine, 13b-1 Following the procedure of synthesis for 13a-1, 18.6 mg (47 vmol) of compound 12b-1 was conjugated with dGMP-morpholidate (10) to give com pound 13b-1. HPLC purification was performed using the same system as for 13a-1 except flow rate was at 8 mLfmin. Compound 13b-1 was eluted at 21.2 min to give 21.5 mg (63o/~ and obtained as a triethylammonium salt with 2- 5% diguanosine diphosphate (dGpp:lG). After removal of solvent a colorless film was obtained. 1 H NMR (500 MHz; CDsOD): 6 8.01 (s, 1H), 7.52 (d, J= 7.4 Hz, 2H), 7.38-7.27 (m, 3H), 6.23 (t, J= 6.8 Hz, 1H), 6.17 (d, J= 9.3 Hz, 1H), 5.03 (dt, JHP = 12.7 Hz, JHF= 46.9 Hz, 1H), 4.71 (m, 1H), 4.25 (m, 1H), 4.15 (m, 1H), 4.12 (m, 1H), 3.56- 3.08 (m, 8H), 2.81 (m, lH), 2.30 (ddd, J= 2.4, 5.4, 12.7 Hz, 1H). s1p NMR (202 MHz; CDsOD): 6 9.46 (dd, Jpp = 17.4 Hz, Jpp= 58.8 Hz, P,), 1.69 (ddd, Jpp = 17.5 Hz, Jpp = 25.5 Hz, JPF = 60.4 Hz, P!l), -10.00 (d, Jpp = 25.5 Hz, Pa). 19F NMR (470 MHz; CDsOD): 6 -220.37 (br, JFH = 47.7 Hz, JFP =60Hz). 2'-Deoxy -5'-0-[( {[ (R)-flu oro{hydroxy[( 1 S)-2-(morpholin -4-y 1)-2-oxo-1-p heny 1- ethoxy]phosphory1}methy1](hydroxy)phosphory1}oxy)(hydroxy)phosphory1] guanosine, 13b-2 Following the procedure of synthesis for 13a-1, 20.5 mg (52 vmol) of compound 12b-2 was conjugated with dGMP-morpholidate (10) to give com pound 13b-2. HPLC purification was performed using the same system as for 13a-1 except the flow rate was 8 mLfmin with 2 - 5% diguanosine diphosphate 98 (dGppdG). After removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz; CDsOD): 6 8.05 (s, 1H), 7.53 (d, J= 7.3 Hz, 2H), 7.38-7.31 (m, 3H), 6.25 (t, J= 6.9 Hz, 1H), 6.19 (d, J= 8.8 Hz, 1H), 5.07 (dt, JHP = 12.8 Hz, JHF = 47.0 Hz, lH), 4.77 (m, 1H), 4.26 (m, 1H), 4.18 (m, lH), 4.12 (m, 1H), 3.62-3.01 (m, 8H), 2.85 (m, 1H), 2.32 (ddd, J= 3.0, 6.4, 13.7 Hz, 1H). s1p NMR (202 MHz; CDsOD): 6 9.51 (dd, Jpp = 17.5 Hz, Jpp= 58.8 Hz, PJ, 1.44 (ddd, Jpp = 17.5 Hz, Jpp = 25.5 Hz, Jpp= 60.4 Hz, P!l), -10.00 (d, Jpp = 25.5 Hz, Pa). 19F NMR (470 MHz; CD 3 0D): 6 -220.00 (br). 5'-0-[( {[Chloro(flu oro ){hydroxy[ ( 1 S)-2-(morp holin -4-y 1)-2-oxo-1-pheny letho xy]phosphoryl}methyl](hydroxy)phosphoryl}oxy)(hydroxy)phosphoryl]-2'-de oxyguanosine, 13c-1 (HPLC, P1) Following the procedure of synthesis for 13a-1, 17 mg (39vmol) of com pound 12c-1 was conjugated with dGMP-morpholidate (10) to give compound 13c-1. The reaction was completed in 7 days. HPLC purification was performed using the same system as for 13a-1 except the flow rate was 8 mLfmin with 2- 5% diguanosine diphosphate (dGppdG). After removal of solvent a colorless f:tlm was obtained. 1 H (500 MHz, D20, pH 10.0): 6 8.03 (s, 1H), 7.53 - 7.39 (m, 5H), 6.31 (t, J= 7.0 Hz, 1H), 6.25 (d, JHP = 7.7 Hz, 1H), 4.75-4.73 (m, 1H), 4.22 (m, lH), 4.18- 4.14 (m, lH), 4.11 - 4.07 (m, lH), 3.73- 3.51(m, 8H), 2.76 (m, lH), 2.49- 2.44 (m, 1H); 19F NMR (470 MHz, D 2 0, pH 10.0): 6 -141.46 (t, JFP = 73.2 Hz); s1p NMR (202 MHz, D20, pH 10.0): 6 5.34 (dd, JFP = 72.0 Hz, Jpp = 37.6 Hz, PJ, -3.31 (ddd, JFP = 74.2 Hz, Jpp = 37.7 Hz, Jpp = 30.1 Hz, PJl), -10.38 (d, Jpp = 30.2 Hz, Pa). 99 5'-0-[( {[Ch1oro(flu oro ){hydroxy[ ( 1 S)-2-(morp holin -4-y 1)-2-oxo-1-pheny 1etho xy]phosphory1}methy1](hydroxy)phosphory1}oxy)(hydroxy)phosphory1]-2'-de oxyguanosine, 13c-2 (HPLC, P2) Following the procedure for synthesis of 13a-1, 13.4 mg (31 vmol) of compound 12c-2 was conjugated with dGMP-morpholidate (10) to give com pound 13b-2. The reaction was completed in 7 days. HPLC purification was performed using the same system as for 13a-1 except the flow rate was 8 mLfmin with 2 - 5% diguanosine diphosphate (dGpp:lG). After removal of solvent a colorless f:tlm was obtained. 1 H (500 MHz, D20, pH 9.9): 6 8.04 (s, 1H), 7.52- 7.38 (m, 5H), 6.30 (t, J = 7.2 Hz, 1H), 6.26 (d, JHP = 7.7 Hz, 1H), 4.76- 4.74 (m, 1H), 4.23 (m, lH), 4.16 (m, 2H), 3.64-3.54 (m, 8H), 2.78-2.74 (m, 1H), 2.49- 2.44 (m, 1H); 19F NMR (202 MHz, D 2 0, pH 9.9):6 -138.97 (JFP =- 73 -74Hz); s1p NMR (202 MHz, D 2 0, pH 9.9): 6 5.18 (dd, JFP = 71.1 Hz, Jpp = 37.9 Hz, P,), -3.41 (ddd, JFP = 74.6 Hz, Jpp = 37.8 Hz, Jpp = 30.2 Hz, P!l), -10.39 (d, Jpp = 30.4 Hz, Pa). 5'-0-[( {[( S)-Ch1oro{hydroxy[( 1 S)-2-(morpholin -4-y 1)-2-oxo-1-p heny1ethoxy ]ph osphory1}methy1](hydroxy)phosphory1}oxy)(hydroxy)phosphory1]-2'-deoxy adenosine, 13d-1 Following the procedure of synthesis for 13a-1, 15 mg (36vmol) of com pound 12a-1 was conjugated with dGMP-morpholidate (10) to give compound 13d-1. HPLC purification was performed using the same system as for 13a-1 except the flow rate was 8 mL/ min. After removal of solvent a colorless f:tlm was obtained. 1 H (500 MHz, D20, pH 9.8): 6 8.48 (s, 1H), 8.25 (s, 1H), 7.52-7.36 (m, 100 5H), 6.50 (t, J= 6.9 Hz, 1H), 6.21 (d, JHP = 8.3 Hz, 1H), 4.28 (m, 1H), 4.16 (m, 2H), 4.07 (t, JHP = 16.7 Hz, 1H), 3.62-3.55 (m, 8H), 2.77 (m, 1H), 2.60-2.56 (m, 1H); s1p (202 MHz, D 2 0, pH 9.8): 6 10.76 (d, Jpp = 8.8 Hz, PJ , 2.43 (dd, Jpp = 8.9 Hz, Jpp = 26.9 Hz, Pil), -10.55 (d, Jpp = 26.9 Hz, Pa). 5'-0.[( {[(R)-Chloro{hydroxy[( 1 S)-2-(morpho lin -4-y 1)-2-oxo-1-pheny lethoxy ]ph osphoryl}methyl](hydroxy)phosphoryl}oxy)(hydroxy)phosphoryl]-2'-deoxy adenosine, 13d-2 Following the procedure of synthesis for 13a-1, 15 mg (36vmol) of com pound 12b-2 was conjugated with dGMP-morpholidate (10) to give compound 13b-2. HPLC purification was performed using the same system as for 13a-1 except the flow rate was 8 mL/ min. After removal of solvent a colorless f:tlm was obtained. 1 H (500 MHz, D20, pH 10.0): 6 8.48 (s, 1H), 8.25 (s, 1H), 7.52- 7.36 (m, 5H), 6.50 (t, J = 6.7 Hz, 1H), 6.20 (d, JHP = 8.4 Hz, 1H), 4.28 (m, 1H), 4.20 (m, 1H), 4.16 (m, 1H), 4.10 (t, JHP = 16.6 Hz, 1H), 3.61 (m, 8H), 2.61-2.56 (m, 1H); s1p (202 MHz, D 2 0, pH 10.0): 6 11.17 (d, Jpp = 8.3 Hz, PJ, 2.51 (dd, Jpp = 8.7 Hz, Jpp = 27.1 Hz, PJl), -10.57 (d, Jpp = 27.1 Hz, Pa). 5'-0-[({[(S)-Chloro(phosphono)methyl](hydroxy)phosphoryl}oxy)(hydroxy)ph osphoryl]-2'-deoxyguanosine, (S)-jl,y-CHCl-dGTP analogue, 14a-1 The triethylammonium salt of compound 13a-1, 5.7 mg (7.7 vmol, de termined by UV) was dissolved in 5 ml of 0.1 N TEAB:MeOH (1:1, pH 8), fol lowed by addition of 10 wt. %Pd/C (2.8 mg, 34 mol o/~ and a stir bar. The pH of the solution was re-adjusted to 8 by bubbling in C02. The reaction mixture was f:trst frozen (dry icefacetone), and then degassed by alternating application of 101 vacuum and fluslting N2 over 30 minutes. The system was then flushed with H2 gas several times. After the last fill of H2 gas, the solution was allowed to melt. The mixture was stirred under rt under H2 for 3 h. Completion of reaction was confirmed by mass spectrometry by monitoring the peak at 538 mfz and by s1p NMR. Purification was performed on a Varian Microsorb C,s HPLC column (5 vm, 250 mm x 21.4 mm) eluted isocratically with 3.5% CHsCN in 0.1 N tri ethylammonium bicarbonate (TEAB) buffer pH 7.4 at a flow rate of9.0 mLfmin (Table B1). Compound 14a-l was eluted at 14.4 min. After removal of solvent a colorless film was obtained, 2.6 mg (62o/~ as a triethylammonium salt. 1 H NMR (400 MHz; D20; pH 10.6): 6 8.05 (s, 1H), 6.32 (dd, J = 7.8, 6.5 Hz, 1H), 4.27- 4.12 (m, 3H), 3.90 (dd, JHP = 16.7 Hz, JHP = 15.4 Hz, 1H), 2.85- 2.75 (m, 1H), 2.53- 2.47 (m, 1H). s1p NMR (202 MHz; D20; pH 10.6): 6 9.24 (d, Jpp = 5.9 Hz, P,), 7.32 (dd, Jpp = 28.2 Hz, Jpp = 6.0 Hz, P!l), -10.05 (d, Jpp = 28.2 Hz, Pa). 5'-0-[({[(R)-Chloro(phosphono)methyl](hydroxy)phosphoryl}oxy)(hydroxy)ph osphoryl]-2'-deoxyguanosine, (R)-jl, y-CHCl-dGTP analogue, 14a-2 Following the procedure for synthesis and purification of 14a-l, 5.4 mg (7.3 vmol, determined by UV) of compound 13a-2 was deprotected by hydro genolysis. HPLC purification gave compound 14a-2 (eluted at 14.2 min) as a triethylammonium salt (2.48 mg, 63o/~. After removal of solvent a colorless film was obtained. 1 H NMR (600 MHz; D20; pH 10.3): 6 8.09 (s, 1H), 6.32 (dd, J = 8.0, 7.0 Hz, 1H), 4.26-4.15 (m, 3H), 3.90 (dd, JHP = 16.7 Hz, JHP = 15.5 Hz, 1H), 2.85-2.80 (m, 1H), 2.52 - 2.48 (m, 1H). s1p NMR (202 MHz; D20; pH 10.3): 6 9.21 (d, Jpp = 6.1 Hz, P,), 7.19 (dd, Jpp = 28.1 Hz, Jpp = 5.9 Hz, PJl), -10.08 (d, Jpp = 28.2 Hz, Pa). 102 2'-Deoxy-5'-0-[({[(S)-fluoro(phosphono)methyl](hydroxy)phosphoryl}oxy)(hy droxy)phosphoryl]guanosine, (S)-jl, y-CHF-dGTP analogue, 14b-l Following the procedure for synthesis of 14a-l, 13 mg (17.9 vmol, de termined by UV) of compound 13b-l was deprotected by hydrogenolysis to give compound 14b-l. Purification was performed using the same system as for 14a-l except the flow rate was 8 mLfmin. Compound 14b-l was eluted at 14.1 min to give 8.2 mg (88o/~ of the product as a triethylammonium salt. After removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz; D20; pH 10.3): 6 8.06 (s, lH), 6.32 (dd, J= 6.4, 7.8 Hz, lH), 4.83 (m, 1H), 4.77 (m, 1H), 4.25 (m, lH), 4.21-4.12 (m, 2H), 2.80 (m, lH), 2.49 (ddd, J= 3.5, 6.4, 14.2 Hz, 1H); 19 F NMR (470 MHz; D20; pH 10.3): 6 -216.24 (ddd, JFH = 45.3 Hz, JFP = 56.0 Hz, JFP = 65.6 Hz); s1p NMR (202 MHz; D20; pH 10.3): 6 6.80 (dd, Jpp = 14.3 Hz, JPF = 55.7 Hz, P,), 4.50 (ddd, Jpp = 14.3 Hz, Jpp = 28.6 Hz, JPF = 65.2 Hz, P!l), -11.07 (d, Jpp = 30.2 Hz, Pa). Lit?: 19F (D 2 0; pH 10): 6 -218.61 (calculated from 19F NMR of -1:1 synthetic mixture by NMR simulation). 2'-Deoxy-5'-0-[({[(R)-fluoro(phosphono)methyl](hydroxy)phosphoryl}oxy)(hy droxy)phosphoryl]guanosine, (R)-jl, y-CHF-dGTP analogue, 14b-2 Following the procedure for synthesis of 14a-l, 15.2 mg (20.9 vmol, de termined by UV) of compound 13b-l was deprotected by hydrogenolysis to give compound 14b-2. Purification was performed using the same system as for 14a-l except the flow rate was 8 mLfmin. Compound 14b-2 eluted at 14.5 min to give 10 mg (92o/~ of the product as a triethylammonium salt. After removal of solvent a colorless f:tlm was obtained. 1 H NMR (500 MHz; D20; pH 10.5): 6 8.09 (s, 1H), 6.31 (dd, J= 6.4, 7.8 Hz, 1H), 4.83 (dt, JHP = 12.7 Hz, JHF= 45.5 Hz, 1H), 103 4.80 (m, 1H), 4.24 (m, 1H), 4.21-4.13 (m, 2H), 2.83 (m, lH), 2.49 (ddd, J= 3.4, 6.4, 13.7 Hz, 1H); 19F NMR (470 MHz; D 2 0; pH 10.5): 6 -216.30 (ddd, JFH = 46.5 Hz, JFP = 56.1 Hz, JFP = 66.8 Hz); s1p NMR (202 MHz; D 2 0; pH 10.5): 6 6.85 (dd, Jpp = 14.3 Hz, Jpp= 55.6 Hz, PJ, 4.51 (ddd, Jpp = 14.3 Hz, Jpp = 30.2 Hz, Jpp= 65.2 Hz, P!l), -11.07 (d, Jpp = 30.2 Hz, Pa). Lit?: 19F (D20; pH 10) -218.67 (calcu lated from 19F NMR of -1:1 synthetic mixture by NMR simulation). 5'-0-[({[Chloro(fluoro)phosphonomethyl](hydroxy)phosphoryl}oxy)(hydroxy) phosphoryl]-2'-deoxyguanosine, }3, y-CFCl-dGTP analogue, 14c-l (HPLC Pl) Following the procedure for synthesis of 14a-l, 5.8 mg (7.6 vmol, deter mined by UV) of compound 13c-l was deprotected by hydrogenolysis using 10 wt. % Pd/C (2 mg, 25 mol o/~ to give compound 14c-l. The total reaction time was 7 hrs. Purification was performed on a Varian Microsorb C,s HPLC column (5 vm, 250 mm x 21.4 mm) eluted isocratically with 3.5% CHsCN in 0.1 N triethylammonium bicarbonate (TEAB) buffer pH 7.4 at a flow rate of 8.0 mLfmin. Compound 14c-l eluted at 10.6 min to give 3.10 mg (73o/~ of the product as a triethylammonium salt, containing 5% of the other diastereomer determined by 19F NMR. After removal of solvent a colorless film was obtained. 1 H NMR (600 MHz, D20, pH 9.8): 6 8.05 (s, 1H), 6.31 (dd, J = 6.4, 7.4 Hz), 4.25 (m, 2H), 4.18- 4.13 (m, 1H, 2.86 - 2.74 (m, 1H), 2.52- 2.46 (m, 1H); 19F NMR (470 MHz, D 2 0, pH 9.8): 6-138.92 (dd, JFP = 78.9 Hz, JFP = 65.5 Hz); s1p NMR (202 MHz, D20, pH 9.8): 6 6.45 (dd, JFP = 65.3 Hz, Jpp = 33.0 Hz, PJ, 0.015 (dt, JFP = 79.7 Hz, Jpp = 33.0 Hz, PJl), -10.14 (d, Jpp = 32.4 Hz, Pa). Lit?: 19F (D 2 0; pH 10): 6 -136.51 (calculated from 19F NMR of -1:1 synthetic mixture by NMR simulation). 104 5'-0-[({[Chloro(fluoro)phosphonomethyl](hydroxy)phosphoryl}oxy)(hydroxy) phosphoryl]-2'-deoxyguanosine, }3, y-CFCl-dGTP analogue, 14c-2 (HPLC P2) Following the procedure for synthesis of 14a-l, 7.98 mg (10.5 vmol, de termined by UV) of compound 13c-2 was deprotected by hydrogenolysis using 10 wt. % Pd/C (2.8 mg, 25 mol o/~ to give compound 14c-l. The total reaction time was 7 hrs. Purification was performed on a Phenomenex C,s HPLC column (5 vm, 250 mm x 21.4 mm) eluted isocratically with 3.5% CHsCN in 0.1 N triethylammonium bicarbonate (TEAB) buffer pH 7.4 at a flow rate of 8.0 mLfmin. Compound 14c-2 eluted at 20.5 min to give 4.28 mg (73o/~ of the product as a triethylammonium salt, containing 5% of the other diastereomer determined by 19F NMR. After removal of solvent a colorless film was obtained. 1 H NMR (500 MHz, D20, pH 9.8): 6 8.08 (s, 1H), 6.33 (t, J = 6.4, 7.4 Hz, 1H), 4.27 - 4.16 (m, 3H), 2.82 - 2.77 (m, 1H), 2.53 - 2.84 (m, 1H); 19F NMR (470 MHz, D20, pH 9.8): 6 -138.81 (dd, JFP = 79.0 Hz, JFP = 65.3 Hz); s1p NMR (202 MHz, D20, pH 9.8): 6 6.42 (dd, JFP = 65.4 Hz, Jpp = 33.1 Hz, P,), 0.046 (dt, JFP = 79.2 Hz, Jpp = 32.0 Hz, Pil), -10.05 (d, Jpp = 31.6 Hz, P;j. Lit?: 19F (D 2 0; pH 10): 6 -136.49 (calculated from 19F NMR of -1:1 synthetic mixture by NMR simula tion). 5'-0.[({[(S)-Chloro(phosphono)methyl](hydroxy)phosphoryl}oxy)(hydroxy)ph osphoryl]-2'-deoxyadenosine, (S)-}3, y-CHCl-dATP analogue, 14d-l Following the procedure for synthesis of 14a-l, 12.4 mg (17.1vmol, de termined by UV) of compound 13d-l was deprotected by hydrogenolysis to give compound 14d-l. Purification was performed on a Phenomenex C,s HPLC column (5 vm, 250 mm x 21.4 mm) eluted isocratically with 6% CHsCN in 0.1 N 105 triethylammonium bicarbonate (TEAB) buffer pH 7.4 at a flow rate of 8.0 mLfmin. Compound 14d-l eluted at 15.4 min to give 7.57 mg (85o/<j of the product as a triethylammonium salt. After removal of solvent a colorless film was obtained. 1 H NMR (600 MHz, D20, pH 9.7): 6 8.48 (s, 1H), 8.23 (s, 1H), 6.51 (t, J= 6.8 Hz, 1H), 4.31 (m, 1H), 4.24-4.15 (m, 2H), 3.91 (dd, JHP = 16.6 Hz, JHP = 15.3 Hz, 1H), 2.85-2.81 (m, 1H), 2.63-2.59 (m, 1H); s1p NMR (243 MHz, D20, pH 9.7) :6 9.12 (d, Jpp = 6.2 Hz, PJ, 7.28 (dd, Jpp = 6.4 Hz, Jpp = 27.2 Hz, P!l), -10.25 (d, Jpp = 27.2 Hz, Pa). 5'-0-[({[(R)-Chloro(phosphono)methyl](hydroxy)phosphoryl}oxy)(hydroxy)ph osphoryl]-2'-deoxyadenosine, (R)-jl, y-CHCl-dATP analogue, 14d-2 Following the procedure for synthesis of 14a-l, 9.8 mg (13.5 vmol, de termined by UV) of compound 13d-2 was deprotected by hydrogenolysis to give compound 14d-2. Purification was performed on a Phenomenex C,s HPLC column (5 vm, 250 mm x 21.4 mm) eluted isocratically with 5% CHsCN in 0.1 N triethylammonium bicarbonate (TEAB) buffer pH 7.4 at a flow rate of 8.0 mLfmin. Compound 14d-2 eluted at 21.0 min to give 5.63 mg (80o/<j of the product as a triethylammonium salt. After removal of solvent a colorless film was obtained. 1 H NMR (500 MHz, D20, pH 9.8): 6 8.49 (s, 1H), 8.23 (s, 1H), 6.51 (t, J = 6.8 Hz, 1H), 4.31 (m, 1H), 4.26- 4.14 (m, 2H), 3.90 (dd, JHP = 15.1, JHP = 16.5 Hz, 1H), 2.85 - 2.80 (m, 1H), 2.63 - 2.58 (m, 1H); s1p (202 MHz, D 2 0, pH 9.8): 6 9.00 (d, Jpp = 6.0 Hz, PJ, 7.05 (dd, Jpp = 6.0 Hz, Jpp = 27.4 Hz, P.il), -10.27 (d, Jpp = 27.3 Hz, Pa). 106 Trimethyl (1S)-1-phenylethyl (chloromethandiyl)bis(phosphonate), 15 Monosodium salt 3a, 458 mg (1.67 mmol) was dissolved in 1 mL of MeOH, loaded onto a column of strong cation exchange DOWEX resin in acidic form (5 mL) and then eluted from the column using MeOH. The eluate was concentrated under vacuum and dried by repeated co-evaporation with anhy drous dioxane until the total weight of the flask remained constant. The prod uct was then redissolved in 2 mL of anhydrous dioxane, followed by sequential addition of PPhs (5 mmol, 1.312 g) and (Rj-(+)-1-phenylethanol (5 mmol, 612 mg, 605 vL). Distilled anhydrous diisopropylazodicarboxylate (DIAD) (2.5 mmol, 508 mg, 495 vL) was dissolved in 1 mL of anhydrous dioxane and added to the reaction mixture dropwise under N2. The reaction mixture was stirred under N2 at rt overnight. After reaction was complete (monitored by s1p NMR), volatiles were removed under vacuum. The crude product was purified by column chro matography on silica gel (10% MeOHfether) to yield 346 mg (65o/~ of 15 as a mixture of four diastereomers (31 P NMR). After removal of solvent a colorless oil was obtained. s1p NMR (202 MHz; CDCls): 6 16- 15 (m). Tetramethyl (bromomethanediyl)bis(phosphonate), 29 (Bromomethanediyl)bis(phosphonic acid) 27, 1.00 g (3.92 mmol) was dis solved in 10 mL of trimethyl orthoformate (91 mmol, 9.7 g). The reaction mix ture was set to reflux for 1 h. Warm water (65'C) was circulated in the conden ser to allow evaporation of the byproducts, MeOH and trimethylformate, in order to drive the reaction to completion. Excess trimethyl orthoformate was removed under vacuum to yield 0.98 g (80o/~ of compound 2a, which was obtained as a colorless oil. The compound was used for the next step without 107 further purification. 1 H NMR (500 MHz; CDCb): 6 4.08 (t, JHP = 17.8 Hz, 1H), 3.91 (m, 12H); s1p NMR (202 MHz; CDCb): 617.99 (s). 108 2.5 References 1. Kowalska, J.; Lewdorowicz, M.; Zuberek, J.; Grudzien-Nogalska, E.; Bojarska, E.; Stepinski, J.; Rhoads, R. E.; Darzynkiewicz, E.; Davis, R. E.; Jemielity, J., Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eiF4E and are resistant to the decapping pyrophosphatase DcpS. RNA-Pub!. RNA Soc. 2008, 14 (6), 1119- 1131. 2. Kowalska, J.; Lukaszewicz, M.; Zuberek, J.; Darzynkiewicz, E.; Jemielity, J., Phosphoroselenoate dinucleotides for modification of mRNA 5' end. ChemBioChem2009, 10 (15), 2469-2473. 3. Kowalska, J.; Zuberek, J.; Darzynkiewicz, Z. M.; Lukaszewicz, M.; Darzyukiewicz, E.; Jemielity, J., Synthesis and properties of boranophosphate mRNA cap analogues. Collect. Symp. Ser. 2008, 10 (Chemistry of Nucleic Acid Components), 383-385. 4. Lin, J. L.; Porter, K. W.; Shaw, B. R., Synthesis and properties of novel triphosphate analogues: Ribonucleoside and, deoxyribonucleoside (a- P-borano, a-P-thio)triphosphates. Nucleosides Nucleotides Nucleic Acids 2001, 20 (4-7), 1019-1023. 5. Lin, J. L.; Shaw, B. R., Synthesis of a novel triphosphate analogue: Nucleoside a-P-borano, a-P-thiotriphosphate. Chem. Commun. 2000, (21), 2115-2116. 6. Chamberlain, B. T.; Upton, T. G.; Kashemirov, B. A.; McKenna, C. E., a Azido bisphosphonates: synthesis and nucleotide analogues. J Org. Chem. 2011, 76 (12), 5132-5136. 7. Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.; Goodman, M. F.; McKenna, C. E., Halogenated p,y-methylene and ethylidene-dGTP-DNA temary complexes with DNA polymerase p: Structural evidence for stereospecific binding of the fluoromethylene analogues. JAm Chem Soc. 2010, 132 (22), 7617-7625. 8. Blackburn, G. M.; Kent, D. E.; Kolkmann, F., The synthesis and metal binding characteristics of novel, isopolar phosphonate analogues of nucleotides. J Chem. Soc.-Perkin Trans. 1 1984, (5), 1119-1125. 9. McKenna, C. E.; Kashemirov, B. A.; Peterson, L. W.; Goodman, M. F., Modifications to the dNTP triphosphate moiety: From mechanistic probes for 109 DNA polyrnerases to antiviral and anti-cancer drug design. Biochim. Biophys. Acta2010, 1804 (5), 1223-1230. 10. McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H., (RJ-P,J"Fluoromethylene dGTP-DNA temary complex with DNA polymerase p. J Am Chem. Soc. 2007, 129 (50), 15412-15413. 11. McKenna, C. E.; Khawli, L.A.; Ahmad, W. Y.; Pham, P.; Bongartz, J.P., Synthesis of a-halogenated methanediphosphonates. Phosphorus Sulfur Silicon Relat. Elem 1988, 37(1-2), 1-12. 12. Marma, M. S.; Khawli, L. A.; Harutunian, V.; Kashemirov, B. A.; McKenna, C. E., Synthesis of a-fluorinated phosphonoacetate derivatives using electrophilic fluorine reagents: Perchloryl fluoride versus 1-chloromethyl-4- fluoro-1, 4-diazoniabicyclo[2. 2.2]octane bis(tetrafluoroborate) (Selectfluor®). J Fluor. Chem. 2005, 126(11-12), 1467-1475. 13. McKenna, C. E.; Sherr, P.-D., Fluorination of methanediphosphonate esters by perchloryl fluoride. Synthesis of fluoromethanediphosphonic acid and difluoromethanediphosphonic acid. J Org. Chem. 1981, 46 (Copyright (C) 2012 American Chemical Society (ACS). All Rights Reserved.), 4573-6. 14. Hutchinson, D. W., Correction. J Organomet. Chem 1987, 319 (2), C39- C39. 15. Hutchinson, D. W.; Semple, G., Relative reactivities oftetraalkyl esters of methylene bisphosphonic acid. J Organomet. Chem 1986, 309 (1-2), C7-C10. 16. Nicholson, D. A.; Cilley, W. A.; Quimby, 0. T., A convenient method of esterification ofpolyphosphonic acids. J Org. Chem. 1970, 35 (9), 3149-3150. 17. Goldstein, J. A.; McKenna, C.; Westheimer, F. H., a Diazobenzylphosphonate dianions JAm. Chem Soc. 1976, 98 (23), 7327-7332. 18. McKenna, C. E.; Kashemirov, B. A.; Roze, C. N., Carbonylbisphosphonate and (diazomethylene)bisphosphonate analognes of AZT 5'-diphosphate. Bioorganic Chem 2002, 30 (6), 383-395. 19. Campbell, D. A., The synthesis of phosphonate esters, an extension of the mitsunobu reaction. J Org. Chem 1992, 57 (23), 6331-6335. 20. McKenna, C. E.; Higa, M. T.; Cheung, N.H.; McKenna, M. C., The facile dealkylation of phosphonic acid dialkyl esters by bromotrimethylsilane. TetrahedronLett. 1977, (2), 155-158. 110 21. McKenna, C. E.; Schrnidhauser, J., Functional selectivity in phosphonate ester dealkylation with bromotrimethylsilane. J Chem. Soc. -Chem Commun. 1979, ( 17), 739-739. 22. Moffatt, J. G., General synthesis of nucleoside 5'-triphosphates. Can. J Chem 1964, 42 (3), 599-604. 23. Moffatt, J. G.; Khorana, H. G., Nucleoside polyphosphates. X. The synthesis and some reactions of nucleoside 5'-phosphoromorpholidates and related compounds. Improved methods for the preparation of nucleoside 5' polyphosphates. JAm Chem. Soc. 1961, 83 (3), 649-658. 24. Rabinowitz, R., The reactions of phosphonic acid esters with acid chlorides. A very mild hydrolytic route. J Org. Chem 1963, 28 (11), 2975-2978. 25. Vepsiilfunen, J.; Nupponen, H.; Pohjala, E., Bisphosphonic compounds V. Selective preparation of (dichloromethylene)bisphosphonate partial esters. Tetrahedron Lett. 1993, 34 (28), 4551-4554. 26. Vepsiiliiinen, J.; Nupponen, H.; Pohjala, E., Bisphosphonic compounds VIII. A facile and selective one-pot synthesis of P,P-dialkyl (dichloromethylene)bisphosphonate partial esters. Tetrahedron Lett. 1996, 37 (20), 3533-3536. 27. Vepsiiliiinen, J. J.; Kivikoski, J.; Ahlgren, M.; Nupponen, H. E.; Pohjala, E. K., An improved synthetic method and the first crystal structures for (dihalomethylene)bisphosphonate partial esters. Tetrahedron 1995, 51 (24), 6805-6818. 28. Ahlmark, M. J.; Vepsiiliiinen, J. J., Strategies for the selectivesynthesis of monosubstituted (dichloromethylene)bisphosphonate esters. Tetrahedron 1997, 53 (47), 16153-16160. 29. Saady, M.; Lebeau, L.; Mioskowski, C., Convenient "one-pof' synthesis of chlorophosphonates, chlorophosphates and chlorophosphoramides from the corresponding benzyl esters. Tetrahedron Lett. 1995, 36 (27), 4785-4786. 30. Marma, M. S.; Kashemirov, B. A.; McKenna, C. E., Synthesis and stability studies of phosphonoformate-amino acid conjugates: A new class of slowly releasing foscarnet prodrugs. Bioorg. Med. Chem Lett. 2004, 14 (7), 1787-1790. 31. Grison, C.; Coutrot, P.; Comoy, C.; Balas, L.; Joliez, S.; Lavecchia, G.; Oliger, P.; Penveme, B.; Serre, V.; Herve, G., Design, synthesis and activity of bisubstrate, transition-state analogues and competitive inhibitors of aspartate transcarbamylase. Eur. J Med. Chem. 2004, 39 (4), 333-344. 111 32. Oertell, K.; Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Shock, D. D.; Beard, W. A.; Wilson, S. H.; McKenna, C. E.; Goodman, M. F., Effect of p,y-CHF and p,)"'CHCl dGTP halogen atom stereochemistry on the transition state of DNA polymerase p. Biochemistry 2012, 51 (43), 8491-8501. 33. Gottlieb, H. E.; Kotlyar, V.; Nudelman, A., NMR chemical shifts of common laboratory solvents as trace impurities. J Org. Chem 1997, 62 (21), 7512-7515. 34. Quin, L., A guide to organophosphorus chemistry. Wiley-Interscience: 2000; p 426. 35. Otwinowski, Z.; Minor, W., Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276 (Macromolecular Crystallography, Part A), 307-326. 36. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E., UCSF chimera - a visualization system for exploratoryresearchandanalysis. J Comput. Chem 2004, 25(13), 1605-1612. 112 Chapter 3 : ./3, y-C(sp2) nucleotide analogues 3.1 Introduction Analogues of nucleotide triphosphate in which a natural pyrophosphate 1s substituted by a non-hydrolyzable bisphosphonate motif have been of con tinuing interest in probing the structure and function of naturally occurring biomolecules such as polymerases. A series of jl, y-CXY dGTP' and jl, y-CXY dTTP analogues2 have been previously synthesized as a powerful "tool-kif' to probe the structure, function, kinetics, mechanism and fidelity of DNA poly merasejl. Replacement of a pyrophosphate bridging oxygen of nucleoside triphos phate by a chemically reactive and electrophilic carbon has been of interest as a basis for the design of novel nucleotide analogues. Introduction of sp2 carbon through carbonyl and diazo functions at the a,jl-bridging position was previous ly studied by McKenna et al. s and Yanachkov et al. 4 The first example of a jl, y carbonyl triphosphate analogne was studied by Yanachkov et al., 4 showing the potential to further explore the spectrum ofjl, y-C(sp2) triphosphate analognes. The common bisphosphonates with sp2 bridging carbon include ethenyli denebis(phosphonate), carbonylbis(phosphonate) and diazobis(phosphonate) (Figure 3.1). Carbonylbis(phosphonic acid) (COBP) is known for its inhibitory activity towards many enzymes having a pyrophosphate binding site,s-7 particu larly with respect to a large number of viral and cellular nucleic acid polymer- 113 ases,s-12 such as HIV-1 reverse transcriptase,1o and DNA polymerase 6" and £.12 Tetraalkyl esters of ethenylidenebis(phosphonate) have long been studied as Michael acceptors due to the presence of the reactive a,~-unsaturated vinyli- dene functional group.1s-16 Systematic studies of nucleophilic addition reactions of ethenylidenebis(phosphonic acid), previously named vinylidenedi(phosphonic acid), were previously performed with various types of nucleophiles,17-21 The tetraalkyl ethenylidenebis(phosphonate) and the acid have been used as syn- thetic precursors for bisphosphonate derivatives.22-24 (Diazomethylene)bis- (phosphonate)s have been proposed as possible biochemical photoprobes.2s oJLo II II RO-P P-OR I I OR OR o 0 o ll)lll RO-P P-OR I I OR OR O N 20 R=H II A 11 alkyl RO-P P-OR I I OR OR Ethenylidenebis(phosphonate) Carbonylbis(phosphonate) Diuomethylenebis(phosphonate) Figure 3.1 Common bisphosphonates with an sp 2 bridging carbon. The influence of the molecular geometry (trigonal pyramidal vs. trigonal planar) at the j3, y-bridging carbon of the corresponding dNTP analogues re- mains to be fully investigated in biochemical experiments. The potential of interaction between the highly electrophilic sp2 bisphosphonate motif of the triphosphate analogues and nucleophiles in the enzyme active site suggests a new mode of inhibition for drug discovery. Here detailed synthetic procedures and characterization data for the j3, y-(C=CH2) analogue of 2'-deoxythymidine 5'- triphosphate (dTTP) (8) are provided. An attempt to synthesize the j3, y-(C=O) analogue of 2'-deoxyguanosine 5'-triphosphate (dGTP) (13) is also described. 114 3.2 Results and discussion 3.2 .1 Synthesis of tetraethyl ethene-1, 1-diylbis(phosphonate), 3 Tetraethyl ethene-1, 1-diylbis(phosphonate) (3) was conveniently prepared from tetraethyl methylenebis(phosphonate) (1) via a two-step, single flask method (Scheme 3.1).26 During the first step, the reaction of 1 with paraform aldehyde was efficiently conducted by using a 1:1:5 molar ratio of 1:diethylamine:paraform-aldehyde with the initial concentration of 1 being about 0.35 M to afford the intermediate compound 2.26 Acid-catalyzed elimina tion of 2 by refluxing in toluene with a catalytic amount of p-toluenesulfonic acid eliminated methanol and afforded the target compound 3.26 Purification of compound 3 was conducted by vacuum distillation allowing large scale prepa ration(> 5 g). Small scale purification of compound 3 by column chromatrogra phy on silica gel was difficult to perform due to the reactive nature of the a,~ unsaturated vinylidene functional group, a well-known Michael acceptor.n-21 Silica-catalyzed nucleophilic addition to compound 3 of H20 or MeOH in the column chromatography system was observed, as byproducts were found in the product after purification. Shortening the time of exposure to silica gel by using preparative TLC eluted with dry solvent dramatically improved the purity but the scale of purification was limited to 100-200 mg. Dealkylation of 3 by BTMS also gave difficulty due to the side reaction of the reactive vinylidene group with bromide present in the reaction mixture. It was therefore essential to use dry solvent and to pre-distill BTMS in order to eliminate HBr. The reaction time was controlled under 15 hrs to achieve complete dealkylation and minimize the 115 formation of side-product. Methanolysis of the silyl ester followed by crystalliza- tion with 1 M methanolic NaOH afforded the tetrasodium salt of ethenyli- denebis(phosphonate) 4 in 90%purity. Scheme 3.1 Synthesis of )3,y-(C=CH2) dTTP analogue, 8. CH 3 0 ' CH 2 ii 0 0 EtO-~.....-....~-OEt ' I olo 11..)....11 Eto-r ~-OEt --- Eto OEt Eto OEt 1 2 5 I) Paraformaldehyde, E~N. reflux. II) p-Tolueneaulfonlc acid, dry toluene, reflux. oJLo II II EtO-P P-OEt ' I EtO OEt 3 iii oJLo II II + -o-r ~-o- 4 Na -0 0- 4 vi 0 'CNH iii) 1). Bll'IIIS, dry DCM; 2). MaCH; 3). NaOH/MoOH iv) (CF 3 C0) 2 0, Et,N, MoCN. v) N-methyllmldazole, MaCN. DJlO 0 N):.O HO~~ ~-0-~-0-d I I 1 0 OH OH OH Yl) 1). DOWEX H•; 2). n-8U..N0H, MeCN; 2). N!40Ac (250 mM). OH 8 A new coupling approach based on N-methylimidazole activated nucleo- side monophosphate27 was explored during the synthesis of the jl, y-EBP-dTIP analogue, 8. Acylation of 2'-deoxythymidine 5'-monophosphate (5) with excess trifluoroacetic acid anhydride (TFAA) in the presence of triethylamine in ace- tonitrile provided the intermediate compound, 6, which had both the hydroxyl group and the phosphate group acylated. Nitrogen protection was required during removal of the volatile components under reduced pressure to prevent 116 decomposition of intermediate 6. Following addition of N-methylimidazole, the electrophilic dTMP-N-methylimidazole intermediate, 7, was prepared. Coupling of 7 with tetra-n-butylammonium salt of ethenylidenebis(phosphonate) pre pared from the sodium salt, 4, afforded the desired triphosphate analogue, 8, which was then isolated by dual-pass preparative HPLC purification (SAX then RP-C,s). The efficiency of this method (2 hrs, -70% isolated yield) is more desirable than the conventional morpholidate coupling method, especially for the coupling with electron deficient bisphosphantes such as dihalogenated bisphosphonates. 1 However, application of this method to the 2'-deoxyguanosine 5'-monophosphate monosodium salt was not as successful with only 10-20% yield initially, due to low solubility of dGMP sodium salt in acetonitrile. The solubility problem was resolved by using anhydrous DMF or converting the sodium salt of the dGMP to triethylammonium salt, but the yield was not improved due to the formation of multiple triphosphate products with similar s1p shifts. It was possible that acylation of the amino group on guanine with trifluoroacetic acid anhydride introduced unexpected side reactions. 3.2.2 Attempts to synthesize thej3,y-(C=O) dGTP analogue, 13 Tetraisopropyl (dichloromethylene)bis(phosphonate) (9) was hydrolyzed by reflux in 12M HCl for 2 hrs to afford (dichloromethylene)bis(phosphonic acid) (10), which was then dissolved in 14.5% aqueous NaOH and refluxed for 2 hrs to provide tetrasodium carbonylbis(phospohnate), 11 (Scheme 3.2). Synthesis of the )3, y-(C=O) dGTP analogue from carbonylbis(phosphonic acid) was at- 117 tempted using conventional DCC-mediated morpholidate coupling with COBP in excess. After 48 hrs of reaction, formation of the desired product was not observed by analytical HPLC but the dGMP-morpholidate (12) was found to be hydrolyzed to dGMP. Yanachkov et al. defmed the half-life of jl, y-(C=O) BuPdGTP analogue as 3 hrs at rt. 4 Compounds IJl, y-(C=O) dNTP analogues] with such a short half-life require a fast coupling method carried out at low tempera- ture, such as TFAA mediated imidazolidate coupling.27 Scheme 3.2 Synthesis of tetrasodium carbonylbis(phosphonate), 11. 0 0 II II iPr-0-,P-...,....P,-0-iPr IPr-0 I\ 0-IPr Cl Cl 9 3.3 Conclusion 0 0 ii II II HO-P P.-OH HdcXI'oH 10 I) 12 M HCI, reflux. II) 14.5% NaOH, reflux. o 0 o - u)l_11 - + 0-P P-0 4Na I I _o o_ 11 Trifluoroacetic anhydride mediated N-methylimidazolidate coupling was utilized to synthesize the jl, y-(C=CH2) dTTP analogue, 7. This compound has a complete different molecular geometry from the previously synthesized jl,y-CXY dNTP analogue "took-kif' members at the jl, y-bridging carbon. It will be inter- esting to see if DNA polymerase jl is capable of incorporating this compound and to compare its Kd and kpo1 with the sp 3 analogues (tl,y-CXY dNTP analogues). The potential of interaction between the chemically reactive vinylidene func- tional group and nucleophilic centers at the enzyme active site would be of interest from a drug discovery perspective. 118 3.4 Experimental 3.4.1 Materials and methods All reagents were purchased from Sigma-Aldrich, Inc. and used as re ceived. HPLC was performed using a Varian ProStar equipped with a Shimadzu SPD-lOA UV detector (0.5 mm path length) with detection at 267 nm. Analytical HPLC analysis was conducted on a Varian PureGel SAX 10 mm x 100 mm 7 vL column eluted with A: H20, B: 0.5 M (0-50% linear) LiCl gradient over 30 min at 4 mLfmin flow rate. Strong Anion Exchange (SAX) HPLC chromatography was performed using a Macherey-Nagel 21.4 mm x 250 mm SP15/25 Nucleogel column eluted with A: H20, B: 0.5 M TEAB pH 7.5 using a gradient that in creased from 0-55% over 10 mins, was level at 55% from 10-15 min, and then increased to 100% from 15-25 mins using an 8 mLfmin flow rate. RP-C,s HPLC purification was conducted using a Varian 21.4 mm x 250 mm Microsorb 100-5 Cl8 column eluted with 0.1 M triethylammonium bicarbonate (TEAB) pH 7.5 buffer containing 3.5% acetonitrile pumped at 8 mLfmin. The fmal product, obtained as the triethylammonium salt, was quantitated by UV absorbance using the extinction coefficient (Am= = 267 nm, £. = 9600) of the natural dTTP compound at pH 7.2s 'H and s1p NMR spectra were obtained on Varian 400-MR and Bruker AMX-500 2-Channel spectrometers. Multiplicities are quoted as singlet (s), doublet (d), triplet (t), unresolved multiplet (m), doublet of doublets (dd), doublet of doublet of doublets (ddd), doublet of triplets (dt) or broad signal (br). All chemical shifts (6) are in parts per million (ppm) relative to residual CHCls in 119 CDCh (6 7.26, 'H NMR), HDO in D20 (6 4.79, 'H NMR),29 extemal85%HsP04 (6 0.00, s1p NMR). s1p NMR spectra were proton-decoupled, and 'H and s1p cou pling constants (J values) are given in Hz. The concentration of the NMR sam ples was in the range of 2 - 5 mgfmL. Compound IUPAC names were assigned using ACD/Labs, Release 12.00, Product Version 12.0 1. Images of the 'H NMR spectra (2, 3, 8) and 31p NMR spectra (2- 4, 6 - 8, 10, 11) are presented in Appendix C. 3.4.2 Synthetic procedures Tetraethyl (2-methoxyethane-1, 1-diyl)bis(phosphonate), 2. In 50 mL of MeOH, 2.625 g (87.4 mmol) paraformaldehyde and 1.26 g (17.1 mmol, 2.08 mL) diethylamine were added. The mixture was heated to reflux until paraformaldehyde completely dissolved. The heat was then removed to cool down the reaction mixture. Following addition of 5.8 g (20.1 mmol, 5 mL) tetraethyl methylenebis(phosphonate) 1, the reaction mixture was kept at refluxing for 24 hrs. Completion of the reaction was confirmed by s1p NMR. The solvent was removed under vacuum. The crude was re-dissolved in 10 mL of MeOH and then the solvent was removed. The crude was dissolved in 10 mL of anhydrous toluene and the solvent was again removed to obtain 6.68 g of 2 (quant.) as a colorless oil with 95% purity (confirmed by s1p NMR). The com pound was used in the next step without further purification. 1 H NMR (400 MHz, CDCh): 6 4.17 - 4.13 (m, OCH2CHs x 4), 3.86 (dt, J = 16.3 Hz, 5.4 Hz, PCH(CH2)P), 3.34 (s, OCHs), 2.66 (tt, J = 5.4 Hz, JHP = 23.9 Hz, PCH(CH2)P), 120 1.31 (t, J= 7.1 Hz, OCH2CHs x 4); s1p NMR (162 MHz, CDCb): 6 21.53 (s). Lit26; 1 H (90 MHz, CDCb): 6 4.02 (m, 8H, OCH2CHs, J = 37.7 Hz), 3.63 (m, 2H, CHsOCH2, J= 15.5 Hz, J= 5.4 Hz), 3.20 (s, 3H, CHsO) 1.18 (t, 12H, CH2CHs, J = 7.1 Hz); s1p (36 MHz, CDCb): 6 21.0. Tetraethyl ethene-1, 1-diylbis(phosphonate), 3. Compound 2 (6.68 g, 20.1 mmol) was dissolved in 25 mL of anhydrous toluene. The catalyst, p-toluenesulphonic acid, 5 mg was added into the reac tion mixture. The Soxhlet extractor filled with activated 4 A molecular sieves was attached to the round bottom flask containing the reaction mixture. On top of the Soxhlet extractor, another condenser was attached. The reaction mixture was heated to reach a steady reflux and allowed to react for 14 hrs. Completion of the reaction was confirmed by s1p NMR. The solvent was removed under vacuum. The crude was dissolved in 50 mL of chloroform. The solution was washed with 10 mL H20 twice and then dried using MgS04. The solvent was then removed under vacuum to afford 6.24 g of compound 3 (quant.) as a colorless oil with 95% purity (confirmed by s1p NMR). The compound was used for the next step without further purification. 1 H NMR (400 MHz, CDCb): 6 7.07 -6.89 (m, P2CCH2), 4.20-4.06 (m, OCH2CHs x 4), 1.34 (t, J= 7.1 Hz, OCH2CHs x 4); s1p NMR (162 MHz, CDCb): 6 13.11 (s). Lit26; 'H NMR (90 MHz, CDCb): 6 6.98 (distorted dd, 2H, H2C=, J = 33.8 Hz, J = 37.7 Hz), 4.32 - 4.00 (m, 8H, OCH2CHs), 1.32 (t, 12H, CH2CHs, J= 7.1 Hz); 31 P NMR (36 MHz, CDCb): 6 12.8. 121 Tetrasodium ethane-1,1-diylbis(phosphonate), 4. Compound 3, 500 mg (1.66 mmol) was dissolved in 15 mL of dry DCM and 2 g ofBTMS (13.05 mmol) was added. The reaction was stirred at rt for 15 hrs. The solvent was removed under vacuum. The crude product was dissolved in MeOH and the reaction mixture was stirred for 30 min. The solvent was again removed under vacuum. The crude product was dissolved in 5 mL MeOH. To this solution, a saturated solution ofNaOH in methanol was added dropwise till the pH was adjusted to 8.9. A white precipitate formed immediately. The white precipitate was collected by filtration to afford 218 mg of compound 4 (48o/~ in 90% purity, confirmed by s1p NMR. s1p NMR (162 MHz; D 2 0; pH 8.9): 6 10.81. 5'-O.(hydroxy{[hydroxy( 1-phophonoethenyl)phosphoryl]oxy}phosphoryl)thy midine, ~.y-EBP-dTTP analogue, 8. 2'-Deoxythymidine 5'-monophosphate, dTMP, (84 mg, 24.7 mmol) was dissolved in 1 mL of anhydrous CHsCN. Under N2, 420 vL (3.0 1 mmol) anhy drous triethylamine was added by a gas tight syringe and the reaction mixture was cooled to 0 'C. In a separate vial, a solution of 420 vL (3.01 mmol) tri fluoroacetic acid anhydride (TFAA) in 300 vL anhydrous CHsCN was prepared and cooled to 0 'C. The TFAA solution was added dropwise into the flask con taining dTMP at 0 'C by a gas tight syringe. The reaction mixture was then stirred at rt for 10 min. Formation of the first intermediate, the mixed anhy dride 6, was confirmed by s1p NMR (Appendix C, Figure C6). After the reaction was completed, solvent was removed under reduced pressure with N2 bubbling. 122 In a separated flask, a solution of 61 vL (78.4 mmol) N-methylirnidazole and 280 vL (1.98 mmol) anhydrous triethylamine were dissolved in 300 vL anhy drous CHsCN was prepared and cooled to 0 'C. The solution was added drop wise into the flask containing the reaction mixture that was chilled on ice. The reaction mixture was then stirred for 15 min at rt. Formation of the second intermediate, the N-methylirnidazolidate 7, was confmnd by s1p NMR (Appendix C, Figure C7). Compound 4, 110 mg (53.1 mmol) was dissolved in water and exchanged on DOWEX H+ column from the sodium salt to the acid. To the collected acid eluate solution, aqueous tetra-n-butylammonium hydroxide (30% w fw) was added dropwise to adjust the pH to 10. The solvent was then removed under vacuum. The n-Bu4N+ salt of compound 4 was dried by repeated co evaporation with anhydrous CHsCN. The n-Bu4N+ salt of compound 4 was then dissolved in 500 vL anhydrous CHsCN and some activated 4 A molecular sieves were added. The reaction mixture containing the N-methylirnidazolidate 7 was then slowly added into the flask containing compound 4 at 0 'C. The reaction mixture was stirred at rt for 15 minutes. 200 vL of the reaction mixture was removed and quenched with 600 vL 250 mM aqueous ammonium acetate buffer to prepare for s1p NMR. After the reaction was confrrmed to be completed by s1p NMR, the reaction mixture was then quenched with 3 mL 250 mM aque ous ammonium acetate buffer. The solvent was removed under vacuum and the crude was then purified by dual preparative HPLC purification (SAX then reverse phase C1s) to yield the fmal compound 8 (-30 o/~ as a triethylammonium salt. After removal of the solvent a colorless film was obtained. 1 H NMR (400 MHz, D20, pH 9.8): 7.74 (s, 1H), 6.59-6.40 (m, PC=CH2P), 6.34 (t, J= 7.0 Hz, 123 lH), 4.65- 6.62 (m, 1H), 4.23- 4.16 (m, 3H), 2.42- 2.30 (m, 2H), 1.92 (s, 3H); s1p NMR (162 MHz, D 2 0, pH 9.8): 6 8.36 (d, Jpp = 51.3 Hz, 1P), 6.93 (dd, Jpp = 28.3 Hz, Jpp = 51.4 Hz, 1P), -9.94 (d, Jpp = 28.2 Hz, 1P). (Dichloromethylene)bis(phosphonic acid), 10 Tetraisopropyl (dichloromethylene)bis(phosphonate), 4.96 g (12 mmol) was dissolved in 20 mL of 12 M HCl. The reaction mixture was refluxed for 2 hrs. After removal of solvent under reduced pressure, 2.90 g (quant.) compound 10 was obtained as a colorless oil. s1p NMR (162 MHz, D 2 0): 6 7.04 (s). CITE Tetrasodium carbonylbis(phosphonate), 11 A solution of 14.5% aqueous NaOH was prepared by dissolving 5 g NaOH into 30 mL water. (Dichloromethylene)bis(phosphonic acid) 2.90 g was dissolved in the aqueous NaOH. The reaction mixture was refluxed for 2 hrs. 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E., Nucleoside carbonyl(di- and triphosphates). J. Chem. Soc.-Perkin Trans. 1 2001, 3080-3084. Zhang, Y.; Hudock, M. P.; Krysiak, K.; Cao, R.; Bergan, K.; Yin, F.; Leon, A.; Oldfield, E., Activity ofsulfonium bisphosphonates on tumor cell lines. J. Med. Chem 2007, 50 (24), 6067-6079. 139 Appendix A. Chapter 1 supporting data N 10 0 10 N 0 "' "'"' .,r .,r .,r \/ ( OFO II I II iPr-O-P-'i'P.-0-iPr I F \ iPr-0 0-iPr 2 I ~---)~~\_~----· 4~~4~~ -~9~~-'s'~ ~o• 's '~~~ l A C8 7 6 5 4 3 Figure Al. lH NMR spectrum (600 MHz; CDCb) of2. OFO II I II iPr-o-P-'i'P.-0-iPr I F \ iPr-0 0-iPr 2 ' ' -123 .0 -1 23 .5 -1 24.0 -1 24 . 5 -125.0 PPM 0 -50 -100 Figure A2. 19F NMR spectrum (564 MHz; CDCb) of 2. 10 <::! 10 l'- N ---- 1 .44 1.4 2 1.40 1.38 PPM lt l _j, 2 1 PPM -150 PPM 140 OFO II j II iPr-o-P-1' P.-0-iPr I F \ iPr-0 0-iPr 2 \I ( I )• .~ ..... ~.., ......... .......... J\~, \.~."',..~P"\ ..... ~ 4.0 3.5 3.0 2.5 2.0 1.5 PPM 40 20 0 -20 Figure A3. 31 P NMR spectrum (243 MHz; CDC h) of 2. (OC')C:O't"""LOf'o.r....O "'d'C"")"f"""C')LON't"""O C""lC""lC""lNNNNN ~~~~~~~~ OFO II All iPr-o-r ~-O-iPr iPr-0 0-iPr 3 ~~~~ 1'-:1'-:1'-:'-: "'1"""'1"""'1"""""'" 5.35 5.30 5.25 5. 20 PPM -40 l PPM 1 .861 . 841.821 .801.78\ .761 .74 PP M 8 7 6 5 4 3 2 1 {PPM Figure A4. 1 H NMR spectrum (243 MHz; CDCb) of3. 141 OFO u_,.lu iPr-o-r ~-o-iPr iPr-0 0-iPr 3 T"" CO C') LO CD C"") a> OONNtoto C"! <""! ""':~ ~ t.C! 00 00 00 00 00 00 N NNNNN y ~--.--...___..~- ..,_~....) ~'~-· - · . •. e . · .... l -;·~...,) A.-.... -.-J.--~~--_,-,o.,.__,~.,._ ' -22 7.8 -228.0 -226.2 -22 8.4 -228 .6 -228 . 8 -229.1Jl PM 00 -150 -200 Figure AS. 19F NMR spectmm (470 MHz; CDCh) of 2. OFO II All iPr-o-r ~-o-iPr iPr-0 0-iPr 3 00 00 N 0 N a> oai -250 ---------~---- _ __...______________ - ----- - 10 . 6 10.4 10.2 10 . 0 9. 8 PPM 40 20 0 -20 Figure A6. 3 1 P NMR spectmm (202 MHz; CDC h) of 3. PPM -40 PPM 142 o Clo II l II iPr-O-P-1' P.-0-iPr I Cl \ iPr-0 0-iPr 4 N 0 .... 0 ""':""': ----~--------------~AL_ ____________________ ~~~--------------------- 7 6 5 4 3 2 Figure A7. 1 H NMR spectrum (500 MHz; CDCh) of4. o Clo II f II iPr-o-p-1' P.-0-iPr I Cl \ tPr-0 0-iPr 4 0 a> N r-.: 40 30 20 10 0 Figure AS. 3 1 P NMR spectrum (202 MHz; CDCh) of 4. 1 0 -1PPM -10 -20 PPM 143 o Clo II All iPr-o-r ~-O-iPr iPr-0 0-iPr 5 \I \/ ( 7 6 5 4 3 2 Figure A9. lH NMR spectrum (500 MHz; CDCb) of5. o Clo II All iPr-o-r ~-O-iPr iPr-0 0-iPr 5 LO ""' .... c-.i .... 40 30 20 10 0 Figure AlO. 31 P NMR spectrum (202 MHz; CDCh) of 5. .... 00 ' a; ("") 1 -10 OPPM PPM 144 \I o Bro II j II iPr-O-P-'i'P.-0-iPr 1 Br \ iPr-0 0-iPr 6 ~ ~------~A~--------~ ~ ~--~ 7 6 5 4 3 2 Figure All. lH NMR spectrum (500 MHz; CDCb) of6. o Bro II j II iPr-0-P --1' P.-0-iPr 1 Br \ iPr-0 0-iPr 6 40 30 20 1 0 0 -10 -20 Figure A12. 31 P NMR spectrum (202 MHz; CDCh) of6. PPM PPM 145 o Bro N N CIO r- CIO r-- ..,f ..,f \I u_..,l,, iPr-o-r ~-o-iPr iPr-0 0-iPr 7 ("') en ( M ./ 7 6 5 4 3 2 Figure A13. lH NMR spectrum (500 MHz; CDCb) of7. ("') I(') .,.... N .,.... \! 1 o Bro II All iPr-o-r ~-O-iPr iPr-0 0-iPr 7 40 30 20 10 0 -10 -20 -30 Figure A14. 31 P NMR spectrum (202 MHz; CDCh) of7. PPM -40 PPM 146 0 0 II F II iPr-0-P'f P.-0-iPr ' \ iPr-0 0-iPr Cl 8 ~~ _ _____ _ ;J lt.. _ _ __________ _ 5 . 00 4.95 4.90 4.8 S' P M C.CT"""'d'C:O...::t 'I"'""'"'" 00') C') "<~:"<~:"<~:<-:<-: 'I"'""'"'""'"'" T"" 'I"'" 0 0 1.461.44 1.4 2 1.40 1.3i'PM _l ..t ------------~----------~~A~----------------~~~~------~---- 8 7 6 5 4 3 2 Figure A15. lH NMR spectrum (500 MHz; CDCb) of8. 0 0 II F II iPr-0-P'f P.-0-iPr ' \ iPr-0 0-iPr Cl 8 00 .., r-- "<1" ..... r-- oo 0 ..... u:) r--: r--: '<I" '<I" '<I" 1 = · .. J .- -~ ---- ---- ·~ -146.0 -146.5 -147 .0 -147 .5 -148.0 PPM 0 -50 -100 -150 -200 -250 Figure A16. 19 F NMR spectrum (564 MHz; CDCb) of 8. OPPM PPM 147 \( 0 0 II F II iPr-0-Pi'P.-0-iPr I \ iPr-0 0-iPr Cl 8 5 .4 5 .2 5 . 0 4 . 8 4 _ 6 4 .4 PPM 40 20 0 -20 Figure A17. 3lp NMR spectrum (243 MHz; CDCh) of8 . ...::tOONMf"o-.T"" ...::f'T"""C')I.ONO en en oo oo oo oo -.:t-.:t-.:t-.:t-.:t-.:t 4 _ 96 4 .9 4 4.92 4 .90 4 _ 88 4 . 86 4 . 84 4 . 82 4 .8CP PM -40 ~~~~ ~ vl~l--------~ 5.5 5.0 4.5 4.0 3.5 Figure A18. 1 H NMR spectrum (500 MHz; CD30D) of9. PPM PPM 148 OFO II All HO-r ~-OH HO OH 9 00 r-- 00 '<I" r-- .., "'~""" "'~""" a.n 1,(') C') en N M M"<t '<I"'(') cri cri cri cri cri cri N NNNNN y _ t ~JJL I''' -228.9 -229.0 -2 29 .1 -229 .2 -229 .3 -229 .4 -229 .5 -229 .6 -229.7 -229 .13PM -210 -220 -230 -240 Figure A19. 19 F NMR spectrum (470 MHz; CDsOD) of9. OFO II All HO-r ~-OH HO OH 9 \! 122 12.0 11 . 8 11 .6 11 .4 1 1 f'JJM 40 30 20 10 0 -10 Figure A20. 3 1 P NMR spectrum (202 MHz; CDCls) of9. -250 -20 PPM PPM 149 .... 0) (D .... 1'- '<!' 0 0) 0) '<!' .., .., \I ( o Clo II All HO-P P.-OH I \ HO OH 10 ~ J J \ __ , _ ..... _ _ ..~ ------- 4 .04 4 . 02 4 . 00 3 .9 8 3 .96 3 . 94 3 . 9:PPM ~ L l 6 5 4 3 2 Figure A21. lH NMR spectrum (500 MHz; D20) of 10. N N 1'- N .... l 30 20 10 0 Figure A22. 1 H NMR spectrum (202 MHz; D 2 0) of 10. 1 PPM o Clo II All HO-r ~-OH HO OH 10 -10 PPM 150 \1 ( o Bro II All HO-r ~-OH HO OH 3.88 3 . 86 3.84 3 . 82 3 .8 0 3 .78 3 .76 3 .74 PP M 5 4 3 2 Figure A23. lH NMR spectrum (500 MHz; D20) of 11 . o Bro II All HO-r ~-OH HO OH 11 .... M M N .... 11 40 30 20 10 0 Figure A24. 3 1 P NMR spectrum (202 MHz; D 2 0) of 11. 1 0 PPM -10 PPM 151 0 0 II F II HO-P,],. F!-OH HO I bH Cl 12 1'- ON a> C"") (D (D 00 a> "<i "<i "<i """ """""" r rr \ I ( -138 -140 -142 -144 -146 -148 Figure A25. 1 9F NMR spectrum (564 MHz; D20) of 12. 0 0 II F II HO-,P...J.,. f\-OH HO ] OH Cl 12 $ 40 20 0 -20 Figure A26. 3 1 P NMR spectrum (243 MHz; D20) of 12. -150 -152 PPM -40 PPM 152 .... 1'- 0 N 00 a> 00 (0 0 N N N oO !D!D!D ~ 0 I(') -~ -~ - ..... N ..- 0 to"<t 1'-"<t..- .... (0 a> a> a> N..- ..,f..,f..,f ..,f..,f ~ ~ 00 0 -....; 8 7 6 5 4 3 2 Figure A27. 1 H NMR spectrum (400 MHz; D 20; pH 10.5) of 15a/b. OMNC.CMC')...:;tC:ONC')CDOT""N Of'..N...::tC:OT"""...::tC.CC')NC.CC')LO't""" C"'>C"'>'<t'<t'<tl(')l(')l(')l(')(j)(O(OI'-00 !D!D!D!D!D!D!D!D!D!D!D!D!D!D T""T""T""'I"""'I"""T""T""'I"""T""T""'I"""T""T""T"" ~~C';'C';'C';'C';'C';'C';'C';'C';'C';'C';'C';'C';' ........_.._..,.___~----- ' .=-.-.--,-.--~,.-,---.-,--~.,.,--,~-,---,--~,--.---.-,--_;:._.._;::c;- - 215.8 -216 . 0 -216 .2 -216.4 -216.6 - 21 6 .8 -217 .0 -21 7 .2 PP M 90 -200 -210 -220 -230 Figure A28. 19 F NMR spectrum (376 MHz; D 20; pH 10.5) of 15a/b. PPM -240 PPM 153 ,_... U1 ...f:>. ~ ~· ... ell ()() ~ ? - ::C-..~ ~ ~ rJJ '"d ($ (") en ~ ~ 0(11 ~ s·-J tj tv 0"'- '"d ::c ,_... 0 .S:S(..) 0 ........ ..... 0'1 Ill - ~I\) 11 1.00 1.05 ; LOB I 1.08 :J: --- 8.052 0 o-..C=o :J: rn 0--c=O :I: b o-..C=o :J: b h(f_ / 6.328 - - 6.310 z.A::-z ~ 6.293 z._>:=<ro J-~ z :J: ., -lfi :::~ 3.895 =~ 3.855 2.717 JC- 2.702 2.530 2.522 2.515 2.507 2.495 2.41!7 79 72 = ~ .... ~ ... ell > ~ ..... IOo . "' - ""tl z ~ ~ 1.00 rJJ '"001 (\) (") a s ----- ~~~~~~ ~- ""; 1.06 - ,_... 0\ t0 ~0 ::c ~t'l tj tv 0 '"d ::co. ,_... 0 U1 - 0 ........ 1.03 ""0 ""0 :s: :I: p 0--c=O :J: ~~., 0-"ll=O :J: b o-..C=o i~~b lLRO J-~ z :J: ., 7.019 6.933 6.681 6.595 5.031 5.000 ~ '" 4.937 4.906 4.859 --4.827 ~ 4.764 " \_ 4.733 4.631 4.599 4.536 4.505 4.458 4.426 4.364 4.332 -10.997 -11.013 -11.170 -11.186 I ~ I J . / \ N't"""f"'--CDLOOOOf"'-. LO't"""~enLOf"'-.NOO "<1'"<!'001'-1'-(D(DI(') ai ai r-: r-: r-: r-: r-: r-: ("") 0 .... .-- - --"'-........... ...., ..... _ ... _ "'> _ _.., 6 . 0 7 .9 7.8 7 .7 7 . 6 PPM I II ! I / \ 01'-<D"<t 1'- a> ("") (D en en 't""" 't""" t\V 0 -~ .... ( ~ .. l'; I 1 1 · wrY,"''r; _J I '\;/~ l~f~-.'O,{'k;\.1.1"~-;t' -9.7 -9 . 8 .,<J . 9 -10.0 -1 0 .1 -1 0 .2 -10.3 PP M 15 10 5 0 -5 -1 0 PPM Figure A31. 3lp NMR spectrum (162 MHz; D20; pH 10.3) of 16a/b. a> a> 00 r-: 0"<!'01(') 1'- I(') I(')("") 't""" 't""" 't""" 't""" tt)tt)tt)tt) ~NI.:NH o Bro o N NANH HO ll_.l_n 0 II 0~ 2 -~ ~- -~- 0 HO OH OH OH 17a/b N 1'- 1'- 00 0 OCD't"""f"'-.""'d' 't"""enf"'-.CDCD ...t<"'i<"'i<"'i<"'i 4 .1 4 . 0 3 .9 3 .8 3.7PP M (D 0 ----~ --~ ("") .... 1'- 1'- I(') (D (DC') (DO (D I(') ("") ("") NNNN 2 .652.60 2 .55 2 .502.452.40 2 .35 PPM 8 7 6 5 4 3 2 Figure A32. 1 H NMR spectrum (400 MHz; D 2 0; pH 10.0) of 17a/b. 1 PPM 155 r C')NMC')"'d'OOC.CT""I"'- MT"""d'OcnC.OC:O"d'C"")C') OOOO"d'"d'MMNNNT"" 01)01),..:,..:,..:,..:,..:,..:,..:,..: ~ ~~---------=-- 1'-/NH o Bro o N NANH HO u_j__u 0 II 0-d 2 -~ ~- -~- 0 HO OH OH OH 17alb o en.., o ,..... 'I"'" ("") co 0 T"" N N 0000 "<!' I ~" ~~ - ~~.~ 1ft~~ . ' ' ' ' ' f1f; ~~J~ -~ 9 .01!.9$ .9C8 .8~ .8C8 .7~PM 7 .55 7 .50 7 .4 5 7 .40 7.35 7 .30 7 .25 7 .20 PPM -9 .9 ·10. 0 ·10.1 ·10.2 -10 .3 PPM 15 10 5 0 -5 -10 PPM Figure A33. 3lp NMR spectrum (162 MHz; D 20; pH 10.0) of 17afb. ... (D 0 oO ~) tl:NH 0 Cl 0 0 N NANH Ho 11+11 O 11 O-d 2 -p p- -p- I I I 0 HO F OH OH OH 18a/b \\ "<!'OOLOOO C') M T"" CD r-.r-.LO"<t NNNN \1 v 8 7 6 5 4 3 2 PPM Figure A34. 1 H NMR spectrum (500 MHz; D 2 0; pH 10.0) of 18a/b. 156 "<t MMCIOMO)CIO ,..... 'I"'" "'"'" "d' co ,..... "'"'" "'~""" N ("")("") ("") "d' 1,(') ai ai ai ai ai ai ai "'"'"'"'"'"'"' ~T NJ ~ J.. .. -; NH 0 Cl 0 0 N NANH Ho 11+11 0 II 0-d 2 -p p- -p- I I I 0 HO F OH OH OH 18a/b I _ _ _ _ _ _ ___ _ _ _ _ ___ ) \) ·------ ------- ---· , , .~~~~~~~~~~~~ -138. 6 -138. 8 -1 39.0 -139.2 -1 39 .4 -1 39 .6 -1 39.8 -1 40 -50 -100 -150 -200 PPM Figure A35. 19 F NMR spectrum (470 MHz; D20; pH 10.0) of 18a/b. (D MOO"<t N (D 0 "<t 0) 1'- (D "<t !D !D !D !D I"--C.OC"")0C')"''"""C01,(')N"'""" CDLOOI.OMOOC.OT""CDLO 1'-1'-!D"<t"<tMMNOO 0000000000 "<to(')l'-0 CD I'- 'I""" M cn cn '~""" "~""" t1l! li:NH 0 Cl 0 0 N NANH I Ho 11+11 0 II 0-d 2 -p p- -p- I I I 0 HO F OH OH OH 18a/b 0 --~ ~ l )~ ,.-...-------· .....,..._,....,..._.,. - -....,.._..,.__,_ '" -9.80 -9.85 .fl.90 -9 .95-10.00-10.0510 1010 1 S.1 0 2 (JIPM /~A_A_M_)lA_ § ~- 0.8 0 .7 0.6 0 .5 0 .4 0.3 0.2 0.1 PPM !I 15 10 5 0 -5 -10 PPM Figure A36. 3 1 P NMR spectrum (202 MHz; D 2 0; pH 10.0) of 18a/b. 157 a 376 MHz b 470 MHz ll.o (26.6 Hz) M~Hz) ~ ~ F N v u b \ ,_) 0_\_ '1 1 8.0 -21 1 8.5 -21 1 9 -21 1 8.0 -21 1 8.2 -21 1 8.4 Figure A37. 19F d~astereomer resonances of 15a/b acqmred at 376 l\tlHz (a) and 470 l\tlHz (b) (D20, pH 9.8). One of the calculated individual 19F NMR (red) is shown in (b). a 162 MHz b 202 MHz ll.o (6.4 Hz) ll.o (7.4 Hz) J(26.8 Hz) Figure A38. Pa diastereomer resonances of 16a/b acquired at 162 l\tlHz (a) and 202 l\tlHz (b) (D20, pH 9.8). 158 a 162 MHz b 202 MHz tJ.o (8.4 Hz) tJ.o (6.8 Hz)::: J (6. 7Hz) J(6.5 Hz) 8.0 7.8 Figure A39. P.fl diastereomer resonances of 16a/b acquired at 162 MHz (a) and 202 MHz (b) (D20, pH 9.8). a 162 MHz b tJ.o (7.8 Hz) ~ Jpp (26.4 Hz) -10.0 -10.2 -1 -10.0 tJ.o (9 .3 Hz) -10.2 202 MHz ldG-CHBrl ~ -10.4 Figure A40. Pa diastereomer resonances of 17a/b acquired at 162 MHz (a) and 202 MHz (b) (D20, pH 9.9). 159 31 P NMR t=O pH 10.1 t = 5 hr pH 9.9 poiJI DNAtnJ subatralll dGMP Fnm hyllrolysls of 151lllt + OFO HO,~A~~OH l l OH OH 15 10 5 0 -5 -10 PPM Figure A41. 31 P NMR spectra ( 162 MHz; D20) of the reaction mixture of 15a/b incorporated by DNA pol j3 at t = 0 hr (pink) and t = 5 hr (blue). N:Co (( I NH 0 F 0 0 N NANH HO, u,.!..u~O, u ~O'd 2 ~ <Rl 'a\ ~ 0 OH OH OH 31 P NMR (PR) t = 0 pH 10.1 t = 5 hr pH 9.9 ./'vi lj"VV"VJV 15b OH 6.2 6.0 5.8 5.6 5.4 PPM Figure A42. PJl resonance of 3 1 P NMR spectra (162 MHz; D20) of the reaction mixture of 15a/b incorporated by DNA pol./3 at t = 0 (pink) and t = 5 hrs (blue). 160 N=Co (( I NH o c 1 o o N NANH2 H0,11-411,0,11,0'd ~ ( ~p ~ 0 OH OH OH 31 P NMR (Pp) t = 0 pH 10.1 I'M OH 16b N=Co (( I NH Cl ~ 0 = 0 0 N N~NH HO,p"6 p n,O,pn,O'd 2 I (S) l p I 0 OH OH OH OH 18a 8.0 7.8 7.6 7.4 PPM Figure A43. PJl resonance of 3lp NMR spectra (162 MHz; D20) of the reaction mixture of 16a/b incorporated by DNA polj3 at t = 0 (pink) and t = 8 hrs (blue). t=Oh pH 10 '<I' .... ' "<f a> -~ .... ~ .... LO 0 0 · r--: LO dGMP ~ Fnm hydrelysls of 17•111 t = 12 h pH 9.8 ~:"~ I ~~ 11 15 1 0 5 0 -5 -10 PPM Figure A44. 3 1 P NMR spectra (202 MHz; D20) of the reaction mixture of 17a/b incorporated by DNA polj3 at t = 0 (pink) and t = 12 hrs (blue). 161 31 P NMR (Pp) t=Oh pH 10 t = 12 h pH 9.8 fV \{"'. 'V u N£0 ~ I NH 0 BrO 0 N N~NH HO, " .).,_ "~0, II ~O'd 2 ~ * ~ ~a 0 OH OH OH OH 17b -9.5 -10.0 -10.5 -11.0 PPM Figure A45. P a resonance of 3 1 P NMR spectra (202 MHz; D 2 0) of the reaction mixture of 18a/b incorporated by DNA polfi at t = 0 (pink) and t = 12 hrs (blue). 31 P NMR (Pp) t=Oh pH 10 7.8 7.6 7.4 7.2 7.0 6.8PPM Figure A46. P.Jl resonance of 3 1 P NMR spectra (202 MHz; D20) of the reaction mixture of 18a/b incorporated by DNA polfi at t = 0 (pink) and t = 12 hrs (blue). 162 Appendix B. Chapter 2 supporting data 0 0 II II Meo-;y~-OMe MeO Cl OMe 2a N to~to~..- 00 "'':tN"''"""O)OO 0 CllCilCilOOOO ..t C"i C"i C"i C"i C"i 00 00 r·M .... 5.5 5.0 4.5 4.0 3.5 Figure B 1. 1 H NMR spectrum (500 MHz; CDCb) of 2a. 0 0 II II Meo-;y~-OMe MeO Cl OMe 2a 30 25 20 15 10 Figure B2. s1 P NMR spectrum (202 MHz; CDC b) of 2a. 3.0 5 PPM PPM 163 (0 r-- o (0 en o """ -.::t T"" en r..n No en 1'- 1'- (0 (0 (0 (0 00 .,..; .,..; .,..; .,..; .,..; .,..; C"'i ~v 0 0 II II Meo-; 1 ~-0Me MeO OMe N F 00 ·~ 2b .... X y l l\_____, L__--------~~~--- 6 5 4 3 2 Figure B3. 1 H NMR spectrum (500 MHz; CD 3 0D) of 2b. X= HDO; Y = CHD20D ovl(')enenM cnOONT""LOLO 1"--000')00"'1""" ~~~NNN .., .., .., .., .., .., \~Hl? 0 0 II II Meo-;"'v' ~-OMe MeO I OMe F 2b -230.0 -230.5 -231.0 -231.5 -232.0 -232.5 -233.0 -233.5 Figure B4. 19 F NMR spectrum (470 MHz; CD 3 0D) of2b. PPM PPM 164 \! 0 0 II II Meo-ry~-OMe MeO OMe F 2b 14.0 13.5 13.0 12.5 PPM 30 25 20 15 10 5 Figure BS. 3 1 P NMR spectrum (202 MHz; CD 3 0D) of2b. 0 0 II F II MeO-,P--J....P,-OMe MeO l OMe Cl 2c t-- t-- NIO N 0 T"" 0 0 C') C') co 0 0 OO'l a> a> ~ ~ ~r'i r'i r'i ~ 4.05 4.00 3 . 95 3 .90'PM X 8 7 6 5 4 3 2 Figure B6. 1 H NMR spectrum (500 MHz; CDCb) of 2c. X = CHCb; Y = CHD20D; Z = HDO z 1 PPM PPM 165 0 0 II F II MeO-f-.J.... ~-OMe MeO [ OMe Cl 2c ·-------~~~ - -------~~-------~---------~-· I " " ~144.5 - 1 45 .0 -14 5 .5 -148 . 0 .146.5 -1 47 . 0 -1 47 .5 -148 .0 -1 48. 5 PPM 0 -50 -100 -150 -200 Figure A7. 19F NMR spectrum (470 MHz; CDCb) of2c. 0 0 II F II MeO-f-.J.... ~-OMe MeO [ OMe Cl 2c 0 (0 .... .., (0 N oO oO \( -250 9 .0 8 . 8 8 .6 8 .4 8. 2 8 . 0 7 .8 7 .6 pp~ 40 20 0 -20 Figure B8. 3 1 P NMR spectrum (202 MHz; CDCh) of2c. -40 PPM PPM 166 0 0 00 ..,f X \\I 0 0 .--"- . _.,- 'I"'" LOOMOOenr-. oooor--enr- en en oo oo <D <D C"'iC"'iC"'iC"'iC"'iC"'i \If !I 0 0 II II + Meo-,PyP,-c) Na MeO OMe Cl 3a 5.5 5.0 4.5 4.0 3.5 3.0 Figure B9. lH NMR spectrum (500 MHz; D 20, pH 7) of 3a. X=HDO en o en oo LO LO N N N N y 0 -~ .,.... 00 0 .,.... cO .,.... LO 0 ,- . ·- 0 jx en o N.,.... (D (D aiai 30 25 20 15 10 0 0 II II + Meo-,P;--.,..--P,-c) Na MeO II ? a oMe Cl 3a 5 Figure BlO. 3 1 P NMR spectrum (202 MHz; D 2 0, pH 7) of3a. U = unidentified byproduct; X = 2a PPM PPM 167 J . - - ---/ T""NON"'d'I.(')N"'d'OOf'..C.Of'o.C')O C"")T"""00C.0NOC')f'.."'d'T"""00C.0NT""" 1'-1'-(D(D(D(DI(')I(')I(')I(')'<t'<to:to:t NNNNNNNNNNNNNN NNNNNNNNNNNNNN ~~ ~l ~-------~· 0 0 r~ 23 .0 22 . 8 22 .6 22 4 22 2 PPM C.OMI.(')f'o.I.(')OT"""MI.(')M"'d' C"")N00C.0C"")T"""C')I.(')C"")000 1'-1'-(D(D(D(DI(')I(')I(')I(')'<t aiaiaiaiaiaiaiaiaiaiai ~ ,---------------, a ...__ _ ___ _ ___ , 0 0 II II + Meo-,P:--v-P..-o Na MeO IJ ~ a oMe Cl 3a 10.0 9.9 9 .8 9 .7 9 .6 9 .5 9 .4 9 .3 PP M A 30 25 20 15 10 5 PPM Figure Bll. lH-coupled 3lp NMR spectrum (202 MHz; D20, pH 7) of3a. C') NOOT""NO 0) 001'-(DO)I'- 0) 0)0)0)1'-1'- ...; ...; ...; ...; ...; ...; 4.04 4 . 02 4 .00 3 .9 8 3 .96 3 .94 3 . 92 PP M 0 0 ...; 0 0 II F II + Meo-,P...J.....P,-c) Na MeO ~ OMe Cl 3c 3 . 86 3 .8 4 3 .8 2 3 . 80 3 .78 3 .76 3.74 3.72 3 .1RP M --------------~~--~~~ 1~j~----~ ~~--~~~j ________ __ 7 6 5 4 3 2 1 PPM Figure B12. 1 H NMR spectrum (500 MHz, CD30D) of3c. 168 0 0 II F II + Meo-,P-.J.... P,-c) Na MeO ~ OMe Cl 3c M T"" LO M CION o(')O'l <D CIO CIO a> <"'i <"'i <"'i <"'i "<t "<t "<t "<t ~~ -143 .0 -143 .2 -1 43 .4 -143.6 -143 . 8 -144.0 -144.2 -144_ 4 -144_ 6 PPM 0 -50 -1 00 -150 -200 Figure B13. 19 F NMR spectrum (470 MHz, CDsOD) of3c. C.OMMT""C"")Of"o-.V O'lNO'lNt--O"<tt- f"o-.C.OMNT""OCOC.O NNNN...t...t<"'i<"'i a I -250 0 0 II F II + Meo-,P-.J....P,-c) Na Meo P ~ a.oMe Cl 4c 13 .0 1 2 . 8 12.6 12 4 12.2 PP M ...... ~ ..... 1"--.r ' .!" l y ' ...................... 'Y'(":"';: ............... "' ... "-"" 4~ 4~ 4 1 2 4 1 0 3 1 8 3~6 3~4 3 1 2 P~M 40 20 0 -20 -40 Figure B 14. 3 1 P NMR spectrum (202 MHz, CD sOD) of 3c. PPM PPM 169 0 0 ...... 0 0 I~ a 4 .6 4 _ 4 ' 42 ' 4 .0 0 0 II F II + Meo-,P...J.....~-o Na MeO IJ ~ a oMe Cl 4c 3 .8 3 .6 p p ~ w 0 ~ ~ p~ Figure B15. lH-coupled 3lp NMR spectrum (202 MHz, CD30D) of3c. ON'<t<D'<I'O)MOLOOMNLOI'-LOO VOI"'--'t"""NC')f"'-.CDCDNOOMf'-'t"""T""f' NNOOOOI"--,...._1"--CDCDC"")C"")NNT""O u:)u:)u:)u:)~~~~~~~~~~~~ T""T""T""'I"""T""T""'I"""T""T""T""T""T""T""'I"""T""'I""" 17.5 17.0 16.5 16.0 15.5 15.0 14.5 14.0 13.5 PPM Figure B 16. 31 P NMR spectrum (202 MHz; CDC h) of 4a ( 4 diastereomers). 170 X en "" r-- oO N I ~ en 0 ~O~~~~~N~WN~~N~M~~M~M~ N~O<"'~N~O~N~N~<"'<"'~ONN~N~ ~~~~~~~~~~MMNN~OO~~~~~ ~~~~~~~~~~~~~~~~~NNNNN ~~~~~~~~~~~~~~~~~~~~~~ 0 0 0 II~ Meo-r~~-o oMe MeO F OMe 4b 14.0 13 .8 1 3 . 6 13.4 1 3 .2 1 3 . 0 1 2 .8 12.6 PP M I I 30 25 20 15 1 0 5 PPM Figure B17. 31p NMR spectrum (202 MHz; CD30D) of4b (4 diastereomers). X= PPh,O NT""""'d'""'d'...:::t'CDC:OOLO T""Mf'o-.C')MLONC')LO <"'<"'~~~~enON !D!D!D!D!D!D!Dr-:r-: ~~~~~~~~~ 4c 40 -142 -144 -146 -148 -150 -152 PPM Figure B18. 19 F NMR spectrum (470 MHz, CDCb) of4c (4 diastereomers). 171 O~MNM~M~~~N~~~~OO~~MMO~~~ ~~~~O~M~~~N~NON~M~~~~~~M~ ~~MMMNN~~oo~~~~~~~MM~~o~~ ~~~~~~~~~~~~~~~~~~~~~~~~~ 20 15 10 5 0 -5 -10 -15 PPM Figure B19. 3lp NMR spectrum (202 MHz, CDCh) of4c (4 diastereomers). \I \I 0 0 0 ..... 00 - c:i 0 }La HO-~ P.-0 ( ~ OH Ho);bH Ba-1/a-2 ~ ~ ~~ ~ ~ ~~ 00 1'- 1'- (D aiaiaiai \If 15 14 13 12 11 1 0 PPM Figure B20. 3lp NMR spectrum (202 MHz; D 2 0; pH 10.0) of diastereomer mixture of6a-1/6a-2. U =unidentified byproducts 172 ;J rt·-·-~ . __ ,., "' ,, " 1) P<::'"'·llw•;',·•·: '''<"F:•<'C.'f j ' ' . ' ' ! : ; 6a-1: 10.5 min I i\. I \ I '1 6a-2: 11.5 min i /·' 'I I ' ('\ } I \ I ', I \ I 1 .1 '.} ·. \. \ . ·--- --- ,, p ' 1~ Figure B21. Preparative HPLC separation of diastereomers 6a-1 and 6a-2. For conditions see Table Bl. en N co 0) 0 0) ..... ..... ""'! 1() ~ '<t N ""'!""'! .... ..... '<t 1'-:U!U! ..... ..... 1() 1() I') I') I') \ \ ~ \/( ~0 ~ ~ ~ HO-r'f~-0 OH HO Cl OH llt-1 X y z w 1() <'! 0 N 1() . ~ ~ .... .... u .. h ,w ~l Jl Jt W \G. 8 7 6 5 4 3 2 PPM Figure B22. lH NMR spectiUm (500 MHz; D20; pH 10.3) of6a-1. U =unidentified impurities; X= HDO; Y, W = EbN; Z =acetone. 173 $ 0 0 ... 1 5 . 65 15.60 15 .551 5.5015 .4 5 PP M 0 () 0 II~ HO-r-..®-~-0 OH HO I OH Cl 6a·1 1 il ji llj il ijl Il l 1 9 .8 5 9.80 9.7 5 9.70 PPM \( 16 15 14 13 12 11 10 PPM Figure B23. 31p NMR spectrum (202 MHz; D 2 0; pH 10.3) of6a-1. 00 to- "<<'LO LO C""l r--:r--: I I 0) ... to LO LO LO .,..;.,..; ~ ~ 0 0 HO-r~~-O(S) OH HO = OH Cl 6a·2 N 0 ... LO"<t.- 0) (0 ("") (0 (0 (0 C"'iC"'iC"'i \/( X y z 7 6 5 4 3 2 PPM Figure B24. 1 H NMR spectrum (202 MHz; D20; pH 9.8) of6a-2 (- 85%purity). There is some impurity of6a-1. M =machine artifact; U =unidentified; X= HDO; Y, Z = EtsN. 174 .,... N '<t N 0 0 ..t ..t .,... .,... $ I 0 -~ .,... 0 ~ 0 HO-~~~-O(SJ OH HO Cl OH 6a-2 LO ----~ 16 14 12 10 8 PPM Figure B25. 3lp NMR spectrum (202 MHz; D 20; pH 9.8) of6a-2 (~85%purity). X, Y = 6a-1. l,t') NT"' "d' l.t') 0 T"' 00 N 1'- T"' ""d" 0 ('I") "d" "d' T"' T"' 0 co"""'" C"i C"i C"i C"i C"i C'i C'i T"' T"' T"' T"' T"' T"' T"' II 0 ~ 0 HO-~ P.-0 r ~ OH ,-...,......, HO ~ OH &b-1/b-2 / X 0T""tctc('I")T""('I")0 ,.._0,...00)("')0)("') OOOO,.._,.._LOLO'<t'<t ,..:,..:,..:,..:,..:,..:,..:,..: I I I I I I I I ~~ ~F 14 12 10 8 6 PPM Figure B26. 3 1 P NMR spectrum (202 MHz; CD 3 0D) of diastereomer mixture of 6b-1/6b-2 in reaction mixture. X= excess lb. 175 en T""l""--cnl""o-cni.OI""- t.c N C N 1.0 1""-- t.C T"" N M '<!" '<!" '<!" '<!" <n 10 ai ai ai ai ai ai ai ai M MMMMMMM r rrrrrrr ~>>~((1~/ 0 () 0 11F~ HO-,P...J.,..f\-0 OH HO ~ OH Cl 6c-1/6c-2 -138.0 -138.5 -139.0 -139.5 -140.0 -140.5 PPM Figure B27. 19F NMR spectrum (202 MHz, D 20, pH 10.2) of diastereomer mixture of6c-1/6c-2 in reaction mixture. ('I")Q('I")O)OOI.OIII:t"f"""I.OT"" T"u:l'<tCOf'o-NOl'<tf'o-N COOt.CIII:tNT""I.O"'I:tNT"" aic:Oc:Oc:Oc:Oc:Or--:r--:r--:r--: \\\II ( \I/ ( 12 10 8 0 0 ,- . ' .... Figure B28. 31 P NMR spectrum (202 MHz, mixture of6c-1/6c-2 in reaction mixture. U = unidentified impurities 0 () 0 IIF~ HO-,P...J...-P,-0 OH HO ~ OH Cl 6c-1/6c-2 u 6 4 PPM D 20, pH 10.2) of diastereomer 176 \\ ON ~COl "''"""O)~"'':t NT"" 'I""""'"'" tOtO tOtO no 1\ ~ ~ 0 N-P P.-0 N\ \_/HoY tJH t ; Cl \._0 0 0 7a-1/a-2 ,- . ./ It") "<t 0 --~ ('..'l"""('o. "':ti.O MNN lt"lM cor-..~ '1""""'1""" C'"iC'"iC'"i C'"iC'"i \\1 ~ 8 7 6 5 4 3 2 1 PPM Figure B29. lH NMR spectrum (500 MHz; D20; pH 9.8) of diastereomer mixture of 7a-lf7a-2. r-----=----------o \I no 1\ ~ ~ 0 N-P P.-0 N\ \_/HoYbH I J Cl \._0 7a-1/a-2 0 .. ~ ... ,..._"''"""T"'C:O('I") t--lt"lOliO"<t r--r--~e~e~e ..... \\ I f 00 r- r . I ... 13.5 13.0 12.5 12.0 11.5 11.0 10.5 PPM Figure B30. 3 1 P NMR spectrum (202 MHz; D20; pH 9.8) of diastereomer mixture of 7a-1/7a-2. 177 ... :ss 1·s:~s:x 'JJ ~ m 0 cf'N-~ ~-OlSJ N "' \_/HoYbH \_J 60 Cl 0 7a-1/a~ ~ oll "' '" u --· 7a·1: 14.3 min I\ 7a l t 15.5 min I ', I I, I ' \ ' I ' \ ' \ ' I ' I ' I \ \ ··- u ,. '· ,, • • •• '' ,, ~ \iifll t~t. Figure B31. Preparative HPLC separation of diastereomers 7a·l and 7a-2. For conditions see Table B 1. = ; ;! -=--O')QCQCQCQ NCO'>CQI.nMI.n•M ~~":~":~~~'roo: MMMMMMMMM a 7 s 5 4 3 Figure B32. 'H NMR spectrum (500 MHz; D 2 0; pH 9.8) of 7a-1. 178 ,...._ "'~""" N ~ 00 (0 (0..., ,... ,... (0 (0 . . . . "'"'" "'"'""'"'""'"'" "'"'" "'"'""'"'""'"'" \/ \ ( 12.5 12.0 11.5 11.0 1 0.5 PPM Figure B33. 31 P NMR spectrum (202 MHz; D 2 0; pH 9.8) of7a-1. Assignment of phosphorus signals is based on 1 H-3 1 P gHMBC spectrum. 179 . 1. ~ t . ~\ . J J_. I u _ 11.55 I 11.&0 11.!)5 {3.16,11.7\1~ -11.10 -e <•·'···~,.~- 11.75 ~ :;: {.: .. u,u .~·. - U .BO I 1.85 + 11.90 • ~·-~ ,--..-,~·--.-- r- •--r-•- r--r- •-;-•- r--r---r- •- r-..-• S.O ?.S 6.S 6.f J 5.5 S.O 4.5 4.0 3.5 3.1J 2.S 2.0 l .S 1.0 O .S Uttpm) Figure B34. 'H-31p gHMBC NMR spectrum (500 MHz; D 2 0; pH 9.8) of7a-l. 180 100 50 S#: 4575 IT: 4.39 ST: 1.66 #A: 10 Negative ion mode Calcd for: C17H24CIN20sP2- m/z = 481.0702 285.0 NL: 3.88e+007 481.1 0 . '';o-17:~10_ ,,=J'J_ .. ~ ... .. s,o"IT " ··~ ,L . h '. 586.7 ' _.5s:-~ __ . 1_ ,.. 637 150 200 250 300 350 400 450 500 550 600 Figure B35. MS (ESI) [M-1]- spectrum of 7a-1. 181 \\ 8 \! "~ ~0 0 N-r~~-0 N\ \.___/ HO I OH ( ) Cl \.__0 7a-2 7 0 ~ .... 6 5 OOOONcncn -:tl'-o(')<"'lo(')N COI"--CDC.0"1""""'1""" <"'i<"'i<"'i<"'i<"'i<"'i \1 v \( 4 3 2 PPM Figure B36. 1 H NMR spectrum (500 MHz; D20; pH 10.0) of7a-2 . ~0 0 1\ p 11 a ( ~ 0 N-Ff~-0 N) \._/HO OH \____ Cl 0 7a-2 0 --~ .... I ·-'"") .... .... .... .... \( .... . -~ .... 14.0 13.5 13.0 12.5 12.0 11.5 11.0 10.5 PPM Figure B37. 3lp NMR spectrum (202 MHz; D 2 0; pH 10.0) of7a-2. Assignment of phosphorus signals is based on lH_3lp gHMBC spectrum. 182 a$< HO Cl bH <.....) 7~ ~~-----L------4~--- · i .- '• ' ; I l i I i r- - .. , .... .. ~ -r--- - I (3. 1>.1 .:1 ~).ii-l,lJ:.t!} f- ·- - - - f- 8.0 ?.~ 1.0 .:,.~ 6.0 S.S ~.fJ 4.~ <1,0 3.5 .l-0 2.5 2.0 t.S 1.0 !.J.~ fl \ 1.1KII) ' • U . .! - ll.2 .. 1:!:1 -u.s - ll.b - ll.7 - J.J.S -11.9 u.o ~ - !:Z.l g ::: - 12.2 12.3 12.-1 - 12. !:- - 12.6 12.1 r-11.5 - 10:.9 Figure 838. 1H-31p NMR gHMBC spectrum (500 11/IHz; D 2 0; pH 10.0) of7a-2. 183 LCQ Instrument Control 03 Nov 2010 03:32 PM S#: 4508 IT: 17.46 ST: 1.64 #A: 10 100 481 .1 50 284.9 Negative ion mode 1\ ~ ~ (S) o N-r~~-o \.__/ 0 1 OH Cl 7a-2 Calcd for: C 17 H24CIN20 8 P£ m/z: 481.0702 962.6 NL: 1.67e+007 1738.5 251.1 J 686.6 0 .-Jll. ··-L .. . lL~~ I 893.9 1255.7 1490.3 146~.7 1671.6 [ 1949.9 200 400 600 800 1000 1200 1400 1600 1800 2000 Figure B39. MS (ESI) [M- 1]- spectrum of7a-2. 184 C:~ca liburi .. .\Dimorpholldate5 411112011 5:3533 PM RT 0 00 -33 32 SM 118 16 21 5000 1\ 4000 2 91 3.27 3000 \ 2000 4 35 1000 ! \ ~ -1000 '~ l I' -2000 WI -3000 -4000 -5000 1 0 1 2 18 T 1 me (m1 n) Negative ion mode 100 39612 0 0 50 100 50 100 50 250 Calcd for: C 13 H 17 FNO, P; _ \1 _t~ m/z: 396.0419 ~6''(~H Q IDb-IMGb-2 2851 6 311 . 04 328. 54 350 .16 39064 418.11 455. 66 Calcd for: C 17 H 24 FN 2 0 8 P,- 0 9 ° m/z: 465.0997 cf'N-~~-.((!1~-~--t) \_/O=OH / - F \__ 71>-1 Calcd for: C 17 H 24 FN 2 0 8 P,- 0 0 m/z: 465.0997 0/\N~~- 300 350 \_/ 0 I OH - F 711..2 400 mil 450 23 55 1\ I \ \ I \ \ 20 30 544 3 2 580J 2 598_73 563 07 5~ .98 487 II 5 Q}I3 5]2 93 57061 59QO I 500 550 600 32 NL 5. 91 E3 Channel A uv D 1 morpholl dateS NL: 1. 14E7 Dlmorphohdate5# 1708 RT 16 76 A V I T - c ES Full ms [250 00-600 00] NL 119E7 D lmorphohdate5#251 0 RT 24 . 12 AV I T- c ES Full ms [250.00-600.00] NL I 83E7 Dlmorphohdate5#2904 RT 2746AV I T - c ES Full ms [250.00-600.00] Figure B40. Analytical LC-MS (ESI) [M-1]- spectra of incomplete reaction mix ture of dimorpholidation, compounds 7b-1 and 7b-2. For analytical HPLC conditions see Table B 1. 185 :I J J 10· 10 12 14 Mnutes 7b-1 : 16.6 min 16 I 7b-2: 17.7 min I 1 8 20 22 Figure B41. Preparative HPLC separation of diastereomers 7b-1 and 7b-2. For conditions see Table Bl. VOOMC:OOLOf'-C:OT"" C.O"'d'MT""T"""0C')C')00 '<t'<t'<t'<t'<t'<t<""lOO r--:r--:r--:r--:r--:r--:r--:~DID ~r~ 0 () 0 /\11 N 0 N-;~~-0 N\ \......../ HO = OH ( ) F \__0 7b-1 T""NNNI"'--f'o.OMCD"'d'T""MM o:t<""lNOOOt--t--<0'<1"<"">00)00 r--r--r--~D<O<O<O<O<O<O'<t<""l<""l <""i<""i<""i<""i<""i<""i<""i<""i<""i<""i<""i<""i<""i u u ~ 8 7 6 5 4 3 2 Figure B42. 1 H NMR spectrum (500 MHz; D 20; pH 10.3) of7b-1. U =Unidentified peaks. 24 PPM 186 -218 . 0 ·218 .1 -218 .2 -218 .3 ·218 .4 -218 .5 -218.6 ·218 .7 P P M "'d' 0 T""O M C') CO 00T"""T""""'d'M N C""l "':!' 1/') 1/') to oO oO oO oO oO oO T"" T"" T"" T"" T"" T"" C')l C')l C')l C')l C')l C')l ~((V 05 -210 -215 -220 Figure B43. 19F NMR spectrum (470 MHz; CD30D) of7b-l. 10 .2 10 .0 9 .8 9 .6 9.4 PP M 01'-(0("")("")01/')C"") LOCOT""LO"'d'CONCD O'lOOOOI'-tol/')1/')"':1' aiaiaiaiaiaiaiai PPM ~~~~ ~ ~~ 25 20 15 1 0 5 PPM Figure B44. 3 1 P NMR spectrum (202 MHz; D20; pH 10.3) of7b-l. 187 -=:t 1"'--1"'--NNOO C')'t""" :.0 "'ld"N't"""OOO 001"'-- o:t 'o:t 'o:t'o:t 'o:t ("") 00 .....: ro-.: ro-.: ro-.: ro-.: ro-.: u:) u:) u u ~ ~ 8 7 6 5 4 3 2 Figure B45. lH NMR spectrum (500 MHz; D 20; pH 10.3) of7b-2. U =Unidentified p eaks. ·217 .9 ·2 18 .0 ·21 8 .1 .218.2 · 21 8 .3 .21 8 .4 ·218 .5 PPM 0) LOOOI'-1'-'o:t 00 00 't""" 't""" ...::t ...::t 0 't""" N~ ~'lid" oO oO oO oO oO oO 't""" 't""" 't""" 't""" 't""" 't""" ~JP -208 -210 -212 -214 -216 -218 Figure B46. 19 F NMR spectrum (470 MHz; CD30D) of7b-2. -220 PPM PPM 188 M00)(00)(0000 'l"""r..ncnC"'),...._T""oo"''""" "<!'M000000o(')o(') ooooaiaiaiai 25 20 1 0 5 Figure B47. 31p NMR spectrum (202 MHz; D20; pH 10.3) of7b-2. LOOOcnNT""OOcnN o(')O)Qo(')o(')O)Qo(') T""NC"")"d'OT""NC"") oOoOoOoOaiaiaiai ...,...,...,...,...,...,...,..., 0 () 0 O IIF~ N-,P--J...-P,-o N\ HO ~ OH I J Cl \.__0 7c-1nc-2 -134 -135 -136 -137 -138 -139 -140 -141 -142 PPM Figure B48. 19F NMR spectrum (470 MHz; D20; pH 9.8) of diastereomer mix ture of7c-1/7c-2. 189 Nt--WM"<t"<t"<I"IONNCilt--MOOIOIO cnC.ON'I"""I,(')C"")C')I"--"'Id"COOI"--"d''l""""d'CO CDCDI,(')I,(')C"")C"")'~""""'~"""I,(')C"")N"''"""OOOOCD r--:r--:r--:r--:r--:r--:r--:r--:u:>u:>u:>u:>u:>u:>..-i..-i 7 .8 7 _ 7 7 .6 7 . 5 7 _ 4 7 .3 7 2 7.1 PPM 6 .6 .,.. 6.4 0 a 0 O IIF~ N-r-.J._.~-0 N\ HO ~ OH ( ) Cl \._0 7c-1nc-2 6 .2 6 .0 5 .8 15 1 0 5 0 -5 PPM Figure B49. 3lp NMR spectrum (202 MHz; D 20; pH 9.8) of diastereomer mix ture of7c-1/7c-2. :::> <( = Tll114i : 34 .0318 Mnutu • Anph tude: ··mAll SPD-20A Ch1-256nm - 16CHC113_a na3_1 00u l 140 j j 16CHCII3_ana3_ 100ul.dat 1~1 1001 ~1 ] J 7c-1/c-2 10 12 14 7c-2: 22.0 min I (P2) 7c-1: 20.6 min (P1) I 16 18 ~ 22 24 26 Minutes 28 30 Figure BSO. Preparative HPLC separation of diastereomers 7c-1 and 7c-2. For conditions see Table Bl. 32 34 190 I(')..., (00 NM -:t..., OO'l -:t-:t I(') -:t N..- (0 I(') r-: r-: u:)u:) C"'iC"'i \ I v \I 0 ~ 0 0 II F () N-,P'tP,-0 N) HO OH \___ Cl 0 7c-1 (P1) 0 N N N 0 C! - 0 - .o .,.... .,.... 1 j II ~ u~ , l .1. w ~ ll I' 1 I I I I I I I I I 8 7 6 5 4 3 2 PPM Figure BSl. 1 H NMR spectrum (500 MHz; D20; pH 10.0) of7c-1 (P1). N en 'I"'",...._ -:t co 0 -:t en o 'I"'" N oO ai ai ai ..., ...,..., ..., \ ~ .... ------ ....... ~ '.~~~~~~~~~~~~ - 1 38 .8 -1 38.9 ·1 39 .0 -1 39.1 -139 .2 -1 39.3 pp~ 0 () 0 O IIF~ N-y .. ,J_..,f\-0 N\ HO I OH ( J Cl \._0 7c-1 (P1) -130 -135 -140 -145 PPM Figure B52. 19 F NMR spectrum (470 MHz; D20; pH 10.0) of7c-1 (P1). 191 00 (0 00 (0 1'- ""' a> (0 1,(') "'ld"N "~""" r--: r--: r--: r--: \ I \ I 1'- 0 ...... NC') f"o.. T"" '<1'0 ""0 'lll:tC"") 'I"'" 0 u:) u:) u:) u:) I \ \ I I(') ---~ 0 9 8 7 6 5 PPM Figure B53. 31 F NMR spectrum (202 MHz; D20; pH 10.0) of7c-1 (P1). \ I I(') a> 1'- I(') NN u:)u:) 0 () 0 O nF~ N-,P..J....P,-0 N\ HO J OH ( ) Cl \._0 7c-2 (P2) (oo 1'- 00 N N (0 a> N a> C.O 1,(') N '~""" ...; ...; ...; ...; \ ( \( 1 '.1 0 - 4'.02 8 7 6 5 4 3 2 PPM Figure B54. 1 H NMR spectrum (500 MHz; D 2 0; pH 10.0) of7c-2 (P2). 192 -1 38 .0 -1 38 .1 -138.2 -1 38 .3 -138.4 PP M 't""" f"'-.1.(') N 0 -:t '(') 0 't""" N N -.::t oO oO oO oO "'"'"'"' ~(vJ Ill 30 -132 -134 -136 -138 -140 -142 -144 PPM Figure B55. 19F NMR spectrum (470 MHz; D20; pH 10.0) of7c-2 (P2). 0 () 0 O IIF~ N~P...J....F\-0 N\ HO I OH ( J Cl \._0 7c-2 (P2) I,() "'d''t""" "'d' 0 C')'t""" 0 r--. 't""" "'d' r--. 't""" "'d' I,() C') 00 1'-o(')M -:tNOOO r-..: r-..:r-..: r-..: u:) u:)u:) ~ \ \(( \\ \! 0 0 ~ 12 10 8 6 4 PPM Figure B56. 3lp NMR spectrum (202 MHz; D20; pH 10.0) of7c-2 (P2). 193 0 "<I"N N 1'-<"' to 1'- a> N NN "<!" "<!" "<!" ~} ~l. \.L jllll l'll'lll 1" 11111 1 " 1"" 1 " 1"" 1" 1 -142.;J42 .4142 .il42 .8142 .l142.i142.Q4 H J43 .1 PP M -60 -80 -100 -120 -140 -160 -180 -200 PPM Figure B57. 19F NMR spectrum (470 MHz; D20; free acid) of 12c-1 (Pl). "<!" to .... 1'- ""a> 1'- a> 0 N N <"'i "<!" "<!" "<!" ·--·----------~-··--- · .·o-J ~ ..... " ..._.,., • ..__...__ ,. _,_ _ _ ,. . ____ . I ' r;cr=;=r;=r.;=,=rr=r;c,--rr;-~.-r,=rc;=;=r;=r;=; -14 2. 0 -1 42.2 -1 42 .4 -142.6 -142. 8 -143.0 -143.2 -143 . .PPM -50 -100 -150 -200 PPM Figure B58. 19 F NMR spectrum (470 MHz; D20; free acid) of 12c-2 (P2). 194 ··~ ., dGMP-morph I I~. \ t '\ . .. _ .. -· .... "-. .... / \._ dGppdG } \ ...,_-- - _ _ ; I ' ' ' ' ' v '· Figure 859. Preparative HPLC purification of lla-1. For conditions see Table 81. "' "' "' '!')<DMC<Oc::> ''" ~ ... "'~ =~~<ct "' ~ :;lli ,..;,..; ... ceCft<"'.l.- :2~~~~~ :::!; ... .,., "' ... ...... "' "' .... "' MM ~:~~ ~~~~ \ \\ ~~ ~ ~ \ \ \ \ \1 v 0 6 .... W .. 0 ~LArd OH 1 ..... .... ~ "' "! ... ., - "' "' / "' ., .... - "! ... - !I'R "! _r.; ~-~ ~ """ ~ ~ ~ ~ JL JL l II I I I I I' I I \ ' ·- ~ .~ ~ I 8 7 6 5 4 3 2 PPM Figure 860. tH NMR spectrum (500 MHz; C20; pH 9.8) of 13a-1. 195 \ j I I I \ . .i \ V""r ""\ 0 0 .... "'d'T""T""T"" toN<"'> a> 1'- 1'- to I(') .... N NN N )~~\ \..1/V'..A,.f ~\11.\_.,..··· ·v .., 2.9 2.8 2.7 2.6 2.5 PPM 10 5 0 -5 Figure B61. 3lp NMR spectrum (202 MHz; D20; pH 9.8) of 13a-1. X= dGpplG Timt: 21.7« hohJtts · Pmp liu.O.: ·· mAU UV Detector Ch1-253nm 200 - runfi runG.dat - 175 dGMP-morph dGMP dGppdG N \ ~X \. j I \'"' PPM 13a-2:18.8 min I -25 Minutes Figure B62. Preparative HPLC purification of 13a-2. For conditions see Table Bl. 196 (0 0 (0 "<!' 0 "<tO I(')"<!' oO r-:r-: ~I \~1 13a-2 I(') 0 "<!' ----~ -~ -~ T"" f"o-. LON C.O 0 N NOOcnT""O (0 M MM N NN r-- u:)u:)u:)u:)u:)u:) ..,f ~~ OM "<!'to NO ..,f..,f I \ Nto t-- 0 a>M 0 (0 t--t--1(')"<!' NNNN \1 v 8 7 6 5 4 3 2 PPM Figure B63. 1 H NMR spectrum (500 MHz; D 20; pH 10.0) of 13a-2. (0 0 (0 "<!' "<tO 0 I(')"<!' oO r-:r-: ~ I ~~ I I 13a·2 I(') - - --~ --<~ .... T"" I'- 1.0 N CD 0 N N 00 C') T""O (0 M MM N NN r-- u:)u:)u:)u:)u:)u:) ..,f ~v~ = r-- ---~ c.- . "'! .... OM "<!'to NO ..,f..,f I I I I Nto t-- 0 a>M 0 (0 t--t--1(')"<!' NNNN \1 v 8 7 6 5 4 3 2 PPM Figure B64. 3 1 P NMR spectrum (202 MHz; D 2 0; pH 10.0) of 13a-2. X= dGpp:lG 197 ~---------------------------------~ 220] - dGMP-morph f ~- 0 1 80 1 t:CNH f 1 801 /' 6o cs oJtL~ ... oJ? ... o~N N).._NH2 t 1 I N ~(SJ~ ~ 0 f 140·1 0~ 9" OH OH OH f 1 20 l1 ~ I OH I 13b-1 r 10 12 14 16 18 20 22 24 Minlies Figure B65. Preparative HPLC purification of 13b-1. For conditions see Table Bl. V ~~~M~M~~V~~~~~~M N~~~~ ~ M~~~NO~~MN~~O~~~~~O~~~ 0 ~~MMMMNNNN~~~ooom~~N~~ ex) ,..:,..:,..:,..:,..:,..:,..:~D~DIDIDID ....;....;....;....; ·~~~~~ 00 - "<tO') ...... 0 CIC! = oC! / ...... C! 0 _,..·_.~ _,....~N ...... / ...... 8 7 6 5 4 3 2 PPM Figure B66. 1 H NMR spectrum (500 MHz; CD30D) of 13b-l. 198 ' !\ r I J\ fJ ~ _ ,./ """ _.,..;,...,..,..,,., .MJ/l'<" ·-~~~ ,.o. ... f'W"'t -21 9. 8 -220 . 0 -220 .2 -220 .4 -220 .6 -220 .!PPM T"" 00 C') .....,. 0 NN I(') N C""l"'::' I(') 0 0 0 0 N NN N C')l C')l~ ~ ~~ {) -205 -210 -215 -220 -225 Figure B67. 1 9F NMR spectrum (470 MHz; CD30D) of 13b-l. T"" l,(') T"" -.::t I(') (D (D ,.._ <D I(') C""l N ai ai ai ai ...:::tcnONI.OT""NM ...::t...::tT"""M...::ti.OT"""M ~~~"":~~~""': 'I"""T""T""T""'I"""T""T""T"" 10 5 0 -5 Figure B68. 31 P NMR spectrum (202 MHz; CD30D) of 13b-l. X= dGpp:lG PPM ...... (D 0) 0) ...... (D 0) ...... ,.._ ~~' -10 PPM 199 Minlies Figure B69. Preparative HPLC purification of 13b-2. For conditions see Table Bl. ~ N~N~N~~~~o~oo~~~~~~~~~ ~ ~N~~~N~~~~~~~~~~N~~~~~ 0 ~~~~~~~NNN~~~~ooo~~N~~ ~ ~~~~~~~~~~~~~~~~~~~~~~ N£0 ~ I NH O 0 F 0 0 N NANH (' 6 011 f 110110~ 2 N 'pfRJ"p/ 'p' I I I I 0 o.......,.... 17 OH OH OH I OH :::,... 13b-2 ~M~~~~~~N ~~~MNN~OO C""lCIOC""lC""lC""lC""lC""lC""lC""l NNNNNNNNN 9 8 7 6 5 4 3 2 Figure B70. 1 H NMR spectrum (500 MHz; CD30D) of 13b-2. PPM 200 N "<t 0 N a> 0 ai c:i ..- N ~ ~ I I \( -214 -220 Figure B71. 1 9F NMR spectrum (470 MHz; CD 3 0D) of 13b-2. 00 NOO't""" C') T"" ON (0 (0 "<t ("') ai ai ai ai Ntoi'-O"<ti'-CON O'lOtoCOO'lOtoCO t.C!<..C!~~co:~~""": T""'I"""T""T""T""'I"""T""T"" 15 10 5 0 -5 Figure B72. 31 P NMR spectrum (202 MHz; CD30D) of 13b-2. X= dGpplG .... (0 1'- 0'l..-"<t a> .... co ~~ '!J.~/x 01 -10 PPM 201 ,,.,, >.\-t: Vlt"'' ·~·l ·•··· <ooA I It lltitOol ::~ 1 X>L_ ________ ~a.~t~~~I __________ __J () ,, I ' . I - __ : • . .. / II " 1' Figure 873. Preparative HPLC purification of 13c-1. For conditions see Table 81. ., ,.._ "' 01.0 N (") r- ;;; 1'-1'-I.O<:t.-(O<:t N N a> N 0 0) 1.0 (") .-r-M.-r-N.- q I.QC'1 C'1C'1(\,f(\,f(\,f I"'; N.-.-..-or-1.0 ., ,.._ ,.._ (0 (0 (0 (0 (0 ... ...;..:;..:;...;...;rnrn \ \\ ~rV ~~~~ 0 ot*~ .,.,., (1011 ... "' "' "' "' 0 0 0 ~ :!; ..g _r~ II.' II~ q ., 13c-1 19.8 min I /\ . '· ! ' .............. / ., "' 0 "' 00> ... . I': <::J: <::!; NNN ~ \1 N <D ~ 0 /~/~ \ ,,_- 8 7 6 5 4 3 2 PPM Figure 874. •H NMR spectrum [500 MHz; DoO; pH 10.0) of 13c-1. 202 NJ (" Jl .. -;: NH 0 0 Cl 0 0 N NANH 6 11+1101100 2 /' -p p- -p- I N , I I 0 O~ HOFOH OH OH :::,. 13c-1 (P1) 0 '<I" 0) .,.... (D .,.... ": -.:1: t.C! .,.... .,.... .,.... '<I" '<I" '<I" I'' ' .... - .... .J \.wl ... J l.J.~-'----·· ·140 .&140 .8-141 .01 4 1. 2-141 . 4-141 .&141 .6-142. 0 PPM -50 -100 -150 -200 PPM Figure B75. 19 F NMR spectrum (470 MHz; D 20; pH 10.0) of 13c-l. (D ON!D 0 N I(") (D (D '<I" N 0 .0 .0 .0 .0 NJ !D'<I"NONO(D(D I(")O'<talNfoo-01(") C')T"""T""NM"'d'l.OCD ~<?<?<?<?<?<?<? ~~ (" Jl .. -;: NH 0 0 Cl 0 0 N NANH 6 11+1101100 2 /' -p p- -p- I N , I I 0 O~ HOFOH OH OH :::,. 13c-1 (P1) ,ll~tJ~~ 5.7 5.8 5.5 5.4 5.3 5.2 5.1 PPM I(") 0) ( ci r 00 0 J . 1 .,.... / dJ.~ -1 0.-UI,lO_~~ -J III ,3G _-411l.4:fii~O.SS'P M 0 0 .,.... 15 10 5 0 -5 -10 PPM Figure B76. 3 1 P NMR spectrum (202 MHz; D20; pH 10.0) of 13c-l. 203 lo •· J I.WIII>'I..W. · "''""',_· ··II~ ~ ..-u '!tv \ <..:ht '!t-fnll'l * .l$111,il ll .. " ... "'·' <I~J . 'Ofl~ - ... , ., .. I I I ' ,, 'I ' II\ I I / \13c-2 19 4 min I . I . I (\ I ,' \ I , , ;a, .~ '"\, ,I , \ I ' - ·' ' . .,! .... / ·. _ __ '·-- .. __ _ .: ' I \\ "-.... _. / '·- " ,., " " ., .. , Figure 877. Preparative HPLC purification of 13c-2. For conditions see Table 81. 3 .. ~ M<O 'l)t".!""' 5 ~ "' .... ~~ ~1 n "' .. ~0) <0 ..... '4') .., "' ~~ ::l ~~ :2~~:;~ "'~ ~~ oioi ...... "' NN \ \\ ~ ~ I \I \\ \ \I N:l ~ I H 0 tf-0 0 N ;. .... oz-Hb to-~ ,-~ "' .. "' 0: ... -~ .. ~ "' ... ~ ::! "! ..; - "' ~ -~ ~ .. "' ~ ~ "! ~ u ULNV J: .1! 1 ~ _ jo lJ_ 8 7 6 5 4 3 2 PPM Figure 878. •H NMR spectrum [500 MHz; D 2 0;pH 9.9) of13c-2. 204 NJ {( JL .. -:: NH 0 0 Cl 0 0 N NANH 6 11+1101100 2 /' -p p- -p- I N I ' ' 0 o--.) HO F OH OH OH 13c-2 (P2) LO 0 "<!" ..- ,._ N 00 a> ..... oO oO cri .., .., .., ..... \!} I I )q, - ...... -~-" \~~ .. ............ _ ___ _ Jl i 1" '1 1"'1"'1 " '1"'1' 1111 11 -1 4GI11J 0. 1 1101440 1SIOm1-DH .J1.4 PPM .go -90 -100 -110 -120 -130 -140 -150 -160 PPM Figure B79. 19 F NMR spectrum (470 MHz; D 20; pH 9.9) of 13c-2. 00 Nl'-00 000<.D"''"""C0C')I,(')I,(') "<!" <Da>O LO"''"""...::tONr...T""C.C "<!" N 0 a> ONN"<t"<I"LO<DI'- ...-) ...-) ...-)..,: <?<?<?<?<?<?<?<? ~~ ~~ NJ {( Jl .. -:: NH 0 0 Cl 0 0 N NANH 6 "+"01100 2 /' -p p- -p- I N I ' ' 0 o-._) HO F OH OH OH 13c-2 (P2) N ..- 0 0 r ,...:. / ,...:. 0000"<1" ..... (D 00 M"<I"LO 000 T~\ I \ I l .JJVU \A ./ ....... , ->(') .... .....~ -2 .9 ..3. 0 ..3.1 -3.2 -3 .3 -3 .4 -3 .5 -3.6 -3 .7 PPM 5.5 5.4 5.3 5.2 5.1 5.0 4 !Jl PM • • + 4 1'4 4 I 4 po 15 10 5 0 -5 -10 PPM Figure B80. 3 1 P NMR spectrum (202 MHz; D20; pH 9.9) of 13c-2. 205 ~ ~ ;x IX 1Y >: - ''· ~0 '( '(""''" .. . -~"' ···~ ~,, l'.,~l:lol l'6olo"" - II:Uil\'1_~-._t. fb eolt l ,ol,. ----' I \\ I • , ., ··- - _,1 ' ., r~lltUI~ I 'I I I\ I I \I ., " ., Figure 881. Preparative HPLC purification of 13d-1. For conditions see Table 81. I I \\ "' .. .. .. ~ <"! "' "' ~ "! :;j "' ... ~ .. ~ ''""" ~ ~ ~ "! 13d-1 16.5 min I '• 'I /'\ · , "·--·-- __ / \, - ... __ - " ., " " ~ 9 8 7 6 5 4 3 2 PPM Figure 882. •H NMR spectrum [500 MHz; D 2 0; pH 9.8) of 13d-1. 206 I \ 0 (0 00 ("") r-- r-- 0 0 ~ 0 C! .... ~f'4'-....,o/J'.I •, 4...._ .... ...,_. 11 1 U:~~~~6~P~ 2.6 2.5 2 4 2.3PPM LO 0 N t-- "'"'" !"-- co C"") LO'<tC""lC""l NNNN \jf 0 C! .... o... oo .... '<I'W 00 0 C! .... ~'4 + I 4 I I 4 I I -1 QlDU64D4lli.WE.60P PM 15 10 5 0 -5 -10 PPM Figure B83. 3lp NMR spectrum (202 MHz; D 20; pH 9.8) of 13d-1. T IITle : 1 0.m 3 Mootes • Jmplitude : •• mALJ ~I ] J J ,.1 SPD-20A Ch1-2S6nm - Run-J_4SOuL Run-t_ 450ul.dat 10 12 Minutes Figure B84. Preparative HPLC purification of 13d-2. For conditions see Table Bl. 13d -2: 16.8 min I 14 16 18 20 207 0 0 00 00o(') N!D "<!' N o(')("') oO oO r--: r--: II \\ ("')0(D(D0 "'"'"0 co 0 C') l,(')l,(')'lll:tNT"" u:)u:)u:)u:)u:) \V /( NH 2 N-AN (I Jl .. ,; 0 0 Cl 0 0 N N_J /' 6s O-~_l_~-0-~-0-d 1 N r (R) 1 1 0 0~ HO OH OH 17 I OH ~ 13d-2 ........ j "": _r "'"": ........ T""f'.. 0 C')L(') T"" 1,(') OOO!DNO'l!DO NN 'I"'" "'1"""0 0 C.O ...;...;...;...;...;...;C"'i 9 8 7 6 5 4 3 2 PPM Figure B85. 1 H NMR spectrum (500 MHz; D20; pH 10.0) of 13d-2. 00 (D 00 "<!' ""'": ""'": ~ 0 ~ .... )l ~*'""" ~~~~ .. ........ jMjii. jii 111~ SIBPM o(')("') 0 a> r--o C') 1,(') c.o T"" a>("') o(')o(')'<t'<t '<t(D c-.ic-.ic-.ic-.i 00 ~f .... ~ NH 2 N-AN (I Jl .. ~ 0 o Cl o O N N_J /' 60-~_l_~-0-~-0-d 1 ~ r (R) 1 1 0 0 ....._...... HO OH OH fJ~M ~of,Ji) \ \....--; .... ·; . ·;~ .'~''l . 1 ~~ . 's~ .~a . 1 ~9 1 • 1 4~ ~~·~ ' 0 ~ .... 17 I OH ~ 13d-2 . ... 15 10 5 0 -5 -10 PPM Figure B86. 3 1 P NMR spectrum (202 MHz; D20; pH 10.0) of 13d-2. 208 ~ ., JX : IY 1 .;o: ''<~ .,..,.,.,, . ., Chi : •:•••oo . • ,. ........... ··~ dGppdG i -- ""-·----~ I ' I \ ,, __ Figure 887. Preparative HPLC purification of 14a-l. For conditions see Table 81. ... .... ~ It')=,. ... C) MC"too-Q MMMM t.D<D~<D ~~~~~~ ("<oj-OJQJO)O) 'ffli~MMMM \ ~ \\\~ ~:lNH o~o o A~ ~~LA~ 1<1&'1 .... ... .. "" M ~ "l "l - " - ./..,; ~~~ '1\ \1 ·"' ~/~ - \_.... ... L H~ ~ J ~h ~ 8 7 6 5 4 3 2 PPM Figure 888. tH NMR spectrum (400 MHz; C20; pH 10.6) of 14a-l. 209 't""" M CO 't""" C')N I(') N 0 00 to -:t N N -:t C""l NN ai ai ,..: ,..: ,..: ,..: 14a-1 7 .7 7 .6 7 .5 7 .4 7 .3 7 .2 7 .1 7 .1J'PM f \ w. ... --t' -......... ..................... . I' .$_7 -9 8 -9.9 - 10 . 01 0 :1 .10 .21 0 .3 PP M 0 0 15 1 0 5 0 -5 ·1 0 PPM Figure B89. 3lp NMR spectrum (202 MHz; D20; pH 10.6) of 14a-1. 200 150 100 50 rme: 22.74n Mnutes -~udt : - m.AU UV Deteetoe Ch1-2S6n m - runl runl .dat Nome 14a-2 dGppdG 14a-2: 14.2 min I J .soL------------------------------------------------------------------------·__J Minutes Figure B90. Preparative HPLC purification of 14a-2. For conditions see Table Bl. 210 LO 00 0 oO ~) N£0 (( I NH o c 1 o o N N.J__NH HO 11 I 11 o 11 o'd 2 'pfR)'p~ 'p~ I I I 0 OH OH OH 0 ~ ...... OH 14a-2 N J ' ~ ...... Mt--LOOOON ID"<!'NOC'lt- N"''"""cnC')OOCO ..,f..,fC"'iC"'iC"'iC"'i r-- N ,..-" C'! 0 ("') ---~ O"<!'NM LOONOO OOOOLO"<t NNNN \\ \1 00 "' '" ~ -- ~ ...... ...... 8 7 6 5 4 3 2 PPM Figure B91. 1 H NMR spectrum (600 MHz; D 20; pH 10.3) of 14a-2. 9 .4 9 .3 9.2 9 .1 9.0PPM ' ,, ,. r-- (D "<!' (D "<!' r-- N a> t-- '<I'M 0 N "'~""" N NT"""'~""" ai ai ~~~~ NJNH Cl (( JL .. A HO ~ I ~ 0 ~ O'dN N NH2 'pfR'J'p~ 'p~ I I I 0 OH OH OH ...... ---~ ...... ...... OH 14a-2 r~ M ~~~t~ 7 .5 7 .4 7 .3 7 .2 7 .1 7 . 11PPM .lL ·9.8 ..S .9 .10 . 0 .10.1 ·10 .2 ·10. 3 P P M 0 0 ...... 15 1 0 5 0 -5 -1 0 PPM Figure B92. 3 l p NMR spectrum (202 MHz; D20; pH 10.3) of 14a-2. 211 ..., o oor--<DvM ~ 0 C') 1,(') 'I"'" 1,(') "'"'"co """ """ ""' ""' ""' (D (D I(') ai ai r-: r-: r-: r-: r-: r-: 0 ~ .,.... 9.5 5 9. 50 9.45 9 .40 9 .3SlPM 'I""" ...... 1,(') 't""" 000'1""""d' C') 0 'I""" 'I"'" ttp 15 10 5 0 -5 -1 0 PPM Figure B93. 31p NMR spectrum (202 MHz; D 20; pH 10.2) of mixture of 14a- 1: 14a-2 (2: 1). 0~------------------~~ >------~~~~~~~~~~~~~--~--~~--~---' 0 2 4 6 8 10 1 2 14 16 18 20 22 24 26 28 30 32 34 Minlies Figure B94. Preparative HPLC purification of 14b-1. For conditions see Table Bl. 212 N (D 0 oO NOI'-"<t C"") NT"" 0 ("") ("") ("") ("") u:)u:)u:)u:) 14b-1 Nf"o.. NT"" C'l(D'<tl'- 0000001'- "<i"<i"<i"<i 0 a> LO 1' r..n 0 C.OT"" N N "'"""'I"'" . "<i "<i "<i "<i C')f"o-.T"""""'d'OONMC.OO"'d' f"o..C.ON'1"""00C')0000,...._ 1'-1'-LOLOLOLO"<t"<t"<t"<t .NNNNNNNNNN 00 00 ...... 0 . .....-·~---~ 8 7 6 5 4 3 2 Figure B95. lH NMR spectrum (500 MHz; D 20; pH 10.3) of 14b-1. 14b-1 ~v~U · · '""~ ' ' ' .l.~ -21 5.7 -21 5.8 -21 5.9 - 2 16 .0 - 2 16 .1 -216 .2 -216.3 - 21 6 .4 - 21 6 .5 -216 .6 PP M T"""f"o..OOf"o-.f"o-.OCD CDLOC:OOf"o-.C')NT""" 0T""'I"""NNNM"'Id" u:)u:)u:)u:)u:)u:)u:)u:) T""T"""'""""'"""T""T"""'"""T"" ~~ -204 -206 -208 -210 -212 -214 -216 -218 Figure B96. 19 F NMR spectrum (470 MHz; D20; pH 10.3) of 14b-1. PPM -220 PPM 213 N't"""f'o.CDN't"""'t"""NO't"""'t"""O ,.._OO'lN,.._O<"'lo(')o('),.._O<"'l O'la'l(D(D,.._,.._(Do(')'<t<"'l<"'lN <D<D<D<D"<i"<i"<i"<i"<i"<i"<i"<i .,.... N -~ I~ _/ 7 .1 7 .0 6.9 6 .8 6 .7 PPM 10 5 0 -5 -10 PPM Figure B97. 31p NMR spectrum (202 MHz; D20; pH 10.3) of 14b-l. .00 1 - . ---------~4 1 b-2:14.5mm _______ l ] , rl:r: I I o-ko o N N NH2 I HO,II II,.O,II,.O'd 2 501 ~ (R ~ ~ 0 I OH OH OH 2 001 OH I 14b-2 150 -1 I 1 00 -1 dGppdG I 50 MiniJes Figure B98. HPLC purification of 14b-2. For conditions see Table Bl. 214 LO 00 0 oO CD 't""" LO 't""" C') O'lf'-. LO-:tto COCO f"o-.N"''""" ..,f ..,f • ..,f ..,f ..,f N:Co ~ I NH 0 F 0 0 N NANH HO n I n~O,n~O'd 2 '~1R)"~ ~ 0 OH OH OH OH 14b-2 8 7 6 5 4 3 2 Figure B99. lH NMR spectrum (500 MHz; D 20; pH 10.5) of 14b-2. 14b·2 ·215.05-216.1 021 6 .15-216.20.216 .25216 .30216 .35216 .40.216 .45 PPM QC')C')NI.OC:OT""f"o-. MN-:tt---:ttoa>OO 'I"""NNN~~C"")'III:t !D!D!D!D!D!D!D!D 't"""'t"""T""T""'t"""T""T""T"" ~ PPM -200 -205 ·21 0 ·215 -220 PPM Figure BlOO. 19 F NMR spectrum (470 MHz; D 20; pH 10.5) of 14b-2. 215 cnoovMcncnoocnr--cnoor- 'l""""d'"d'~"--~"--OC"")r..nr..nr--..oC"") ocnr--tor-.1'-tol(')vMMN r-o:u:iu:iu:i..,f..,f..,f..,f..,f..,f..,f..,f 14b-2 to I(') r~ ~~ I ~MWML j" I I ' 1 : .;,.-! I ~M 7 .2 7 .1 7 .0 6 .9 6 . 8 6 .7 PPM 4.9 4.8 4 .7 4.6 4.5 4.4 4. 3 4.2 PPM _) 10 5 0 -5 -10 PPM Figure BlOl. s1p NMR spectrum (202 MHz; D20; pH 10.5) of 14b-2. 0 l~NH F NJl .. A HOl .. i._g .. o,g,o'd N NH2 I I I Q OH OH OH OH 14b-1 :14b-2 (1:3) T"" 1"--f'..C:OC:OCD...:::t"'d'I"--OMMMCDC') CD NI.Of'..C')N....::tCDC')N...:;:tCDC:O'I"""I'- 0 T""'I"""'I"""T""NNNNC"")C"")C"")C"")"d'"d' u:j u:ju:ju:ju:ju:ju:ju:ju:ju:ju:ju:ju:ju:ju:j T"" T""'I"""T""T""T""'I"""T""T""'I"""T""T""T""'I"""'I""" \~\\1/lYP? """ 0 ---- "'"' -215.4 -215.6 ·215.8 ·216.0 -216.2 -216.4 PPM Figure B102. 19F NMR spectrum (470 MHz; D20; pH 10.5) of mixture of 14b- 1: 14b-2 (1:3). 216 NNI'-1'-0)0)IDLO"<tLO"<t"<tNM -.:tr--.c.ocncnNc.Dcnc.oc:ol,(')'l"""l,(')l"- oO)!'-<DO)O)OOI'-1'-<D<D<DLO"<t ,._:ujujuj..;..;..;..;..;..;..;..;..;..; 0 N 0 0 /~ I _j 1 0 5 0 -5 -1 0 PPM Figure B103. s1p NMR spectrum (202 MHz; D20; pH 10.5) of mixture of 14b- 1: 14b-2 (1:3). Tim@ : 25 .1211 Mnut@S • .Pmplitude: •• mAIJ SPD-2GA Ch1-256nm - l_lOOuL 4501 Narne3_300uL.dat 400 50 14c-1: 10.6 min I 10 12 Minutes Figure B 104. HPLC purification of 14c-1. For conditions see Table Bl. Ni.:o ~ I NH oxF.Cb 0 N N.J__NH HOII IIOIIO'd 2 'p p~ 'p~ I I I 0 OH OH OH OH 14c-1 (P1) 14 16 18 20 22 24 217 "' I(') 0 oO 00 N 0 M N '~""""~""" C') M MM N u:)u:)u:)u:) ti:NH 0 Cl 0 0 N NANH Ho 11+11 0 II 0-d 2 -p p- -p- I I I 0 HO F OH OH OH 14c-1 (P1) S .3551 .34QJ::fi.3~.31 ii .J0fl2~280PPM I(') - C! .... \V 4.30 4 .25 4 .2 0 4 .15 PPM ..-en o -:t..-10 f'-.1(')-:t .NN N \\1 .A}JJJ~ ,II~~ " " . . .._ _ __ ____ ___ _ _ ... .., _ _ 2 .85 2 .80 2 .75 2 .7 2 . 65 2 .60 2 .55 2 .50'f' M 8 7 6 5 4 3 2 PPM Figure BlOS. lH NMR spectrum (600 MHz, D20, pH 9.8) of 14c-l. Sample contains 5% the other diastereomer, 14c-2. T"" T"" cor--. I'- T""M f"'-. r--cncno ociociociai "'"'"'"' .... $/) ,---------------------~----, ti:NH 0 Cl 0 0 N NANH Ho 11+11 0 II 0-d 2 -p p- -p- I I 1 0 HO F OH OH OH 14c-1 (P1) -100 -120 -140 -160 -180 PPM Figure B106. 19 F NMR spectrum (470 MHz, D20, pH 9.8) of 14c-l. Sample contains 5% the other diastereomer, 14c-2. 218 't""" CO 't""" CD a> N ,.._ 0 (D I(')<"'> N !D !D !D !D a> 00 I(') ,... (D a> IDO'<tNOO"<t ~NOO't"""~ oooqqq NJ (( JL .. -:: NH 0 Cl 0 0 N NANH Ho 11+11 0 II 0-d 2 6 .8 6 .7 6 . 6 6.5 6 .4 6.3 6.2 PPM -p p- -p- I I 1 0 HO F OH OH 1 0 0 { .,.; OH 14c-1 (P1) ,...,..! $ \,, ",....,... '" -9.8 -9 .9 -1 0 .01 0 .1 -1 0 .2-W .:i'P M 15 10 5 0 -5 -10 PPM Figure B107. 3lp NMR spectrum (202 MHz, D20, pH 9.8) of 14c-1. ~i :1 ~J ::> ~J ~ 4 J = ~J ~J 100 0 Sample contains 5% the other diastereomer, 14c-2. SPD-20A Ch1-2S6nm - 4_SOuL 4_SOuL.dat Name 14c·2 (P2) 14c-2: 20.5 min I 0.0 2.5 5 .0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 Minutes Figure B108. HPLC purification of 14c-2. For conditions see Table Bl. 219 .., 00 0 oO N_)l ~ Jl .. -:: NH 0 Cl 0 0 N NANH Ho 11+11 0 II 00 2 -p p- -p- I I I 0 HO F OH OH 0 - ~ ..... OH 14c-2 (P2) \\ j ii jiiij I jiii j iijliij ijiilj I 4.304 .284 .264 .244 .224.2~ .1 84 .18 PM ...:::t'C')"t"""...:;t NtoMOO 001"-1(")-.:1" NNNN \1 \( ili j i I I I I I I 2 .80 2.75 2 .70 2.65 2 .60 2 .55 PPM 8 7 6 5 4 3 2 PPM Figure B109. 1 H NMR spectrum (500 MHz, D20, pH 9.8) of 14c-2. Sample contains 5% the other diastereomer, 14c-l. 0 0)001(") to O)Nto (0 1"-000) oO oO oO oO .., .., .., .., ..... ~V? ~----------------------~ N_)l ~ JL .. -::NH 0 Cl 0 0 N NANH Ho 11+11 0 II 00 2 -p p- -p- I I I 0 HO F OH OH OH 14c-2 (P2) -13 8 .4 -13 8 .5 ·138 .6 ·1 38 .7 ·1 38 .8 ·138 .9 ·139. 0 PP M 125 -130 -135 -140 -145 -150 PPM Figure BllO. 19 F NMR spectrum (470 MHz, D20, pH 9.8) of 14c-2. Sample contains 5% the other diastereomer, 14c-1. 220 N 0 "<!' 00 (D 0 "<!'"" (0 1,(') ~ T"" !D !D !D !D co 0) 1,(') "'"'" "'"'" C') C')M00T"""L00 MNOOT""M ooooqq 0 ~N£NH 0 Cl 0 0 N NANH Ho 11+11 0 II 0-d 2 -p p- -p- I I I 0 HO F OH OH OH 14c-2 (P2) j 0 0 r . r ..... 15 10 5 0 -5 -10 PPM Figure B111. 3lp NMR spectrum (202 MHz, D 2 0, pH 9.8) of 14c-2. Sample contains 5% the other diastereomer, 14c-1. Ttm• : 21.1 1173 MootfiS - lmplilude: ·• mAU SPD·20A Ch1-259nm - Run3_150uL 250 Run l.da.t 14d-1: 15.39 min I 200 ~ 150 14c:l-1 100 50 ~------------------------~~~ 10 12 14 16 18 20 Minutes Figure B 112. HPLC purification of 14d-1. For conditions see Table B1. 221 M N 00 ..., "<!' N oO oO \ I N C! 0 .... ro- o NH2 N:CN ~ I 0 (:I 0 0 N N_) Ho II : II O II Ob -p----p- -p- I (S) l l 0 HO OH OH OH 14d-1 T""T""OMOOLOO ...::tOOC:OC') "'1"""-.::f'L(')C"")OOCO lrr.(")ONC:O C"")N"'"""cncnC')oo c:oooc.olrr.(") ..t..t..tC"'iC"'iC"'iC"'i NNNN \\1 \~ \\ \! 00 (0 /~/~ \..____ ,____, .. ~ . .._.._______.,lj~ l 9 8 7 6 5 4 3 PPM Figure B113. 1 H NMR spectrum (600 MHz, D20, pH 9.7) of 14d-l. N "<!' 00 "<!' 1'- N M 0 "'d'N M T"" 'I"'" 'I"'" ("")("") N N ai ai r--: r--: r--: r--: ~ ~~- I ------------------~NH~2 ~N+N 0 C! .... N 0 0 91 0 0 NJlN_) Ho u : nonob -p----p- -p- I (S) 1 1 0 HO OH OH OH 14d-1 .... ..., en o .... ..., 00 JUt ,) 0 , J~~ 7 .40 7 .35 7 .30 7 .25 7 .2!FPM -10 .1 0-10.15-10 .20-1 0 .25-10.30 PP M N C! 15 10 5 0 -5 -1 0 PPM Figure B114. 3 1 P NMR spectrum (243 MHz, D20, pH 9.7) of 14d-1. 222 ~ 1 r ..... VI ::: .,..,.,, • ..,.,.,"'" ' ""' - IO e•l ._,.,, R:I. .. (.UOIIL~'It ;x 14d-2 21.0 min 1,, 1 .. 1:0: IX " >: " I' I • I --~·--------/'-___) v \ " I i\ I I I I ' I ' I ' ' \ , Figure 8115. HPLC purification of14d-2. For conditions see Table 81 . "' ~ .. ... :;; .... ~ ~~~ <D<O,._<Ditt'M Qltt'MM<:::I,._ M<'ool.-cr»O'J<O ~<ti'C#MMM 'CfO')<OC Itt'~ ('o,j <0 <0 ,._ CD Itt) ~~~~ I I ~ \\I If \\ $ NJ; a t/J HO~~~o~j~o~ N Hb ~ Ott Ott ~)L___ftL OH 14H .~_A_ ., ., '·' " ·- :n :;,o :.~ va :n :•- ~ ... "' ~= .... .... .... "! :l ~ - .. /~~ ~ ~ .J ('o,j/~ ,, _____ ~~ IL ' ~ I L_ 8 7 6 5 4 3 2 PPM Figure 8116. •H NMR spectrum [500 MHz 1 Do0 1 pH 9.8) of 14d-2. 223 N N NN<DI' T""" co ("")00')(0 0 0') T"" T""C') 0') ai ex) r--:r--:~DID $ ~~--~ NH 2 0 0 ..... 0 0 ..... ~N+N 0 Cl 0 0 NJLN) H0-~_1_~-0-~-0b I (R) I I 0 HO OH OH ~~ OH 14d-2 ..... 00 0 ("") N C""l 0 0 0 0 ..... 9 .1 0 9.05 9 .00 8 . 95 8 .91J'PM J .~ .~ 7 .20 7 .15 7 .1 0 7 . 05 7 .00 6 .95 PPM .JJ~ -1 0 _ gm .tiW .liD .2[1) .;2J{I .l"OO .J{I .40P PM " " "'""1' 15 1 0 5 0 -5 -10 PPM Figure B117. s1p NMR spectrum (202 MHz, D20, pH 9.8) of 14d-2. ~ T"' CD lll:tOIII:t('I")OtcT"'CD tci'-OCD CD l,t') COa.t')lll:tT""a.t')T"'T"'I'- tcl'-00 T"' T"' "'l:t "'l:t "'l:t "'l:t ('I") ('I") ('I") N T"' T"' ('I") ('I") ai ai ,..: ,..: ,..: ,..: ,..: ,..: ,..: ,..: c::i c::i c::i c::i ~~~--~ NH2 ~N-rN 0 Cl 0 0 NJLN) HO-~_l_~-0-~-0~ I I I 0 HO OH OH ..... ~ 0 14d-1:14d-2 OH ..... Cll Cll c::i ,,___-MA~"oJ... ~ ..... - .....,_..,_ • .., 9 . 1 3. 1 "B. 1 2l. 1 2il. 1 1i:. 1 1!£ 1 1~. 1 n:,M 1 7 .50 7 .45 7.40 7 .35 7 .30 ' 7 .25 PP M -10 .1 0 -1 0 .1 5 -1 0.20 -1 0 .25 -1 0 .30 -10.35 PP M I 10 5 0 -5 PPM Figure B 118. 3 1 P NMR spectrum (202 MHz; D 2 0; pH 9. 7) of mixture of 14d- 1: 14d-2 (2.5: 1). 224 0 0 0 II II = Meo-,PyP.,_-o--=--- MeO 0Md 5 ) Cl 15 I .J 0 (COlOlOl'<t(C 'I"'" t.CLOCLOMcn LO "'l:tMMNN"''""" ...; ...; ...; ...; ...; ...; ...; 'I"'" T"' T"' T"' 'I"'" T"' 'I"'" LOLO .-(a COl Nor= O)'I"""L()T"' (CMf'-M Ill Ill"':"': 'I"'" 'I""" T"' T"' T"' 'I"'" T"' T"' r-- M '<tO 00 00 00 16 15 14 13 12 11 PPM Figure B 119. 3lp NMR spectium (202 MHz; CDCh) of the mixture of four diastereomers of 15. 0 C! .... Ol 0 00 0 .... 20 18 16 14 12 10 8 6 PPM Figure B 120. 3 1 P NMR spectium (202 MHz; D 2 0) of the diastereomer mixture of 15a/b without ~-cyclodextrin. 225 0 0 0 II II = Meo-,P......,..... R,_-o--ts;-- 0 , 0 +Na- Cl - Na+ 1611/b with ~-cyclodextrin r- oo 0 0 .... 17 16 15 14 13 12 11 10 9 8PPM Figure B 121. 3 1 P NMR spectrum (202 MHz; D20) of the diastereomer mixture of 15a/b with ~-cyclodextrin. 0 0 II II MeO-~-...,....~-OMe MeO bl OH 17 0 0 II II MeO-~y~-OH MeO :· OH Cl 19 0 0 II II Mao-ry~-oMa HO Cl OH 0 .... r-: 18 N 0 ..t 25 20 15 10 PPM Figure B122. H-coupled 3 1 P NMR spectrum (202 MHz; D 20 capillary) of the reaction mixture of 18 after silyl demethylation by BfMS and methanolysis. 226 0 0 II II MeO-P P.-OMe I '-.,/ \ MeO I OMe Br 29 35 30 25 20 15 10 Figure B123. 3 1 P NMR spectrum (202 MHz; CDCh) of29. I o(') ("") 00 o(') 0 r- N 00 r-- ~...;...; N N N \Y 0 II 0 II Meo-r......_...~-oMe MeO OMe 31 0 ("") ...; 0 0 0 II II + '==! Meo-r......_...~-o- Na .... MeO OMe 32 l~ I IV 0 0 II II Meo-r 1 ~-oMe MeO OMe Br 29 ("") r-- C'oi 0 0 II II + N MeO-P P.-0- Na ']\ 0 MaO OMe !...: Br 30 J I I 5 a> ""' to ("") LO M T"" LO a.a. oor-- ~~ ~~ y \( a> a> 0 '==! ! • .... .... I J j I PPM 25 20 15 1 0 5 PPM Figure B124. 3 1 P NMR spectrum (202 MHz) of the reaction mixture of monocle methylation of29 with EtaN in CH3CN. 227 Figure B125. Detailed view of the incoming nucleotide 14a-1, (S)-}3, y-CHCl dGTP in the active site of the X-ray crystal structure of its ternary DNA pol ~:DNA complex (PDB ID: 4DOC). The interatomic distance between the Cl (magenta) and N112 of Arg183 is 5.24 A. 228 Figure B126. Detailed view of the incoming nucleotide 14a-2, (R)-13,y-CHCl dGTP in the active site of the X-ray crystal structure of its ternary DNA pol I3:DNA complex (PDB ID: 4DOB). The interatomic distance between the Cl (magenta) and Nr]2 of Arg183 is 3.46 A. 229 Figure B127. Detailed view of the incoming nucleotide 14b-l, (S)-13,y-CHF dGTP in the active site of the X-ray crystal structure of its ternary DNA pol I3:DNA complex (PDB ID: 4DOA). The interatomic distance between the F (green) and N112 of Arg183 is 5.06 A. 230 Table B 1. HPLC conditions a Experiment Column Mobile phase Retention time 6a-1 Varian Microsorb 0.1 NTri- Separation of ethylammonium 10.5 min C,s HPLC column diastereomers (5 vm, 250 mm x bicarbonate, 3.5% of6a CHsCN, pH 7.2, 6a-2 21.4 mm) 15.0 mLfmin 11.5 min 7a-1 Varian Microsorb 0.1 NTri- Separation of ethylammonium 14.2 min diastereomers C,s HPLC column bicarbonate, 15% of7a (5 vm, 250 mm x CHsCN, pH 7.4, 8.0 7a-2 21.4 mm) mLfmin 15.2 min 7b-1 Varian Microsorb 0.1 NTri- Separation of ethylammonium 14.3 min diastereomers C,s HPLC column bicarbonate, 15% of7b (5 vm, 250 mm x CHsCN, pH 7.4, 8.0 7b-2 21.4 mm) mLfmin 15.5 min Analytical 7b-1 0.1 NTri- HPLC for LC- Varian Microsorb ethylammonium 23.6 min MS analysis of C,s HPLC column bicarbonate, 10% reaction (5 vm, 250 mm x CHsCN, pH 7, 1.0 7b-2 mixture of 7b- 4.6 mm) mLfmin 1/7b-2 27.2 min 13a-1 18.5 min (9 mLfmin) 0.5 N 13a-2 Purification of Macherey-Nagel Triethylammonium 18.8 min (9 Nucleogel SAX bicarbonate, pH mLfmin) 11a-1, 11a-2, 1000-10 (150 mm 7.4. Gradient: (0-10 13b-1 llb-1, llb-2 x 25 mm) min, 55% 10-16 min, 55% 16-25 21.2 min (8 min, 100o/<j mLfmin) 13b-2 19.8 min (8 mLfmin) 231 14a-l 14.4 min (9 mLfmin) 14a-2 Purification of Varian Microsorb 0.1 NTri- 14.2 min (9 C,s HPLC column ethylammonium mLfmin) 12a-l, 12a-2, (5 vm, 250 mm x bicarbonate, 3.5% 14b-l 12b-l, 12b-2 21.4 mm) CHsCN, pH 7.4 14.1min(8 mLfmin) 14b-2 14.5 min (8 mLfmin) a. Detection wavelength 256 nm. 232 Table B2. Crystallographic statistics of 14a-1, 14a-2, 14b-1 and 14b-2. 14a-1 14a-2 14b-1 14b-2 PDB ID 4DOC 4DOB 4DOA 4D09 Data Collection Space Group P21 P21 P21 P21 a (A) 50.69 50.60 50.64 50.69 b (A) 80.02 80.40 79.93 79.91 c (A) 55.72 55.63 55.52 55.61 J3 (0 ) 107.68 107.73 107.70 107.66 dmin (A) 1.95 2.05 2.05 2.05 Rrnerge ( 0 /~a, b 0.088 0.080 0.083 0.102 (0.430) (0.368) (0.395) (0.427) Completeness (o/~ 98.1 (86.5) 94.5 (68.3) 93.5 (65.2) 94.5 (68.3) Unique Reflections 25195 24826 24769 25195 ( 1817) ( 1663) (1717) (1817) Total Reflections 89573 86622 86288 89573 1/ cr 15.2 (1.93) 13.5 (2.5) 13.8 (2.5) 11.1 (2 .0) Refinement r.m.s. deviations Bond lengths (A) 0.012 0.014 0.013 0.007 Bond angles n 1.086 1.491 1.303 1.131 Rwork (o/¥ 19.98 20.93 19.49 18.84 Rfree (o/~ 24.90 26.28 24.33 24.31 ~verage B Factors '"' Protein 27.14 29.45 30.29 26.50 DNA 40.96 43.18 42.07 38.39 Analogue 19.64 19.73 20.17 16.26 Ramachandaran Analysis Favored 98.2 99.4 98.2 98.2 Allowed 100 100 100 100 ~umbers in the parentheses refer to the highest resolution shell of data (10%). CRvork = 1 00 X L IF obs I -IF ca!clfLIF obsl 233 Appendix C. Chapter 3 supporting data ""001.(')~0)1'-~CDI.(')T"""d'T"'('I")OOO toll')ll')ll')'<t'<tC")NOOltoll')N.-C") T"'T"'T"'T"'T"'T"'T"'T"'CDOOOOOOOOOO('I") ..t..t..t..t..t..t..t..tC"'iC"'iC"'iC"'iC"'iC"'iC"'i 1'--C")Oll'--'<tOOO"<t. ('I")NO,..._tca.nT""OCD r--r--r--tototototo.n NNNNNNNNN ?"~ 0 I CH2 ~)J? Eto-r ~-oEt 3 .92 3 . 90 3 .88 3 .86 3 .8 4 3 .8::PPM C") 0 C"'i 2.75 2.70 2. 65 2. 60 PPM 0 -----~ ~5 ~0 15 10 25 Figure Cl. 1 H NMR spectrum (400 MHz; CDCb) of2. ;;!) Ill ... N I EtO OEt 2 2.0 ?Ha 0 I CH 2 ~~~ Eto-r ~-OEt EtO OEt 2 ~ ~ ~ 0 Figure C2. 3 1 P NMR spectrum ( 162 MHz; CDCb) of 2. 1.5 -20 PPM PPM 234 en o o 10 oo en o en oo r-: !D !D \\I NT"" T""M 0 O'<!'C""lNID N "'~""""'"""'I"'" 0 ..,f..,f..,f..,f..,f ~Jl~ Eto-r ~-OEt 7 .10 7 .05 7 .00 6. 95 6 . 90 PPM .... r~ I J EtO OEt 3 7 6 5 4 3 Figure C3. lH NMR spectrum (400 MHz; CDCh) of 3. (D 0 .... ...; .... ~Jl~ Eto-r ~-OEt EtO OEt 3 40 30 20 10 0 Figure C4. sl P NMR spectrum ( 162 MHz; CDCls) of 3. 2 -10 \V .... .... PPM PPM 235 -~Jl~ - 0-P P-0 I \ + 0 0 4Na 4 0'> 0 00 0 .... 40 30 20 10 0 Figure CS. slp NMR spectrum (162 MHz; D 20; pH 8.9) of4. 20 0 "CNH 0 N~O R0-~-0-d I 0 OR 10 OR 6 0 -10 -20 -10 PPM -30 PPM Figure C6. slp NMR spectrum (202 MHz; D20 capillary) of6 in reaction mixture. 236 20 JNH '+ l .. ~ N='\ 0 N 0 ~N-~-0-, .o. I OR~ OR 7 10 0 N N a> 0 1 -10 -20 -30 PPM Figure C7. 3 1 P NMR spectrum (202 MHz; D20 capillary) of 7 in reaction mixture. 1'-a'l"<<"l'-a'l CO C') LO M 't""" I(')("')("')("')("') !D!D!D!D!D \IV 0 a> ""' ..,; 0 N (0 ..,; I I \I (0 ~ ("') (0 N ~ ..,;..,; I I \\ 0 ("')I(') ~N N 00 (0 NO N ~~ "<!"<"'> .,.; .,.;.,.; NN I I \~ 4.6 4.5 4.4 4.3 4. 2 PPM 2.50 2 .45 2 .40 2.35 2.3Cf'PM I(') a 0 ( .-i ) I(') 0 ( N 0 j 0 ( . } ~ I 8 7 6 5 4 3 Figure C8. 1 H NMR spectrum (400 MHz; D 2 0 ; pH 9.8) of8. ~ ""'" a> I(') C"! .C"! ~ ~ ""' 0 I ' C"'i 2 PPM 237 0 Y.x oJLo o N o HO,II "~0,11~00 ~ ~ ~ 0 U _ _ ~, HO OH OH OH 8 u J • - . {1.>4,1.74}~ {2.17,2.19} ' I • f4m..•.J7il.w; Q.'Il;J.'Il) (4A&,M6} ~ "" ) ' t {U4,1.15} ~ I {1.52,7.52}~ I U U ~ ~ ~ ~ ~ ~ ~ ~ ~ U ~ U U M ~ fZ(ppm) Figure C9. gCOSY spectrum (162 MHz; D20; pH 9.8) of 8. 238 N 0 "' c::i LO toi'-Nal"<t T""" cn,...._OI,(')CO 1,(') "''"""T""OOOC.O oO oO ,..: ,..: u:) u:) \\ \1 1/ 0 .... ~ ~~ J 8 10 5 0 -5 Figure ClO. 31p NMR spectrum (162 MHz; D 20; pH 9.8) of8. MOOMI'-LOtoNOON"<tOOl"<tON "d'OT""Nf"'-.C')C')CDCDcnLONf'-T"""f"o-.0 cnoo,...._c.o"'"'...:::tC"")NC.O"d'C"")C"")"f"""OCO,...._ ,..:,..:,..:,..:,..:,..:,..:,..:u;u;u;u;u;u;.,.;.,.; 0 00 (0 "'1'- 0"' 00 0 (0 (0 1'- 00 c::ic::ic::ic::i ~""';"""';""';'" 0 .... 0 -~ "Cx oJLo o N o HO~II 11,..0~11,..00 p p p 0 r~ 1 .... I I I HO OH OH I f ~ OH 8 - I -10 00 0 / . ' .... PPM 10 5 0 -5 -10 PPM Figure Cll. lH decoupled 31p NMR spectrum (162 MHz; D20; pH 9.8) of8. 239 0 0 II II HO-}'/(~-OH HOCI CIOH 10 25 20 15 1 0 5 0 -5 -1 0 Figure C12. 31p NMR spectrum (162 MHz; D 2 0; pH 9.5) of 10. o 0 o - ll)lll - + 0-P P-0 4Na I I _o o_ 11 en CIO ("') 0 40 30 20 10 0 -10 -20 -30 Figure C13. 31p NMR spectrum (162 MHz; D 2 0; pH 9.5) of 11. s -15 PPM -40 PPM 240

Abstract (if available)

Abstract 2’-Deoxynucleoside 5’-triphosphate analogues with β,γ-bridging oxygen replaced by a CXY group are useful chemical probes to investigate DNA polymerase catalytic and base‐selection mechanisms. A long‐standing limitation of such probes has been that conventional synthetic methods generate the analogues as mixtures of two diastereomer components in equal amounts when the bridging carbon substitution is nonequivalent (X ≠ Y). X‐ray structural studies of DNA polymerase β (pol β) carried out with ~1:1 mixture of β,γ-CXY dGTP diastereomers revealed stereospecific binding exclusively with the fluorine‐containing diastereomer analogues. These findings provided strong impetus in investigating the effect of stereochemical interactions of the β,γ-diastereomers on pol β kinetics, the stereochemical effect that could not be addressed in the previous linear free energy relationship (LFER) correlation plotting log(κpol)of the ~1:1 mixed diastereomers vs. the pKₐ₄ of the prochiral bisphosphonate leaving group. ❧ A general solution to the long‐standing synthetic challenge was developed with eight examples of β,γ-CXY dNTP diastereomers: (S)- and (R)-CHCl, (S)- and (R)-CHF, and (S)- and (R)-CFCl dGTP diastereomers, as well as (S)- and (R)-CHCl dATP diastereomers. Central to their preparation was the synthesis of the HPLC separable diastereomeric bisphosphonate synthons, the P,C-dimorpholidate derivatives of the (R)-mandelic acid monoesters of the parent prochiral bisphosphonic acids. Selective acidic hydrolysis of the P-N bond afforded "portal" diastereomers, which were readily coupled to morpholine‐activated dGMP or dAMP. Removal of the chiral auxiliary by hydrogenolysis gave the individual diastereomeric nucleotides, of which the diastereomeric purities were confirmed by ³¹P and/or ¹⁹F NMR spectroscopy. After treatment with Chelex‐100 to remove traces of paramagnetic ions, at pH ~10 the diastereomer pairs exhibit discrete Pα and Pᵦ ³¹P resonances, and discrete ¹⁹F resonances for fluorine-containing diastereomer pairs. The more upfield Pα and more downfield Pᵦ resonances, as well as the more upfield ¹⁹F NMR resonances for the β,γ-CHF dGTP diastereomer, were assigned to the R configuration at the Pᵦ-CHX-Pᵧ carbons, based on the absolute configurations of the individual diastereomers as determined from the X‐ray crystallographic structures of their ternary complexes with DNA and polymerase β. ❧ An NMR method that was developed to identify the individual diastereomers in their mixture solutions was successfully applied to analyze kinetic assays in which the (R)- and (S)-β,γ-CHX dGTP (X = F, Cl, Br) diastereomers were competing for pol β incorporation. Selective incorporation by pol β was observed for all three diastereomer pairs, with stereospecificities of 3 (β,γ-CHF), 7 (β,γ-CHCl) and 2-3 (β,γ-CHBr). The (R)-stereoisomer was preferentially inserted for both the β,γ-CHF and β,γ-CHCl analogues. In the case of β,γ-CHBr analogue, although the absolute configuration of the preferred stereoisomer has not been identified yet, the preferred isomer also showed more upfield Pα and more downfield Pᵦ resonances, similar to those of the (R)-CHF and (R)-CHCl stereoisomers. ❧ Pre‐steady‐state kinetics of pol β were performed with the fully characterized individual diastereomers of (R)- and (S)-β,γ-CHF dGTP and (R)- and (S)-β,γ-CHCl dGTP. For both diastereomer pairs, the (R)-stereoisomer was favored over the (S)-stereoisomer for G•C correct incorporation, with stereospecificities [(kpol/Kd)R/(kpol/Kd)S] of 3.8 and 6.3, respectively, and also for G•T misincorporation with stereospecificities of 11 and 7.8, respectively. The pre‐steady state kinetics and direct competition assays showed similar stereospecificities with β,γ-CHCl and β,γ-CHF diastereomer pairs for G•C correct incorporation.

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Asset Metadata

Creator Wu, Yue (author)

Core Title Novel stereochemical probes for DNA polymerases: nucleoside triphosphate beta,gamma-CXY analogues

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 12/13/2013

Defense Date 10/31/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag analogue,bisphosphonate,chiral,diastereomer,nucleotide,OAI-PMH Harvest,polymerase

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles E. (committee chair), Goodman, Myron F. (committee member), Qin, Peter Z. (committee member), Thompson, Barry C. (committee member)

Creator Email yuew@usc.edu,yuewu.joy@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-359801

Unique identifier UC11296554

Identifier etd-WuYue-2224.pdf (filename),usctheses-c3-359801 (legacy record id)

Legacy Identifier etd-WuYue-2224.pdf

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Document Type Dissertation

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Type texts

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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