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Substituent effects on acetal hydrolysis
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Substituent effects on acetal hydrolysis
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SUBSTITUENT EFFECTS ON ACETAL HYDROLYSIS by Lucy Ke-Ying Jao A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In P a rtia l F u lfillm e n t of the Requirements fo r the Degree MASTER OF SCIENCE (Biochemi s try ) August, 1965 U N IV E R S IT Y O F S O U T H E R N C A L IF O R N IA T H E G R A D U A T E S C H O O L U N IV E R S IT Y P A R K L O S A N G E L E S 7 , C A L IF O R N IA 3 ' \ e > J 3 ^ This thesis, written by Luch Ke-Ying Jao under the direction of h$X....Thesis Committee, and approved by all its members, has been pre sented to and accepted by the Dean of The Graduate School, in partial fulfillment of the requirements for the degree of Master of Science .............. ' ^ Dean D ate..... m g h s i j l s & S . . . . : . . . . . . . . . . . . ... THESIS C O M M ITTEE .... I \ • * . J /-) Chairm an y t? ., / ^ 3 £ ■ 2.-63— 2M— G 4 GRATEFULLY DEDICATED TO MY PARENTS ACKNOWLEDGEMENTS I am deeply indebted to Dr. Thomas H. F ife , under whose supervision the present work was done, fo r stim ulation and tra in in g in bio-organic chemistry. I p a r tic u la r ly wish to extend my sincere appreciation for his kind patience, thoughtful guidance, and encouragement throughout th is work. I would also lik e to thank the other members of my committee, Drs. Visser and Fluharty, fo r th e ir helpful c r itic is m and coopera tio n . I wish to express my sincere g ratitu d e to my colleagues, Miss Terry Laico and Miss S ally Howard, fo r t h e ir friendship and help during my stay at the U n iv ers ity of Southern C a lifo r n ia . TABLE OF CONTENTS PAGE INTRODUCTION ................................... 1 HISTORI CAL BACKGROUND.................................................................... 3 Acetal Hydrolysis .............................. . . . . . . 3 S tructural S im ila r it y of Glycosides .................................................. 5 Glycoside Hydrolysis. 6 Chemical Hydrolysis of Glycosides....................................................... 6 Acid Hydrolysis of Glycosides ........................ 6 A lk alin e Hydrolysis of Glycosides • ................................... 10 Enzymatic Hydrolysis of Glycosides . . . . . .................... 11 Design of A Model System fo r Glycosidase Hydrolysis. . 15 EXPERIMENTAL PROCEDURE AND RESULTS ................................................................. 18 Preparation of 2 - (Substituted PhenoxyJ-Tetrahydropyrans . 18 Preparation of 2-Phenoxy-Tetrahydropyran . . . . . . . 18 Preparation of 2- (ja-Ch 1 orophenoxy) -Tetrahydropyran • . 18 Preparation of 2- (jD-Methyl phenoxy) -Tetrahydropyran . . 18 Preparation of 2- (jD-Methoxyphenoxy)-Tetrahydropyran. . 19 Preparation of 2 - (o-Ch1orophenoxy)-Tetrahydropyran . . 19 Preparation of 2 - (ci-Methy 1 phenoxy)-Tetrahydropyran . • 19 Preparation of 2 - (o-Methoxyphenoxy)-Tetrahydropyran. . 19 Preparation of 2-(j>-Ni trophenoxy)-Tetrahydropyran . . 19 Preparation of Substituted Benzaldehyde Diethyl Acetals . 20 Preparation of Benzaldehyde Diethyl Acetal . . . . . . 20 Preparation of £-Methylbenzaldehyde Diethyl Acetal . . 20 iv V PAGE Preparation of p-Methoxybenzaldehyde Diethyl A ce tal. . 20 Preparation of p-Chlorobenza1dehyde Diethyl Acetal . . 20 Preparation of p-N itrobenza1dehyde Diethyl A cetal. . . 21 Preparation of m-Methy1 benza1dehyde Diethyl Acetal . . 21 Preparation of m-Methoxybenzaldehyde Diethyl A ce tal. . 21 Preparation of m -Nitrobenza1dehyde Diethyl A cetal. . . 21 Preparation of 2 - (Substituted Phenyl) - 1 ,3-D?oxolanes. . . 21 Preparation of 2 - (Phenyl) - 1 ,3-Dioxolane .............................. 22 Preparation of 2 - (p-Ch1oropheny1 }-1 ,3-Dioxolane . . . 22 Preparation of 2 - (p-Methylphenyl) - 1 , 3-Dioxolane . . . 22 Preparation of 2 - (p-Methoxypheny1 ) - 1 ,3-Dioxolane . . . 22 Preparation of 2 - (p -N itro p h en yl) - 1 ,3-Dioxolane . . . . 22 Preparation of 2 - (Substituted Phenyl) - 1 ,3-0xathiolanes . 22 Preparation of 2 - (Pheny1 ) - 1 ,3-0x ath io lan e . . . . . . 23 Preparation of 2 -(p -C h 1oropheny1 ) - 1 ,3-0xathiolane . . 23 Preparation of 2 - (p-Methoxypheny1 ) - 1 ,3-0xath io lan e . . 23 Preparation of 2 -(p -N itro p h e n y l)-l,3 -0 x a th io la n e . . . 23 Preparation of 2-(p-M ethylpheny1 ) - 1 ,3-0xath io lan e . . 23 Preparation of 2 - (m-Methy1pheny1 ) - 1 ,3-0xath io lan e . . 24 Preparation of 2-(m -M ethoxyphenyl)-l,3-0xathiolane . . 2 b K in e tic Measurements ............................................................ 24 Results . . . .........................................................................................................29 DISCUSSION........................................................................................................................42 SUMMARY AND CONCLUSION ...............................................................................................52 BIBLIOGRAPHY ....................................................................................................................... 54 LIST OF TABLES TABLE PAGE 1. Rates of Hydrolysis of 2 - (Substituted phenoxy)- Tetrahydropyrans .................................................................................... 29 11. Rates of Hydrolysis of Substituted Benzaldehyde Diethyl A c e t a l s .......................................................... 30 111. Rates of Hydrolysis of 2-(S u b s titu te d phenyl)- 1. 3-Dioxolanes . . ............................................ 31 IV. Temperature Dependence of Rates for Hydrolysis of Substituted Benzaldehyde Diethyl Acetals and 2 - (Substituted pheny1 )-1 ,3-Dioxolanes .................... 32 V. A c tivatio n Parameters fo r Substituted Benzaldehyde Diethyl Acetals and 2 - (Substituted phenyl)- 1.3-Dioxolanes ........................ 33 V I. Rates of Hydrolysis of 2 - (Substituted phenyl)- 1.3-0xathiolanes in HC1 ................................... . . . . . . 3^ V I 1. Rates of Hydrolysis of 2 - (Substituted phenyl)- 1. 3 - 0xath i ol anes in H C I O ^ ............................. 35 vi LIST OF FIGURES A Plot of log kr Values vs♦ cr Constants for the 2 - (Substituted phenoxy)-Tetrahydropyrans ......................... A Plot of log k^ Values vs. a Constants fo r the Diethyl Acetals of Substituted Benza1dehydes and the 2 - (Substituted pheny1 ) - 1 ,3-Dioxolanes . . . A Plot of log k^j vs_. cr+ fo r Diethyl Acetals of Substituted Benza1dehydes . ................................................. A Plot of log k^ vs_. cr+ fo r 2-(Substi tuted phenyl)- 1,3-0xath i olanes in HC1 ....................................... '“ jh / A Plot of log k^ vs. cr Constants fo r 2 - (Substi tuted phenyl) - 1, 3- 0xathiolanes in HCIO^ ........................................ '4 “ / A Plot of log k + Hq v s . cr Constants fo r 2 -(S u b s ti- tuted pheny1) - 1, 3- 0xathiolanes in HCIO^ . . . . . . INTRODUCTION Ever since Emil Fischer^ demonstrated in 189^ that yeast ex- 2 3 tra c ts hydrolyzed cv-methy 1 -g 1 ucoside and maltose, many studies have been done on the p u rific a tio n of g 1ycosidases, s p e c if ic it ie s of these enzymes and t h e ir enzymatic mechanisms. Although the study of glycosidases has continued fo r more than h a lf a century, th e ir mech anisms of action are s t i l l obscure. There are two known approaches to the study of mechanisms of enzymes: ( 1) the d ire c t study of enzymatic reactions and (2 ) through chemical model systems. Model systems seem to be a highly rational approach, employing the assumption that the mechanism of enzyme action does not involve any special chemistry but rather a combination of ordinary non-enzymatic chemical mechanisms. It would seem reasonable that before the mechanistic p o s s ib ilitie s by which an enzyme catalyzes a reaction can be considered, the basic chemistry of the non-enzymatic reaction must be known. Although i t has been found that acetals hydrolyze via an A -l^ mechanism, this mechanism cannot be applied to the enzyme-catalyzed hydrolysis of glycosides. Enzymes are proteins; th e ir c a t a ly tic functional groups are weak acids and bases, the side chains of amino acids at the a ctive surface. Optimum a c t iv it y of glycosidases is at n e u tr a lit y and a bell shaped pH-rate p r o file is obtained, which might indicate that two ionizable groups are involved in the c a t a ly tic a ctio n . I t is conceivable that these enzymes might act as bifunc tio n a l c a ta ly s ts , and catalyze the hydrolysis of glycosides via a general acid and general base mechanism. 2 A study of the e ff e c t of various substituents on acetal hydro lyses might shed lig h t on th e ir tra n s itio n sta te s. From th is in fo r mation an ideal model might then be designed to act as general acid and general base c a ta ly s t and so catalyze a reaction with the same e ffic ie n c y as an enzyme. Four series of substituted acetals were studied: ( l ) para- and ortho-subst?tu te d -2-phenoxv-tetrahydropyrans. (2 ) para- and meta- substituted benzaldehyde diethyl a c e ta ls , (3) 2- (substituted phenyl)- c 1, 3~dioxolanes, and (4) 2 -(s u b s titu te d p h e n y l)-!,3 -o x a th io la n e s . I I . HISTORICAL BACKGROUND A. Acetal Hydrolysis The fa c t that acetal hydrolysis takes place through acid cata lysis has been known generally fo r some time; however, Bronsted and 4 5 Wynne-Jones * did a series of experiments to study the nature of th is c a ta ly s is . In p a r tic u la r , they studied the e ffe c ts of a series of acid types and strengths on acetal hydrolysis. They found that c ataly sis by acids other than the hydronium ion is undetectable, and th is type of c a ta ly s is was termed " s p e c ific acid c a ta ly s is " to d i s t i n guish i t from general acid c a ta ly s is , th at is, c a ta ly s is by any acid other than 1 yoniurn ion. The same type of work has been carried out by Kreevoy and Taft^ oh d if f e r e n t acetals hydrolyzed in a series of acids and buffers; they came to the same conclusion, i . e . , that hydro lysis of acetals is a s p e c ific acid-catalyzed reaction. There are two p o s s ib ilitie s fo r the mechanism of acid-catalyzed acetal hydrolysis. E ith e r a fa s t p re-eq u ilib riu m protonation step is followed by a rate-determ ining uni molecular breakdown of the protonat- ed interm ediate, a mechanism termed A - l , or an A-2 mechanism involv ing a tta c k by a solvent molecule on the protonated acetal takes place. Ingold^ has proposed an "A -l" mechanism for acid-catalyzed hydrolysis of simple a c e ta ls , as follow s: + H R O R1 H O R^ ^ O R 1 R \ r / 3 C ——i — » / C . slow step c — OR* '''OR1 ' fa s t R 'OR' There are fiv e lines of evidence that support th is mechanism: ( l ) The point of cleavage was proven to be between the aldehyde carbon and alcohol oxygen.®»^>10 po|_ exarnp je> j n ^ g studies of O'Gorman and 8 Lucas, an o p tic a lly a ctive d i-s -b u ty l acetal was used and its hydro!- ysis in d ilu te aqueous phosphoric acid was studied. The s p e c ific ro ta tio n of the product alcohol recovered from the reaction was e s s e n tia lly identical w ith that o f the s ta rtin g alcohol. I f the mechanism involved formation of a carbonium ion at the butyl carbon, i there would be considerable racem ization. (2) The logarithm of the rate constant for acetal hydrolysis in concentrated ,acid solution was found to be proportional to the Hammett A c id ity F u n c t i o n . ^ ^ 13 14 Zucker and Hammett ’ had previously obtained results which showed that in reactions involving a unimolecular breakdown of a conjugate acid, the log of the rate constant is proportional to the Hammett A c id ity Function HQ; on the other hand, i f a reaction involves a water molecule in the tra n s itio n s ta te , then the logarithm of the rate constant w i l l be proportional to the stoichiom etric acid concentration + . . ^ 3 0 * (3) The rate constant for acetal hydrolysis is three times fa s te r in D2O than in H2 0 . ^ ’ ^ Values ^ of the r a t io of ^ r e la t iv e rates of reactions catalyzed by acid or base in D2O and H2O 5 have been used as a c r ite r io n to d istinguish between s p e c ific and general acid or base c a ta ly s is . I t has been found e m p iric a lly that the rate constants fo r s p e c ific acid c a ta ly s is are 1.4 - 3 .0 times fa s te r in D2O than H2O. Furthermore, A-l reactions are characterized by rate constants 2-3 times fa s te r in D2O , than in H2 O, while rate constants fo r A-2 reactions are 1 .4 -1 .7 times fa s te r in D2O than in H2O . ^ Rate constants for general acid c a ta ly s is are 0 .3 - 1 .4 times fa s te r in D2O than H2O. (4) Entropy of a c tiv a tio n v a lu e s ^ j2 0 ,2 1 fo r acetal hydrolyses have been found to be small p o sitive values. Long 22 e_t aj[. have suggested that the entropy of a c tiv a tio n for the A-2 mechanism must be less than that of the A -l mechanism, since the water molecule involved loses its tra n s ia tio n a I and ro tatio n a l fr e e dom. Typical values for the a c tiv a tio n entropies of acetal hydrolyses 9 fi 07 are near zero or p o s itiv e . (5) Volumes of a c tiv a tio n ’ J fo r acid- catalyzed acetal hydrolyses were found to have small positive values. 20 K o s k ik a llio and Whalley have concluded that the volume of a c tiv a tio n would be small and p o sitive fo r unimolecular reactions and negative fo r bimolecular reactions. B. S tructural S im ila r it y to Glycosides The structures of glycosides are very s im ila r to those of simple a c e ta ls . The glycosidic linkage can be regarded as an acetal bond in the following formula: R=H fo r aldose sugars and Ch^OH fo r 2-ketose sugars; the group CR1R1 'R11 1 is the aglycone. Glycosidases catalyze hydrolysis to the 6 parent sugar (hem iacetal) and the alcohol RI R"R,nC0H. The glycosidic linkage features many in te re s tin g points fo r the 2 h study of enzyme mechanisms 1. An asymmetric carbon atom forms part of i t . I t is well known th at only certain types of reactions involving an asymmetric carbon w i l l change the absolute configuration about that atom. In th is respect, any configurational changes or lack of them found in bio chemical transformations of a glycosidic bond may favor or rule out c ertain chemical mechanisms fo r the action of an enzyme. For example, i f the reaction proceeds v ia an SN2 mechanism (nucleophi1ic s u b stitu tio n second o r d e r )» an o p t ic a lly active product w i l l be ob tained in which Walden inversion has occurred at the asymmetric center; i f the reaction proceeds v ia an SN| mechanism (nucleophi1ic i su b stitu tio n f i r s t o rd e r), a racemized product might be obtained. i • ' • 2. The aglycone ra d ic a l, C R ^ 'R " 1, may be anything from a simple a lk y l or aryl radical to another long chain of glycosyl residues. The s p e c ifitie s of the glycosides toward chemical or enzymatic hydrol ysis are dependent upon both the sugar residue and the aglycone. C. Glycoside Hydrolysis 1) Chemical Hydrolysis of Glycosides The chemical hydrolysis of the glycosidic linkage can take place under two d if f e r e n t sets of conditions and by two separate mechanisms. The acid -catalyzed hydrolysis of glycosides is very s im ila r to that of simple a ce ta ls; the a 1ka1i-cataly sed hydrolysis is apparently unique fo r glycosides. a) Acid Hydrolysis of Glycosides 7 Generally, the glycosidic linkage is re a d ily broken by acid hydrolysis, the ease depending on both the nature of aglycone and the sugar. I t was found, as a ru le, that glycosides w ith a lip h a tic aglycone groups (methyl and ethyl glycosides) are more resistan t to acid hydrolysis than those with aromatic aglycone groups ’ Although various glycosides have a considerable d ifferen ce in the a c tiv a tio n energies, th is d iffere n ce does not seem to be related 27 e n t ir e ly to the aromatic or a lip h a tic character of the aglycone group . 25 In the acid hydrolysis of D-g1ucopyranosides with various aglycones, the rate increases in the order e th y l, methyl, benzyl, phenyl. The structure of the pyranose ring also plays a large role in 28 the hydrolysis of the glycosides. I t has been shown by Paul that 2- 0-methyltetrahydropyran (methyl 2 , 3 , 4 , -trideoxyaldopentopyranoside) is ra p id ly hydrolyzed by d ilu te acid at room temperature. The rates of hydrolysis of the ethyl 2 , 3 ,-d id e o xy -, and ethyl 2-deoxy-, and 29 ethyl Qf-D-glucopyranoside decrease in the order named . I t is, th e re fo re , obvious th at the su b stitu tio n of hydroxyl groups on the tetrahydropyran structure g re atly decreases the ease of hydrolysis 27 of the glycosidic linkage. Overend e t a l. concluded that e le c tro n ic e ffe c ts undoubtedly contribute to the g re a tly enhanced l a b i l i t y to acid of 2-deoxyglucosides, removal of the inductive e ffe c t of the 2 - hydroxyl group enhancing both the formation and the decomposition of the conjugate acid in the acid-catalyzed hydrolysis. 30 Reeves found that methyl o-D,-glucopyranoside, which has the most stable conformational structure of the hexopyranosides, is also 8 the most slowly hydrolyzed, the resistance to hydrolysis in members o f the series becoming less marked as more in s t a b ilit y factors are introduced. The s ta b iliz in g e ffe c t of su b stitu tio n on carbon 5 is illu s t r a t e d by the increasing resistance to hydrolysis of the series of methyl glycosides of cz-D-lyxose, Q'-L-rhamnose, o-D-mannose and D- Glycer-o'-manno-heptose, having the o-D-mannopyranose structure in which the substituted groups on carbon 5 are: -H, -CH^, -CH2OH, -CHOH-CH2OH. 31 The acid-catalyzed hydrolysis of glycosides has been found to 32 proceed via an A -I mechanism. Evidence includes: ( l ) p a ra lle lis m between the rates of hydrolysis of methyl and phenyl 01- and 3-D-gluco- pyranosides and the Hammett A c id ity Function H0 » (2) isotopic tracer experiments33 which demonstrated that the D-glucosyl-oxygen bond i s ruptured in the h ydrolysis, and (3 ) the values of entropies of a c tiv a - 2c 34 tio n , ’ which f a l l between +10 and +20 e . u . , indicating a unimole- 22 cu lar mechanism. There are two possible A -l mechanisms for acid-catalysed hydrol ysis of glycopyranosides involving glycosyl-oxygen bond fis sio n and •34 intermediate carboniurn ions:J t j 9 GLYCOSIDE H HO slow h eterolysi % A-l (B) mechajyk 0 > s 1ow heterolys i s A r 1(A) mechan i sm + h2 o OH +0H -0H ;t OH -ROH +H--.0 H, OH H ,0H sugar or > substituted sugar These two schemes are k in e t ic a lly indisting uishable from each other, 35 although scheme A is associated w ith an oxygen isotope e f f e c t . Shafizadeh and T h o m p s o n ^ ’ 37 have drawn a tte n tio n to much circum s ta n tia l evidence in favor of a mechanism involving ring-opening. p Vernon^ ’ 38 ancj coworkers present d ire c t evidence, i . e . stereo- | i chemical inversion in acid-catalyzed methanolysis and an oxygen isotope- i e ffe c t in acid hydrolysis of glucosides, which leads to the general j } conclusion th at acid-catalyzed solvolyses of a - and P -D -g1ucosides do . j not involve ring-opening at any stage. Scheme A is being accepted as 10 a basis fo r the discussion of the experimental results here. b) A lk alin e Hydrolysis of Glycosides 39 I t is usually considered v that the glycosidic linkage* is highly resistan t to a lk a lin e hydrolysis to a point where simple alk yl glucosides remain unattacked unless extremes of temperature and a lk a li concentration are used. However, the property of a l k a l i - s e n s itiv it y is c le a r ly a function of the aglycone, and is only s lig h t ly affected by the sugar residue. The a lk a 1i-s e n s itiv e glycosides may be c la s s ifie d into three types: ( 1) glycosides of phenols, (2 ) glycosides of enols conjugated with a carbonyl group, (3 )glycosides of alcohols substituted in the "3" position by a negative group. It is evident that the aglycone which imparts a 1ka1i - s e n s i t i v it y to the glycosidic linkage is one which may act to withdraw electrons from the glycosidic linkage. For example, the n it r o group is a strong j i electron withdrawing group. When i t is present in a phenyl glycoside,! such as p-nitrophenyl glycosides, i t makes the glycoside about equally! la b ile to acid and a l k a l i ; dinitrophenyl glycosides are unstable in I neutral s o lu tio n . | The mechanism of the a lk a lin e hydrolysis of glycosides Is con- ! sidered to be an SN2 "backside displacement". The hydroxyl groups on j C-2 and C-6 (in a hexoside) become involved; the C-2 group normally j must be trans to the glycosidic linkage for the a jk a lin e hydrolysis to! i occur. I 11 HO HO \,OH ( CH20H 0 I CH20H ch2— 0 O H £l 0 L 1 Lindberg ’ studied a series of trans- and ci s-methy1 pyranosides HoNyH / in highly a lk a lin e so lu tio n . He found that the tra n s -methyl pyrano sides hydrolysed much fa s te r than c? s-methyl pyranosides. These results are in favor of the "backside a tta c k ". . 2) Enzymatic Hydrolysis of Glycosides hydrolysis of glycosidic bonds, i . e . bonds in which the reducing group of the sugar is involved, but i t includes not only enzymes acting on simple glycosides, but also enzymes such as the amylases, which hydrolyze glycoside bonds in polysaccharides. There are two k 2 types of glycosidases: exoglycosidases and endoglycosidases; the former remove the terminal non-reducing sugar from the chain and the la t t e r cleave internal glycosidic linkages;, Most substrates fo r th is class of enzymes have no charged groups. In general, each enzyme is s p e c ific fo r a p a rtic u la r monosaccharide ring, but the attached aglycone group may have a more or less marked influence, and in some cases the enzyme may be as s p e c ific fo r the A ll the enzymes in th is c la s s if ic a tio n are concerned w ith aglycone as fo r the sugar. 12 The point of rupture of the glycosidic linkage in the enzymatic- 43 44 catalyzed hydrolysis ’ has been found to be at the glycosyl-oxygen bond rather than at the oxygen-aglycone bond. 24 The configuration of the products re su ltin g from glycosidase- catalyzed hydrolysis is, as a ru le, the same as the substrates; the exceptional case is o-amylase, which inverts the configuration of the products. 45 Glycosidases give b ell shaped pH-rate p ro file s , with V at max • approximately neutral pH. A bell shaped pH-rate p r o file is charac t e r i s t i c of a reaction which depends on two ionizing groups. The apparent rate constants for the reactions decrease sharply on both sides of the pH optimum. There are many mechanisms postulated fo r the action of g lyco si dases. A good example of the m ultistep approach necessary to propose p lausible enzyme mechanisms is that fo r a-amylase. a-Amylases, I analogous to endopeptidases, catalyze the hydrolysis of internal glycosidic linkages in a polysaccharide polymer, to give the product 46 with retention of configuration . The carboxy1-anion and protonated h is tid in y l imidazole have been proposed to be at the active s it e . The 47 follow ing mechanism for Q'-am'ylase was proposed: 13 :=o c-=o r 0H /N CHoOH OH HO HO HO h2o =0 H HOr HO OH H - N ^ TTTTTTT1 At the a ctive s ite of or-amylase, the protonated h is tid in y l imidazole acts as a general acid c a ta ly s t by protonating the glycosyl oxygen, w hile the carboxyl anion acts as a nucleophile, attacking the C-l atom. This mechanism fo r enzymatic hydrolysis can be described as general acid-general base c a ta ly s is . General acid c a ta ly s is , that is, c a ta ly s is by any proton donor 48 other than the hydroniurn-ion, includes three cases Case 1: a proton tran sfers from the general acid to the sub s tra te in the rate determining step, then the protonated intermediate rap id ly gives the fin a l product. S + HA * SH + A (slow) SH -* products (fa s t) 14 Case 2: hydrogen bonding takes place - between the general acid and the substrate in a fa s t eq u ilib riu m step, then th is intermediate breaks down in a slow step not so lely involving a proton tra n s fe r. S + HA —..... > S * HA (fa s t) S * HA + R ------> products (slow) Case 3: a fa s t p re-eq u ilib riu m protonation between the general acid and the substrate is followed by a rate-determ ining step involv ing the conjugate base of the general acid. S + HB < > SH* + B (fa s t) SH+ t B < — products (slow) In a ll cases the rate of general acid-catalyzed reactions can be expressed as the follow ing: Rate = (substrate) 21 kj (HA.) Case 1 and case 3 can be used to describe the mechanistic role of the carboxyl anion and the protonated h is tid in y l imidazole in the o-amylase catalyzed hydrolysis. In case 1, slow protonation of the substrate by general acid is analogous to the protonated h is tid in y l imidazole protonating the glycosyl oxygen. In case 3, the protonated intermediate reaction with base is indisting uishable from the carboxyl i anion acting as a nucleophile in attacking the protonated glycoside. j I There is at present no known analogy in organic chemistry fo r j i any mechanism of simple acetal or glycoside hydrolysis involving j • I i general acid-general base c a ta ly s is , and therefore there is no chemicalj i basis for any proposed enzymatic mechanism u t i l i z i n g th is type of i i c a ta ly s is . I t is thus apparent that before progress can be made in j i the e lu cid a tio n of the enzymatic mechanism, jthe jphemistry of_a_cetal_____ j 15 hydrolysis must be fu rth e r studied so that the p o s s ib ilitie s for the enzymatic reaction may be known. 3) Design of A Model System for Glycosidase Hydrolysis Since a-amylase appears to involve the hydrolysis of a p a r tic u la r g 1ycosidic 1inkage u t i l i z i n g a carboxyl anion and a proto nated imidazole as c a ta ly s ts , studies have been in it ia t e d to in v e s ti gate the c ataly sis of acetal hydrolysis by each group. Since i t has been proposed that n u cleophilic attach by carboxyl anion is involved k~] in the rate determining step of a-amylase hydrolysis , i t was of in te re s t to study nu cleo p h ilic p a rtic ip a tio n in acetal hydrolysis. Such p a rtic ip a tio n would be optimal i f i t were intram olecular. The reason that acetal hydrolysis is normally an A-l reaction is that the energy gain from s t a b iliz a tio n of a carbonium ion in a bimolecular reaction is not enough to compensate for the loss of tra n s la tio n a l entropy in going from a unimolecular to a bimolecular reaction. Trans it g la tio n a l entropy is not lost in intram olecular reactions. F ife studied the hydrolysis of Y .y -d ie th o x y b u ty ric acid in acid and found some evidence for nu cleo p h ilic p a rtic ip a tio n by the carboxyl anion a t neutral pH. The pH-rate p r o file showed that s p e c ific acid c a ta ly s is took place at low pH and th at nu c leo p h ilic p a rtic ip a tio n might take place close to the pK of the carboxyl group. The second order rate constant for hydrolysis of the anionic species is 35 times larger than the rate constant for the s p e c ific acid-catalyzed hydrolysis of the acid species. A mechanism was proposed to account for th is rate enhancement. > ° C2H5 J h c h 2 xo c 2 h c h 2 \no c 2 h c h2 \ c h 2 I r -H+ .> | \ * I ,0 » j CH ^ p ^ O " *---------- CH2 _ r J ) - C H ^ p / CH„ 2 5 6 2 I co oh Even though th is rate enhancement of 35 times is l i t t l e compared 6 10 to the enzymatic ra te , which is about 10 -1 0 times greater, never theless an intram olecular p a rtic ip a tio n of carboxyl anion in this case is s ig n if ic a n t, in showing th is type of mechanism to be chemically fe a s ib le . Knowledge of the tra n s itio n state for acetal hydrolysis is very important for the design of more e f f i c i e n t model compounds. The two p o s s ib ilit ie s for the nature of the tra n s itio n state in acid-catalyzed acetal hydrolyses are a carbonium ion and a protonated a c e ta l. The present work was undertaken to study e le c tro n ic e ffe c ts on a c id -c a ta lyzed acetal hydrolysis to obtajn knowledge of whether the tra n s itio n state resembles a carbonium ion or a protonated a c e ta l. To in te rp re t the results i t was found necessary to use the Hammett e q u a tio n .^ > 5 1 Hammett equation is used to study the polar e ffe c t th at substituent groups exert on the rate of the reaction. The equation log k /k Q = po was developed for the reactions of benzoic acid d e riv a tiv es and has found most success in applications where the reaction center is conjugated with a meta or para-substituted benzene ring, but the re la tio n has been found to hold for other cases also. I f the tra n s itio n state fo r a reaction has carbonium ion character, then resonance in te ra c tio n would be expected between the substituent and the reaction center; a curved po p lo t would then be obtained. In order to compensate fo r this resonance in teractio n between a substi- 17 tuent and the reaction center, and to a tta in a s tra ig h t lin e r e la - + 52 tio n sh ip , o’ constants were developed. J III. EXPERIMENTAL PROCEDURE AND RESULTS A. Preparation of 2 - ( Substituted Phenoxy)-Tetrahydropyrans The procedure of Woods and K ra m e r^ >5^ WaS followed: 2 , 3 , - dihydropyran (0.15 mole) containing 2 drops of concentrated hydro c h lo ric acid was slowly added to the appropriate phenol ( 0 .15 mole) in an ice bath. The mixture was allowed to stand a t room temperature fo r 24 hours and then tran sferred in to a separatory funnel containing NaOH (10% by weight, 50 ml) s o lu tio n , and shaken w e ll. The organic m aterial was extracted in to eth e r and then dried over anhydrous sodium s u lfa te . The ether was removed by flash evaporation, and the residue d i s t i l l e d under vacuum. 2-Phenoxv-Tetrahydropyran Properties: colorless liq u id , b.p. 90-92 (3.5mm), 22 nD 1.5219 Lit. : b.p. 103 (4mm) np 1.5228 2 - (p-chlorophenoxy)-Tetrahvdropyran Properties: white c ry s ta ls , m.p. 48-49°C L i t . 55 : m.p. 48.5-49°C 2 - (p-methylphenoxy)-Tetrahydropyran Properties: colorless liq u id , b.p. 98-99 (3mm) 21 nQ 1.5176 Anal. : calcd. fo r C12H16O2 : C, 75.00; H, 8.33 found: C, 75.48; H, 8.52 i i 19 2 - ( p-methoxyphenoxy)-Tet rahydropyran P roperties: colorless liq u id , b.p. 120 (1.5mm) 24 nD 1.5244 Anal. : calcd. fo r c > 69.30; H, 7.69 found: C, 6 9 . 16; H, 7.76 2 - (o -ch 1orophenoxy)-Tetrahydropyran Properties: colorless liq u id , b.p. 114-115 (3.5mm) 23 nD 1.5341 Anal. : calcd. forCj ^H|^C10 2 : C, 62.11; H, 6.12 found: C, 62.00; H, 5.95 2 - ( o-me thy 1phenoxy)-Tet rahydropy ran Properties: colorless liq u id , b.p. 95~96 (4mm) 23 np 1 .5 1 5 0 Anal. : calcd. fo r C, 75.0; H, 8.33 found: C, 75.29; H, 8.18 2 - ( o-me thoxyphen oxy) -T e t rahyd ropyran P roperties: colorless liq u id , b.p. 106 ( . 8mm) 23 nD 1.5299 Anal. : calcd. for C^HjgO^: C, 69.23; H, 7.69 j found: C, 6 9 . 15; H, 7.60 J 2 - ( p-n i trophenoxy)-Te t rahyd ropyran i 1 The preparative method for th is compound is a m odification i i | of the general method. 2 , 3 -dihydropyran ( 0.075 mole) and p - n itr o - ; | phenol (0.075 mole) in 50 ml of benzene was s tirre d in a dry oxygen j j | j free nitrogen atmosphere fo r two hours, and then a pinch of p-toluene- i | * — . J : ^ s u lfo n ic acid was added as c a ta ly s t. The reaction mixture was s t ir r e d . 20 fo r three more hours and then washed w ith NaOH (2% by w eight, 50 ml) so lu tio n . The organic m aterial was extracted into ether and then dried over sodium s u lfa te . The ether was removed by flash evapora tio n , and the residue was c ry s ta liz e d from a mixed solvent of eth er and 1igroin ( 1: 1) . B. Preparation of Substituted Benzaldehyde Diethyl Acetals 56 The procedure of Claisen was followed: The appropriate benz aldehyde d ieth yl acetal was synthesized by tre a tin g the commercially obtained benzaldehyde (0.15 mole) and absolute ethanol (0 .3 mole) with ethyl orthoformate (0.15 m ole). Two drops of e th an o lic HC1 was added as a c a ta ly s t. A fte r allowing the mixture to stand 24 hours | i at room temperature, anhydrous potassium carbonate was added to I I n e u tra liz e the excess acid . The excess ethanol was removed by I ! I 1 i flash evaporation and the residual liq u id was d i s t i l l e d under vacuum. ; j Benzaldehyde Diethyl Acetal j or Properties: colorless liq u id , b.p. 89 (7mm), nD 1.4771 L i t . 5 6 ,5 7 : b.p. 93-95 (11mm) pgeMethylbenzaldehyde Diethyl Acetal 25 Properties: colorless liq u id , b .p . 68-69 (2mm), np 1.4785 58 22 I L i t . : b.p. 105-6 (22mm), np 1.484-5 | p-Methoxybenzaldehyde Diethyl Acetal i 25 i p ro p e rties : colorless liq u id , b .p . 104 (3 . 5mm), np 1.4899j L i t . ' ^ ’ : b.p. 125 (41 mm), np ^ * ” * 1.4890 1 I p-Ch1orobenzaldehyde Diethyl Acetal . 2 5 P ro p erties: colorless liq u id , b .p . 92-93 (3mm), nD 1.4922| L i t .^1 : b.p. 108 (3mm)_______________________ _______ 21 p-N itrobenza1dehyde Diethyl Acetal P roperties: lig h t green liq u id , b.p. 135.5 (3.4mm), 25 nD 1.5082 L i t . ^ : b.p. 153-5 (6mm) m-Methylbenzaldehyde Diethyl Acetal 25 P roperties: colorless liq u id , b.p. 75 (3mm), nD 1.4838 r O n r L i t . : b.p. 190 (750.5mm), np 1.4841 m-Methoxybenzaldehyde Diethyl Acetal 25 P roperties: colorless liq u id , b.p. 90 (1.8mm), n^ 1.4896 Anal. : calcd. fo r C^HjgO^: C, 68.57; H, 8.57 found: C, 6 8 . 6 8 ; H, 8.54 m-NitrobenzaIdehyde Diethyl Acetal Properties: lig h t green liq u id , b .p . 114-5 (2mm), 25 nD 1.5053 62 L i t . : b.p. 145-146 (6mm) C. Preparation of 2 - (Substituted Phenyl) - 1 ,3-Dioxolanes 2 - (Substituted Phenyl) - 1 ,3-Dioxolanes were prepared by reac- , tin g the appropriate benzaldehyde ( 0.15 mole) with ethylene glycol (0.15 mole) in re flu x in g benzene (100 m l). A trace of p-toluenesu1 - fonic acid was added as a c a ta ly s t. Water was continuously removed from the reaction by azeotropic d i s t i l l a t i o n with benzene. At the conclusion of the reaction 2 ml of water had collected in the rece i ver. The reaction mixture was washed with NaOH solution (10% by weight, 50 m l). The ether e x tra c t was dried over anhydrous sodium s u lfa te . The benzene and ether were removed by fla sh evaporation _____ 22 and the residual liq u id was p u rifie d by d i s t i l l a t i o n through a Nester-Faust spinning-band column. 2 - (Phenyl) - 1,3-Dioxolane 25 Properties: colorless liq u id , b .p . 61-2 (1mm), np 1.5258 L i t . 63 : b.p. 106-7 (limm), n^° 1.5270 2 - (p-Ch1oropheny1 ) - 1 ,3-Di oxolane P roperties: colorless liq u id , b.p. 123.5 (11.5mm), np7 1.5373 L i t . 63 : b.p. 136-139 (13mm), n22 1.54.11 2-(p-Methy 1 phenyl)-!,3-D ? oxolane 25 Properties: colorless liq u id , b.p. 88 (2.4mm), np 1.5213 L i t . 64 : b.p. 123-5 ( 4 - 5mm), np° 1.5144 2 -(p -M eth o xyp h en yl)-l,3-D i oxolane P ro p erties: colorless liq u id , b.p. 97-98 (1.5mm), n25 1.5341 L i t . : b.p. 158-60 (17mm), np0 1.5362 2 -(p -N i trophenyl)-!,3^D ioxolane P roperties: white c ry s ta ls , m.p. 90 . • 64 __ r L i t . : m.p. 93.5 D. Preparation of 2-(S u b s titu te d pheny1 ) - 1 ,3-0xathiolanes These were prepared by reacting the benzaldehyde (0.255 mole) w ith P-marcaptoethanol ( 0.25 mole) in re flu xin g benzene (100 m l). A small amount of p-toluenesulfonic acid (0 .4 g .) was added as a c a ta ly s t. Water was continuously removed from the reaction by azeotropic d i s t i l l a t i o n w ith benzene. At the conclusion of the reaction, 2 ml of water had collected in the re c e iv e r. The reaction 23 mixture was washed with aqueous NaHCO^ solution and then thoroughly with d i s t i l l e d w ater. The ether e x tra c t was dried over anhydrous sodium s u lfa te . The benzene and ether were removed by flash evap oration and the residual liq u id was p u rifie d by d i s t i l l a t i o n through a Nester-Faust spinning-band column. 2 - (Phenyl) - 1 ,3-0xath i olane 24 P roperties: colorless liq u id , b.p. 83 (2.5mm), n^ 1.584-3 L i t . : b.p. 86-87 (5mm) 2 - (p-Ch1oropheny1)- 1,3-0xathi olane 23 Properties: colorless liq u id , b .p . 120 (1.7mm), np 1.5916 L i t . ; b.p. 124 (0.9mm) 2- (p-Methoxypheny1) - 1, 3- 0xath i olane 24 Properties: colorless liq u id , b .p . 123 (0.8mm), n^ 1.5824 Anal. : calcd. fo r C]o^1292^: 61.22; H, 6.12 found: C, 61.34; H, 6.18 2- (p-Ni tropheny1) - 1, 3- 0xathiplane Properties: yellow c ry s ta ls , m.p. 74°C L i t . 67 : m.p. 73-77°C 2- ( p-Methy1phenyl)- 1, 3 - 0xath i olane This compound was prepared according to the general proced ure except that NaOH (0.5% by weight 50 ml) was used to n e u tra liz e the reaction m ixture. Properties: colorless liq u id , b.p. 100 (0.75mm), np 1.5744 Anal. : calcd. for C, 6 6 . 6 6 ; H, 6 .6 6 found: C, 66.01; H, 6.54 24 2-(m -M eth ylp h en yl)-1, 3- 0xathiolane This compound was prepared according to the general procedure except toluene was used as solvent instead of benzene and the reaction mixture was washed w ith NaOH (2% by weight 50 ml) instead of NaHCO^ and water. Properties: colorless liq u id , b.p. 9 5 -5 _95*9 (0.75mm), np3 1.57H Anal. : calcd. fo r CjqH^OS: C, 6 6 . 6 6 ; H, 6 .6 6 found: C, 6 6 . 6 8 ; H, 6 .6 8 2- (m-Methoxyphenyl) - 1, 3- 0xath i plane This compound was prepared by the same method as 2-(m- Methy1 phenyl) - 1 ,3-0x ath i olane. , , r ^ 2 if Properties: colorless liq u id , b.p. 116 (0.65mm), n^ 1.5792 Anal. : calcd. for C|qH^202S: C, 61.22; H, 6.12 found: C, 61.09; H, 5.9^ E. K in e tic Measurements 1. The rates of hydrolysis of the 2 -(s u b s titu te d phenoxy)- tetrahydropyrans were measured in 47*5% ethanol-h^O ( v . / v . ) at a constant ionic strength of 0.1 M made up with KC1. At the observed pH value of 2.9 constant pH was maintained by excess hydrochloric aci d . The 2 - (substituted phenoxy)-tetrahydropyran was weighed into a 50 ml volumetric fla s k and the fla s k was f i l l e d to the mark with the acid solution and shaken vigorously u n til the m aterial was completely in so lu tio n . The solution was then allowed to come to constant 25 temperature (30 + 0.04) by thermostating in a constant temperature bath. At each time in te r v a l, 2 ml of sample solution was pipetted in to a culture tube (150 x 20mm) containing 5 ml of a 0.1 M K0H solu tio n , containing 50% ethanol, on the ice bath. The tube was shaken vigorously in order to insure n e u tra liz a tio n of the acid by K0H. The reaction mixture was then tran sferred to a stoppered cuvette and placed in the thermostated spectrophotometer. Rates were measured by follow ing the increase in optical density at the wavelength of maximum absorption fo r each phenolate ion. The reactions were followed to 50-70% completion. The pH of each k in e tic run was ro u tin ely checked at " i n f i n i t e time" i . e . , approximately 48 hours a fte r the apparant end po in t. The pseu d o -first order constants (k . , ) were obtained from the slopes of plots of log (O.D. oo - 0 . D . , ) / obsd. I ( O . D . ^ -0 .D . ) vs. time. ' oo t A ll spectrophotometric studies were conducted on a Model PM Q , 11 Zeiss spectrophotometer. A ll pH measurements were made on a Model 22 radiometer pH meter at the same constant temperature employed in the k in e tic runs. The k in e tic results are presented in Table 1. A s tra ig h t line was obtained when loq k . was plotted vs. o, the Hammett substi- 3 obs. ^ tuent constant, w ith a p value equal to - 0 . 8 2 , as shown in fig u re 1. 2. The rates of hydrolysis of the substituted benzaldehyde dieth yl acetals and 2- (substituted phenyl) - 1, 3 “dioxolanes were 68 measured in 50% dioxane -water ( v . / v . ) at a constant ionic strength of 0.1 M made up w ith KC1. The rates were measured spectrophoto- m e tric a lly w ith a Zeiss PM Q. 11 spectrophotometer by follow ing the in- 26 crease in optical density due to the aldehyde product, at the wave length of maximum absorption fo r each compound. The rates were g enerally followed to 75% completion. I n f i n i t y points were taken at 10 h a l f - l i v e s and again at 20 to 30 h a lf - liv e s and were s ta b le . The p seu d o -first order constants (k0bsc(.) were obtained from the slopes of plots of log (O.D. ^ -0 .D . j ) / ( 0 . D . <*> " 0 . 0 . t ) vs. time. Constant temp eratu re was maintained in the k in e tic runs by c ir c u la tin g water at 3 0 + 0. 1 , from a Haake Model F.constant-temperature c ir c u la tin g bath, through a Zeiss Constant-temperature c ell holder. The apparent pH of each solution was measured on a Model 22 Radiometer pH meter standardized with aqueous b u ffers . At pH values less than 3 .0 , pH was held constant by excess HC1. At pH values above 3 .0 , formate and acetate buffers were employed. Varying the concen tra tio n of b u ffer at constant pH had no e ffe c t on ra te . The change in rate of hydrolysis produced by varying the substituent group was so large th at i t was not possible to study a ll of the compounds at one pH value. In order to make a comparison, th e re fo re , a second-order rate constant was calculated fo r each compound by d ivid in g k0ksd> by a^|. The glass electrode gives the correct pH reading in concentrated 69 dioxane-water mixture . I f the pH meter reading in th is mixed solvent d iffe re d from the true pH there would be an e rro r in the absolute values of the second-order constants, but the r e la tiv e rates fo r the compounds studied would be c o rre c t. Hydrolysis of acetals in 50% dio- xane-water over the pH range studied gives plots of log k0(jSC ) vs * apparent pH which are s tra ig h t lines with slopes of - 1 . 0 . In work u t i l i z i n g 9 9 . 8% D2O as solvent, the glass electrode correction formula 27 70 71 of F ife and Bruice was employed in the determination of ap The a c tiv a tio n parameters were calculated from the following 72 equations : 1. Free Energy of A c tivatio n (AF*) = -RT In • h kT 2. Enthalpy of A ctivatio n (AH*) = -RT^ din kt dT 3. Entropy of A ctiv atio n (AS*) - A h* AF* T Where -1 -1 k^ = rate constant = 1. mole sec -27 h = Planck constant = (6624 + 0.002) x 10 erg sec. k = Boltzmann constant = (1.380) x 10 ^ erg deg. ^ R = Gas constant per mole = 1.98 c a l. T = Absolute temperature = 303 °K = 30°C Enthalpies of a c tiv a tio n were obtained from the slopes of plots of log k0ksc| vs. 1/T (Equation 2 ) , and points were obtained at four temperatures (20, 30, 40 and 5 0 °C ). The rates were measured in t r i p lic a te at each temperature with an average deviation of 2% in the rate constants. The k in e tic results are presented in Table 11 - V and in figures 2 - 3 » 3. The rates of hydrolysis of compounds [in the series of 2- (subst?tuted pheny1) - 1, 3- oxathiolanes were measured in 40% dioxane- water ( v . / v . ) at various acid concentrations. I t was not possible to maintain constant ionic strength in these rate measurements due 28 to the wide range of acid concentrations involved. The rate measure ments were performed in exactly the same way as in the case of the d i oxolanes. The k in e tic results are presented in Tables Vl-Vlf and in figures 4 -6 . Table 1 Rates o f H y d ro ly s is o f S u b s titu te d 2 - (P h en o x y)-te tra h yd ro p yran s in 47.5% Ethanol Water3 k x 102 Compd. X, ro jjL 'P obsd min , X=0CH| Y=H Y 307 4.66 X=CH3 Y=H 297 4.213 X-H Y=H 289 2.46 X=C1 Y-H 301 1.94 X=N02 Y=iH 400 0.565 X=H Y=0CH3 289 10.52 X=H Y=CH3 290 1.738 X=H Y=C1 295.5 1.890 3 T = 30+ 0 .1 °, |i.=0,1M., apparent pH= 2.90. ^ Wave length at which appearance of product was followed. Table I I Rates o f h y d ro ly s is o f S u b s titu te d Benzaldehyde D ie th y l A c e ta ls in 50% D ioxane-W ater or 50% Dioxane -Deuterium 0?cidea Compd. * 0 -CH(0C2H5 ) 2 A, PH kobsd min."^ k ^ ,1.mole"' min."^c kp, 1.mo min.” 1 Y-H V X=0CH3 - 283 5.79® 0.0473 29,200 11 Y=H X-CH3 258 4 .7 6 f 0.0685 3,937 111 Y=H X=H 280 4 . 12f 0.0549 723.3 IV Y=H X-CI 257.5 2.67 0.388 181.3 541.1 V Y=H X=N02 267 1.30 0.0923 1.84 5.01 VI y=och3 X=H 254.5 5 . 00f 0.00453 453.1 VI 1 “< II o □c X=H 252 4 .4 6 f 0.0406 1,170 VI 11 y=no2 X=H 250 2.36 0,0108 2.48 a . - o b P= -3.35 3 T=30 * 0 .1 °, p,=0.1M. Wave length at which appearance of product was followed. C kH=kobsd/aH d|<D = kobsd/aD S Acetate b u ffe r- f Formate b u ffer. 31 Table 11 I Rates of Hydrolysis of 2 - (para-Substituted Phenyl)-1,3~Dioxolanes in 50% Dioxane-Water 3 or 50% Dioxane-Deuterium Oxide Compd. X ‘ mmb pH ^obsd k^,l mole""^ kg, 1 .mol« IX x=och3 283 4.09s 0.0670 824.1 X x=ch3 258 2.99 0.122 119.2 335.9 XI X=H 280 2.36 0.111 25.4 70.1 XI 1 X=C1 257.5 1.72 0.118 6.19 17.8 XIII x=no2 267 1.30 0.00271 0.0541 0.177 P=-3.35 aT=30 + 0 .1 °, [i=0.lM. ^Wave length at which appearance of product was followed. 1 < H = kobsd/aH. ^D = ^obsd/aD. e Formate buffer. Table IV c Temperature Dependence of k ^ - j for Hydrolysis of and 2-(para-Substituted p h en ylJ-l,3” Dioxolanes in Substituted Benzaldehyde Diethyl 50% dioxane-Watera ^obsd> min.” ^ ! Compd. pH pp. 198-220. 32. Long, F . A ., Proc. Chem. Soc., 220 (1957). 33. Bunton, C .A ., Lewis, T .A ., L lew llyn , D.R. and Vernon, C .A ., J . Chem. Soc., 4419 (1955$, 34. Semke, L .K ., Thompson, N.S. and W illiam s, D.G., J. Org. Chem., 2 S L » 1041 (1964). 35. Banks, C.B ., Meinwald, Y ., R h in d -tu tt, A . J ., S heft, I . and Vernon, C .A ., J. Chem. Soc., 3240 (1961). 36. Shafizadeh, F. and Thompson, A ., J. Org. 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Jao, Lucy Ke-Ying
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Substituent effects on acetal hydrolysis
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Graduate School
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Master of Science
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Biochemistry
Degree Conferral Date
1965-08
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chemistry, biochemistry,OAI-PMH Harvest
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