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Studies of bisphosphate-conjugated fluorescent imaging compounds and 8-oxo-dGTP derivatives

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Studies of bisphosphate-conjugated fluorescent imaging compounds and 8-oxo-dGTP derivatives

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Content i STUDIES OF BISPHOSPHONATE-CONJUGATED FLUORESCENT IMAGING COMPOUNDS AND 8-OXO-dGTP DERIVATIVES by Yiying Zheng ______________________________________________________________________________ A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) May 2020 Copyright 2020 Yiying Zheng ii DEDICATION To my family. iii ACKNOWLEDGMENTS First and foremost, I would like to thank Professor Charles McKenna for accepting me to his research group, suggesting the research topics to me, providing me the chance to pursue my independent research, and giving me the opportunity to collaborate with great coinvestigators (Drs. Ichiro Nishumura, Akishige Hokugo, Kenzo Morinaga and Hiroko Okawa from Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry and Prof. Myron Goodman from Department of Molecular Biology, USC). Your advice is always welcomed and appreciated. I would like to take this opportunity to thank our UCLA collaborators for their kind permission to include their biological data in my thesis. Second, I would like to thank Dr. Boris Kashemirov for not only for his help in chemistry on a regular basis, but also for the great conversations throughout the years. It was a pleasure to learn chemistry from you, while discussing work ethics and life perspectives from different angles. Thank you for all the support and encouragement. Third, I would also like to thank Dr. Eric Richard for his significant contributions in the Osteoadsorptive Fluorogenic Substrate (OFS) work and guidance while handing over the project. Our research was funded by U19CA177547 and Dornsife College of Letters, Arts and Sciences. My journey through graduate school would not be the same without good rapport from former and present McKenna lab members. The contribution from outside the group is equally important from the inside. I would like to thank my screening, qualifying and defense committee members*: Profs. Andrey Vilesov, Chao Zhang, Katherine Shing, Peter Qin, and Ralf Haiges for their insightful feedbacks. As well as the teaching and lab staff who I have worked with for the last five years*: Drs. Jennifer Moore, Rebecca Broyer, Thomas Bertolini, Zhang Xiaojun, and Mr. Thuc Hoc Do. And the students who I have encountered from all walks of life. The personal stories that you shared will forever be sealed in my heart. I appreciate the help given from the USC Chemistry iv staff for their great work on instrument maintenance and administration*: Allan Kershaw, Inah Kang, Magnolia Benitez, Michele Dea, and VWR staff Daryl. Thanks to the Women in Chemistry (WiC), where I have launched the Twitter page (@usc_wic), and the Women in Science and Engineering (WiSE) for always putting in so much thoughtful efforts to support women graduate students. Next, I would like to thank my family for their unwavering support and patience they demonstrated throughout my PhD journey. Letting me travel halfway across the world alone to pursue my dream was a constant worry for them. I could not be more appreciative and grateful of my family who takes long-haul flights every year to visit and be always available for call or text, whenever I need someone to talk to. Last but not least, a special shout-out to the Singaporean and Malaysian community in Los Angeles for always organizing local events and opening up your places for gatherings so that each and every one of us could have a taste of home away from home. *Listed in alphabetical order (and not in order of preference). v TABLE OF CONTENTS DEDICATION ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES LIST OF SCHEMES ABSTRACT CHAPTER 1 Bisphosphonates and their Conjugates for Bone-Specific Drug Targeting 1.1 Introduction 1.2 Bisphosphonates 1.3 Structure Activity Relationship Studies of Bisphosphonates 1.4 Bisphosphonate Conjugates 1.5 Conjugate Design 1.6 Cancer Drug Conjugates 1.7 Osteoporosis 1.8 Imaging 1.9 Our Osteoadsorptive Fluorogenic Substrate (OFS) Design 1.10 Alternatives to Bisphosphonates 1.11 Conclusions 1.12 References CHAPTER 2 Osteoadsorptive Fluorogenic Substrate-1 (OFS-1) 2.1 Introduction 2.2 Chemical Synthesis 2.3 Experimental Section 2.4 Enzyme Kinetics Measurements 2.5 In Vitro Activation 2.6 In Vivo Live Imaging 2.7 Molecular Docking 2.8 References CHAPTER 3 Osteoadsorptive Fluorogenic Substrate-3 (OFS-3) 3.1 Introduction 3.2 Chemical Synthesis 3.3 Experimental Section 3.4 Enzyme Kinetics Measurements ii iii vii viii x xi 1 1 1 3 5 8 9 13 16 18 22 24 25 26 47 47 47 49 51 56 62 65 75 78 84 84 84 86 88 92 vi 3.5 Analysis of Probe Efficiencies 3.6 Mouse Periodontitis Models 3.7 Imaging Results 3.8 Conclusions 3.9 References CHAPTER 4 Synthesis of 8-Oxo-dGTP and its β,γ-CH2-, β,γ-CHF-, and β,γ-CF2 Analogues 4.1 Introduction 4.2 Results and Discussion 4.3 Conclusions 4.4 Experimental Section 4.5 References BIBLIOGRAPHY APPENDICES APPENDIX A: Chapter 2 Supporting Data APPENDIX B: Chapter 3 Supporting Data APPENDIX C: Chapter 4 Supporting Data 93 94 98 100 102 104 104 104 107 112 112 127 139 158 171 179 vii LIST OF TABLES Table 1.1. Anticancer bisphosphonate conjugates. Table 2.1. Experimentally determined kcat and Km values derived using GraphPad Prism 7.04 obtained from the kinetic analysis of Z-FR-AMC with human cathepsins B, L, K and S at 25 ºC. Table 2.2. Experimentally determined kcat and Km values derived using GraphPad Prism 7.04 obtained from the kinetic analysis of Z-FR-AMC with human cathepsins B, L, K and S at 37 ºC. Table 2.3. Experimentally determined kcat and Km values derived using GraphPad Prism 7.04 obtained from the kinetic analysis of OFS-1 with human cathepsins B, L, K and S at 37 ºC. Table 2.4. Calculated log D values (MarvinSketch 15.12.14.0). Table 3.1. Components that make up OFS-1, OFS-2 and OFS-3. Table 3.2. Experimentally determined kcat and Km values derived using GraphPad Prism 7.04 obtained from the kinetic analysis of OFS-3 with human cathepsins B, L, K and S at 37 ºC. Table 3.3. Calculated log D values (MarvinSketch 15.12.14.0). Table 4.1. HPLC conditions. 11-13 57-58 58 59 78 85 93 94 127 viii LIST OF FIGURES Figure 1.1. Chemical structures of pyrophosphoric acid and bisphosphonic acid derivatives. Figure 1.2. Bisphosphonates bind to bone by chelation of calcium ions. Binding strengths displayed by bisphosphonates vary with varying side groups, which indicate further contribution to binding by those atoms. Figure 1.3. Chemical structures of clinically used bisphosphonic acid derivatives. Figure 1.4. Schematic diagram of bisphosphonate IQF probes (OFS-1, OFS-2, OFS-3) potentially selective to ctsk. Figure 2.1. Chemical structure of OFS-1 and major ctsk cleavage site determined by the LCMS analysis of fragments. Figure 2.2. Synthesis of OFS-1. Figure 2.3. LC-MS of OFS-1 after incubation with ctsk. Figure 2.4. Calcium phosphate-coated plates were pre-incubated with OFS-1 probe (5.00 µM) and human osteoclasts were seeded. Figure 2.5. Human osteoclasts cultured on calcium phosphate-coated plates pre-treated with OFS-1 (5.00 µM). Figure 2.6. OFS-1 for in vivo live imaging of multiple myeloma-induced aberrant osteoclastogenesis. Figure 2.7. Tooth extraction-induced osteoclastic jawbone resorption detected by OFS-1. Figure 2.8. Imaging Data for OFS-1. Figure 2.9. Chemical structure of Abz-HPGGPQ-EDN2ph. Figure 2.10. Computer modelling was performed to analyze the OFS probe efficiencies. Figure 2.11. One of the best conformations of OFS-2 predicted by ICM (score -42). 4 5 6 22 48 50 61 63 64 67-68 70-71 73-74 75 76 77 ix Figure 3.1. Synthesis of OFS-3. Figure 3.2. Experimental periodontitis generated in mice by placing a silk suture around the maxillary left second molar (M2). Figure 3.3. Gingival swelling (dotted line) developed on the ligature-induced periodontitis side of maxilla. Figure 3.4. Expression of pro-inflammatory cytokines. Figure 3.5. Relative distributions of bacteria at the phylum and genus levels identified by 16S rRNA gene sequencing. Figure 3.6. A micro-CT radiographic study revealed noticeable bone loss on Day 7. Figure 3.7. Imaging Data for OFS-3. Figure 3.8. IHC data of ctsk activity. Figure 3.9. OFS-3 Signal tissue penetration. Figure 4.1. Structures of 8-oxo-dGTP 17, β,γ-methylene (CH2)- 18, β,γ-fluoromethylene (CHF)- 19, and β,γ-difluoromethylene (CF2)-8-oxo-dGTP 20. Figure 4.2. 31 P NMR spectra of 17, 18, 19 and 20. 88 95 95-96 96 97 98 99 99 99-100 106 111 x LIST OF SCHEMES Scheme 1.1. Synthesis of RIS-linker using the “magic-linker” approach. Scheme 4.1. One-pot-three-step synthesis (Method A) of 8-Oxo-dGTP 17. Scheme 4.2. Synthesis of 8-oxo-dGTP 17, β,γ-methylene (CH2)- 18, β,γ-fluoromethylene (CHF)- 19, and β,γ-difluoromethylene (CF2)-8-oxo-dGTP 20 using Khorana’s morpholidate method (Method B). Scheme 4.3. Synthesis of 8-oxo-dGTP 17, β,γ-methylene (CH2)- 18, β,γ-fluoromethylene (CHF)- 19, and β,γ-difluoromethylene (CF2)-8-oxo-dGTP 20 using a combination of Bogachev’s and Jakeman’s methods (Method C). 20 108 109 110 xi ABSTRACT Bisphosphonates are stable analogues of pyrophosphate that contain two phosphonate groups bound to a central carbon atom. The presence of two phosphonate groups enables bisphosphonates to chelate calcium ion, which is found in bone mineral. Bone affinity and antiresorptive abilities appear to be two separate properties of bisphosphonates. The bone-binding properties of these compounds may serve to localize these drugs at the site of osteoblast and osteoclast activity, thereby inhibiting osteoclast-mediated bone resorption. The high bone affinity as well as the specific binding of bisphosphonates are attractive features that could be exploited for targeted delivery of drugs to the bone. This idea was further explored in the work presented in this thesis for various applications, including targeted delivery of therapeutics to the bone and bone imaging. Two categories of projects are covered in this thesis: 1) imaging-related studies and 2) non-imaging-related studies. The first chapter provides an overview of the general characteristics of bisphosphonates and their structure-activity relationship studies, an outline of the bisphosphonate conjugate designs and their applications to cancer, osteoporosis and imaging, as well as our osteoadsorptive fluorogenic substrate (OFS) design and bisphosphonate alternatives. The two subsequent chapters focus on work related to the synthesis, enzyme kinetics measurements, in vitro and in vivo studies of our two novel imaging probes, OFS-1 and OFS-3. The last chapter centers on the synthesis of 8-oxo- 2’-deoxyguanosine-5’-triphosphate (8-oxo-dGTP) and its β,γ-methylene (CH2)-, β,γ- fluoromethylene (CHF)-, and β,γ-difluoromethylene (CF2)-8-oxo-dGTP analogues. A tool kit consisting of 8-oxo-dGTP and its three analogues will be used to study the effects of leaving group on the nucleotidyl transfer mechanism as well as the fidelity of DNA polymerases (pols). 1 CHAPTER 1 Bisphosphonates and their Conjugates for Bone-Specific Drug Targeting 1.1 Introduction Bone metastases are secondary cancers that are extensively investigated since they strongly reduce the possibility of cure and affect the quality of life of cancer patients. The bone microenvironment supports tumor survival and growth, which facilitates the formation of metastases in bone. 1,2 Consequently, the interactions between bone and cancer have been studied. The most common primary tumors that metastasize to the bone include lung, breast, prostate, thyroid, renal and oral cancers as well as multiple myeloma. 3,4 Nearly 75% of patients with advanced breast or prostate cancer develop bone metastases, which greatly decreases the likelihood of survival. 5,6 Tumor development in bone has been shown to result from mutually stimulatory interactions between tumor and bone cells, specifically osteoblasts and osteoclasts. This has been described as the vicious cycle of bone metastasis. 7 Several studies have confirmed that bone metastases involve complex interactions between multiple cell types such as fibroblasts, immune cells like macrophages, Natural Killer (NK) cells, T and B cells, in addition to osteocytes, osteoblasts and osteoclasts. 4,8,9 This intricate interplay of interactions disrupts osteoclast- and osteoblast-mediated bone homeostasis. Eventually, the disturbance of the balanced communication between osteoclasts and osteoblasts leads to either osteolytic or osteoblastic lesions resulting in decreased or increased bone formation, respectively. The mechanism of formation of bone metastasis involves several steps which are like tumor metastasis into other non-osseous organs. 10 Briefly, primary tumor cells enter the bone microenvironment assisted by proteolytic enzymes, thereby escaping the host-surveillance 2 mechanism. Cancer cells with migration ability invade the bone marrow stroma and induce their own blood supply within the endosteal bone surface. Subsequently, these cancer cells can stimulate the osteoclastic or osteoblastic activity depending on the tumor nature. 10 Skeletal integrity is usually maintained by a moderate balance between osteoblasts (forming bone) and osteoclast (resorbing bone). In metastatic bone lesions, bone surfaces are often surrounded by both osteoclasts and tumor cells, where osteoclasts induce bone resorption in the presence of tumor cells. Biological factors that enhance the function of osteoclasts include parathyroid hormone- related peptide (PTHrP) in breast cancer and receptor activator of nuclear factor kappa-B ligand (RANKL) as expressed by osteoblasts. These factors stimulate osteolysis, thereby releasing several growth factors from the extracellular bone matrix that support tumor survival. Hence, a vicious cycle is established that promotes tumor growth and accelerates bone destruction. 3,10 Numerous methods have been exploited to interrupt this vicious cycle by interfering with osteoclast-mediated bone resorption. Based on the hypothesis that skeletal diseases can be treated by improving bone integrity and preventing the growth and survival of tumors, osteoclast inhibitors have been utilized as powerful tools to prevent bone metastases. 11 Even though current treatment modalities do not cure metastatic bone cancers, they considerably interfere with the progression of skeletal-related events and bone destruction. Recent publications have reviewed various molecular-targeted therapies for treatment of osteosarcoma 12 and novel therapeutic agents for treatment of bone metastases. 13 Among the novel therapies developed for bone metastases, bisphosphonate-based treatments are particularly attractive, since these drugs have a long clinical history as anti-resorptive agents and are extensively applied in combination therapies with other anti-cancer agents. 3 It is well known and reported that bisphosphonates are particularly effective in decreasing or preventing bone damage. These chemicals have been refined and altered to become drugs for clinical treatment of chronic diseases such as Paget’s disease, osteoporosis, fibrous dysplasia and osteogenesis imperfecta. 14–16 Currently, bisphosphonates have become a standard treatment for patients with malignant bone disease. Both preclinical and preliminary clinical data confirm that bisphosphonates can avoid cancer-induced bone loss and prevent bone metastasis in patients with early-stage cancer. 17,18 However, apart from their obvious therapeutic efficacy, bisphosphonates are also associated with serious adverse effects. Extensive use of bisphosphonates may lead to side effects such as gastrointestinal (GI) irritation, osteonecrosis of jaw (ONJ) and femoral fractures, 19 which has prompted the development of advanced alternatives. 20,21 Hence, bone-targeted therapeutics are particularly attractive to minimize undesired side effects and for advanced treatment of bone tumors, in view of their potential to locally increase drug concentrations to impart a maximum therapeutic effect and to possibly target multiple drugs to resistant tumors. 22 1.2 Bisphosphonates Bisphosphonates are analogues of inorganic pyrophosphate 23 that contain two phosphonate groups bound to a central carbon atom. The central oxygen atom in pyrophosphate is replaced with a carbon atom in bisphosphonates that allows for two side chains designated R1 and R2 (Figure 1.1). This P-C-P bond is resistant to enzymatic hydrolysis as opposed to the hydrolytically labile P-O-P bond in pyrophosphate. 4 Figure 1.1. Chemical structures of pyrophosphoric acid and bisphosphonic acid derivatives. Bisphosphonates inhibit bone resorption by binding to hydroxyapatite in the bone extracellular matrix (Figure 1.2). This phenomenon is further enhanced at high bone turnover rates. The specific binding of bisphosphonates to bone mineral helps to avoid unwanted side effects and improve their safety profile. 24 They are highly stable in biological systems and are secreted from the kidney in their original form. This high selectivity for mineral deposits and corresponding low systemic toxicity have inspired the clinical applicability and translation of bisphosphonates. 25 Bisphosphonates in general, have a higher affinity for hydroxyapatite than for other calcium-based minerals such as oxalate, carbonate or pyrophosphate. 26 This selective affinity makes it possible to target imaging and therapeutic agents. Metabolic bone diseases like bone metastases allow enhanced exposure of hydroxyapatite, which give rise to enhanced accumulation of bisphosphonate-conjugated agents at these pathological sites. These sites may include pathological calcifications in non-osseous soft tissues, which can be effectively targeted using bisphosphonate- conjugated imaging or therapeutic agents. 14,27 5 Figure 1.2. Bisphosphonates bind to bone by chelation of calcium ions. Binding strengths displayed by bisphosphonates vary with varying side groups, which indicate further contribution to binding by those atoms. 1.3 Structure Activity Relationship Studies of Bisphosphonates Generally, bisphosphonates are classified based on their generation and their chemical structure. Structurally, bisphosphonates are either nitrogen-free (non-amino-bisphosphonates; first generation) or nitrogen-containing (amino-bisphosphonates). Nitrogen-free bisphosphonates which are the first generation of bisphosphonates include etidronate (EHDP), clodronate (CLO) and tiludronate (TLN) (Figure 1.3). Nitrogen-containing bisphosphonates which are the newer generation of bisphosphonates include alendronate (ALN), pamidronate (APD), risedronate (RIS), zoledronate (ZOL) and ibandronate (IBN) (Figure 1.3). Therapeutic effects of bisphosphonates such as high bone binding affinity and anti-resorption are highly correlated to three structural factors. These include the nature of the R1 and R2 groups, as well as the P-C-P backbone motif. 6 Name R1 R2 Etidronate (EHDP) OH CH3 Clodronate (CLO) Cl Cl Tiludronate (TLN) H Alendronate (ALN) OH Pamidronate (APD) OH Risedronate (RIS) OH Ibandronate (IBN) OH Zoledronate (ZOL) OH Minodronate (MIN) OH Figure 1.3. Chemical structures of clinically used bisphosphonic acid derivatives. The presence of a hydroxyl group at the R1 position has a substantial effect on the binding affinity of bisphosphonates to the bone mineral. With R1 being either a hydrogen or halogen atom, bisphosphonates form a bidentate interaction with the metal atom formed by the two phosphonate groups. 28 The α-OH group at R1 position in bisphosphonates increases their chelating ability by creating a third interaction with the metal atom, in addition to the regular bidentate bond observed in bisphosphonates. 29 This tridentate binding exhibits enhanced affinity to hydroxyapatite. 30,31 The R2 group mainly influences the anti-resorptive activity which is the inhibition of bone resorption, observed for bisphosphonates. 32,33 R2 groups containing a primary amine that is 7 connected to bisphosphonate backbone by an alkyl chain, such as in APD and ALN, help increase potency by 10-100 fold in comparison to nitrogen-free bisphosphonates. 23,34 As a result, nitrogen- containing bisphosphonates emerged as a new class of bisphosphonates. This effect is not limited to primary amine nitrogen-containing bisphosphonates, but is observed in tertiary nitrogen- containing bisphosphonates, such as IBN. 35 The most potent bisphosphonates, including RIS, ZOL, and MIN share a common structural feature. They all contain nitrogen-containing heterocyclic rings. In addition to its presence in the R2 group, the nitrogen atom needs to have a critical distance from the bisphosphonate groups. 36 Bisphosphonates containing nitrogen in the R2 group exhibit a higher binding affinity as compared to nitrogen-free bisphosphonates. 23 The difference in bone-binding affinity between amino and non-amino bisphosphonates was attributed to changes in the zeta-potential of hydroxyapatite surfaces. The presence of positively charged R2 groups leads to positive charging of mineral surfaces upon binding of these bisphosphonates, thereby increasing subsequent binding to negatively charged phosphonate groups due to electrostatic interactions. 31 In addition, the higher affinity for hydroxyapatite of amino bisphosphonates compared to non-amino bisphosphonates is related to the formation of hydrogen bonds between the amino group of bisphosphonates and the hydroxyapatite surface. For instance, the amino moieties in alendronate allow additional interactions due to the formation of N-H-O hydrogen bonds on the hydroxyapatite surface. 37,38 These interactions indicate the strong affinity of bisphosphonates for bone and their potential applications for bone-targeting purposes. Besides the R1 and R2 groups, the P-C-P backbone is also crucial in conferring to bisphosphonates with their therapeutic activity. Many studies on the stability against hydrolysis have been performed for various classes of phosphorous compounds, including monophosphonates, P-N-P and P-C-C-P, and P-C-P compounds. 39 Phosphonic acids and P-C-C-P compounds are non- 8 hydrolysable. Only compounds with a P-C-P motif are stable and both phosphonate groups are also required for the pharmacological activity of bisphosphonates. 39 1.4 Bisphosphonate Conjugates As a result of the high bone affinity as well as the specific binding of bisphosphonates, bisphosphonate conjugates have been created to deliver drugs specifically to the bone. Bisphosphonate conjugate designs and their applications to cancer, osteoporosis and imaging will be discussed in subsequent sections. The classes of molecules that have been conjugated for bone delivery include anticancer and small molecule drugs, proteins, antibiotics, and imaging agents. 40– 44 The variety of conjugated molecules, combined with a range of possible linkers to the bisphosphonates, yields a wide array of potential treatments. Conjugation can target the molecule to the bone, reducing systemic exposure and increasing drug half-life and exposure at the site of disease. Conjugates also have the potential for combination therapy, as their bisphosphonate moiety provides the abovementioned activities, in addition to increasing bone mineral density (BMD), while delivering a second drug to the bone. Bisphosphonate conjugates and complexes are not new, as bisphosphonate complexes with radiolabeled ligands like 99m Tc have been in clinical practice for bone imaging for many years 45–49 and estrogen conjugates were tested in rats as early as 1996 50 . Decades have passed, since conjugate investigation began. Most applications remain at the preclinical stage without advancing to human trials or clinical use, despite demonstrations of bone localization, safety and/or efficacy. Consequently, the promise of this approach can only be realized with its move into drug development and clinical trials. 9 1.5 Conjugate Design Conjugates can have different designs that are controlled by three important factors: (i) the drug payload, (ii) the bisphosphonate, and (iii) method of conjugation. 51 The choice of the drug payload is defined by the target disease, and is typically a compound with proven clinical activity. The chemical properties and potential attachment sites of a drug payload as well as the choice of bisphosphonate moiety will influence the methods of conjugation. Further, different bisphosphonates provide a variety of biological activities, bone affinities and anti-resorptive mechanisms of action. The method for conjugation provides expansion and honing of conjugate function. Generally, the following design standards should be considered when conjugating bisphosphonates to other compounds: a) The bone-targeting affinity of bisphosphonates and biochemical efficacy of compounds such as drugs and fluorophores might be reduced upon chemical conjugation with bisphosphonates; b) The chemical stability of the linker between bisphosphonates and the compound of interest ultimately determines the biochemical functionality in vivo; c) Excessive consumption of the bone-binding groups of bisphosphonates through conjugation with other compounds might stimulate their accumulation in the liver or spleen, thereby resulting in systemic and non-specific accumulation; d) Delivery of biomolecules at the target site requires active release of conjugated drugs once the target site is reached. Drug and bisphosphonate can be directly conjugated with no linker (Table 1.1, 1-6), or small to large linker may be used to separate the individual drugs (Table 1.1, 7-12). The linker must be stable enough to prevent separation before bone localization, but excessive stability may prevent release of active drug moieties after bone binding. Linkers can be directly from a single bisphosphonate to a single drug moiety or can attach a bisphosphonate to a nanoparticle or polymer structure. The different bisphosphonate side chains attached to the geminal carbon as well as the 10 phosphates allow diverse linker chemistry. Linkers between conjugates can include amides, esters, thioesters, or phosphoesters, which are non-specifically cleaved in vivo. Proteins conjugated to bisphosphonate with disulfide linkers earlier on demonstrated in vitro targeting with loss of bone binding upon cleavage by physiological thiols. 52 However, disulfide-linked conjugates were shown not to be released from a mineral matrix in vivo despite cleavage in vitro, indicating the importance of confirmation of linker cleavage in animal models. 53 Linkers can also have target- specific cleavages, which further refine localization of drug release. These include osteoclast- specific linkers that are sensitive to cathepsin K (ctsk) or matrix metalloprotease (MMP), enzymes secreted by osteoclasts during resorption, or acid hydrolysable for release in the resorptive pit formed by osteoclasts. 54 Hydrolysable linkers can release a drug from the bisphosphonate conjugate, though released products must be ensured of full function of both moieties without any toxic byproducts. Conversely, design of a non-hydrolysable connection of functional moieties can ensure against diffusion of drug from the bone after bisphosphonate binding. Cathepsin-sensitive linkers allow release only upon arriving in an environment with the desired enzyme, as demonstrated in vitro with a bisphosphonate-doxorubicin conjugate with quick release of drug. 55 Agents with cathepsin-sensitive linkers between a bisphosphonate and chemotherapeutic molecule also demonstrate increased efficacy against tumor growth 56,57 in animal models. The length of linker may also have effects on drug binding and separation, though this is still unclear as greater conjugate binding rates was reported for shorter linkers, others have reported no effect on binding rate. 58,59 The possible combinations of bisphosphonate molecules, drugs and linkers seemingly allow accurate drug delivery systems to diverse targets while preventing systemic toxicity. 11 No. Ref(s) Structure 1 60 Methotrexate-Bisphosphonate Conjugate 2 61 PS-341 3 62–64 5-Fluoro-2'-Deoxyuridine-Alendronate Conjugate (5-FdU-Ale) 4 65 Cis-Diammine (P,P’-Diethyl Methylenebisphosphonato) Platinum(II) (DEBP-Pt) 5 66 Camdronate 12 6 43,67,68 Bisphosphonate Diethylenetriaminepentaacetic Acid (DPTA) Conjugate (DPTA/BP) 7 43,67,68 Bisphosphonate 5-Fluorouracil Conjugate (5-FU/DP) 8 43,67,68 Gemcitabine (Gemzar) 9 44,69 Cytarabine-Etidronate Conjugate 10 66 Trypdronate 13 11 55 Acid-Sensitive Doxorubicin Bisphosphonate Prodrug Cathepsin B (Ctsb)-Cleavable Bisphosphonate Prodrug 12 70 Pullulan-Poly(ethylene Glycol)-Alendronate (Pull-(PEG-ALN)) Color code: Red – anticancer parent drug; blue – bisphosphonate; black – linker. Table 1.1. Anticancer bisphosphonate conjugates. 1.6 Cancer Drug Conjugates Bone is an attractive target for cancer drug targeting as systemic anti-cancer treatment involves high toxicity and causes widespread adverse side effects, especially for bone neoplasms where higher doses are required to achieve the necessary concentrations at the site of disease. Bone- targeted cancer chemotherapy has been investigated in multiple studies from in vitro studies to 14 animal models. The anti-cancer properties of bisphosphonates may offer bifunctional treatment with further anticancer drug conjugation, even though bisphosphonate uptake is limited in most cells due to the charged nature of the bisphosphonate. Numerous studies do not fully explore effects from bisphosphonates on their conjugate beyond bone targeting, and they do not explore whether toxicity is reduced by the targeting action, yet many studies demonstrate increased efficacy with a bisphosphonate-anticancer conjugate. Several in vitro studies show increased efficacy when conjugating a traditional cancer chemotherapeutic with a bisphosphonate. However, many also lack convincing data for conjugate function. An earlier study showed that bisphosphonate and methotrexate were linked by a peptide bond and was successfully localized to bone (Table 1.1, 1). 60,71 A decade later, another bisphosphonate-methotrexate conjugate was shown to induce apoptosis in osteosarcoma (OS) cells in vitro but at a rate like the standard methotrexate OS treatment. 72 Bisphosphonate-conjugated proteasome inhibitors (Table 1.1, 2) exhibit strong toxicity on multiple myeloma cell lines. 61 A 5- fluoro-2’-deoxyuridine- alendronate conjugate (Table 1.1, 3) showed increased hydroxyapatite binding and cytotoxicity to cancer cells, but did not demonstrate separation of the two drug moieties. 63 The same conjugate was tested in a gastric adenocarcinoma cell line and showed slightly higher efficacy against cancer than non-malignant cells, but less sensitivity than to separate drug components, again indicating lack of release of individual components from the conjugate. 64 Another in vitro study created a paclitaxel, alendronate, and pullulan conjugate with a ctsk- sensitive linker (Table 1.1, 12) which assembled into a colloidal sphere and showed higher antiproliferative activity with the bisphosphonate than without in metastatic breast cancer and OS cells. 70 A dialkylbisphosphonate platinum complex (Table 1.1, 4) was created and tested in vitro with the idea to bring the antitumor effects of platinum to bone metastases. 65 A doxorubicin- 15 bisphosphonate conjugate with ctsb or acid-sensitive linkers (Table 1.1, 11) showed stability in plasma with quick release of drug, but only one of the compounds showed a higher mouse maximum tolerated dose (MTD) in mice than doxorubicin alone and efficacy has not been investigated. 55 Besides cell-based studies, numerous studies went beyond cell culture to pre-clinical animal models to ensure bone localization and antitumor efficacy. A 5-fluorouracil (5-FU) bisphosphonate conjugate (Table 1.1, 3) showed accumulation in bone with rapid clearance of unbound conjugate, 73 and labeling this 5-FU or diethylenetriaminepentaacetic acid (DTPA) bisphosphonate conjugate with 188 Re (Table 1.1, 6) showed bone accumulation for potential combination therapy. 74 A gemcitabine/bisphosphonate conjugate (Table 1.1, 8) labeled with 99m Tc or 188 Re was created by the same group, which was demonstrated to bind bone in vitro and localize to bone in vivo. The amide linkage allowed potential cleavage to release local concentrations of the drug in attempt to reduce the toxicity of gemcitabine and 188 Re when administered systemically. 43,67 This gemcitabine-bisphosphonate conjugate reduced the size and number of bone metastases in a mouse metastatic breast cancer model. 68 A esterolytic bisphosphonate- camptothecin conjugate (Table 1.1, 5) was created, which bound hydroxyapatite and hydrolyzed under physiological conditions to release the free drug. 66 Another structural design of the conjugates, where phosphate group of bisphosphonate was used to link to nucleoside-5’- monophosphate, thus providing an analog of nucleoside triphosphate capable of releasing both components at the bone, had been reported. 44 The etidronate-cytarabine compound MBC-11 (Table 1.1, 9) increased BMD and reduced incidence of bone metastases in mouse breast cancer and multiple myeloma models, 44 significantly outperforming the control groups treated with free cytarabine, free etidronate or the combination of free cytarabine and etidronate. These types of 16 conjugates were demonstrated to hydrolyze in mouse and human serum with a half-life in a scale of hours, suggesting time for bone binding with a release of the anticancer drug in the bone microenvironment. 69 Osteodex (ODX) and MBC-11 are two examples of bisphosphonate conjugates that have advanced to oncology clinical trials. ODX is a poly-bisphosphonate containing dextran, alendronate and guanidine with reported preclinical efficacy. 75–77 The phase I trial (NCT01595087) and phase IIb (NCT02825628) trial to evaluate efficacy and tolerability of ODX show that ODX acts as a brake medicine in metastatic castration-resistant prostate cancer. The ODX treatment slowed down the course of the disease in the skeleton of most patients who underwent the entire treatment for 5 months, thereby indicating stabilization of bone turnover markers. Further, the phase IIb indicates a high tolerability of the patients for ODX without serious side effects. MBC-11 reported reduction of cancer-induced bone lesions in several patients. The phase I study for patients with cancer-induced bone disease treated with MBC-11 is completed with maximum tolerated dose and indications of efficacy. 78 These examples have brought the approach to clinic and if successful, will be the first therapeutic bisphosphonate conjugates approved for human use. 1.7 Osteoporosis Even though degenerative bone disease is often treated with bisphosphonates, conjugating bisphosphonates to further inhibit bone resorption in pre-clinical studies have taken place. Conjugated molecules include those involved in the bone turnover balance. One example is a bisphosphonate conjugate with osteoprotegerin (OPG), a RANKL inhibitor preventing formation of osteoclasts, which accumulated in bone with twice the concentration of non-targeted OPG, and to an even greater extent in an osteoarthritis rat model with active bone remodeling. 79 Another 17 important regulator of bone turnover is estrogen, but systemic treatment may increase risk of breast or uterine cancers. Estradiol-bisphosphonate conjugates were also explored with one compound showing successful estrogenic activity in bones and not uterus, while others showed no effect. 50,80 Calcitonin, which acts through calcitonin receptors on osteoclasts to reduce resorption, is also taken up by other cells and has a short biological half-life resulting in minimum effect from systemic administration. Calcitonin retained activity and bound hydroxyapatite when conjugated, 81 and the conjugate showed superior ability to maintain bone volume and density in an ovariectomized rat model compared to free calcitonin. 82 A PEGylated calcitonin- bisphosphonate conjugate further improves stability in circulation and bone targeting. 83 Another regulatory molecule that lacks efficacy when administered systemically is parathyroid hormone (PTH). A PTH peptide was conjugated to a bisphosphonate molecule at its N-terminus and was shown to maintain its activity in cells. 59 To stimulate bone regeneration after osteoporotic losses, a bisphosphonate was conjugated with prostaglandin E2 (PGE2) for its anabolic effects on bone. This conjugate was designed with different linkers, which allowed the desired release of free PGE2, and the end result showed increased rates of bone growth over free PGE2. 84 A bisphosphonate conjugate with a PGE2 EP4 receptor subtype agonist was later developed for its increased stability compared to PGE2, and without the systemic side effects of the agonist alone. 85 The agonist-bisphosphonate conjugate reversed osteopenia in an ovariectomized rat model. 86 A proto-oncogene tyrosine-protein kinase Src kinase inhibitor was discovered with anti-resorptive activity, and by adding a targeting moiety showed in vivo protection against hypercalcemia. 87,88 Most recently, a conjugate drug named C3 which has a synthetic, stable EP4 agonist (EP4a) covalently linked to an inactive ALN, produces significant anabolic effects that reverse osteopenia, allows bone remodeling, and has the potential to treat postmenopausal osteoporosis. 89 18 1.8 Imaging The use of bone targeting for medical imaging has been successfully established for clinical use. High contrast bone imaging is important for the identification of bone metastases and diagnosis of metabolic and other bone disorders. Technetium-99m ( 99m Tc)-linked bisphosphonates are actively used in bone scintigraphy to image areas of high bone turnover. The ability of bisphosphonates to be linked to a γ-emitting technetium (Tc) isotope and their affinity for sites of high bone turnover with quick elimination from soft tissue has supported their use in imaging, and radiopharmaceuticals coupled to bisphosphonates are widely used in bone scans to identify and evaluate bone issues. 90 Clinically available conjugates include 99m Tc hydroxyethylidene diphosphonate ( 99m Tc-HEDP), 99m Tc methylene diphosphonate ( 99m Tc-MDP), 99m Tc hydroxymethylene diphosphonate ( 99m Tc-HMDP) 14,54 and 99m Tc-(bis)alendronate- diethylenetriaminepentaacetic acid (DTPA) conjugates are still in development 73,91 . Bisphosphonate conjugates with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) core were also designed for imaging and allow easy radiolabeling by complexation of a metal isotope 92 , and other chelation agents are being investigated as well 14 . A bisphosphonate- 68 Ga imaging system for Position Emission Topography (PET) 93 was developed, indicating that more imaging systems are still in progress and can further advance current techniques. Fluorescent conjugates for research applications and for greater resolution than scintigraphy have also been created. Fluorescent imaging allows a longer signal life than scintigraphy but is limited by depth of signal in tissue in humans, suggesting the desired application should determine the technique. 94–96 19 For instance, the McKenna group has developed a new synthetic approach known as the “magic linker” to obtain novel fluorescently labeled derivatives of RIS and its analogues, including its phosphocarboxylate and deoxy forms. 95,97 This method not only allows an active amino group that readily reacts with N-hydroxysuccinimide (NHS)-activated fluorescent dyes but also provides a great spot for further modifying the linker length between nitrogen-containing bisphosphonates and the fluorescent dye. Increasing the distance between the fluorescent dye and the pyridyl moiety of RIS-linker can be achieved by reacting the original RIS-linker with commercially available compounds containing a carboxylic acid and a tert-butyloxycarbonyl (Boc)-protected amino group at the other end. The fluorescently labeled bisphosphonate analogues are prepared by reacting the appropriate “magic” epoxide linker with heterocyclic nitrogen-containing bisphosphonates under mild and aqueous conditions (Scheme 1.1). The reaction progress is monitored using 31 P nuclear magnetic resonance (NMR) spectroscopy to reduce formation of undesired dialkylated product. This reaction introduces a primary amino group that allows for facile conjugation of the nitrogen- containing bisphosphonate linker with a succinimidyl ester-activated fluorescent dye, yielding the desired dye-bisphosphonate conjugate. The final conjugate can be easily purified using reversed phase high-performance liquid chromatography (RP-HPLC) performed on a C18 column. Characterization and purity can be assessed using ultraviolet-visible (UV-Vis), fluorescence and both 1 H and 31 P NMR spectroscopies. These probes enable visualization of the uptake of heterocyclic nitrogen-containing bisphosphonates by bones, bone tissues and cells. Their bone binding affinity and anti-resorption as well as anti-prenylation activity of the parent drugs are still partially retained with effective inhibitory concentration in the micromolar range. 95,98 20 Scheme 1.1. Synthesis of RIS-linker using the “magic-linker” approach. A pamidronate-pullulan conjugate with attached fluorescent or magnetic resonance imaging (MRI) moieties was demonstrated to be successful for binding hydroxyapatite and accumulating in regenerating bone tissue 99 . MRI and PET imaging could also benefit from improved bone imaging, and conjugation to a chelator such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 4-([bis-(phosphonomethyl))carbamoyl]methyl)-7,10-bis(carboxymethyl)-1,4,7,10- tetraazacyclododec-1-yl)acetic acid (BPAMD), or 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA) is required for these imaging techniques. 14 Mammography which is based on the imaging of micro-calcifications with X-rays, is the current gold standard for detection of breast cancer. The sensitivity and specificity can be increased by introducing fluorescent bisphosphonates that target hydroxyapatite. Conjugation of a near-infrared (NIR) fluorophore to pamidronate by an amide linkage resulted in an eight-fold increase in specificity for hydroxyapatite as compared to other calcium salts. 100 Soft tissue-embedded hydroxyapatite crystals were detected with high sensitivity and specificity. This is an important prerequisite for early breast cancer detection. Far-red fluorescent pamidronate (FRFP) has been introduced as a biomarker for the localization of bisphosphonates to monitor site-specific 21 accumulation of bisphosphonates in vivo. FRFP binds to bone tissue but does not compete with standard bisphosphonates. Consequently, measurement of bone surface area gives its quantification and its removal results from increased bone turnover. 101 These agents can also be combined with other fluorescent biomarkers to determine specific drug-cell interactions. Using this similar approach, a recent study described the quantitative evaluation of tumor volume and mineralization in an osteosarcoma mouse model using NIR dual-channel optical imaging. Briefly, 2-deoxyglucose was used along with an infrared (IR) fluorophore as a glucose monitoring agent, whereas pamidronate-IRDye (Osteosense) acted as mineral targeting agent. This dual-imaging system produced high-quality images of osteosarcoma lesions within shorter periods of time than conventional bone scintigraphy. 102 The biodistribution, circulatory half-life, and clearance route of pamidronate-functionalized NIR fluorophores was shown to be strongly influenced by the physicochemical properties of conjugated fluorophores (charge and hydrophobicity). 103 Since bisphosphonate is a negatively charged small molecule, the optimal balance of total surface charge and hydrophobicity after conjugation to NIR dyes is of significant importance to design targeted contrast agents for bone-specific imaging and optimized in vivo performance. Pamidronate- conjugated IR fluorophores localize in bone tissue which allows for real-time intraoperative imaging of bone metastasis, bone growth, and microcalcifications, as opposed to undesired non- specific accumulation in cartilage, liver, spleen and skin tissue. Hence, conjugation strategies allow the development of targeted imaging agents for the imaging technique of choice. Besides conjugation of fluorophores with bisphosphonates, gold nanoparticles were coated with alendronate to increase imaging contrast of damaged bone or microcalcifications associated with breast cancer. 104 22 1.9 Our Osteoadsorptive Fluorogenic Substrate (OFS) Design The use of bisphosphonate-selective fluorescent ctsk imaging agents which we developed based on a human ctsk-cleavable sequence is reported in this section. Our design of bone targeted, ctsk- potentially-selective intramolecularly quenched fluorescence (IQF) probe is illustrated (Figure 1.4). Besides using lysine as the trifunctional backbone, our probes consist of bisphosphonate, ctsk-cleavable peptide, fluorophore and quencher. Figure 1.4. Schematic diagram of bisphosphonate IQF probes (OFS-1, OFS-2, OFS-3) potentially selective to ctsk. Pamidronate and alendronate were chosen as high-bone-affinity bisphosphonates 105 with amino group available for coupling with the carboxy group of lysine. The carboxy group of lysine is first activated with Sulfo-NHS. Sulfo-NHS is used instead of NHS because of its higher solubility in aqueous solution. The half-life of activated ester is known to be 4-5 hours at 0 °C and pH 7, 106 1 hour at 25 °C and pH 8, 107 and 10 minutes at 4 °C and pH 8.6. 108 In our hands, the activation of the lysine, followed by the coupling with the bisphosphonate of our choice, and deprotection of the Boc group give low yield (5-20%), as a result of the poor solubility of the chosen bisphosphonate as well as the competing hydrolysis of the activated ester. 23 Ctsk-cleavable peptide is an important part of this probe. The substrate specificity of ctsk has been extensively studied, as the enzyme is an important drug target for the treatment of osteoporosis. 109 Among the mammalian cysteine cathepsins, ctsk was found to have unique preference for a proline residue in the P2 position, 110 the primary determinant of its substrate specificity. Thus, the histidine-proline-glycine-glycine-proline-glutamine (HPGGPQ) peptide was found as a highly selective ctsk substrate 111 , and was applied in our fluorescent probes. Three bisphosphonate bone-targeted ctsk-specific fluorogenic substrates, OFS-1, OFS-2 and OFS-3 were synthesized. They each contain the peptide sequence glycine-histidine-proline-glycine-glycine- proline-glutamine-glycine (GHPGGPQG), but their ctsk activation techniques vary dramatically. Our results demonstrate that incorporating a peptide sequence known to be cleavable by a specific protease is insufficient to ensure rapid activation. Dye structure, polarity and charge also appear to play a particularly important role. A requirement for efficient Förster Resonance Energy Transfer (FRET) is a good overlap between the emission spectrum of the fluorophore and the absorbance spectrum of the quencher. 112 Fluorophores were chosen for visible-light microscopy (OFS-1) and in vivo imaging in the NIR region (OFS-2 and OFS-3). Black Hole Quencher-1 (BHQ-1) is a quencher optimized to work with carboxyfluorescein (FAM)-based probes and has excellent spectral overlap with it. Sulfo- Cyanine5 (Sulfo-Cy5) and Alexafluor® 647 (AF-647) have very similar emission spectra with maxima at 668 nm, since they share the same pentamethine cyanine chromophore. However, AF- 647 has better solubility due to its two additional sulfonate groups and a better fluorescence quantum yield. Both Black Hole Quencher-3 (BHQ-3) and BlackBerry® Quencher-650 (BBQ- 650) have good spectral overlap with these dyes. 24 The chemical synthesis, enzyme kinetics measurements as well as in vitro and in vivo studies for both OFS-1 and OFS-3 will be elaborated in Chapters 2 and 3, respectively. The chemical synthesis and enzyme kinetics measurements of OFS-2 was done by our postdoctoral scholar, Dr. Eric Richard and hence would not be covered in this thesis. 1.10 Alternatives to Bisphosphonates Even though this topic is not the purpose of this review, it is important to recognize the limitations of bisphosphonates that have prompted investigation into other bone targeting moieties. Long-term inhibition of osteoclasts with bisphosphonates can lead to ONJ, nephrotoxicity, hypocalcemia, and ocular toxicity. 113 Bisphosphonates have low oral bioavailability with less than 1% adsorption from oral dosing, and may cause GI irritation in patients. Low adherence to oral drug regimens is problematic to many patients, whereas bone association of the molecule is rapid and dissociation is low and can take years. 24 Further, long-term effects may not be desirable in some cases, as long- term use can cause bone hardening leading to brittleness and ONJ. Non-osteoclastic bone cells do not take up bisphosphonates bound to matrix. 114 In addition, non-osteoclastic cells experience no detectable protein prenylation effects under conditions which strongly inhibit the same pathway in osteoclasts, 115 suggesting other cells do not take up bisphosphonates as osteoclasts, reducing off- target effects of the drug. The generally low toxicity, well-established mechanism, and availability of bisphosphonates create a simple and straightforward drug development pathway that has discouraged development of other targeting systems which are relatively more expensive and incur high manufacturing and production costs. A few bone-targeting strategies have been developed and undergone preliminary studies. For example, denosumab which is a human monoclonal antibody used for the treatment of osteoporosis, hypercalcemia, bone cancer and cancer-related 25 bone problems, has obtained clinical success. 116 Monoclonal antibodies, 117,118 small-molecule inhibitors, 119,120 RNAi, 121,122 tetracycline derivatives, 54,123,124 acidic peptides, 125–127 and biological and synthetic bone-binding molecules 128–130 have been demonstrated to successfully bind and target therapeutics to the bone. 1.11 Conclusions Bone-targeted therapeutics have the potential to significantly improve treatments for bone- associated diseases and cancers. The high mineral content of the bone hydroxyapatite matrix and tissue-specific cells provide a highly specific environment for multiple drug-targeting strategies. No one other class of molecules has been demonstrated to have all the properties that bisphosphonates bring to bone drug-targeting: high bone affinity, anti-cancer effects, inhibition of bone resorption, pharmacokinetic stability and accessible chemistry for application of conjugates. Substantial progress has been made with the use of bisphosphonates to directly treat degenerative bone disorders together with emerging evidence for improvement in treating bone cancers. Bisphosphonates are now being additionally investigated as targeting moieties to direct conjugated drugs to the bone microenvironment. Multiple drug classes have demonstrated success in specific delivery of drug to bone and improved efficacy of treatment. Hydrolysable and target-specific linkers between bisphosphonate and the conjugated drug allow release of drug upon bone binding. Numerous drugs and conjugation techniques have shown significant promise in drug targeting and efficient treatment of bone maladies such as osteoporosis. The elements of ideal bone-targeting therapies are known, with continuing bisphosphonate conjugate studies. Further advances in clinical trials will be made in the treatment of bone disorders as well as in the development of NIR fluorescent bone-specific contrast agents. 26 1.12 References (1) Sottnik, J. L.; Dai, J.; Zhang, H.; Campbell, B.; Keller, E. T. Tumor-Induced Pressure in the Bone Microenvironment Causes Osteocytes to Promote the Growth of Prostate Cancer Bone Metastases. Cancer Res. 2015, 75 (11), 2151–2158. https://doi.org/10.1158/0008-5472.CAN-14- 2493. (2) Futakuchi, M.; Fukamachi, K.; Suzui, M. Heterogeneity of Tumor Cells in the Bone Microenvironment: Mechanisms and Therapeutic Targets for Bone Metastasis of Prostate or Breast Cancer. Adv. Drug Deliv. Rev. 2016, 99, 206–211. https://doi.org/10.1016/j.addr.2015.11.017. 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In adult bone homeostasis, activated osteocytes are the primary source of the key regulatory cytokine of osteoclastogenesis, RANKL. 1 In contrast, RANKL derived from other cells associated with a number of bone diseases leads to overzealous osteoclastogenesis and bone destruction. For example, bone destruction and pain are a prominent pathological feature of multiple myeloma leading to a risk of skeletal morbidity. The skeleton is the most common organ to be affected by metastatic cancer and the presence of extraosseous disease and the extent of the bone disease are powerful predictors of outcome. Studies have shown a strong correlation between the rate of bone resorption and clinical outcome, both in terms of skeletal morbidity and progression of the underlying disease or death. 2 Therefore, an early detection of tumor-induced osteoclastic activity will aid to reach accurate diagnosis and prognosis. Ctsk is a cysteine protease produced by osteoclasts that is essential for degradation of type I collagen, elastin and gelatin in bone resorption. 3,4 Detection of ctsk activity both in vitro and in vivo offers a powerful strategy for studying the role of this enzyme in disease processes and for evaluating drugs that affect ctsk activity. Several fluorogenic probes activated by ctsk have been developed and tested in vivo. 5–7 They employ an IQF strategy wherein two mutually-quenching fluorophores (e.g. Cyanine 5.5) are connected by a peptide that constitutes a highly specific, cleavable substrate for ctsk. In the intact state, these molecules have minimal fluorescence. Ctsk- 48 catalyzed hydrolysis of the peptide permits separation of the paired chromophores by diffusion, curtailing quenching and thereby ‘turning on’ fluorescence. The scope of these probes in imaging ctsk activity in vivo after injection is limited because the intensity of the fluorescent signal decays due to rapid clearance of the fluorophore. 7 For specific, long-lasting detection of ctsk on bone surfaces populated by osteoclasts, modification of the probe with a bisphosphonate moiety is an attractive option. Bisphosphonates typically exhibit strong affinity for bone mineral, where they may remain adsorbed for weeks to years until released by a resorption process that dissolves the adsorbing hydroxyapatite matrix. 8 They have been conjugated with fluorescent dyes to provide diverse imaging reagents that bind to hydroxyapatite. 9–11 Extension of this approach to create bone-targeted fluorogenic sensors specific for a particular protease such as ctsk is desirable to achieve more specific imaging probes of osteoclast-mediated processes that occur at the bone surface. The synthesis of the first example of such a probe, OFS- 1 (Figure 2.1) and its utility in detecting bone resorption due to early disease in several mouse models of bone neoplasma and surgical injury will be discussed in subsequent sections. Figure 2.1. Chemical structure of OFS-1 and major ctsk cleavage site determined by the LCMS analysis of fragments. 49 2.2 Chemical Synthesis In designing the prototype probe (OFS-1), the orthogonally Fmoc- (which is stable under acidic conditions) and Boc- (which is stable under basic conditions) protected lysine was selected as a trifunctional linker. The carboxy group of the Nα-Fmoc-Nε-Boc-(L)-lysine was first activated with N-hydroxysulfosuccinimide sodium salt (Sodium Sulfo-NHS) and then coupled with excess pamidronic acid (APD) to give 1. APD was selected as the bisphosphonate auxiliary due to its known bone affinity 12 and its conveniently connectable aminoethyl side chain. Excess of APD was used due to the low reactivity of the amino group in the aminoethyl side chain of APD to make it more susceptible for coupling with the sulfo-NHS-activated lysine, which has a half-life of ~3 hours at pH 7.5. Sodium Sulfo-NHS was used instead of NHS to increase the solubility of the activated lysine in water. The coupling reaction with APD did not happen with NHS-activated lysine due to its poor solubility in water. Once APD coupled with lysine, the Boc group was removed using trifluoroacetic acid (TFA). The single-digit yield of 6% was because of the simultaneous hydrolysis of the sulfo-NHS-activated lysine at pH 7.5, rendering the lysine inactive, while coupling to sodium pamidronate formed from the neutralization reaction between sodium carbonate and APD. 1 was attached to 5-carboxyfluorescein (5-FAM), followed by the removal of the 9-fluorenyl-methyloxycarbonyl (Fmoc) protecting group using 20% piperidine in N,N- dimethylformamide (DMF) to give 2. To increase the reactivity of the free amine in 2, Boc-β- alanine (Boc-β-Ala) was used as a linker that allowed the amine to be less sterically hindered by placing it further away from the bulky 5-FAM group as well as the APD. The Boc group in β-Ala was removed with TFA to give 3. The customized peptide consisted of GHPGGPQG, a derivative of the known ctsk-selective sequence HPGGPQ 13 that was modified to avoid racemization. Further, it contained a Boc group to protect the free amine in glycine (G), and Trt groups to protect 50 the nucleophilic amine in histidine (H) as well as the amide in glutamine (Q). The peptide was first activated with N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) as the coupling agent. TSTU was chosen because it has excellent stability in DMF in closed vial and is efficient in converting the hydroxy group to water-compatible succinimidyl ester. 3 was linked to the C-terminus of the Boc- and trityl (Trt)-protected peptide. The Boc and Trt protecting groups were subsequently removed using a mixture of TFA, water and triisopropylsilane (TIS) to give 4. TIS was added in this reaction mixture as a scavenger in peptide synthesis. Black Hole Quencher- 1 (BHQ-1), a non-emitting FRET quencher was coupled to the N-terminus of the peptide to give 5, which is the OFS-1 product (Figure 2.2). Due to the large size of the molecule, the synthesized OFS-1 was only characterized by high-resolution mass spectrometry (Appendix A, Figure A-18). Figure 2.2. Synthesis of OFS-1. 51 2.3 Experimental Section General Experimental Information for Synthesis Nα-Fmoc-Nε-Boc-(L)-lysine, dicyclohexycarbodiimide (DCC) and TIS were purchased from Sigma Aldrich. N-hydroxysulfosuccinimide sodium salt and N-Boc- -Ala succinimidyl ester were purchased from Chem-Impex International, Inc. APD was provided courtesy of Novartis. TFA and TSTU were purchased from Oakwood Chemical. 5-FAM succinimidyl ester was purchased from ChemShuttle. Anhydrous DMF and tetrahydrofuran (THF), acetonitrile, ethyl acetate, methanol and triethylamine (TEA) were purchased from EMD Millipore Corporation and used without further drying. Sodium carbonate and sodium bicarbonate were purchased from Fisher Scientific. Custom synthesized peptide, Boc-GH(Trt)PGGPQ(Trt)G, was purchased from Applied Biological Materials Inc. BHQ-1 succinimidyl ester was purchased from Biosearch Technologies. 1 H and 31 P NMR spectra were obtained on Varian 400-MR 2-Channel NMR, Varian VNMRS- 500 2-Channel NMR and Varian 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 triplets (dt), triplet of doublets (td), doublet of doublet of doublets (ddd) or broad signal (br). All chemical shifts (δ) are in parts per million (ppm) relative to HOD in D2O (δ 4.79, 1 H NMR), external 85% H3PO4 (δ 0.00, 31 P NMR). 31 P NMR spectra were proton-decoupled, and 1 H and 31 P coupling constants (J values) were given in Hz. The concentration of the NMR samples was in the range of 1-3 mg/mL for intermediates. Preparative HPLC was performed using a Shimadzu LC-8A equipped with a LabSolutions Lite Release 5.71 SP2 software and Shimadzu SPD-10A UV detector (0.5 mm path length). Low-resolution mass spectrometry was performed on a Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI source in the negative ion mode. LC-MS was also performed on the Finnigan LCQ Deca XP Max mass spectrometer in 52 negative mode with a Thermo Finnigan Surveyor PDA Plus detector (1 cm path length) and MS Pump Plus, all controlled using Xcalibur software, version 2.0.7. High-resolution mass spectrometry was performed using a Q Exactive TM Plus Hybrid Quadrupole-Orbitrap TM mass spectrometer. 3-(Nα-Fmoc-Nε-Boc-(L)-lysineamido)-1-hydroxypropane-1,1-diyl)bis(phosphonic acid) (1): Nα-Fmoc-Nε-Boc-(L)-lysine (4.68 g, 10.0 mmol) was dissolved in anhydrous DMF (25.0 mL). N- hydroxysulfosuccinimide sodium salt (2.60 g, 12.0 mmol, 1.2 eq) was added. The mixture was sonicated to give a white suspension. DCC (2.06 g, 10.0 mmol, 1.0 eq) was added. The mixture was stirred for 2 hours. Reaction was monitored by TLC (100% ethyl acetate, visualized with UV 254 nm), starting material had a Rf value of 0.2–0.5 and product had a Rf value of 0. The mixture was centrifuged, and the supernatant was collected and concentrated under reduced pressure. In parallel, a suspension of pamidronic acid (7.06 g, 30.0 mmol, 3.0 eq) in water (100 mL) was treated with sodium carbonate until pH was 7.5. A solution of the activated ester in THF (25.0 mL) was added. A white suspension was observed. The mixture was stirred at room temperature overnight. The mixture was diluted with THF (188 mL), and then filtered. The filtrate was concentrated under reduced pressure and partitioned between 0.100 M triethylammonium bicarbonate (TEAB) buffer (pH = 8.5, 100 mL) and ethyl acetate (300 mL). The aqueous layer was extracted with ethyl acetate (300 mL × 3) and concentrated under reduced pressure. A mixture of TFA (9.50 mL) and water (0.500 mL) was added to the residue. The resulting mixture was stirred at room temperature overnight. Solvents were removed under reduced pressure. The residue was co-evaporated with methanol (50.0 mL) and dried in vacuo until the complete disappearance of 31 P NMR resonance signal at 17.62 ppm to give 1 (0.615 mmol, 6% yield), which was used in the next step without further purification. Amount was determined by UV absorbance (PBS buffer,  = 6566 at 300 nm). 53 1 H NMR (400 MHz, D2O)  7.84 - 7.66 (m, 2H), 7.66 - 7.43 (m, 2H), 7.43 - 7.14 (m, 4H), 4.71 - 4.46 (m, 3H), 4.45 - 4.26 (m, 1H), 4.13 (s, 1H), 3.90 - 3.67 (m, 1H), 3.47 - 3.11 (m, 2H), 2.81 (t, J = 7.7 Hz, 2H), 2.11 - 1.78 (m, 2H), 1.63 - 1.30 (m, 2H), 1.19 - 0.85 (m, 2H). 31 P NMR (162 MHz, D2O)  17.81. MS (ESI) calculated for C24H32N3O10P2 [M-H] - 584.2, found 585.3. (S)-5-((5-amino-6-((3-hydroxy-3,3-diphosphonopropyl)amino)-6-oxohexyl)carbamoyl)-2- (6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (2): Compound 1 (340 mg, 0.581 mmol) was suspended in water (5.10 mL) and THF (3.85 mL), sodium bicarbonate was added until the pH was 7.3. 5-Carboxyfluorescein succinimidyl ester (300 mg, 0.634 mmol, 1.1 eq) in THF (10.2 mL) was added. The orange solution was stirred at room temperature overnight. Solvents were removed under reduced pressure. The residue was suspended in DMF (27.2 mL) and treated with piperidine (6.80 mL). The resulting solution was stirred at room temperature overnight. Solvents were removed under reduced pressure. The residue was dissolved in water (680 mL) and acetic acid (1.60 mL) was added. The aqueous layer was extracted with ethyl acetate (680 mL × 3). The aqueous phase was evaporated under reduced pressure to give the crude product 2 (0.201 mmol, 34% yield). Amount was determined by UV absorbance (PBS buffer,  = 73,000 at 493 nm). 1 H NMR (400 MHz, D2O) δ 8.03 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 7.00 (d, J = 9.5 Hz, 2H), 6.50 (d, J = 7.9 Hz, 4H), 3.78 (t, J = 6.5 Hz, 1H), 3.50 (dt, J = 13.1, 6.4 Hz, 1H), 3.42 – 3.27 (m, 3H), 2.03 (tt, J = 13.4, 7.9 Hz, 2H), 1.89 – 1.79 (m, 2H), 1.59 (p, J = 7.5 Hz, 2H), 1.38 (p, J = 7.6, 7.1 Hz, 2H). 31 P NMR (162 MHz, D2O) δ 17.83. MS (ESI) calculated for C30H32N3O14P2 [M-H] - 720.1, found 720.2. (S)-4-((5-(3-aminopropanamido)-6-((3-hydroxy-3,3-diphosphonopropyl)amino)-6- oxohexyl)carbamoyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (3): To a solution of compound 2 (40.0 mol) in THF (274 μL), water (113 μL) and saturated sodium bicarbonate 54 solution (74.0 μL) was added N-Boc- -Ala succinimidyl ester (0.300 M in THF, 267 μL, 80.0 mol, 2.0 eq). The mixture was stirred at room temperature overnight. Solvents were removed under reduced pressure. The residue was dissolved in 0.100 M TEAB buffer containing 5% acetonitrile (pH = 8.5, 400 μL). The crude product was purified by reverse-phase HPLC (Hamilton® PRP-1 column, 21.2 x 250 mm, 7 μm) using 0.100 M TEAB buffers containing 5% and 70% acetonitrile (pH = 8.5, A and B, respectively). Gradient was as follows: 0–2 min, 20% B; 2–20 min, 20–60% B; 20–22 min, 60–100% B; flow rate, 6 mL/min. Target eluted with a retention time of 15 min. Product fractions were pooled and concentrated under reduced pressure to give the Boc-protected intermediate. MS (ESI) calculated for C38H45N4O17P2 [M-H] - 891.2 found 891.3. To the intermediate was added water (50.0 μL) and TFA (950 μL). The yellow solution was stirred at room temperature overnight. Solvents were removed under reduced pressure. The residue was purified by reverse-phase HPLC (Hamilton® PRP-1column, 21.2 x 250 mm, 7 μm) using 0.100 M TEAB buffers, A and B. Gradient was as follows: 0–2 min, 20% B; 2–20 min, 20–60% B; 20–22 min, 60–100% B; flow rate, 6 mL/min. Target eluted with a retention time of around 9 min. Product fractions were pooled and concentrated under reduced pressure to give 3 (10.6 mol, 27%). Amount was determined by UV absorbance (PBS buffer,  = 73,000 at 493 nm). 1 H NMR (400 MHz, D2O) δ 8.06 (d, J = 1.8 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.25 (d, J = 7.9 Hz, 1H), 7.07 (d, J = 9.8 Hz, 2H), 6.65 – 6.51 (m, 4H), 4.13 (dd, J = 9.5, 4.7 Hz, 1H), 3.42 (t, J = 7.2 Hz, 2H), 3.33 (t, J = 6.8 Hz, 2H), 3.15 (dt, J = 13.9, 6.8 Hz, 2H), 2.64 (dt, J = 11.9, 6.6 Hz, 2H), 2.11 – 1.93 (m, 2H), 1.85 – 1.71 (m, 1H), 1.69 – 1.47 (m, 3H), 1.47 – 1.33 (m, 2H). 31 P NMR (243 MHz, D2O) δ 17.60. MS (ESI) calc. for C33H38N4O15P2[M-H] - 791.2, found 791.0. Boc-GH(Trt)PGGPQ(Trt)G-OSu (P1): Protected peptide Boc-GH(Trt)PGGPQ(Trt)G-OH (68.4 mg, 53.0 µmol) was treated with 0.250 M distilled TEA solution in anhydrous DMF (636 55 µL, 159 µmol, 3.0 eq) followed by 0.250 M TSTU solution in anhydrous DMF (233 µL, 58.3 µmol, 1.1 eq). The mixture was shaken vigorously and then left at room temperature for 30 min. Solvent was removed under reduced pressure. The residue was used immediately in the following step without purification. MS (ESI) calculated for C76H83N12O14 [M+H] + 1387.6, found 1387.1. (4): To a solution of 3 (10.6 µmol) in water (108 µL), saturated sodium bicarbonate solution (27.0 µL), and THF (108 µL) was added activated peptide P1 (53.0 µmol, 5.0 eq) in THF (216 µL). The mixture was shaken vigorously overnight. Solvents were removed under reduced pressure. The residue was dissolved in buffer B (2.00 mL) and purified by HPLC (Hamilton® PRP-1column, 21.2 x 250 mm, 7 µm) using 0.100 M TEAB buffers, A and B. Gradient was as follows: 0–2 min, 50% A; 2–15 min, 50–100% B; 15–22 min, 100% B; flow rate, 6 mL/min. The product had a retention time of 16 min. Product fractions were pooled and concentrated under reduced pressure to give the Boc- and Trt-protected intermediate. MS (ESI) calculated for C105H113N15O26P2 [M- 2H] 2- 1030.9, found 1031.2. The intermediate was treated with a mixture of TFA, water and TIS (95:2.5:2.5, 400 µL). The mixture was shaken at room temperature overnight. Solvents were removed under reduced pressure. The residue was dissolved in 0.100 M TEAB buffer B (500 µL) and purified by reverse- phase HPLC (Hamilton® PRP-1 column, 21.2 x 250 mm, 7 µm) using 0.100 M TEAB buffers A and B. Gradient was as follows: 0–2 min, 20% B; 2–20 min, 20–60% B; 20–22 min, 100% B; flow rate, 6 mL/min. The product had a retention time of 8.5 min. Product fractions were pooled and concentrated under reduced pressure to give 4 (4.00 mol, 38%). Amount was determined by UV absorbance (PBS buffer,  = 73,000 at 493 nm). MS (ESI) calculated for C62H77N15O24P2 [M-H] - 1478.5, found 1478.5. 56 (5): To 4 (1.00 µmol) in saturated sodium bicarbonate solution (24.0 μL) was added solid sodium bicarbonate until no more solid was dissolved. A 0.0420 M solution of succinimidyl ester of BHQ- 1 (THF/DMF 2:1, 90.0 µL, 3.78 µmol, 3.8 eq) was added. The mixture was shaken vigorously at room temperature overnight. Solvents were removed under reduced pressure. The residue was dissolved in ammonium acetate buffer containing 70% acetonitrile (50.0 mM, 500 μL). The crude product was purified by reverse-phase HPLC (Hamilton® PRP-1column, 10 x 250 mm, 7 μm) using 50 mM ammonium acetate buffers containing 5% and 70% acetonitrile (pH = 7.4, A and B, respectively). Gradient was as follows: 0–3 min, 10% B; 3–13 min, 10–100% B; 13–32 min, 100% B. The product had a retention time of 14.5 min. Product fractions were combined and evaporated to give 5 (0.325 μmol, 33%). Amount was determined by UV absorbance (PBS Buffer, pH 7.4,  = 34,660 at 534 nm estimated by addition of BHQ-1 and carboxyfluorescein extinction coefficients). HRMS (ESI) calculated for C88H103N21O28P2 [M-2H] 2- 981.8384, found 981.8386. 2.4 Enzyme Kinetics Measurements OMNICATHEPSIN® fluorogenic substrate, Z-Phe-Arg-AMC (Z-FR-AMC), where Z represents benzyloxycarbonyl and AMC represents 7-amino-4-methylcoumarin, procathepsin K (Proctsk), cathepsins B, L and S (ctsb, ctsl and ctss) were purchased from Enzo Life Sciences, Inc. Michaelis-Menten steady state kinetic analysis was used to determine the kinetic constants of the commercially available substrate Z-FR-AMC with ctsb, ctsk, ctsl and ctss. The final concentration of substrates ranged from 0.250 µM to 1.00 mM, and the concentration of dimethyl sulfoxide (DMSO) in the assays was less than 2% (v/v). The concentrations of ctsb, ctsk, ctsl and ctss were 1, 20, 1.37 and 10 nM, respectively. All kinetic assays were performed at 25 ºC and 37 ºC in triplicate. The kinetic constants kcat (s -1 ) and Km (µM) of Z-FR-AMC with human cathepsins B, K 57 and L at 25 ºC were reported 14 and compared to our experimental values which included ctss at 25 ºC (Table 2.1). The hydrolysis of AMC from Z-FR-AMC was monitored fluorometrically using AMC as the calibration standard. The excitation and emission wavelengths were 350 nm and 450 nm, respectively. The analysis buffer was made up of 50 mM sodium acetate/50 mM acetic acid, pH 5.5, 1.00 mM dithiothreitol (DTT), 2.50 mM ethylenediaminetetraacetic acid (EDTA). Z-FR- AMC was poorly soluble in the analysis buffer, and 1.07% DMSO was needed to dissolve it. Enzyme concentrations ctsb, ctsk, ctsl and ctss were 1 nM, 20 nM, 1.37 nM and 10 nM, respectively. Proctsk was activated with a buffer consisting of 32.5 mM sodium acetate/32.5 mM acetic acid at pH 3.5. Substrate concentrations ranged from 0.250 µM to 1.00 mM. The experimental data was fitted to the Michaelis-Menten equation, vo = vmax[S] 𝐾𝑚 +[𝑆 ] , where vo is the initial velocity (µmol/s), [S] is the substrate concentration (µM), vmax is the maximum velocity (µmol/s) and Km is the substrate concentration at half vmax (µM) using GraphPad Prism 7.04 software to calculate the kcat (s -1 ) and Km (µM). The difference in the experimental and reported kcat (s -1 ) and Km (µM) values could be attributed to the different commercial sources of enzymes as well as their preparation methods. The reported ctsb was purchased from Cortex Biochem (which no longer exists today), while enzymes ctsk, ctsl and ctss were prepared by heterologous expression, purification, and active-site titration. 14 Z-FR-AMC Enzyme Experimental Results at 25 °C Reported Values at 25 °C 14 kcat (s -1 ) Km (µM) kcat (s -1 ) Km (µM) Ctsb 217 ± 1.10 56.4 ± 1.06 7.20 ± 0.100 38.1 ± 2.40 Ctsk 7.91 ± 0.0825 24.8 ± 1.11 2.10 ± 0.400 48.5 ± 1.90 Ctsl 2.74 ± 0.0647 8.92 ± 1.05 7.50 ± 0.100 2.20 ± 0.200 Ctss 15.7 ± 0.142 100 ± 2.98 Not Performed 58 Table 2.1. Experimentally determined kcat and Km values derived using GraphPad Prism 7.04 obtained from the kinetic analysis of Z-FR-AMC with human cathepsins B, K, L and S at 25 ºC. Subsequently, the experimental kcat (s -1 ) and Km (µM) values of Z-FR-AMC with human cathepsins B, K, L and S at 37 ºC (Table 2.2) were obtained. These results show that the synthetic substrate Z-FR-AMC was cleaved by all four human cathepsins B, K, L and S, and hence, not selective. Table 2.2. Experimentally determined kcat and Km values derived using GraphPad Prism 7.04 obtained from the kinetic analysis of Z-FR-AMC with human cathepsins B, K, L and S at 37 ºC. Enzyme kinetic measurements were then performed to determine the selectivity of OFS-1 towards ctsb, ctsk, ctsl and ctss. Kinetic enzyme parameters kcat (s -1 ) and Km (µM) were determined from initial rates at 37 ºC and pH 5.5 in a buffer consisting of 100 mM sodium acetate/100 mM acetic acid, 100 mM sodium chloride, 10.0 mM DTT, 1.00 mM EDTA, with the addition of 0.0100% Brij-35 surfactant to boost the activity of the enzymes (Table 2.3). Enzyme concentrations (ctsb, ctsk, ctsl and ctss) were 10.0 nM. Proctsk was activated with a buffer consisting of 32.5 mM sodium acetate/32.5 mM acetic acid at pH 3.5. Substrate concentrations ranged from 0.0781 µM to 2.500 µM. Fluorescence measurements were taken every 1 minute for 60 minutes using a Biotek Synergy H4 plate reader with black Corning 3720 96-well plates. The excitation and emission wavelengths were 485 nm and 528 nm, respectively. Parameters were Z-FR-AMC Enzyme Experimental Results at 37 °C kcat (s -1 ) Km (µM) Ctsb 378 ± 4.53 42.5 ± 1.99 Ctsk 2.64 ± 0.0376 57.3 ± 3.03 Ctsl 94.1 ± 2.38 296 ± 18.3 Ctss 91.8 ± 0.440 117 ± 1.77 59 determined by finding initial velocities and selecting wells where the substrate concentration was below Km and assuming second-order kinetics and pseudo first-order conditions with respect to substrate. Values were averaged over all concentrations within the linear region. Fluorescence calibration was performed using a model compound lacking the quencher in OFS-1. OFS-1 Enzyme Experimental Results at 37 °C kcat (s -1 ) Km (µM) Ctsb No Activity No Activity Ctsk 0.107 ± 0.00249 3.89 ± 0.133 Ctsl No Activity No Activity Ctss 0.184 ± 0.0140 2.83 ± 0.346 Table 2.3. Experimentally determined kcat and Km values derived using GraphPad Prism 7.04 obtained from the kinetic analysis of OFS-1 with human cathepsins B, K, L and S at 37 ºC. In contrast to Z-FR-AMC which is non-selective towards the four human cathepsins tested, these experimental results show that OFS-1 was cleaved by only two human cathepsins K and S. Hence, OFS-1 is ctsk- and ctss-selective. Contents of three triplicate wells of 100 µL each were passed through an Oasis HLB cartridge to adsorb OFS-1 and fragments and separate them from buffer salts. Cartridge was washed with water (1.00 mL x 2) and then 70:30 acetonitrile, water mixture (0.500 mL) was used to elute the products. After concentration in vacuum, the sample was injected for LCMS (Shodex ODP-50 4D column; 50.0 mM TEAB buffer pH 9, A: 5%, B:75% ACN; gradient: 0-1 min: 100% A, 1-11 min 0-100% B; sample dissolved in 15.0 µl of buffer B). Fragments resulting from cleavage at the glutamine (Q)-glycine (G) bond were found to be the major products from the reaction with ctsk (Figure 2.3). Cleavage at the glycine (G)-glycine (G) 60 bond was also observed, but as a minor process (Figure 2.3). Based on the ratio of the Q-G and G-G BHQ-1 side fragments, the rate of G-G cleavage occurs at ~10% the rate of Q-G cleavage. This contrasts with the G-G cleavage site reported for Abz-HPGGPQ-EDN2ph. 13 A reduced form of OFS-1 was also observed with mass-to-charge ratio (m/z) indicating the loss of one oxygen atom, likely from the nitro group on BHQ-1 forming a nitroso group. Absorbance of this compound at 535 nm was diminished relative to OFS-1, indicating that the lost oxygen atom must have come from the BHQ-1 chromophore. BHQ-1 and similar azo dyes are known to be reactive to DTT. 61 Figure 2.3. LC-MS of OFS-1 after incubation with ctsk. 62 2.5 In Vitro Activation In vitro activation was conducted at Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry by Drs. Kenzo Morinaga, Akishige Hokugo and Ichiro Nishimura. OFS-1 can be activated by human osteoclast cells in vitro. Time-lapse photomicrographs obtained after seeding osteoclasts onto calcium phosphate-coated wells preabsorbed with OFS-1 showed increased fluorescence around cells that were resorbing the calcium phosphate substrate. Some osteoclasts can be seen migrating on the surface without completely dissolving the calcium phosphate, leaving a trail of enhanced fluorescence in their wake. This is how the probe is presumed to function in vivo, by fluorescently labeling any regions exposed to osteoclast resorptive activity. Little fluorescence was observed in areas where the calcium phosphate layer had been dissolved completely (solid triangles, Figure 2.4) due to a lack of calcium phosphate for the probe to bind to. The areas of the substrate unaffected by osteoclast activity exhibit weak, even fluorescence as a result of incomplete quenching in the intact probe. Another phenomenon observed was a transient decrease in fluorescence within the boundaries of the osteoclast sealing zone, seen as rapidly moving dark areas around the cells. This effect results from the pH sensitivity of FAM. The pH under an active osteoclast has been reported to be as low as 4.5. 15 FAM cyclizes to a nonfluorescent form when protonated and has a pKa of approximately 6.5. 16 This is further evidence that the probe is exposed to the sub-osteoclast environment as intended. Concentrated fluorescent intensity in the osteoclast cells themselves was also observed, both attached to and detached from the substrate (Figures 2.4 and 2.5). This shows that the probe is being up-taken by the osteoclasts as has been seen with other fluorescently-labeled bisphosphonates in vitro and in vivo. 9,10,17–19 63 Figure 2.4. Calcium phosphate-coated plates were pre-incubated with OFS-1 probe (5.00 µM) and human osteoclasts were seeded. Time-lapse photomicroscopy revealed OFS-1 probe activation around the periphery of the resorption pits (large arrow, 9-hour image). Cytoplasma of osteoclasts also contained fluorescent signal likely due to internalization of activated OFS-1. Osteoclast migration left a fluorescent trail (dotted arrow). Data kindly provided by Drs. Kenzo Morinaga, Akishige Hokugo and Ichiro Nishumura. 64 Figure 2.5. Human osteoclasts cultured on calcium phosphate-coated plates pre-treated with OFS- 1 (5.00 µM) gave a localized fluorescent signal (large arrows) (OFS-1 + Osteoclast). Resorption pits can also be observed as dark areas in the fluorescent images (triangles). When osteoclasts were co-cultured with RPMI-8226-Luc human multiple myeloma cells (small arrows) on OFS-1 pre- incubated calcium phosphate-coated plates, a strong fluorescent signal was observed (OFS-1 + Osteoclast + 8226 Multiple Myeloma). Odanacatib (ODN) applied to this co-culture system significantly attenuated the OFS-1 activation (OFS-1 + Osteoclast + 8226 Multiple Myeloma + ODN). Data kindly provided by Drs. Kenzo Morinaga, Akishige Hokugo and Ichiro Nishumura. This probe can be used in vitro to determine whether osteoclasts are delivering ctsk to the surface of bone and can be used to track their migration on mineral substrates. Since it can easily be applied to calcium mineral substrates at room temperature, OFS-1 is more convenient and physiologically relevant than the traditional approach to measuring osteoclast activity and migration on glass with gold nanoparticles. 20 65 It has been reported that multiple myeloma cells increase osteoclastic bone resorption. Previous studies have demonstrated the effect of multiple myeloma cells through indirect methods such as measuring soluble RANKL 21,22 production. In the present study, the osteoclast behavior on OFS- 1 pre-adsorbed calcium phosphate-coated plates in a co-culture environment with human multiple myeloma RPMI-8226-Luc cells was examined. Time-lapse photomicrography of live osteoclasts and RPMI-8226 cells demonstrated robust activation of OFS-1 fluorescent signals on the calcium phosphate plate surface around the resorption pits and migrating osteoclasts (Figure 2.5). The OFS-1 fluorescent signal was abnormally increased by RPMI-8226 cells (Figure 2.5) and was effectively inhibited by ODN (30 nM) (Figure 2.5). This confirmed that the OFS-1 signal activation was dependent on ctsk. ODN is known to be a highly selective inhibitor of ctsk with an IC50 value in cells of 5 nM for CTSK and 1050, 4083, and 45 nM for ctsb, ctsl, and ctss, respecively. 23 The inhibition of increased fluorescence in osteoclast cultures at 30 nM ODN indicates that activation of OFS-1 by these ubiquitous lysosomal proteases is not responsible for the fluorescent signal observed. 2.6 In Vivo Live Imaging Mice experiments were conducted at Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry by Drs. Kenzo Morinaga, Hiroko Okawa, Akishige Hokugo and Ichiro Nishimura. The use of OFS-1 for in vivo live imaging of disease-induced aberrant osteoclastogenesis was further investigated. Human clonal multiple myeloma RPMI-8226-Luc cells were injected into immunodeficient (NOD/Shi-scid, IL2rγnull) NOG mice. In this model, orthotopic engraftment of RPMI-8226-Luc cells has been documented by luciferase-based bioluminescence in vivo imaging; 24 however abnormal osteoclastic bone resorption induced by 66 multiple myeloma was only demonstrated by in vivo or post-humeral micro-computed tomography (micro-CT) image analysis. 25 Immunocompetent mice from NOD-scid IL2rγnull (NSG) mice that received transplantation of human embryonic CD34+ hematopoietic stem cells, liver and thymus (NSG-BLT mice) was developed. After 8~10 weeks, over 90% of circulating immune cells were of human origin in NSG-BLT mice, 26 to which RPMI-8226-Luc cells were injected via tail vein and one day later, OFS-1 was injected via retro-orbital venous plexus (Figure 2.6a). After 4~5 weeks of RPMI-8226-Luc cell injection, luciferase-bioluminescent signal was found localized in the area of lumber bone (Figure 2.6b) or femurs and tibia (Figure 2.6c). The micro-CT images of corresponding bone suggested abnormal osteolysis, indicating RPMI-8226-Luc cell orthotopic engraftment in NSG-BLT mice as observed in NOG mice (Figures 2.6b, 2.6c and 2.6d). Using this mouse model, a time-course bioluminescent examination was performed. The initial luciferase bioluminescent signal at the tail vein injection area progressively decreased over 3~4 weeks and eventually disappeared (Figure 2.6e). Then, a luciferase bioluminescent signal appeared at a distant bone site from the injected tail such as cervical bone at 5-weeks. In this series, the fluorescent signal of OFS-1 co-localized with the RPMI-8226-Luc bioluminescent signal (Figure 2.6e) was observed. Further, a weak but distinct fluorescent signal of OFS-1 prior to detecting the RPMI-8226-Luc bioluminescent signal was observed. The mineral affinity of OFS-1 allows it to accumulate on bone surface and leave a lasting fluorescent signal in response to osteoclast activity. In vivo live imaging experiments show that the OFS-1 probe shows enhanced signal, which may be sensitive enough to predict locally increased osteoclast activity due to the orthotopic engraftment of RPMI-8226-Luc cells. 67 68 Figure 2.6. OFS-1 for in vivo live imaging of multiple myeloma-induced aberrant osteoclastogenesis. a. After NSG/BLT humanized mice were established, human RPMI-8226-Luc cells were injected via tail vein. One week later, OFS-1 was injected via retro-orbital venous plexus. Mice were then imaged using an In-Vivo-analysIS (IVIS) Lumina II in vivo imaging system and micro-CT. b. Bioluminescence in vivo images (bottom) and 3-dimensional (3D)-reconstructed ex vivo micro-CT images (top) of representative mice after 4 weeks of injection of RPMI-8226- Luc cells or vehicle solution. As a reference, human RPMI-8226-Luc cells were injected into immunodeficient NOG mice (right). Human multiple myeloma cells were engrafted in both NSG/BLT and NOG mice. c. In vivo bioluminescence and fluorescent images of representative mice 3, 4, and 5 weeks after the RPMI-8226-Luc cell injection. There was a bioluminescent signal near the tail vein injection site until week 4, suggesting the presence of RPMI-8226-Luc cells. However, this bioluminescent signal near the tail vein injection site disappeared at week 5, while a new bioluminescent signal was detected at the cervical/thoracic bone (arrow). Fluorescent in vivo imaging revealed a localized signal superimposed on the bioluminescent signal at the cervical/thoracic bone (arrow), suggesting osteoclastic activity induced by orthotopic-egrafted RPMI-8226-Luc cells. d. Post-mortem micro-CT images of cervical region of the above mice revealed extensive osteolytic lesions (arrows), which corresponded to the sentinel OFS-1 signal. e. In vivo live imaging (left) of an RPMI-8226-Luc cells-engrafted NSG/BLT mouse with OFS-1 injection identified the strong fluorescent signal in femurs and tibias. Post-mortem micro-CT imaging (right) revealed decreased trabecular bone structure in the RPMI-8226-Luc cells- engrafted NSG/BLT mouse as compared to a control mouse. Data kindly provided by Drs. Kenzo Morinaga, Akishige Hokugo and Ichiro Nishumura. 69 The localized osteoclastic activity induced by tooth extraction was also detected by OFS-1 in vivo. Tooth extraction is a commonly prescribed oral surgery and the wound in the jawbone socket heals with active bone remodeling. However, the alveolar bone socket never reaches its previous dimensions because the remaining jawbone structure undergoes progressive bone resorption 27 (Figure 2.7a). OFS-1 with an “always-on” analogue lacking the quencher moiety (Compound 4) referred to as PFC-1 (for positive fluorescent control) (Figure 2.7b) was compared. Mice treated with the “always-on” PFC-1 compound demonstrated fluorescent signal throughout the maxilla and femur bones. In contrast, OFS-1-treated mice revealed disproportionate fluorescent signal at and around the tooth extraction site (Figure 2.7c). 70 71 Figure 2.7. Tooth extraction-induced osteoclastic jawbone resorption detected by OFS-1. a. C57Bl6J mice underwent tooth extraction of one of maxillary first molars (M1) resulting in a surgically created extraction socket (Soc) in alveolar bone flanking palatal bone (Pal Bone). After tooth extraction, a cluster of osteoclasts (arrowheads, red by tartrate-resistant acid phosphatase staining) were induced on the surface of palatal bone immediately adjacent to the extraction socket. b. Time-course experiment. Mice were administered either OFS-1, 5-FAM-bisphosphonate (“always-on” fluorescently tagged bisphosphonate) or saline vehicle solution 1 day prior to tooth extraction. Maxillary tissues and femur bones were harvested 10 days after tooth extraction. c. Tooth extraction wound (arrow) of oral mucosa and maxillary bone was well healed in all mice. OFS-1-derived fluorescent signal was disproportionately detected at and around the tooth extraction site, whereas 5-FAM-bisphosphonate signal was observed throughout the maxilla and femur bone. Faint OFS-1 signal at the non-extraction side of maxilla and femur indicated physiological bone remodeling. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. A fluorescent signal in mice pretreated (one day prior to ligation) with OFS-1 was detected at the periodontitis lesion site as early as Day 3 and persisted until Day 14 (Figure 2.8a). After removal of the disease-causing ligature on Day 7, the signal decreased (Figure 2.8b). Quantitative analysis indicated that highest fluorescent signal was on Day 3, indicating significant secretion of ctsk during the initial stage of ligature-induced periodontitis. The quantitative polymerase chain reaction (qPCR) of mouse gingival tissue confirmed a high level of ctsk expression on Day 3 (Figure 2.8c). A micro-CT radiographic study revealed noticeable boss loss on Day 7, which progressively increased by Day 14 (Figures 2.8d and 2.8e). A strong fluorescent signal was observed at the periodontitis side of maxilla in mice with ligature-induced periodontitis as early as 72 Day 3 and throughout the experimental period (Figure 2.8f). The activation of OFS-1- and Abz- HPGGPQ-EDN2ph-ctsk-sensitive FRET probes was compared in the model on Day 3. A strong fluorescent signal from OFS-1 was consistently observed, however no fluorescent signal from Abz-HPGGPQ-EDN2ph was detected (Figure 2.8g). Details about Abz-HPGGPQ-EDN2ph will follow in the next section. 73 74 Figure 2.8. Imaging Data for OFS-1. a. Ligature-induced periodontitis induced in C57BI6J mice by placing a silk suture around the maxillary left second molar (M2). Gingival swelling (dotted line) was developed on the ligature-induced periodontitis side of maxilla. b. The area of palatal gingival swelling was measured as standardized by the circumferential area of the first molar (M1). c. Expression of cytokine genes was assessed by qPCR of palatal gingiva harvested from control side (blue bars) and periodontitis side (orange bars). d. Periodontitis-induced alveolar bone resorption was evaluated by micro CT imaging. Bone loss in the M2 area of ligature-induced periodontitis was not observed until Day 7 and Day 14. e. Alveolar bone loss was evaluated by micro-CT imaging, which also demonstrated the progressive bone loss. f. Mice with ligature- induced periodontitis were treated with intravenous (IV) injection of OFS-1 24 hours prior to the ex vivo examination. IVIS data revealed a strong fluorescent signal at the periodontitis side of maxilla (white arrows) as early as Day 3 and throughout the whole experimental period. g. A separate study compared the activation of OFS-1- and Abz-HPGGPQ-EDN2ph-ctsk-sensitive FRET probes in the model on Day 3. Localized activation of OFS-1 was consistently observed, however no fluorescent signal from Abz-HPGGPQ-EDN2ph was detected. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. This study demonstrated highly specific and sensitive fluorescent signal activation by OFS-1 in in vitro and in vivo live imaging models, responding to aberrantly increased osteoclastic activity. OFS-1 and its derivatives may become not only a useful compound for investigating the pathological mechanisms of disease induced bone resorption, but more importantly, a powerful diagnostic and prognostic tool for a range of osteolytic diseases such as multiple myeloma, bone metastases from solid tumors including breast and prostate cancers as well as osteoporosis, osteoarthritis, rheumatoid arthritis, osteomyelitis and periodontitis. 75 2.7 Molecular Docking Abz-HPGGPQ-EDN2ph (Figure 2.9), where Abz represents o-aminobenzoic acid and EDN2ph represents N-(2,4-dinitrophenylethylenediamine) was found as an efficient and selective probe for ctsk with catalytic efficiency of 426, 000 M -1  s -1 . 13 Figure 2.9. Chemical structure of Abz-HPGGPQ-EDN2ph. Comparison of this probe with our bisphosphonate-containing probes shows that only OFS-1 was cleaved by ctsk with relatively similar rate, whereas OFS-2 was cleaved much slower. To understand these differences, the molecular docking approach was used. Molecular docking becomes increasingly difficult for more flexible ligand. Considering the number of rotatable bonds in OFS-1 (66) and in OFS-2 (79), the model compound Abz-HPGGPQ-EDN2ph (31 rotatable bonds) was docked in ctsk. A crystal structure of human ctsk with ODN as the inhibitor at 1.4-Å resolution (5TDI) 28 was used with ICM-Pro 29 3.8-6a modeling software to explore OFS-1 binding modes versus binding of a model substrate peptide (Figure 2.10). Docking of a model compound shows good fit of the probe in the active side of ctsk. 76 a b Figure 2.10. Computer modelling was performed to analyze the OFS probe efficiencies. a. Best conformation predicted by ICM 29 (thoroughness 8, score -39; desirable score criterion < -32) of a model ctsk substrate, Abz-HPGGPQ-EDN2ph, in the active site of ctsk. Electrostatic charge areas: red, negative; blue, positive; white, neutral. Yellow = Cysteine 25 (C25). b. One of the best conformation of OFS-1 predicted by ICM 29 (score -46). The distance between sulfur of C25 which is nucleophile in peptide hydrolysis and carbonyl carbon of glycine where cleavage happens 30 is noted to be 3.1 Å. Due to the large size of the OFS-1 molecule, the whole protein as active site was defined, using thoroughness parameter equal to 8. The molecule was docked several times. One of the multiple conformations with a good score is shown (Figure 2.10b). In this conformation, the position of the peptide portion of the probe in the active site is like Abz-HPGGPQ-EDN2ph with distance between C25 sulfur and Gly-carbonyl carbon equal to 3.4 Å. 77 Despite multiple docking of OFS-2 in ctsk, conformations with a good score where peptide is in the active site of the enzyme were not found. The typical conformation of OFS-2, as predicted by ICM 29 is displayed (Figure 2.11). This happens where negatively charged sulfonic groups are stabilized by positively charged arginines and lysines including R8 and K9 located opposite to the active site of ctsk. Figure 2.11. One of the best conformations of OFS-2 predicted by ICM 29 (score -42). Being on the edge of the capabilities of the technique (due to the large molecular size of our probes), molecular docking suggests that the difference in OFS probes behavior is a very unique feature of ctsk, which is mainly due to the presence of positively charged amino acid clusters residing opposite to the active site of the enzyme. 31 Hence, electrostatic interactions of these clusters with negatively charged groups of the ligand could prevent alignment of the molecules for selective peptide-bond cleavage. Taking into account that every OFS probe has negatively charged (at pH 5.5, optimal pH for ctsk 32 ) bisphosphonate, stabilities in the presence of ctsk correlates well with the number of sulfonic groups: 4 in OFS-2 (very slow cleavage) and 0 in OFS-1 (efficient cleavage). The presence of sulfonic groups also dramatically changes the lipophilicity of probes. 78 The calculated distribution coefficient (log D) values of the two OFS probes together with the reference probe, Abz-HPGGPQ-EDN2ph are shown (Table 2.4). Probe Number of Sulfonic Groups Log D Efficiency of Cleavage OFS-1 0 -5.45 Efficient OFS-2 4 -10.49 Very slow Abz-HPGGPQ-EDN2ph 0 -3.51 Very efficient Table 2.4. Calculated log D values (MarvinSketch 15.12.14.0). With the goal of obtaining OFS probes with fluorescence approaching the infrared spectrum for optimal in vivo performance, a NIR imaging bisphosphonate conjugate OFS-2 was designed in which the internally quenched fluorescence (IQF) dye pair was Alexa Fluor 647-Black Hole Quencher-3 (AF647-BHQ-3). OFS-2 was activated by ctsk more slowly than OFS-1 and failed to generate an active-site pose scoring below the criterion value, with preferred binding to an exterior positive region including arginine8 (R8) and lysine9 (K9). This led to a redesigned probe (OFS-3) of an alternative, more neutral dye pair which consisted of Sulfo-Cy5 as the fluorophore and BBQ650 as the quencher. 2.8 References (1) Divieti Pajevic, P.; Krause, D. S. Osteocyte Regulation of Bone and Blood. Bone 2019, 119, 13–18. https://doi.org/10.1016/j.bone.2018.02.012. (2) Terpos, E.; Ntanasis-Stathopoulos, I.; Gavriatopoulou, M.; Dimopoulos, M. A. Pathogenesis of Bone Disease in Multiple Myeloma: From Bench to Bedside. Blood Cancer J. 2018, 8 (1), 7. https://doi.org/10.1038/s41408-017-0037-4. 79 (3) Brömme, D.; Okamoto, K.; Wang, B. B.; Biroc, S. Human Cathepsin O2, a Matrix Protein-Degrading Cysteine Protease Expressed in Osteoclasts: FUNCTIONAL EXPRESSION OF HUMAN CATHEPSIN O2 IN SPODOPTERA FRUGIPERDA AND CHARACTERIZATION OF THE ENZYME. J. Biol. Chem. 1996, 271 (4), 2126–2132. https://doi.org/10.1074/jbc.271.4.2126. (4) Novinec, M.; Lenarčič, B. Cathepsin K: A Unique Collagenolytic Cysteine Peptidase. Biol. 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Pathol. 2008, 173 (1), 161–169. https://doi.org/10.2353/ajpath.2008.070494. 84 CHAPTER 3 Osteoadsorptive Fluorogenic Substrate-3 (OFS-3) 3.1 Introduction As mentioned in the previous chapters, ctsk is a protease secreted by osteoclasts that is central to normal and disease-related bone resorption. 1,2 Imaging probes of ctsk activity on bone tissue surfaces in vitro and in vivo would be useful to elucidate the role of osteoclastic ctsk in skeletal disorders, wherein the enzyme is also a potential marker of abnormal bone resorption. IQF imaging systems incorporating a peptide substrate of ctsk have been reported to generate a fluorescent signal when the enzyme cleaves the peptide linker. 3 However, these reagents are limited by relatively short tissue half-lives due to rapid clearance and most importantly, are not bone-specific. As mentioned in Chapter 1, bisphosphonates are well-known structural analogues of pyrophosphate that exhibit high affinity for bone mineral. The evolution of novel OFS imaging probe components towards the infrared region, extending OFS-1 (5-FAM-BHQ-1, 528-nm emission) to two new far-red fluorophore-quencher IQF dye pair, OFS-2 (AF647-BHQ-3, 665-nm emission) and OFS-3 (Sulfo-Cy5-BBQ-650, 662-nm emission) is presented (Table 3.1). The creation and utility of these bone-targeting IQF systems further leverage our previous successes in constructing bisphosphonate-conjugated fluorescent dyes for bone imaging in our group. 4,5 85 Name Bisphosphonate Fluorophore Quencher Peptide Boc-β-Ala OFS-1 Pamidronate 5-FAM BHQ-1 GHPGGPQG Yes OFS-2 Alendronate AF647 BHQ-3 GHPGGPQG No OFS-3 Pamidronate Sulfo-Cy5 BBQ-650 GHPGGPQG Yes Table 3.1. Components that make up OFS-1, OFS-2 and OFS-3. The paradigm shift from visible to NIR region in our design allows the creation of bone-specific imaging agents that can be used for preclinical studies of bone growth or real-time fluorescence guided surgery. The NIR window that extends from 650 to 900 nm is especially useful because of several special features such as minimal tissue autofluorescence and low light scattering that lead to higher sensitivity and optical contrast. Compared with visible wavelengths, NIR fluorescence light is invisible, thereby allowing highly sensitivity real-time image guidance in surgery without changing the surgical field. 6 NIR fluorescence imaging is a rapid and cost-effective tool for monitoring bone diseases and to quantitatively assess bone regeneration in living tissue. It is an emerging field that allows noninvasive monitoring of biological processes in vivo and enables testing at intervals to demonstrate how bone tissues develop and respond to bone-targeting agents. Developing NIR fluorescent imaging agents targeted to metastatic bone tumors will significantly lead to novel possibilities for real-time diagnosis and treatment of bone metastasis in patients. Since a bone scan provides the less accurate diagnosis of osteolytic bone metastatic tumor than osteogenic, the target-specific NIR fluorescence imaging of osteolytic lesion on metastatic tumor 86 could assist surgeons for intraoperative image-guided surgery. The future of bone metastasis imaging by using the structure-inherent targeting of NIR imaging agents involving bisphosphonates will likely involve the development of increasingly more powerful imaging agents that will greatly improve diagnostic accuracy. Hence, rational design of NIR imaging agents by considering molecular properties (dye structure, polarity and charge), target specificity, biodistribution, and targeting mechanism is essential to overcome the limitations for clinical use of NIR imaging agents. This chapter will focus mainly on OFS-3. An analysis of probe efficiencies comparing the three OFS probes to the reference probe, Abz-HPGGPQ-EDN2ph will be performed. A preliminary evaluation of OFS-3 that demonstrates their early detection of periodontitis in a mouse model will be discussed later, followed by the imaging results. 3.2 Chemical Synthesis In designing the prototype probe (OFS-3), the orthogonally Boc- (which is stable under basic conditions) and Fmoc- (which is stable under acidic conditions) protected lysine was selected as a trifunctional linker. The carboxy group of the Nα-Boc-Nε-Fmoc-(L)-lysine was first activated with sodium sulfo-NHS and then coupled with excess APD to give 1. Nα-Boc-Nε-Fmoc-(L)-lysine was used instead of Nα-Fmoc-Nε-Boc-(L)-lysine in the synthesis of OFS-3. This is because unlike in the synthesis of OFS-1 where the cost of quencher (BHQ-1) is more expensive than the cost of fluorophore (5-FAM), the cost of fluorophore (Sulfo-Cy5) is more expensive than the cost of quencher (BBQ650) in OFS-3. Hence the coupling of Sulfo-Cy5 took place in the last step after basic removal of the Fmoc protecting group on the lysine. As seen in both syntheses, the cost of starting materials greatly affects the order of reaction. APD was selected as the bisphosphonate 87 auxiliary due its known bone affinity 7 and its conveniently connectable aminoethyl side chain. Excess of APD was again used due to the low reactivity of the amino group in the aminoethyl side chain of APD to make it more susceptible for coupling with the sulfo-NHS-activated lysine. Once APD coupled with lysine, the Boc group was removed using TFA. The single-digit yield of 5% was due to the simultaneous hydrolysis of the sulfo-NHS-activated lysine at pH 7.5, while coupling to sodium pamidronate. 1 was linked to Boc-β-Ala, followed by the deprotection of Boc group using TFA to give 2. β-Ala acts as a spacer to link the TSTU-activated GHPGGPQG ctsk-cleavable peptide, followed by the deprotection of Boc and Trt groups using a mixture of TFA, water and TIS to give 3. The cost of customized peptide [Boc-GH(trt)PGGPQ(trt)G-OH] is lower than the cost of the quencher (BBQ650), so the peptide coupling took place before the quencher coupling. 3 was coupled to the succinimidyl ester of BBQ-650 to give 4. The Fmoc-protected amino group was deprotected using 20% piperidine in DMF, to connect the fluorophore which is Sulfo-Cy5 to the ε-amino function of the lysine to give 5 which is the OFS-3 product (Figure 3.1). Compounds 1 to 5 were only characterized by mass spectrometric analysis (Appendix B). 88 Figure 3.1. Synthesis of OFS-3. 3.3 Experimental Section General Experimental Information for Synthesis Nα-Boc-Nε-Fmoc-(L)-lysine and DCC were purchased from Sigma Aldrich. N- hydroxysulfosuccinimide sodium salt and N-Boc- -alanine succinimidyl ester were purchased from Chem-Impex International, Inc. PAM was provided courtesy of Novartis. TFA and TSTU were purchased from Oakwood Chemical. Sulfo-Cy5 succinimidyl ester was purchased from Lumiprobe. Anhydrous DMF and THF, acetonitrile, methanol and TEA were purchased from EMD Millipore Corporation and used without further drying. Sodium carbonate and sodium bicarbonate were purchased from Fisher Scientific. Custom synthesized peptide, Boc- GH(Trt)PGGPQ(Trt)G, was purchased from Applied Biological Materials Inc. BlackBerry Quencher 650 (BBQ-650) succinimidyl ester was purchased from Berry & Associates, Inc. Preparative HPLC was performed using a Shimadzu LC-8A equipped with a LabSolutions Lite Release 5.71 SP2 software and Shimadzu SPD-10A UV detector (0.5 mm path length). Low- 89 resolution mass spectrometry was performed on a Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI source in the negative ion mode. LC-MS was also performed on the Finnigan LCQ Deca XP Max mass spectrometer in negative mode with a Thermo Finnigan Surveyor PDA Plus detector (1 cm path length) and MS Pump Plus, all controlled using Xcalibur software, version 2.0.7. (S)-(3-(6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-aminohexanamido)-1- hydroxypropane-1,1-diyl)bis(phosphonic acid) (1): Nα-Boc-Nε-Fmoc-(L)-lysine (468 mg, 1.00 mmol) was dissolved in anhydrous DMF (2.50 µL). N-hydroxysulfosuccinimide sodium salt (260 mg, 1.20 mmol, 1.2 eq) was added. The mixture was sonicated to give a white suspension. DCC (206 g, 1.00 mmol, 1.0 eq) was added. The mixture was stirred for 2 hours. Reaction was monitored by TLC (100% ethyl acetate, visualized with UV 254 nm), starting material had a Rf value of 0.2– 0.5 and product had a Rf value of 0. The mixture was centrifuged, and the supernatant was collected and concentrated under reduced pressure. In parallel, a suspension of pamidronic acid (706 mg, 3.00 mmol, 3.0 eq) in water (10.0 mL) was treated with sodium carbonate until pH was 7.5. A solution of the activated ester in THF (2.50 mL) was added. A white suspension was observed. The mixture was stirred at room temperature overnight. The mixture was diluted with THF (40.0 mL), and centrifuged. Supernatant was collected and solids were washed with THF:water = 10:1 (12.5 mL × 3). Centrifugation was used to remove solids. Combined supernatants were concentrated under reduced pressure. Methanol (30.0 mL) was added. The mixture was stirred at room temperature for 15 minutes. The white solids were collected by centrifugation and washed with methanol (30 mL × 2). Solids were collected, dried in vacuo and used without further purification. A mixture of TFA (0.950 mL) and water (0.0500 mL) was added to the residue. The resulting mixture was stirred at room temperature overnight. Solvents were 90 removed under reduced pressure. The residue was co-evaporated with water (5.00 mL) and dried in vacuo to give 1 (0.0500 mmol, 5% yield), which was used in the next step without further purification. Amount was determined by UV absorbance (PBS buffer,  = 6566 at 300 nm). MS (ESI) calculated for C24H32N3O10P2 [M-H] - 584.2, found 584.3. (S)-(3-(6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(3- aminopropanamido)hexanamido)-1-hydroxypropane-1,1-diyl)bis(phosphonic acid) (2): To a solution of compound 1 (33.1 mol) in THF (1.02 mL), water (0.340 mL) and saturated sodium bicarbonate solution (86.0 μL) was added N-Boc- -alanine succinimidyl ester (18.9 mg, 66.2 mol, 2.0 eq). The mixture was stirred at room temperature overnight. Solvents were removed under reduced pressure. The residue was dissolved in 50.0 mM TEAB buffer containing 5% acetonitrile (pH = 8.5, 100 μL). The crude product was purified by reverse-phase HPLC (Hamilton® PRP-1column, 10 x 250 mm, 7 μm) using 50.0 mM TEAB buffers containing 5% and 75% acetonitrile (pH = 8.5, A and B, respectively). Gradient was as follows: 0–1 min, 0% B; 1– 11 min, 0–100% B; flow rate, 4 mL/min. Target fractions were pooled, acidified to pH < 6 by addition of acetic acid and concentrated under reduced pressure to give the Boc-protected intermediate (11 µmol, 33% yield). Amount was determined by UV absorbance (PBS buffer,  of α-Boc-ε-Fmoc lysine = 18,600 at 263 nm). MS (ESI) calculated for C32H46N4O13P2 [M-H] - 755.3 found 755.3. To the intermediate was added water (50.0 μL) and TFA (950 μL). The yellow solution was stirred at room temperature overnight. Solvents were removed under reduced pressure. The residue was used without further purification. MS (ESI) calculated for C27H37N4O11P2 [M-H] - 655.2, found 655.3. 91 Boc-GH(Trt)PGGPQ(Trt)G-OSu (P1): Protected peptide Boc-GH(Trt)PGGPQ(Trt)G-OH (89.0 mg, 69.0 µmol) was treated with 0.250 M distilled TEA solution in anhydrous DMF (690 µL, 173 µmol, 2.5 eq) followed by 0.250 M TSTU solution in anhydrous DMF (304 µL, 76.0 µmol, 1.1 eq). The mixture was shaken vigorously and then left at room temperature for 30 min. Solvent was removed under reduced pressure. The residue was used immediately in the following step without purification. MS (ESI) calculated for C76H83N12O14 [M+H] + 1387.6, found 1387.7. (3): To a solution of 2 (10.0 µmol) in water (84.4 µL), saturated sodium bicarbonate solution (26.0 µL), and THF (219 µL) was added activated peptide P1 (69.0 µmol, 7.0 eq) in THF:water = 5:1 (106 µL). The mixture was shaken vigorously overnight. Solvents were removed under reduced pressure. The intermediate was treated with a mixture of TFA, water and TIS (95:2.5:2.5, 500 µL). The mixture was shaken at room temperature overnight. Solvents were removed under reduced pressure. The residue was dissolved in water (500 µL) to give a yellowish suspension. Dichloromethane (500 µL) was added to dissolve insoluble trityl deprotection byproduct. The organic layer was carefully decanted from the bottom with a glass pipette. This procedure was repeated twice more leaving the aqueous layer only slightly cloudy with no visible particles. The aqueous phase was purified by reverse-phase HPLC (Shodex Asahipak ODP-50 4D column, 4.6 x 150 mm, 5 µm) using 50.0 mM TEAB buffers A and B. Gradient was as follows: 0–1 min, 20% B; 1–11 min, 20–100% B; flow rate, 1 mL/min. The product had a retention time of 6.3 min. Product fractions were pooled, acidified to pH < 6 by addition of acetic acid and concentrated under reduced pressure to give 3 (2.00 mol, 20%). Amount was determined by UV absorbance (PBS buffer,  = 19,400 at 263 nm). MS (ESI-) calc. for C56H77N15O20P2 [M-H] - 1341.5, found 1342.5. 92 (4): To a solution of 3 (2.88 µmol) in water (8.60 µL), saturated sodium bicarbonate solution (8.60 µL), and THF (43.0 µL) was added succinimidyl ester of BBQ-650 (4.39 mg, 6.15 µmol, 2.1 eq). THF (43.0 µL) was added to raise the THF:water = 5:1. The mixture was shaken vigorously at room temperature overnight. Solvents were removed under reduced pressure. The residue was suspended in DMF (0.800 mL) and treated with piperidine (0.200 mL). The resulting solution was stirred at room temperature overnight. Solvents were removed under reduced pressure to give 4 (1.04 µmol, 36%). Amount was determined by UV absorbance (PBS buffer,  = 40,667 at 598 nm). MS (ESI) calculated for C73H102N21O24P2 [M-H] - 1718.7, found 1718.5. (5): To 4 (1.00 µmol) in DMF (10.0 µL) was added TEA (0.360 µL, 2.58 µmol, 2.6 eq). A 0.01 M solution of succinimidyl ester of sulfo-cyanine5 in DMF (300 µL, 3.00 µmol, 3.0 eq) was added. The mixture was shaken vigorously at room temperature overnight. Solvents were removed under reduced pressure. The residue was dissolved in DMF (100 μL). The crude product was purified by (Shodex Asahipak ODP-50 4D column, 4.6 x 150 mm, 5 µm) using 50.0 mM TEAB buffers A and B. Gradient was as follows: 0–1 min, 0% B; 1–11 min, 0–100% B; flow rate, 1 mL/min. The product had a retention time of 8.1 min. Product fractions were pooled, acidified to pH < 6 by addition of acetic acid and concentrated under reduced pressure to give 3 (0.340 mol, 34%). Amount was determined by UV absorbance (PBS buffer,  = 40,667 at 598 nm). MS (ESI) calculated for C105H138N23O31P2S2 [M-2H] 2- 1170.9, found 1171.3. 3.4 Enzyme Kinetics Measurements Enzyme kinetic measurements were performed to determine the selectivity of OFS-3 towards ctsb, ctsk, ctsl and ctss. Kinetic enzyme parameters kcat (s -1 ) and Km (µM) were determined from initial rates at 37 ºC and pH 5.5 in a buffer consisting of 100 mM sodium acetate/100 mM acetic 93 acid, 100 mM sodium chloride, 10.0 mM DTT, 1.00 mM EDTA, and 0.0100% Brij-35 (Table 3.2). Enzyme concentrations (ctsb, ctsk, ctsl and ctss) were 10.0 nM. Only ctsk was activated with a buffer consisting of 32.5 mM sodium acetate/32.5 mM acetic acid at pH 3.5. Substrate concentrations ranged from 0.0781 µM to 2.500 µM. Fluorescence measurements were taken every 1 minute for 60 minutes using a Biotek Synergy H4 plate reader with black Corning 3720 96-well plates. The excitation and emission wavelengths were 630 nm and 662 nm, respectively. Parameters were determined by finding initial velocities and selecting wells where the substrate concentration was below Km and assuming second-order kinetics and pseudo first-order conditions with respect to substrate. Values were averaged over all concentrations within the linear region. Fluorescence calibration was performed using sulfo-cyanine5 which is the fluorophore of OFS-3. OFS-3 Enzyme Experimental Results at 37 °C kcat (s -1 ) Km (µM) Ctsb No Activity No Activity Ctsk 0.00235 ± 0.0000944 1.57 ± 0.122 Ctsl No Activity No Activity Ctss 0.000807 ± 0.0000490 3.20 ± 0.300 Table 3.2. Experimentally determined kcat and Km values derived using GraphPad Prism 7.04 obtained from the kinetic analysis of OFS-3 with human cathepsins B, L, K and S at 37 ºC. Like OFS-1, these experimental results show that OFS-3 was cleaved by only two human cathepsins K and S. Hence, OFS-3 is ctsk- and ctss-selective. 94 3.5 Analysis of Probe Efficiencies Taking into account that every OFS probe has negatively charged (at pH 5.5, optimal pH for ctsk 8 ) bisphosphonate, stabilities in the presence of ctsk correlates well with the number of sulfonic groups: 0 in Abz-HPGGPQ-EDN2ph (very efficient cleavage), 0 in OFS-1 (efficient cleavage), 4 in OFS-2 (very slow cleavage) and 2 in OFS-3 (slow cleavage). As mentioned in Chapter 2, the presence of sulfonic groups dramatically changes the lipophilicity of probes. The calculated distribution coefficient (log D) values of the three OFS probes together with the reference probe, Abz-HPGGPQ-EDN2ph are shown (Table 3.3). Probe Number of Sulfonic Groups Log D Efficiency of Cleavage OFS-1 0 -5.45 Efficient OFS-2 4 -10.49 Very slow OFS-3 2 -7.67 Slow Abz-HPGGPQ-EDN2ph 0 -3.51 Very efficient Table 3.3. Calculated log D values (MarvinSketch 15.12.14.0). 3.6 Mouse Periodontitis Model Mice experiments were conducted at Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishimura. To examine early diagnosis of periodontitis by OFS-3, a ligature-induced periodontitis model was used. Ligature-induced periodontitis was induced in C57Bl6J mice by placing a silk suture around the maxillary left second molar (M2). 9 After placement of a silk ligature around M2, OFS-3 was injected systemically one day prior to euthanasia after various experimental periods (1, 3, and 7 95 days). In an additional group, the ligature was removed on Day 7. The mouse was euthanized on Day 14, a day after IV injection of OFS-3 (Figure 3.2). Figure 3.2. Experimental periodontitis was generated in mice by placing a silk suture around the maxillary left second molar (M2). The timeline of the ligature-induced mouse periodontitis model: Day 0 – ligature placed; Day 7 – ligature removed; Day 13 – IV injection of OFS-3; Day 14 – tissue collection. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. The maxilla specimens were imaged with the IVIS Lumina II imaging system. Molar ligature and gingival tissue were harvested for 16S ribosomal RNA (16S rRNA) gene sequencing and inflammation-related gene expression respectively. The gingival swelling area surged on Day 3 (Figure 3.3). A micro-CT imaging was performed, and periodontal bone loss height was measured as the distance from the cementoenamel junction to the alveolar bone crest. Figure 3.3. Gingival swelling (dotted line) was developed on the ligature-induced periodontitis side of maxilla. The area of palatal gingival swelling was measured as standardized by the 96 circumferential area of the first molar (M1). A bar chart of gingival swelling area against the timeline of the ligature-induced mouse periodontitis model shows a significant increase on Day 3. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. Relative gene expression for Tumor Necrosis Factor alpha (TNF alpha), RANKL, Interleukin 6 (IL6), Interleukin 17A (IL17A), Interleukin 1b (IL1b) was represented on Day 1, 3, 7 and 14, respectively (Figure 3.4). In addition, 16S rRNA gene sequencing revealed that there are 30 genera that are significantly different between Day 1, Day 3 and Day 7 (Figure 3.5). Figure 3.4. The expression of pro-inflammatory cytokines, particularly IL6 and IL1B, as well as RANKL increased on Days 3 and 7. RANKL which is a cytokine to induce osteoclastogenesis, 10 was also present in the periodontitis legion. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. 97 Figure 3.5. Relative distributions of bacteria at both the phylum and genus levels identified by 16S rRNA gene sequencing. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. A micro-CT radiographic study revealed noticeable bone loss on Day 7, which progressively increased by Day 14 (Figure 3.6). Dental radiography is routinely used to examine periodontal disease. 11 However, positive diagnosis is only available after significant loss of alveolar bone. This study demonstrates that OFS imaging probes can detect ctsk activity much earlier. Ctsk expression is initiated at the preosteoclast stage and continues throughout the mature osteoclast stage. The cellular source of ctsk in gingival tissue from Day 1 to 3 may include preosteoclasts. Hence, these OFS probes can sensitively detect preosteoclast-derived ctsk. 98 Figure 3.6. A micro-CT radiographic study revealed noticeable bone loss on Day 7. After removal of ligature on Day 7, there was a decrease in the bone loss from both the buccal and palatal side. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. 3.7 Imaging Results Mice experiments were conducted at Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishimura. A fluorescent signal in mice pretreated (one day prior to ligation) with OFS-3 was strongly detected at the periodontitis lesion site beginning on Day 1 and continued until Day 14 (Figure 3.7). After removal of the disease-causing ligature on Day 7 (Figure 3.7), the signals decreased. Quantitative analysis indicated that highest fluorescent signal was on Day 3, indicating significant secretion of ctsk during the initial stage of ligature-induced periodontitis. The qPCR of mouse gingival tissue confirmed a high level of ctsk expression on Day 3. Immunohistochemistry data (IHC) further indicates that gingival tissue was strongly ctsk-positive on Day 3 (Figure 3.8). Moreover, the analysis shows deeper tissue penetration in mice without gingiva than with gingiva (Figure 3.9). 99 Figure 3.7. Imaging Data for OFS-3. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. Figure 3.8. IHC data of ctsk activity. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. With gingiva Without gingiva 100 Figure 3.9. OFS-3 Signal tissue penetration. Data kindly provided by Drs. Hiroko Okawa, Akishige Hokugo and Ichiro Nishumura. Patients with periodontitis are often unaware of disease progress due to the lack of pain symptoms and the lack of early detection method. Diagnosis of moderate to severe periodontitis is based on 25%~50% and more than or equal to 50% loss of alveolar bone in dental radiographs, respectively. It must be noted that the periodontal surgical treatment is contraindicated for patients with advanced ages, leaving only tooth extraction as definitive treatment. The present study demonstrated that the strong activation of OFS-3 occurred from the initial stage of mouse ligature- induced periodontitis, suggesting the possibility of early disease detection prior to the radiographic loss of alveolar bone. Furthermore, the unexpected early activation of OFS-3 on Day 1 of the ligature-induced mouse periodontitis may provide a novel evidence of the pathological mechanism of the disease onset. 3.8 Conclusions Our novel OFS imaging probes consist of a fluorophore-quencher IQF dye pair linked by a ctsk- cleavable peptide as previously described, but with the critical addition of a bisphosphonate moiety to anchor the probe to bone surfaces, where it can remain adsorbed until activated by osteoclast- related ctsk activity. Pamidronate was chosen as a high bone-affinity bisphosphonate having an amino group to conjugate to the carboxylic acid group of lysine, which serves as a trifunctional scaffold to construct the probes. The fluorophore dye is connected by the lysine side, and a GHPGGPQG ctsk- cleavable peptide links the quencher to the rest of the molecule via a β-alanine spacer. The design 101 was optimized by molecular modeling of the probe into the active site of ctsk, subject to method constraints imposed by the overall probe size. Our initial probe, OFS-1 was selected for visible-light detection, matching 5-FAM with BHQ-1. As NIR fluorescence would be more readily detected in vivo in deep tissue due to better penetration, minimal tissue autofluorescence and low light scattering, the incorporation of other IQF dye pairs capable of generating fluorescence at longer wavelengths is considered. In our study, the NIR probes which involve AF657 (OFS-2) and Sulfo-Cy5 (OFS-3) as the emitting fluorophores, and appropriately chosen quenchers (BHQ-3 and BBQ-650, respectively) were designed and synthesized. Molecular modeling and ctsk enzyme activation kinetics revealed that larger, more anionic dyes (as well as the omission of the a β-Ala spacer to simplify the synthetic route) in OFS-2 drastically reduced the activation rate. Hence, OFS-2 was not taken forward to the in vitro and in vivo studies. This led to OFS-3, although ~10 times less ctsk-active substrate than OFS-1, proved to be an useful imaging probe. Thus, extension of this approach into the NIR region requires careful consideration of multiple structural and charge factors involving the selected IQF dye pair, linker length and other possible factors such as target specificity, biodistribution, and targeting mechanism. OFS-1 and OFS-3 applied in a murine ligation-induced periodontitis model, resulted in strong fluorescent signals at the expected lesion site much earlier than the detection of periodontal disease by other imaging methods used. OFS-3-treated mice have already started showing a signal on Day 1. This indicates an early onset local release of activatable ctsk probe at the preosteoclast stage. Therefore, evolving OFS probes will be useful to investigate the pathological mechanisms of disease-induced bone resorption. These probes show great potential as powerful diagnostic tools for a wide range of osteolytic diseases, including periodontitis. 102 3.9 References (1) Novinec, M.; Lenarčič, B. Cathepsin K: A Unique Collagenolytic Cysteine Peptidase. Biol. Chem. 2013, 394 (9), 1163–1179. https://doi.org/10.1515/hsz-2013-0134. (2) Connor, J. R.; LePage, C.; Swift, B. A.; Yamashita, D.; Bendele, A. M.; Maul, D.; Kumar, S. Protective Effects of a Cathepsin K Inhibitor, SB-553484, in the Canine Partial Medial Meniscectomy Model of Osteoarthritis. Osteoarthritis Cartilage 2009, 17 (9), 1236– 1243. https://doi.org/10.1016/j.joca.2009.03.015. (3) Kozloff, K. M.; Quinti, L.; Patntirapong, S.; Hauschka, P. V.; Tung, C.-H.; Weissleder, R.; Mahmood, U. Non-Invasive Optical Detection of Cathepsin K-Mediated Fluorescence Reveals Osteoclast Activity in Vitro and in Vivo. Bone 2009, 44 (2), 190–198. https://doi.org/10.1016/j.bone.2008.10.036. (4) Kashemirov, B. A.; Bala, J. L. F.; Chen, X.; Ebetino, F. H.; Xia, Z.; Russell, R. G. G.; Coxon, F. P.; Roelofs, A. J.; Rogers, M. J.; McKenna, C. E. Fluorescently Labeled Risedronate and Related Analogues: “Magic Linker” Synthesis. Bioconjug. Chem. 2008, 19 (12), 2308–2310. https://doi.org/10.1021/bc800369c. (5) Sun, S.; Błażewska, K. M.; Kadina, A. P.; Kashemirov, B. A.; Duan, X.; Triffitt, J. T.; Dunford, J. E.; Russell, R. G. G.; Ebetino, F. H.; Roelofs, A. J.; et al. Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile Imaging Toolkit for Applications in Bone Biology and Biomedicine. Bioconjug. Chem. 2016, 27 (2), 329–340. https://doi.org/10.1021/acs.bioconjchem.5b00369. 103 (6) Owens, E. A.; Henary, M.; El Fakhri, G.; Choi, H. S. Tissue-Specific Near-Infrared Fluorescence Imaging. Acc. Chem. Res. 2016, 49 (9), 1731–1740. https://doi.org/10.1021/acs.accounts.6b00239. (7) Roelofs, A. J.; Stewart, C. A.; Sun, S.; Błażewska, K. M.; Kashemirov, B. A.; McKenna, C. E.; Russell, R. G. G.; Rogers, M. J.; Lundy, M. W.; Ebetino, F. H.; et al. Influence of Bone Affinity on the Skeletal Distribution of Fluorescently Labeled Bisphosphonates in Vivo. J. Bone Miner. Res. 2012, 27 (4), 835–847. https://doi.org/10.1002/jbmr.1543. (8) Dejica, V. M.; Mort, J. S.; Laverty, S.; Percival, M. D.; Antoniou, J.; Zukor, D. J.; Poole, A. R. Cleavage of Type II Collagen by Cathepsin K in Human Osteoarthritic Cartilage. Am. J. Pathol. 2008, 173 (1), 161–169. https://doi.org/10.2353/ajpath.2008.070494. (9) Marchesan, J.; Girnary, M. S.; Jing, L.; Miao, M. Z.; Zhang, S.; Sun, L.; Morelli, T.; Schoenfisch, M. H.; Inohara, N.; Offenbacher, S.; et al. An Experimental Murine Model to Study Periodontitis. Nat. Protoc. 2018, 13 (10), 2247–2267. https://doi.org/10.1038/s41596-018-0035- 4. (10) Yao, Z.; Lei, W.; Duan, R.; Li, Y.; Luo, L.; Boyce, B. F. RANKL Cytokine Enhances TNF-Induced Osteoclastogenesis Independently of TNF Receptor Associated Factor (TRAF) 6 by Degrading TRAF3 in Osteoclast Precursors. J. Biol. Chem. 2017, 292 (24), 10169–10179. https://doi.org/10.1074/jbc.M116.771816. (11) Tugnait, A.; Carmichael, F. Use of Radiographs in the Diagnosis of Periodontal Disease. Dent. Update 2005, 32 (9), 536–542. https://doi.org/10.12968/denu.2005.32.9.536. 104 CHAPTER 4 Synthesis of 8-Oxo-dGTP and its β,γ-CH2-, β,γ-CHF-, and β,γ-CF2 Analogues 4.1 Introduction Oxidative DNA damage constitutes a major threat to genetic integrity, and has been implicated in the pathogenesis of a wide variety of diseases, ranging from cancer, neurodegenerative and neurodevelopmental disorders, inflammatory disorders to aging. 1–6 8-Oxo-2’-deoxyguanosine (8- oxo-dG) is the most common harbinger of oxidative DNA damage 7 , and has been implicated in carcinogenesis both by inducing mutations 8 and by epigenetic modulation of gene expression. 9–11 8-Oxo-dG is also believed to contribute significantly to aging 12–14 and cancer 15 . This mutagenicity occurs due to the conformational shift of the N9-C1’ glycosidic bonds from anti to syn. 8-Oxo-dG functionally mimics a thymidine (dT) in syn conformation. This leads to stable formation of a pro- mutagenic 2’-deoxyadenosine dA (anti):8-oxo-dG (syn) mispair instead of the non-mutagenic 2’- deoxycytosine dC (anti):8-oxo-dG (anti) base pair. 16 As a result of this stable dA:8-oxo-dG Hoogsteen base mispairing, replicative DNA polymerases (pols) often insert the incorrect dA opposite 8-oxo-dG instead of the correct dC. 17–20 Moreover, the dA:8-oxo-dG mispair avoids proofreading, the inbuilt error-detection mechanism found in high-fidelity replicative pols. 21 The correct dC:8-oxo-dG Watson-Crick base pair is instead recognized as a mismatch, which consequently leads to a much lower efficiency of dC incorporation opposite 8-oxo-dG. 22 As essential building blocks for life, deoxyribonucleotides (dNTPs), the precursors of DNA are not exempted from the threat that arises from reactive oxygen species (ROS). Besides attacking DNA bases directly, ROS also cause oxidation of the dNTP pool present in cells. 8-Oxo-2’- deoxyguanosine-5’-triphosphate (8-Oxo-dGTP) is formed from the oxidation of 2’- 105 deoxyguanosine-5’-triphosphate (dGTP) in the cellular nucleotide pool. 23 dGTP is observed to be more prone to oxidation than 2’-deoxyguanosine (dG) in DNA. 24 Nucleotide pools contain sufficient 8-oxo-dGTP to promote mutagenesis even with cellular sanitizing activities. 25,26 There are currently 17 known human pols that polymerize growing chains of DNA by incorporation of dNTPs opposite a DNA template. 27 Each of these pols will intrinsically incorporate damaged nucleotides with a different propensity, depending on its specific properties. Incorporation of 8-oxo-dGTP has been shown to be catalyzed by many different pols across all families and reverse transcriptases into DNA and RNA. The incorporation efficiencies as well as preference for either a templating A or C depend on the individual pol and whether 8-oxo-dGTP adapts a syn or anti conformation during base pairing. 26,28–40 Incorporation of 8-oxo-dGTP by pols has been shown to generate A:T → C:G transversion mutations. 41–44 Some of the questions that have not been addressed in this field include which of the pols leads to incorporation of 8-oxo-dGTP into DNA in vivo, under which circumstance this happens, and whether insertion of an 8-oxo-dGTP by replicative pols triggers proofreading activity or even initiates a pol switch to continuously promote DNA synthesis. Pol β, pol η, REV1, pol ξ, pol κ have all been implicated in catalyzing 8-oxo-dGTP incorporation into DNA in vivo. 22 It thus remains to be addressed how exactly cells deal with such situations to alleviate both the mutational as well as the potentially cytotoxic potential that results from 8-oxo-dGTP insertion. Hence, understanding the basic mechanism of catalysis by pols, including the fidelity of DNA synthesis in the presence of 8-oxo-dGTP analogues, is the first step towards answering these questions. In this study, a set of CXY dNTP probes that includes one known 8-oxo-dGTP 17 45 and three novel 8-oxo-dGTP analogues, namely β,γ-methylene (CH2)- 18, β,γ-fluoromethylene (CHF)- 19, and β,γ-difluoromethylene (CF2)-8-oxo-dGTP 20 (Figure 4.1) was synthesized. 106 Figure 4.1. Structures of 8-oxo-dGTP 17, β,γ-methylene (CH2)- 18, β,γ-fluoromethylene (CHF)- 19, and β,γ-difluoromethylene (CF2)-8-oxo-dGTP 20. A tool kit consisting of the four 8-oxo-dGTP analogues will be used to study the effects of leaving group on the nucleotidyl transfer mechanism as well as the fidelity of pols. 46–48 These β,γ-CXY-8-oxo-dGTP analogues, each having a different leaving group mimicking pyrophosphate in 8-oxo-dGTP 17, can be structurally tuned by varying the X and Y substituents to exhibit a large range of the bisphosphonate leaving group conjugate acid pKa4 values. A leaving- group effect can thus be investigated with the derivatives acting as sensitive chemical probes of relative P-O charge stabilization in the transition state (TS). 46–49 In a simplified manner, as X and Y are more electronegative, the pKa4 decreases, meaning that the bisphosphonate leaving group aptitude increases since it is more stable as an anion. The more stable the negative charge, the better the bisphosphonate acts as a leaving group. If chemistry is rate-determining and the P-O bond breaking is slow relative to a pre- or post-chemical rate-determining step (RDS), then a plot of the log of the catalytic rate constant (kpol) versus pKa4 (Brønsted plot 50 ) is predicted to be linear (linear free energy relationship, LFER 51 ) with a negative slope in which the magnitude reflects the sensitivity of the TS to charge stabilization. 8-Oxo-dGTP 17 β,γ-CXY-8-Oxo-dGTP CXY = CH 2 (18), CHF (19), CF 2 (20) 107 4.2 Results and Discussion Early oxidative methods to prepare 17 in low or unstated yield directly from dGTP were not reproduced by others. 23,52,53 Alternative phosphorylation of 8-oxo-2’-deoxyguanosine-5’- diphosphate (8-oxo-dGDP) by nucleoside diphosphate kinase was reported to give 8-oxo-dGTP in only 9% yield, 54,55 and no yield was given for a 8-step procedure that began from dG. 29 Nampalli and Kumar subsequently described a multi-gram scale synthesis of 17 from 8-bromo-2’- deoxyguanosine (8-bromo-dG) in 36% overall yield via conversion to the 8-benzyloxy derivative by treatment with sodium/benzyl alcohol in DMSO, which was then treated with phosphorous oxychloride (POCl3) in triethyl phosphate followed by bis(tributylammonium) pyrophosphate, tributylamine and hydrolysis of the resulting cyclic triphosphate intermediate in TEAB buffer at pH 7.5. 45 Thus modification of this approach to synthesis of 18 – 20 by substituting the appropriate bisphosphonate for pyrophosphate was examined. 8-Oxo-dG 4 was prepared from dG 1 in 39% yield. 45,56 However, the phosphorylation conditions 45 applied to 8-benzyloxy-2’-deoxyguanosine (8-benzyloxy-dG) 3 did not result in a significant amount of 17 (less than 2%). A first attempt to verify phosphorylation of 8-benzyloxy-dG 3 using the one-pot-three-step synthesis (Method A) described by Nampalli and Kumar 45 was made. However, the starting material 3 remained intact as POCl3 was hydrolyzed to H3PO4. Phosphorylation of 3 with POCl3 to give 8-benzyloxy-2’-deoxyguanosine 5’-monophosphate (8-benzyloxy-dGMP) was explored, but the starting material 3 again remained intact as POCl3 was again, hydrolyzed to H3PO4. An alternate synthesis via intermediate 4 (prepared in 33% from 1) 45,56 using the one-pot-three-step synthesis (Scheme 4.1) developed by Ludwig 57 and others 58 was examined, but obtained an overall 108 yield of only 1%, apparently due to formation of side-products 59–61 , and the difficulties in isolating the desired product 17 on a milligram scale. Scheme 4.1. One-pot-three-step synthesis (Method A) 45 of 8-Oxo-dGTP 17. A further attempt at the bisphosphonylation of 8-oxo-dGMP-morpholidate 6 which was obtained in nearly quantitative yield by standard DCC-promoted coupling of 8-oxo-dGMP 5 (Method B) 62 with morpholine in 1:1 H2O:t-BuOH at 95 °C was made. Reaction of 6 with the tributylammonium salt of the corresponding bisphosphonic acids in DMF at room temperature gave 8-oxo-dGTP 17, β,γ-CH2-8-oxo-dGTP 18, β,γ-CHF-8-oxo-dGTP 19, and β,γ-CF2-8-oxo-dGTP 20 in 4%, 17%, 4%, and 2% yields, respectively (Scheme 4.2). Moreover, it is the first time that the morpholidate coupling of a 2’-deoxynucleoside 5’-monophosphate with the tributylammonium salt of pyrophosphoric acid is described. 109 Scheme 4.2. Synthesis of 8-oxo-dGTP 17, and β,γ-CXY-8-oxo-dGTP analogues where X and Y are H 18; X is H , Y is F and X is F, Y is H 19; X and Y are F 20 using Khorana’s morpholidate method 62 (Method B). More satisfactory results were obtained using a modification of Bogachev’s 63 and Jakeman’s 64 methods (Method C). Thus, a suspension of 5 in a mixture of TEA and excess trifluoroacetic anhydride in DMF (instead of acetonitrile 64 ) was treated with N-methylimidazole to give the corresponding 8-oxo-dGMP-N-methylimidazolide, which was then added to 2 equivalents of the appropriate tributylammonium bisphosphonate in DMF (Scheme 4.3). The yields were in general significantly higher: 26% (17), 26% (18), and 20% (20). However, the yield of 19 was ~8% and was not improved by substituting acetonitrile for DMF as the solvent. 110 Scheme 4.3. Synthesis of 8-oxo-dGTP 17, and β,γ-CXY-8-oxo-dGTP analogues where X and Y are H 18; X is H , Y is F and X is F, Y is H 19; X and Y are F 20 using a combination of Bogachev’s 63 and Jakeman’s 64 method (Method C). The reaction time for the coupling of 8-oxo-dGMP-N-methylimidazolidate 7 and the tributylammonium salt of the bisphosphonic acid, as Jakeman and his co-worker pointed out, 64 is dependent on the nature of the substituent on the methylene group of the bisphosphonic acid. Bisphosphonic acids containing more electronegative substituents in general react more slowly. The reaction took 180 minutes (slowest) to couple for (difluoromethylene)bisphosphonic acid, in comparision to 120 minutes (fastest) for (methylene)bisphosphonic acid. The coupling time for pyrophosphoric acid and (fluoromethylene)bisphosphonic acid only differed by 10 minutes (160 minutes for pyrophosphoric acid and 150 minutes for (fluoromethylene)bisphosphonic acid, respectively), because of the similar electronegativity between oxygen and fluoromethylene group on the bridging carbon. This might be due to the decrease in the nucleophilicity of the phosphoryl oxygen atoms caused by the electron-withdrawing effect of the electronegative substituents on the bridging carbon. In all of these coupling reactions, the reaction (2-3 hours) was significantly shorter than previous coupling methods. 57,58,62 When the tributylammonium salt of the bisphosphonic acid was not dried thoroughly by azeotroping with anhydrous DMF before use, a 111 side reaction which led to the formation of di-8-oxo-2’-deoxyguanosine-5’-5’-pyrophosphate (8- oxo-dGpp8-oxo-dG) occurred as indicated by the characteristic 31 P NMR chemical shifts of this species. 65 Proton-decoupled 31 P NMR spectra 17 – 20 are presented (Figure 4.2). Despite of the difference in the frequency of the spectrometer used, a gradual upfield shift of the Pγ and Pβ resonance signals when a more electronegative substituent (CF2 > CHF > CH2) is introduced to the bridging β- methylene group was observed. The Pβ resonance signal in 17 is most upfield shifted, followed by that in 20, 19 and 18. A similar pattern is observed for Pγ. There is no effect on the Pα resonance signal which remains constant at about -10 ppm. Figure 4.2. 31 P NMR spectra of 17, 18, 19 and 20. In consistency with the previous study of β,γ-fluoromethylene and β,γ-difluoromethylene analogues of dGTP performed in our group, 66 19 and 20 exhibit a multiplet with δ -220.04 ppm, and a pair of dd with δ -121.61 ppm in the 19 F NMR spectrum. 112 Compounds 17 – 20 were purified by dual-pass preparative HPLC (SAX and C18), and characterized by 1 H, 31 P (Figure 4.2), 19 F NMR and HRMS. 4.3 Conclusions In summary, β,γ-methylene- 18, (R/S)-β,γ-fluoromethylene- 19, and β,γ-difluoromethylene-8- oxo-dGTP 20 were prepared by two methods: first DCC-promoted conjugation of 8-oxo-dGMP- morpholidate 6 in anhydrous DMSO with the tributylammonium salt of the appropriate methylenebis(phosphonic acids), second coupling of the tributylammonium salt of the appropriate methylenebis(phosphonic acids) with an activated 8-oxo-dGMP-imidazolidate 7. 8-oxo-dGTP 17 was also synthesized by a one-pot-three-step method in addition to these two methods. The latter method used to prepare β,γ-8-oxo-dGTP analogues is preferred, since it shortened the reaction times owing to the increased reactivity of the electrophilic phosphoryl group, and resulted in higher yields than the former method. 4.4 Experimental Section General Experimental Information for Synthesis dG was purchased from Alfa Aesar. Methylenediphosphonic acid, fluoro- and difluoromethylenebisphosphonic acids were prepared according to literature procedures. 46,47,64,66– 68 All other reagents were purchased from commercial sources and used as obtained, unless specified otherwise. 1 H, 19 F and 31 P NMR spectra were obtained on Varian 400-MR 2-Channel NMR, Varian VNMRS-500 2-Channel NMR and Varian VNMRS-600 3-Channel NMR spectrometers. Multiplicities are quoted as singlet (s), doublet (d), triplet (t), unresolved multiplet 113 (m), doublet of doublets (dd), doublet of triplets (dt), triplet of doublets (td), doublet of doublet of doublets (ddd) or broad signal (br). All chemical shifts (δ) are in parts per million (ppm) relative to CH3OH in CD3OD (δ 3.34, 1 H NMR), CHCl3 in CDCl3 (δ 7.26, 1 H NMR), HOD in D2O (δ 4.79, 1 H NMR), DMSO in DMSO-d6 (δ 2.54, 1 H NMR), 69 external 85% H3PO4 (δ 0.00, 31 P NMR), or external CFCl3 (δ 0.00, 19 F NMR). 31 P NMR spectra were proton-decoupled and proton-coupled, and 1 H, 19 F and 31 P coupling constants (J values) were given in Hz. The concentration of the NMR samples was in the range of 3-5 mg/mL for intermediates and 1-3 mg/mL for final compounds. Preparative HPLC was performed using a Shimadzu LC-8A equipped with a LabSolutions Lite Release 5.71 SP2 software and Shimadzu SPD-10A UV detector (0.5 mm path length) with detection at 293-nm wavelength (Table 4.1). Low-resolution mass spectrometry was performed on a Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI source in the negative ion mode. LC-MS was also performed on the Finnigan LCQ Deca XP Max mass spectrometer in negative mode with a Thermo Finnigan Surveyor PDA Plus detector (1-cm path length) and MS Pump Plus, all controlled using Xcalibur software, version 2.0.7. High-resolution mass spectrometry was performed using a Q Exactive TM Plus Hybrid Quadrupole-Orbitrap TM mass spectrometer. 8-Br-dG (2): A suspension of dG monohydrate 1 (320 mg, 1.12 mmol) in acetonitrile (12.0 mL) and water (3.00 mL) was treated with freshly crystallized N-bromosuccinimide (NBS) (from water) (300 mg, 1.68 mmol, 1.5 eq) in three aliquots (~100 mg each) over 30 minutes, which converted to 10 minutes per addition. The mixture was stirred for an additional 30 minutes at room temperature. The mixture was filtered through a celite pad. The residue was washed with acetone (5.00 mL x 2) to yield off-white flaky precipitates of compound 2. (360 mg, 1.04 mmol, 93%). 1 H NMR (500 MHz, DMSO-d6) δ 10.78 (s, 1H, 1-NH), 6.48 (s, 2H, 2-NH2), 6.15 (t, J = 7.3 Hz, 1H, 114 1’-H), 5.25 (td, J = 3.0, 1.4 Hz, 1H, 5’-OH), 4.85 (td, J = 6.2, 2.3 Hz, 1H, 4’-H), 4.41-4.38 (m, 1H, 3’-H), 3.79 (td, J = 5.7, 2.8 Hz, 1H, 5’-H), 3.62 (dt, J = 11.3, 5.6 Hz, 1H, 5’-H), 3.49 (dt, J = 11.9, 6.1 Hz, 1H, 3’-OH), 3.16 (dt, J = 13.8, 7.1 Hz, 1H, 2’-H), 2.09 (ddd, J = 13.0, 6.5, 3.3 Hz, 1H, 2’- H). Lit 70 : 1 H NMR (400 MHz, DMSO-d6/CDCl3) δ 10.75 (s, 1H), 6.36 (s, 2H), 6.17 (dd, J = 6.6 Hz, J = 8.1 Hz, 1H), 5.18 (d, J = 4.3 Hz, 1H), 4.90 (dd, J = 5.0 Hz, J =7.0 Hz, 1H), 4.42 (td, J = 3.2 Hz, J = 6.6 Hz, 1H), 3.83 (td, J = 2.9 Hz, J = 5.1 Hz, 1H), 3.65 (dt, J = 4.9 Hz, J = 11.6 Hz, 1H), 3.53 (ddd, J = 5.3 Hz, J =7.0 Hz, J = 11.9 Hz, 1H), 3.11 (ddd, J = 6.3 Hz, J = 8.1 Hz, J = 13.2 Hz, 1H), 2.09 (ddd, J = 2.7 Hz, J = 6.5 Hz, J = 13.2 Hz, 1H). 8-Benzyloxy-dG (3): Sodium metal (120 mg, 5.20 mmol, 5.0 eq) was dissolved in benzyl alcohol (3.75 mL) under nitrogen atmosphere at room temperature. 8-Br-dG 2 (360 mg, 1.04 mmol) was added, followed by anhydrous DMSO (2.50 mL) to ensure complete dissolution of 2. The color of yellow solution intensified from pale yellow to bright yellow over time. After heating at 70 °C for 24 hours, the mixture was poured into diethyl ether (100 mL) solvent for precipitation of the desired compound. The precipitates were filtered. The crude product was purified by CombiFlash chromatography, with a gradient of 0 → 20% methanol (plus a few drops of ammonium hydroxide) in dichloromethane. The fractions containing the desired product were checked by TLC analysis and concentrated under reduced pressure to yield white solids of compound 3 (140 mg, 0.375 mmol, 36%). Compound 4 (50 mg, 0.177 mmol, 17%) was also isolated as white solids with CombiFlash chromatography, at 30% methanol (plus a few drops of ammonium hydroxide) in dichloromethane. 1 H NMR (400 MHz, DMSO-d6) δ 10.55 (br, 1H, 1-NH), 7.50 – 7.35 (m, 5H, 8- OCH2-C6H5), 6.31 (s, 2H, 2-NH2), 6.08 (t, J = 7.3 Hz, 1H, 1’-H), 5.40 (s, 2H, 8-OCH2Ar), 5.18 – 5.14 (m, 1H, 5’-OH), 4.78 – 4.75 (m, 1H, 4’-H), 4.25 – 4.20 (m, 1H, 3’-H), 3.73 – 3.67 (m, 1H, 5’-H), 3.45 – 3.36 (m, 1H, 5’-H), 2.85 (dt, J = 13.8, 6.8 Hz, 1H, 2’-H), 2.01 (td, J = 6.8, 3.7 Hz, 115 1H, 2’-H). Lit 71 : 1 H NMR (500 MHz, DMSO-d6) δ 10.98 (br, s, 1-NH, D2O exchangeable), 7.59 – 7.38 (m, 5H, 8-OCH2C6H5), 6.34 (s, 2H, 2-NH2, D2O exchangeable), 6.14 (t, 1 H, 1’-H), 5.42 (s, 2H, 8-OCH2Ar), 5.21 – 4.56 (m, 2 H, 3’- and 5’-OH, D2O exchangeable), 4.40 – 4.12 (m, 1 H, 4’- H), 3.82 – 3.61 (m, 1 H, 3’-H), 2.40 – 2.05 (m, 2 H, 2’-H). Lit 72 : 1 H NMR (500 MHz, DMSO-d6) δ 10.55 (s, 1H, N-H), 7.49 – 7.31 (m, 5H), 6.31 (s, 2H), 6.08 (t, J = 7.2 Hz), 5.40 (dd, J = 17.3, 11.9 Hz, 2H), 5.16 (d, J = 4.3 Hz, 1H), 4.77 (J = 6.0 Hz, 1H), 4.23 (m, 1H), 3.70 (m, 1H), 3.43 (m, 1H), 3.36 (m, 1H), 3.08 (br, 1H), 2.85 (m, 1H), 2.02 (m, 1H), 1.17 (t, J = 7.2 Hz, 1H). 8-Oxo-dG (4): A solution of 8-benzyloxy-dG 3 (140 mg, 0.375 mmol) in methanol (5.50 mL) was treated with 1.00 M HCl (0.550 mL) at room temperature. The clear solution was stirred at room temperature for 1 hour. Removal of water and methanol under reduced pressure gave a yellow residue. The crude product was purified by CombiFlash chromatography, with a gradient of 0 → 30 % methanol to yield white solids of compound 4 (85.0 mg, 0.300 mmol, 80%). 1 H NMR (500 MHz, MeOD) δ 6.26 (dd, J = 8.4, 6.5 Hz, 1H, 1’-H), 4.54 (dt, J = 5.7, 2.5 Hz, 1H, 4’-H), 3.96 (q, J = 3.4 Hz, 1H, 3’-H), 3.85 – 3.66 (m, 2H, 5’-H2), 2.99 (ddd, J = 13.5, 8.5, 6.1 Hz, 1H, 2’-H), 2.12 (ddd, J = 13.3, 6.5, 2.5 Hz, 1H, 2’-H). Lit 45 : 1 H NMR (DMSO-d6) δ 6.55 (br, 2H, NH2, D2O exchangeable), 6.07 (dd, 1H, J = 6.0, 9.0 Hz, 1’-H), 5.18 (br, 1H, 3’-OH, D2O exchangeable), 4.86 (br, 1H, 5’-OH, D2O exchangeable), 4.60 (br, 1H, 8-OH), 3.79 (m, 1H, 3’-H), 3.56 (m, 1H, 4’-H), 3.56 (m, 2H, 5’-H2), 2.97 (m, 1H, 2’-Ha), 2.08 (m, 1H, 2’-Hb). 8-Oxo-2’-deoxyguanosine 5’-monophosphate (8-Oxo-dGMP, 5): 8-Oxo-dG 4 (100 mg, 0.353 mmol) was dissolved in trimethyl phosphate (8.00 mL). Dissolution took place at 110 °C. Water and trimethyl phosphate (~4.00 mL) were removed by vacuum distillation. The clear solution was cooled to room temperature, followed by -78 °C in a dry ice-acetone bath for 10 minutes. POCl3 (49.5 µL, 0.530 mmol, 1.5 eq) was added dropwise. The clear solution was stirred at 0 °C in an 116 ice bath for 5 hours. 0.500 M TEAB buffer (pH = 7.50, 8.00 mL) was cooled to -10 °C, and added to the solution as a slurry. The clear solution was left to stir at room temperature overnight for 16 hours. Solvents were removed under reduced pressure. The white suspension was filtered to remove the white solids and dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50). The desired product was isolated using one-stage preparative HPLC: strong anion exchange (SAX) column. The fractions containing 5 were collected, combined and lyophilized giving 5 as a TEA salt (26.0 mg, 0.00716 mmol, 20 %). 1 H NMR (400 MHz, D2O, pH 7.50) δ 6.02 (t, J = 7.2 Hz, 1H, 1’-H), 3.99 – 3.82 (m, 2H, 4’-H and 3’-H), 3.73 (dt, J = 10.7, 6.1 Hz, 1H, 2’-H), 3.17 – 3.06 (m, 2H, 5’- H2), 2.10 (ddd, J = 13.7, 7.4, 4.4 Hz, 1H, 2’-H) ppm. NH2, NH, 3’-OH, and P-OH protons have been exchanged with D2O. 31 P Proton-decoupled NMR (162 MHz, D2O, pH 7.50) δ 3.85 (s) ppm. 31 P Proton-coupled NMR (162 MHz, D2O, pH 7.50) δ 3.85 (t, J = 6.1 Hz) ppm. MS (ESI) calculated for C10H14N5O8P - [M-H] - 362.05, found 362.20. 8-Oxo-dGMP-morpholidate (6): A solution of 8-oxo-dGMP 5 (20.0 mg, 0.0551 mmol, 1.0 eq) in H2O (1.00 mL) was treated with 1.00 M HCl solution to lower the pH to 2. A cloudy solution was observed upon addition of 1.00 M HCl solution. Distilled morpholine (14.4 mg, 0.165 mmol, 3.0 eq; 1.01 g/mL, 14.2 µL) in t-BuOH (1.00 mL) was then added dropwise using a 100 µL gas- tight syringe to obtain a clear solution. The solution was stirred at room temperature for 15 minutes, and then refluxed at 95 °C. DCC (34.1 mg, 0.165 mmol, 3.0 eq) in t-BuOH (2.00 mL) was added dropwise over 2 hours (0.250 mL/15 min). The mixture was refluxed at 95 °C for another 3 hours. The reaction was monitored by 31 P NMR spectroscopy. A singlet at 7.52 ppm was observed with D2O capillary tube, which indicated the formation of the morpholidate 6. Solvents were removed under reduced pressure. The residue was re-suspended in a minimum volume of cold water (1.00 mL). The suspension was filtered to remove the byproduct, dicyclohexylurea as white solids. 117 Water was removed under reduced pressure to give a light brown oil. The oil was further dried by co-evaporating with DMF (2.00 mL x 3). Solvent was removed under reduced pressure to yield compound 6 (20.0 mg, crude). The morpholidate 6 was directly used in the coupling step without further purification. 8-Oxo-dGMP-imidazolidate (7): The triethylammonium salt of 8-oxo-dGMP 5 (20.0 mg, 0.0551 mmol) was suspended in a mixture of acetonitrile (200 µL) and TEA (38.4 µL, 0.275 mmol, 5.0 eq), cooled to 0 °C in an ice bath and stirred under nitrogen. In a separate flask, a solution of trifluoracetic anhydride (38.3 µL, 0.275 mmol, 5.0 eq) in acetonitrile (50.0 µL) was cooled to 0 °C and added dropwise by a syringe to the flask containing 8-oxo-dGMP 5. The reaction was stirred for 10 minutes at room temperature. A pale-yellow solution was observed. Excess trifluoroacetic anhydride and the in situ TFA produced were then removed under reduced pressure. The mixture was then cooled to 0 °C in an ice bath. In a separate flask, a mixture of N- methylimidazole (13.2 µL, 0.165 mmol, 3.0 eq) in acetonitrile (50.0 µL), and TEA (38.4 µL, 0.275 mmol, 5.0 eq) was prepared, cooled to 0 °C in an ice bath, and then added to the flask containing the trifluoroacyl phosphate. The reaction was stirred for 10 minutes at 0 °C, after which a bright yellow solution was observed. Preparation of the activated 8-oxo-dGMP-N-methylimidazolidate 7 was confirmed by 31 P NMR spectroscopy (δP -10.56 (s)) with D2O capillary tube. Bis(tri-n-butylammonium) pyrophosphate (9): Tetrasodium pyrophosphate decahydrate 8 (1.00 g, 2.24 mmol) was dissolved in water (22.0 mL). The solution was applied to a Dowex 50WX8 (H + ) column (5.00 g). The column was washed with water. Tri-n-butylamine (1.00 mL) in ethanol (9.00 mL) was added to the eluate until pH turned 5.00. The solution was stirred at room temperature for 1 hour. The ethanol/water solution was removed under reduced pressure, co- evaporated with ethanol (5.00 mL x 2) and anhydrous DMF (5.00 mL x 2) and dried in vacuo to 118 yield compound 9 as a white hygroscopic solid (983 mg, 1.79 mmol, 80%). 31 P Proton-decoupled NMR (162 MHz, D2O, pH 5.33) δ -10.89 (s) ppm. Lit 73 : 31 P NMR (121 MHz, D2O) δ -10.05 ppm. Tris(tri-n-butylammonium) pyrophosphate (10): Tetrasodium pyrophosphate decahydrate 8 (1.00 g, 2.24 mmol) was dissolved in water (22.0 mL). The solution was applied to a Dowex 50WX8 (H + ) column (5.00 g). The column was washed with water. Tri-n-butylamine (2.00 mL) in ethanol (8.00 mL) was added to the eluate until pH turned 8.00. The solution was stirred at room temperature for 1 hour. The ethanol/water solution was removed under reduced pressure, co- evaporated with ethanol (5.00 mL x 2) and anhydrous DMF (5.00 mL x 2) and dried in vacuo to yield compound 10 as a white hygroscopic solid (983 mg, 1.79 mmol, 80%). 31 P Proton-decoupled NMR (162 MHz, D2O, pH 5.33) δ -10.10 (s) ppm. Lit 73 : 31 P NMR (121 MHz, D2O) δ -10.05 ppm. Methylenediphosphonic acid (12): A solution of tetraisopropyl methylenediphosphonate 11 (1.00 g, 2.90 mmol) in 12.0 M HCl (10.0 mL) was refluxed at 80 °C for 5 hours. The mixture was brought to room temperature and monitored by 31 P NMR spectroscopy until reaction was complete. The mixture was concentrated under reduced pressure to give a colorless oil. Methanol (5.00 x 2 mL) was added. Volatiles were removed under reduced pressure to yield compound 12 as white solids (300 mg, 1.65 mmol, 57%). 31 P Proton-decoupled NMR (243 MHz, D2O, pH 0.50) δ 18.14 (s) ppm. Lit 64 : 31 P NMR (D2O) δ 16.7 ppm. 31 P Proton-coupled NMR (243 MHz, D2O, pH 0.50) δ 18.13 (t, J = 20.9 Hz) ppm. Tributylammonium salt of methylenediphosphonic acid (13): A solution of methylenediphosphonic acid 12 (100 mg, 0.568 mmol) in water (2.00 mL) was treated with tri-n- butylamine (0.205 mL, 0.863 mmol, 1.5 equiv) in ethanol (2.00 mL) dropwise until pH turned 2.50 at room temperature. The mixture was stirred at room temperature for 1 hour. Solvents were removed under reduced pressure. The mixture was co-evaporated with ethanol (2.00 mL) giving 119 the desired salt which was further dried by co-evaporating with DMF (2.00 mL x 3). Concentration under reduced pressure yielded compound 13 as colorless oil (200 mg, crude). The compound was not characterized and used in the next step without purification. Tetraisopropyl (fluoromethylene)bisphosphonate (14a) and tetraisopropyl (difluoromethylene)bisphosphonate (14b): Sodium hydride (0.260 g as a 60% oil immersion, 2.90 mmol, 2.2 equiv) was treated with anhydrous THF (6.00 mL) and anhydrous DMF (6.00 mL) at 0 °C. A suspension of sodium hydride in THF and DMF was observed. Tetraisopropyl methylenediphosphonate 11 (1.00 g, 2.90 mmol) was added dropwise with a dried 1 mL gas-tight syringe at 0 °C over 10 minutes. The mixture was stirred at 0 °C for 30 minutes. Selectfluor TM (1.54 g, 4.35 mmol, 1.5 equiv) was added. Bubbles of hydrogen gas were produced. The mixture was brought to room temperature and stirred for an additional hour. The reaction was monitored by 31 P NMR spectroscopy. After 2 hours, the mixture was quenched with saturated ammonium chloride solution (20.0 mL, endpoint: colorless solution), and extracted twice with ethyl acetate (40.0 mL x 2). The combined organic layers were washed with water (20.0 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by flash chromatography, liquid load, 0 → 50% ethyl acetate/hexane and 0 → 80% ethyl acetate/hexane, ELSD detector, 40-gram column to yield compound 14a (120 mg, 0.331 mmol, 11%) and compound 14b (350 mg, 0.920 mmol, 32%) as colorless oils. 14a 31 P Proton-decoupled NMR (162 MHz, CDCl3) δ 9.58, 9.19 ppm. Lit 67 : 31 P NMR (neat) δ 10.7 (ddt, JPF = 63 Hz, JPH = 12 Hz, JPH = 3 Hz) ppm. 31 P Proton-coupled NMR (162 MHz, CDCl3) δ 9.59 (dt, J = 13.7, 3.5 Hz), 9.31 – 9.04 (m) ppm. 19 F NMR (470 MHz, CDCl3) δ -226.40 (dt, 1F) ppm. Lit 67 : 19 F NMR (neat) δ -221 (dt, JFP = 63 Hz, JFH = 44 Hz) ppm. 14b 31 P Proton-decoupled NMR (243 MHz, CDCl3) δ 2.33, 1.97, 1.61 ppm. Lit 67 : 31 P NMR (neat) δ 2.80 (tt, JPF = 84 Hz, JPH = 3 Hz) ppm. 31 P Proton-coupled 120 NMR (243 MHz, CDCl3) δ 2.38, 2.03, 1.67 ppm. 19 F NMR (470 MHz, CDCl3) δ -122.11 (tt, 2F) ppm. Lit 67 : 19 F NMR (neat) δ -121 (t, JFP = 85 Hz) ppm. (Fluoromethylene)bisphosphonic acid (15a): A solution of tetraisopropyl (fluoromethylene)bisphosphonate 14a (120 mg, 0.331 mmol) in water (1.00 mL) was treated with 12.0 M HCl (0.276 mL, 3.31 mmol, 10 eq). The clear solution was refluxed at 80 °C for 6 hours. The mixture was concentrated under reduced pressure to yield compound 15a (60.0 mg, crude) as a colorless oil. 31 P Proton-decoupled NMR (162 MHz, D2O) δ 9.99 (d, J = 8.9 Hz), 9.58 (d, J = 9.8 Hz) ppm. Lit 67 : 31 P NMR (neat) δ 10.5 (d, JPF = 64 Hz) ppm. 31 P Proton-decoupled NMR (162 MHz, D2O) δ 9.99 (d, J = 12.9 Hz), 9.58 (d, J = 12.9 Hz). 19 F NMR (470 MHz, CDCl3) δ -227.36 (dt, F) ppm. Lit 67 : 19 F NMR (neat) δ -225 (dt, JFH = 46 Hz, JPH = 63 Hz) ppm. (Difluoromethylene)bisphosphonic acid (15b): A solution of tetraisopropyl (difluoromethylene)bisphosphonate 14b (175 mg, 0.460 mmol) in water (1.00 mL) was treated with 12.0 M HCl (0.383 mL, 4.60 mmol, 10 eq). The clear solution was refluxed at 80 °C for 6 hours. The mixture was concentrated under reduced pressure to yield compound 15b (90.0 mg, crude) as a colorless oil. 31 P Proton-decoupled NMR (243 MHz, D2O) δ 3.30, 2.94, 2.60 ppm. Lit 67 : 31 P NMR (neat) δ 3.7 (t, JPF = 86 Hz) ppm. 31 P Proton-coupled NMR (243 MHz, D2O) δ 3.34 (d, J = 7.7 Hz), 2.98 (d, J = 7.7 Hz), 2.63 (d, J = 7.2 Hz) ppm. 19 F NMR (470 MHz, CDCl3) δ - 124.39 (t, 2F) ppm. Lit 67 : 19 F NMR (neat) δ -121 (t, JFP = 86 Hz) ppm. Tributylammonium salt of (fluoromethylene)bisphosphonic acid (16a): A solution of (fluoromethylene)bisphosphonic acid 15a (60.0 mg, 0.309 mmol) in water (1.00 mL) was treated with tri-n-butylamine (0.110 mL, 0.464 mmol, 1.5 equiv) in ethanol (1.00 mL) dropwise until pH turned 2.50 at room temperature. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and co-evaporated with anhydrous DMF (2.00 121 mL x 3) to yield compound 16a as a colorless oil (100 mg, crude). The compound was not characterized and used in the next step without purification. Tributylammonium salt of (difluoromethylene)bisphosphonic acid (16b): A solution of (difluoromethylene)bisphosphonic acid 15b (90.0 mg, 0.425 mmol) in water (1.00 mL) was treated with tri-n-butylamine (0.151 mL, 0.636 mmol, 1.5 equiv) in ethanol (1.00 mL) dropwise until pH turned 2.50 at room temperature. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, co-evaporated with anhydrous DMF (2.00 mL x 3) and dried in vacuo to yield compound 16b as a brown oil (150 mg, crude). The compound was not characterized and used in the next step without purification. 8-Oxo-dGTP (17) using Method A: A solution of 8-oxo-dG 4 (100 mg, 0.350 mmol) in trimethyl phosphate (8.00 mL) was vacuum distilled to remove water from 8-oxo-dG 4 and trimethyl phosphate (~4.00 mL). The solution was cooled down in a dry ice-acetone bath, and treated with POCl3 (49.5 μL, 0.530 mmol, 1.5 eq) dropwise under nitrogen atmosphere. The solution was stirred in a dry ice-acetone bath for 10 minutes, and then stirred in an ice bath for 5 hours. Bis(tri-n-butylammonium) pyrophosphate 9 (968 mg, 1.77 mmol, 5.0 eq) in anhydrous DMF (3.54 mL) was required to prepare 0.500 M DMF solution of 9. 0.500 M DMF solution of 9 and tri-n-butylamine (0.420 mL, 1.77 mmol, 5.0 eq) were added. The cyclic phosphate formed was stirred at room temperature for 30 minutes, then hydrolyzed with 1.00 M TEAB buffer (30 mL, pH 7.50) for 16 hours. Solvents were removed under reduced pressure. The white suspension was filtered to remove the white solids and dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50), and the desired product was isolated using two-stage preparative HPLC: first pass, SAX column; second pass, C18 column. The fractions containing 17 were collected, combined and lyophilized giving 17 as a TEA salt (1.99 mg, 0.00381 mmol, 1%). 1 H NMR (500 MHz, MeOD) δ 6.21 (t, J = 122 7.2 Hz, 1H, 1’-H), 4.43 (dt, J = 10.8, 7.4 Hz, 1H, 4’-H), 4.17 – 4.04 (m, 2H, 2’-H and 3’-H), 3.29 – 3.17 (m, 2H, 5’-H), 2.12 (ddd, J = 13.5, 7.0, 3.4 Hz, 1H, 2’-H) ppm. 31 P Proton-decoupled NMR (202 MHz, MeOD) δ -10.23 (d, γ-P), -10.86 (d, α-P), -23.23 (t, β-P) ppm. 31 P Proton-coupled NMR (202 MHz, MeOD) δ -10.12 (d, γ-P), -10.81 (d, α-P), -23.17 (t, β-P) ppm. 1 H NMR (400 MHz, D2O, pH 9.15) δ 6.23 (t, J = 7.2 Hz, 1H, 1’-H), 4.33 – 4.30 ( m, 1H, 4’-H), 4.19 – 4.15 (m, 1H, 3’- H), 3.76 – 3.73 (m, 1H, 5’-H), 3.68 – 3.65 (m, 1H, 5’-H), 3.24 – 3.08 (m, 1H, 2’-H), 2.31 – 2.25 (m, 1H, 2’-H) ppm. NH2, NH, 3’-OH, and α-, β-, γ-P-OH protons have been exchanged with D2O. Lit 45 : 1 H NMR (D2O) δ 6.13 (dd, 1H, J = 6.0, 9.0 Hz, 1’-H), 4.22 (m, 1H, 4’-H), 4.05 (m, 1H, 3’- H), 3.18 (m, 2H, 5’-H2), 2.85 (m, 1H, 2’-Hb), 2.16 (m, 1H, 2’-Ha) ppm. NH2, NH, 3’-OH, and 5’- OH, protons have been exchanged with D2O. 31 P Proton-decoupled NMR (162 MHz, D2O, pH 9.15) δ -6.78 (d, J = 20.4 Hz, γ-P), -11.41 (d, J = 20.4 Hz, α-P), -22.91 (t, J = 20.0 Hz, β-P) ppm. Lit 45 : 31 P NMR (D2O) δ -8.1 (d, γ-P), -10.1 (d, α-P), -22.22 (t, β-P). 31 P Proton-coupled NMR (162 MHz, D2O, pH 9.15) δ -6.79 (d, J = 21.3 Hz, γ-P), -11.44 (d, J = 21.3 Hz, α-P), -22.92 (t, J = 20.3 Hz, β-P) ppm. MS (ESI) calculated for C10H16N5O14P3 - [M-H] - 521.983, found 522.008. HRMS (ESI) calculated for C10H16N5O14P3 - [M-H] - 521.98338, found 521.98383. 8-Oxo-2’-deoxyguanosine 5’-triphosphate (17) using Method B: A solution of bis(tri-n- butylammonium) pyrophosphate 9 (317 mg, 0.578 mmol, 5.0 eq) in anhydrous DMSO (1.00 mL) was treated with 8-oxo-dGMP-morpholidate 6 (50.0 mg, 0.116 mmol) in anhydrous DMSO (1.00 mL) dropwise. The mixture was stirred at room temperature in a nitrogen atmosphere for 5 days. The solvent was removed under reduced pressure. The colorless residue was dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50), and the desired product was isolated using two-stage preparative HPLC: first pass, SAX column; second pass, C18 column. The fractions containing 18 were collected, combined and lyophilized giving 17 as a TEA salt (2.09 mg, 0.00400 mmol, 4%). 123 8-Oxo-2’-deoxyguanosine 5’-monophosphate-β,γ-CH2-bisphosphonate (18) using Method B: A solution of tributylammonium salt of methylenediphosphonic acid 13 (96.0 mg, 0.266 mmol, 5.0 eq) in anhydrous DMSO (0.500 mL) was treated with 8-oxo-dGMP-morpholidate 6 (23.0 mg, 0.0532 mmol) in anhydrous DMSO (0.500 mL) dropwise. The mixture was stirred at room temperature in a nitrogen atmosphere for 96 hours; 3 days. Solvent was removed under reduced pressure. The colorless residue was dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50), and the desired product was isolated using two-stage preparative HPLC: first pass, SAX column; second pass, C18 column. The fractions containing 18 were collected, combined and lyophilized giving 18 as a TEA salt (4.75 mg, 0.00912 mmol, 17%). 1 H NMR (400 MHz, MeOD) δ 6.20 (t, J = 7.2 Hz, 1H, 1’-H), 4.44 – 4.38 (m, 1H, 4’-H), 4.11 – 4.02 (m, 1H, 3’-H), 3.34 – 2.96 (m, 2H, 5’- H2), 2.34 (t, J = 20 Hz, 2H, CH2), 2.15 – 2.08 (m, 1H, 2’-H) ppm. 31 P Proton-decoupled NMR (162 MHz, MeOD) δ 13.97 (d, J = 7.5 Hz, γ-P), 8.30 (d, J = 24.7 Hz, β-P), -10.16 (d, J = 25.2 Hz, α-P) ppm. 31 P Proton-coupled NMR (MeOD) δ 13.97 (td, J = 19.4, 7.7 Hz, γ-P), 8.30 (d, J = 23.7 Hz, β-P), -10.17 (dt, J = 25.3, 6.4 Hz, α-P) ppm. MS (ESI) calculated for C 11H17N5O13P3 - [M-H] - 520.099, found 520.004. HRMS (ESI) calculated for C11H17N5O13P3 - [M-H] - 520.00412, found 520.00458. 8-Oxo-2’-deoxyguanosine 5’-monophosphate-β,γ-CHF-bisphosphonate (19) using Method B: A solution of tributylammonium salt of (fluoromethylene)bisphosphonic acid 16a (100 mg, 0.264 mmol, 5.0 eq) in anhydrous DMSO (0.500 mL) was treated with 8-oxo-dGMP- morpholidate 6 (23.0 mg, 0.0532 mmol) in anhydrous DMSO (0.500 mL) dropwise. The mixture was stirred at room temperature in a nitrogen atmosphere for 5 days. Solvent was removed under reduced pressure. The colorless residue was dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50), and the desired product was isolated using two-stage preparative HPLC: first pass, SAX 124 column; second pass, C18 column. The fractions containing 19 were collected, combined and lyophilized giving 19 as a TEA salt (1.19 mg, 0.00222 mmol, 4%). 1 H NMR (500 MHz, MeOD) δ 6.21 (t, J = 7.3 Hz, 1H, 1’-H), 5.04 – 4.94 (m, 1H, CHF), 4.52 – 4.37 (m, 1H, 4’-H), 4.11 – 4.05 (m, 2H, 3’-H and 2’-H), 3.28 – 3.18 (m, 2H, 5’-H2), 2.11 (ddd, J = 13.4, 7.0, 3.4 Hz, 1H, 2’-H) ppm. 31 P Proton-decoupled NMR (202 MHz, MeOD) δ 8.00 (d, J = 60.2 Hz, γ-P), 1.23 (m, β-P), -10.24 (d, J = 26.6 Hz, α-P) ppm. 31 P Proton-coupled NMR (202 MHz, MeOD) δ 8.00 (d, J = 59.4 Hz, γ-P), 1.21 (m, β-P), -10.23 (d, J = 25.9 Hz, α-P) ppm. 19 F NMR (376 MHz, D2O, pH 8.01) δ - 217.32 (dd, 1F) ppm. MS (ESI) calculated for C 11H16FN5O13P3 - [M-H] - 537.995, found 538.012. HRMS (ESI): calculated for C11H16FN5O13P3 - [M-H] - 537.99470, found 537.99524. 8-Oxo-dGMP-β,γ-CF2-bisphosphonate (20) using Method B: A solution of tributylammonium salt of (difluoromethylene)bisphosphonic acid 16b (92.0 mg, 0.232 mmol, 5.0 eq) in anhydrous DMSO (0.500 mL) was treated with 8-oxo-dGMP-morpholidate 6 (20.0 mg, 0.0463 mmol) in anhydrous DMSO (0.500 mL). The mixture was stirred at room temperature in a nitrogen atmosphere for 5 days. Solvent was removed under reduced pressure. The colorless residue was dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50), and the desired product was isolated using two-stage preparative HPLC: first pass, SAX column; second pass, C 18 column. The fractions containing 20 were collected, combined and lyophilized giving 20 as a TEA salt (6.04 mg, 0.00108 mmol, 2%). 1 H NMR (400 MHz, MeOD) δ 6.19 (t, J = 7.3 Hz, 1H, 1’-H), 4.43 – 4.39 (m, 1H, 4’- H), 4.09 – 4.06 (m, 1H, 3’-H), 3.26 – 3.20 (m, 2H, 5’-H2), 2.10 – 2.06 (m, 1H, 2’-H) ppm. 31 P Proton-decoupled NMR (162 MHz, MeOD) δ 2.70 (td, J = 78.1, 57.9 Hz, γ-P), -5.22 (m, β-P), - 10.40 (d, J = 28.9 Hz, α-P) ppm. 31 P Proton-coupled NMR (162 MHz, MeOD) δ 2.70 (td, J = 78.1, 57.9 Hz, γ-P), -5.31 (m, β-P), -10.40 (d, J = 28.7 Hz, α-P) ppm. 19 F NMR (565 MHz, MeOD) δ - 125 120.95 (dd, 2F) ppm. MS (ESI) calculated for C11H15F2N5O13P3 - [M-H] - 555.985, found 556.007. HRMS (ESI) calculated for C11H15F2N5O13P3 - [M-H] - 555.98528, found 555.98572. 8-Oxo-dGTP (17) using Method C: A solution of 8-oxo-dGMP-N-methylimidazolidate 7 (23.6 mg, 0.0551 mmol) was prepared, and then added dropwise by a syringe to a flask containing tris(tetra-n-butylammonium) pyrophosphate (99.3 mg, 0.110 mmol, 2.0 eq) and DMF (100 µL) at 0 °C. The reaction was stirred at 0 °C under nitrogen for 160 minutes, and then quenched with 0.500 M TEAB buffer (1.00 mL, pH 7.50). Solvent was removed under reduced pressure. The colorless residue was dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50), and the desired product was isolated using two-stage preparative HPLC: first pass, SAX column; second pass, C18 column. The fractions containing 17 were collected, combined and lyophilized giving 17 as a TEA salt (7.57 mg, 0.0144 mmol, 26%). 8-Oxo-dGMP-β,γ-CH2-bisphosphonate (18) using Method C: A solution of 8-oxo-dGMP-N- methylimidazolidate 7 (23.6 mg, 0.0551 mmol) was prepared, and then added dropwise by a syringe to a flask containing of tributylammonium salt of methylenediphosphonic acid 13 (99.2 mg, 0.110 mmol, 2.0 eq) and DMF (100 µL) at 0 °C. The reaction was stirred at 0 °C under nitrogen for 120 minutes, and then quenched with 0.500 M TEAB buffer (1.00 mL, pH 7.50). Solvent was removed under reduced pressure. The colorless residue was dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50), and the desired product was isolated using two-stage preparative HPLC: first pass, SAX column; second pass, C18 column. The fractions containing 18 were collected, combined and lyophilized giving 18 as a TEA salt (7.46 mg, 0.0143 mmol, 26%). 8-Oxo-dGMP-β,γ-CHF-bisphosphonate (19) using Method C: A solution of 8-oxo-dGMP-N- methylimidazolidate 7 (23.6 mg, 0.0551 mmol) was prepared, and then added dropwise by a syringe to a flask containing of tributylammonium salt of (fluoromethylene)bisphosphonic acid 126 16a (101 mg, 0.110 mmol, 2.0 eq) and DMF (100 µL) at 0 °C. The reaction was stirred at 0 °C under nitrogen for 150 minutes, and then quenched with 0.500 M TEAB buffer (1.00 mL, pH 7.50). Solvent was removed under reduced pressure. The colorless residue was dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50), and the desired product was isolated using two-stage preparative HPLC: first pass, SAX column; second pass, C18 column. The fractions containing 19 were collected, combined and lyophilized giving 19 as a TEA salt (2.40 mg, 0.00446 mmol, 8%). 8-Oxo- dGMP-β,γ-CF2-bisphosphonate (20) using Method C: A solution of 8-oxo-dGMP-N- methylimidazolidate 7 (23.6 mg, 0.0551 mmol) was prepared, and then added dropwise by a syringe to a flask containing of tributylammonium salt of (difluoromethylene)bisphosphonic acid 16b (103 mg, 0.110 mmol, 2.0 eq) and DMF (100 µL) at 0 °C. The reaction was stirred at 0 °C under nitrogen for 180 minutes, and then quenched with 0.500 M TEAB buffer (1.00 mL, pH 7.50). Solvent was removed under reduced pressure. The colorless residue was dissolved in 0.500 M TEAB buffer (1.50 mL, pH 7.50), and the desired product was isolated using two-stage preparative HPLC: first pass, SAX column; second pass, C18 column. 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Phase 1 study of the bone-targeting cytotoxic conjugate, etidronate-cytosine arabinoside (MBC-11), in cancer patients with bone metastases. JCO. 2017;35(15_suppl):2589-2589. doi:10.1200/JCO.2017.35.15_suppl.2589. 158 APPENDIX A: Chapter 2 Supporting Data 159 Figure A-1. 1 H NMR of 1. Data kindly provided by Dr. Eric Richard. 1 160 Figure A-2. 31 P NMR of 1. Data kindly provided by Dr. Eric Richard. 161 Figure A-3. Mass spectrum of 1. 162 Figure A-4. 1 H NMR of 2. Data kindly provided by Dr. Eric Richard. 2 163 Figure A-5. 31 P NMR of 2. Data kindly provided by Dr. Eric Richard. 164 Figure 1. RP-HPLC profile of crude reaction mixture of Boc-protected 3. Figure A-6. Mass spectrum of 2. Figure A-7. Mass spectrum of Boc-protected 3. Datafile Name:3aprep4.lcd Sample Name:3a 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 min 0 500 1000 1500 2000 2500 3000 3500 4000 mAU 0.00 0.25 0.50 0.75 1.00 1.25 psi(x10,000) B.Conc 493nm,4nm 2 Boc-protected 3 165 Figure A-8. RP-HPLC profile of crude reaction mixture of 3. Figure A-9. 1 H NMR of 3. Data kindly provided by Dr. Eric Richard. Datafile Name:3bprep1.lcd Sample Name:3a 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 0 500 1000 1500 2000 2500 3000 3500 4000 mAU 0.00 0.25 0.50 0.75 1.00 1.25 psi(x10,000) B.Conc 493nm,4nm 3 166 Figure A-10. 31 P NMR of 3. Data kindly provided by Dr. Eric Richard. 167 Figure A-11. Mass spectrum of 3. Figure A-12. Mass spectrum of P1. 168 Figure A-13. RP-HPLC profile of crude reaction mixture of Boc- and Trt-protected 4. Figure A-14. Mass spectrum of Boc- and Trt-protected 4. Datafile Name:4aprep2.lcd Sample Name:4a 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min 0 1000 2000 3000 4000 5000 mAU 0.00 0.25 0.50 0.75 1.00 1.25 psi(x10,000) B.Conc 493nm,4nm Boc- and Trt-protected 4 3 169 Figure A-15. RP-HPLC profile of crude reaction mixture of 4. Figure A-16. Mass spectrum of 4. Datafile Name:4b prep4.lcd Sample Name:4b 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 min 0 500 1000 1500 2000 2500 3000 3500 4000 mAU 0.00 0.25 0.50 0.75 1.00 1.25 psi(x10,000) B.Conc 493nm,4nm 4 170 Figure A-17. RP-HPLC profile of crude reaction mixture of OFS-1. Figure A-18. Mass spectrum of 5 (OFS-1). Datafile Name:5-1-8.lcd Sample Name:5-1 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min 0 500 1000 1500 2000 2500 3000 3500 4000 mAU 0.00 0.25 0.50 0.75 1.00 1.25 psi(x10,000) B.Conc 493nm,4nm 4 OFS-1 BHQ-1 171 APPENDIX B: Chapter 3 Supporting Data 172 Figure B-1. Mass Spectrum of 1. 173 Figure B-2. Mass Spectrum of Boc-protected 2. 174 Figure B-3. Mass Spectrum of 2. 175 Figure B-4. Mass Spectrum of P1. 176 Figure B-5. Mass Spectrum of 3. 177 Figure B-6. Mass Spectrum of 4. 178 Figure 5. Mass Spectrum of 4. Figure B-7. Mass Spectrum of 5. 179 APPENDIX C: Chapter 4 Supporting Data 180 Figure C-1. 1 H NMR (500 MHz, DMSO-d6) of 2. X = CH3OH. 181 Figure C-2. 1 H NMR (400 MHz, DMSO-d6) of 3. U = unidentified impurity; X = CH2Cl2; Y = CH3OH. 182 Figure C-3. 1 H NMR (500 MHz, MeOD) of 4. X = H2O; Y = CH3OH. 183 Figure C-4. 1 H NMR (400 MHz, D2O, pH 7.50) of 5. U = unidentified impurities; X = MeOH; Y, Z = Et3N. 184 Figure C-5. 31 P Proton-decoupled NMR (162 MHz, D2O, pH 7.50) of 5. 185 Figure C-6. 31 P Proton-coupled NMR (162 MHz, D2O, pH 7.50) of 5. 186 Figure C-7. Low-resolution MS (ESI) [M-H] - Spectrum of 5. 187 Figure C-8. 31 P Proton-decoupled NMR (162 MHz, D2O Capillary Tube) of 9. 188 Figure C-9. 31 P Proton-decoupled NMR (162 MHz, D2O Capillary Tube) of 10. 189 Figure C-10. 31 P Proton-decoupled NMR (243 MHz, D2O, pH 0.50) of 12. 190 Figure C-11. 31 P Proton-coupled NMR (243 MHz, D2O, pH 0.50) of 12. 191 Figure C-12. 31 P Proton-decoupled NMR (162 MHz, CDCl3) of 14a. 192 Figure C-13. 31 P Proton-coupled NMR (162 MHz, CDCl3) of 14a. 193 Figure C-14. 19 F NMR (470 MHz, CDCl3) of 14a. 194 Figure C-15. 31 P Proton-decoupled NMR (243 MHz, CDCl3) of 14b. 195 Figure C-16. 31 P Proton-coupled NMR (243 MHz, CDCl3) of 14b. 196 Figure C-17. 19 F NMR (470 MHz, CDCl3) of 14b. 197 Figure C-18. 31 P Proton-decoupled NMR (243 MHz, D2O, pH 0.50) of 15a. 198 Figure C-19. 31 P Proton-coupled NMR (243 MHz, D2O, pH 0.50) of 15a. 199 Figure C-20. 19 F (470 MHz, CDCl3) of 15a. 200 Figure C-21. 31 P Proton-decoupled NMR (243 MHz, D2O, pH 0.50) of 15b. 201 Figure C-22. 31 P Proton-coupled NMR (243 MHz, D2O, pH 0.50) of 15b. 202 Figure C-23. 19 F (470 MHz, CDCl3) of 15b. 203 Figure C-24. 1 H NMR (500 MHz, MeOD) of 17. X = CH3OH; Y, Z = Et3N. 204 Figure C-25. 31 P Proton-decoupled NMR (202 MHz, MeOD) of 17. 205 Figure C-26. 31 P Proton-coupled NMR (202 MHz, MeOD) of 17. 206 Figure C-27. 1 H NMR (400 MHz, D2O, pH 9.15) of 17. V= CH2Cl2; W = HOD; X = CH3OH; Y, Z = Et3N. 207 Figure C-28. 31 P Proton-decoupled NMR (162 MHz, D2O, pH 9.15) of 17. 208 Figure C-29. 31 P Proton-coupled NMR (162 MHz, D2O, pH 9.15) of 17. 209 Figure C-30. Low-resolution MS (ESI) [M-H] - Spectrum of 17. 210 Figure C-31. High-resolution MS (ESI) [M-H] - Spectrum of 17. 211 Figure C-32. 1 H NMR (400 MHz, MeOD) of 18. T = H2O; U = unidentified impurities; W = CH3OH; X = CH3CN; Y, Z = Et3N. 212 Figure C-33. 31 P Proton-decoupled NMR (162 MHz, MeOD) of 18. 213 Figure C-34. 31 P Proton-coupled NMR (162 MHz, MeOD) of 18. 214 Figure C-35. Low-resolution MS (ESI) [M-H] - Spectrum of 18. 215 Figure C-36. High-resolution MS (ESI) [M-H] - Spectrum of 18. 216 Figure C-37. 1 H NMR (500 MHz, MeOD) of 19. V = H2O; W = CH3OH; X = CH3CN; Y, Z = Et3N. 217 Figure C-38. 31 P Proton-decoupled NMR (202 MHz, MeOD) of 19. 218 Figure C-39. 31 P Proton-coupled NMR (202 MHz, MeOD) of 19. 219 Figure C-40. 19 F NMR (376 MHz, D2O, pH 8.01) of 19. 220 Figure C-41. Low-resolution MS (ESI) [M-H] - Spectrum of 19. 221 Figure C-42. High-resolution MS (ESI) [M-H] - Spectrum of 19. 222 Figure C-43. 1 H NMR (400 MHz, MeOD) of 20. V = H2O; W = DMSO; X = CH3CN; Y, Z = Et3N. 223 Figure C-44. 31 P Proton-decoupled NMR (162 MHz, MeOD) of 20. 224 Figure C-45. 31 P Proton-coupled NMR (162 MHz, MeOD) of 20. 225 Figure C-46. 19 F NMR (565 MHz, MeOD) of 20. 226 Figure C-47. Low-resolution MS (ESI) [M-H] - Spectrum of 20. 227 Figure C-48. High-resolution MS (ESI) [M-H] - Spectrum of 20.

Abstract (if available)

Abstract Bisphosphonates are stable analogues of pyrophosphate that contain two phosphonate groups bound to a central carbon atom. The presence of two phosphonate groups enables bisphosphonates to chelate calcium ion, which is found in bone mineral. Bone affinity and antiresorptive abilities appear to be two separate properties of bisphosphonates. The bone-binding properties of these compounds may serve to localize these drugs at the site of osteoblast and osteoclast activity, thereby inhibiting osteoclast-mediated bone resorption. ❧ The high bone affinity as well as the specific binding of bisphosphonates are attractive features that could be exploited for targeted delivery of drugs to the bone. This idea was further explored in the work presented in this thesis for various applications, including targeted delivery of therapeutics to the bone and bone imaging. Two categories of projects are covered in this thesis: 1) imaging-related studies and 2) non-imaging-related studies. ❧ The first chapter provides an overview of the general characteristics of bisphosphonates and their structure-activity relationship studies, an outline of the bisphosphonate conjugate designs and their applications to cancer, osteoporosis and imaging, as well as our osteoadsorptive fluorogenic substrate (OFS) design and bisphosphonate alternatives. The two subsequent chapters focus on work related to the synthesis, enzyme kinetics measurements, in vitro and in vivo studies of our two novel imaging probes, OFS-1 and OFS-3. The last chapter centers on the synthesis of 8-oxo-2’-deoxyguanosine-5’-triphosphate (8-oxo-dGTP) and its β,γ-methylene (CH₂)-, β,γ-fluoromethylene (CHF)-, and β,γ-difluoromethylene (CF₂)-8-oxo-dGTP analogues. A tool kit consisting of 8-oxo-dGTP and its three analogues will be used to study the effects of leaving group on the nucleotidyl transfer mechanism as well as the fidelity of DNA polymerases (pols).

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Creator Zheng, Yiying (author)

Core Title Studies of bisphosphate-conjugated fluorescent imaging compounds and 8-oxo-dGTP derivatives

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 04/21/2020

Defense Date 03/13/2020

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

Tag 8-oxo-dGTP,bisphosphonate conjugates,bisphosphonates,bone,imaging,OAI-PMH Harvest,osteoadsorptive fluorogenic substrate

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles (committee chair), Qin, Peter (committee member), Shing, Katherine (committee member)

Creator Email yiying89sg@yahoo.com,yiyingzh@usc.edu

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