Chemical biology of matrix metalloproteases

CHEMICAL BIOLOGY OF MATRIX METALLOPROTEASES Wang Jun NATIONAL UNIVERSITY OF SINGAPORE 2006 CHEMICAL BIOLOGY OF MATRIX METALLOPROTEASES Wang Jun Under the supervision of Associate Professor Yao Shao Qin A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS I would like to express my greatest gratitude to my supervisor Associate Professor Yao Shao Qin for his novel ideas, patient guidance, and continuous encouragement throughout my studies. His passion in science stimulates me to devote my life to academic research. I would also like to express my appreciation to my lab members, especially my partners, Uttamchandani Mahesh, Li Junqi and Hu Mingyu; it is with their close cooperation that my projects have been able to move smoothly. The contribution from other group members will never be forgotten, a lot of thanks go to Rajavel Srinivisan, Sun Hongyan, Resmi C. Panicker, Sun Liping, Siti Aishah Bte Ahmad, Ng Su Ling, Tan Ching Tian and Li Jiexun in the chemistry lab, Chattopadhaya Souvik, Hu Yi, Huang Xuan, Tan Lay Pheng, Girish Aparna and Grace Y. J. Chen in the biology lab. Discussion with them always stimulates me to learn more in this field. I appreciate the support of the service laboratory staff – Ms Ler Peggy and Mdm Han Yanhui from the NMR lab, Mdm Lai Hui Ngee and Mdm Wong Lai Kwai from the Mass Spectrometry lab – in providing training and technical expertise. Last but not least, my beloved girlfriend, Huang Lijuan, and my family members, for their continuous encouragement and support during my master’s study. i I am also grateful to the National University of Singapore, for providing me the research scholarship. Some parts of the work which are described in this thesis were done by my partners, thus I greatly acknowledge their contributions as listed below: Uttamchandani Mahesh did all the screening work and protein labeling experiments. Li Junqi helped me with the scale up reactions of the warheads synthesis as well as the library construction. Hu Mingyu provided one warhead scale up reaction (6H). Sun Liping instructed me how to use the IRORI system and cooperated with the 400member MMPI library synthesis. Rajavel Srinivisan and Li Jiexun kindly shared with me the twelve azides which they made for the azide library. ii TABLE OF CONTENTS Page Acknowledgements i Table of Contents iii List of Tables vii List of Figures viii List of Schemes x Index of Abbreviations xii Publications xv Abstract xvi Chapter 1 Introduction 1 1.1 Metalloproteases 1 1.2 Matrix Metalloprotease inhibition 2 1.3 Fragment based discovery of metalloprotease inhibitors 6 1.4 Inhibitor Fingerprinting 8 1.5 Activity-based metalloprotease profiling Chapter 2 Developing a Solid Phase Synthetic Strategy for Succinyl 10 13 Hydroxamate-based Matrix Metalloprotease Inhibitors (MMPIs) 2.1 Design of synthesis route of warheads bearing hydrophobic P1’ substitutions 2.2 Design of synthesis route of warheads with P1’ substitution mimicking natural 14 amino acid side chains iii 13 2.3 Spilt and pool synthesis of small molecule library using IRORI 16 2.4 Activity-Based High-Throughput Profiling of Metalloprotease Inhibitors Using 18 Small Molecule Microarrays Chapter 3 Fragment Based Discovery of Non-peptide Based Metalloprotease 23 Inhibitors 3.1 MMPI library design using click chemistry 23 3.2 Rapid assembly and in situ screening of metalloprotease inhibitors 24 Chapter 4 Activity-based Metalloprotease Profiling 29 4.1 Design affinity-based matrix metalloprotease probes 29 4.2 Activity based fingerprinting of metalloprotease 30 4.3 Perspective-Activity based metalloprotease probes 33 Chapter 5 Experimental Section 35 5.1 General Information 35 5.2 Synthesis of succinyl hydroxamate-based warhead bearing hydrophobic 35 side chains 5.3 Synthesis of succinyl hydroxamate-based warheads with P1’ substitution 42 mimicking nature amino acid sides 5.3.1 Synthesis of warheads 6I and 6J 42 5.3.2 Synthesis of warhead 6K 46 5.3.3 Synthesis of warhead 6L 47 iv 5.4 Solid phase synthesis of 400 member library containing 20 amino acids in 50 the P2’ and P3’ positions 5.4.1 Synthesis of Fmoc-Lys(Biotin)-OH 51 5.4.2 Experimental details of the solid phase synthesis 52 5.4.3 Library characterization 53 5.5 54 Solid phase synthesis of 1000 member library containing 10 amino acids in the P2’ and P3’ positions and 10 variations in the P1’ position 5.6 Rapid assembly of metalloprotease inhibitors using click chemistry 55 5.7 Rapid assembly of metalloprotease probes using click chemistry 65 5.7.1 Synthesis of azide 32 65 5.7.2 Construction of MMP probes (A-H) using “Click Chemistry” 67 5.7.3 Synthesis of probes (I-L): 68 5.7.4. LC-MS characterization of the final probes (A-L) 69 Chapter 6 References 78 Chapter 7 Appendices 84 7.1 Side Product 20 84 7.2 Side Product 21 84 7.3 Side Product 22 85 7.4 HNMR and CNMR of Alkyne A 86 7.4 HNMR and CNMR of Alkyne B 87 7.4 HNMR and CNMR of Alkyne C 88 7.4 HNMR and CNMR of Alkyne D 89 v 7.4 HNMR and CNMR of Alkyne E 90 7.4 HNMR and CNMR of Alkyne F 91 7.4 HNMR and CNMR of Alkyne G 92 7.4 HNMR and CNMR of Alkyne H 93 7.4 HNMR and CNMR of Alkyne F5 94 7.4 HNMR and CNMR of Alkyne G6 95 vi LIST OF TABLES Table 1 IC50 (in μM) and Ki (in μM) of selected inhibitors Table 2 Ki/IC50 values and ESI-MS results of 6 selected inhibitors from the 400 MMPI library together with commercial inhibitor GM6001. vii 28 54 LIST OF FIGURES Figure 1 Catalytic mechanism of MMPs 2 Figure 2 Standard nomenclature for substrate residues and their corresponding 3 binding sites Figure 3 Binding of a hydroxamic inhibitor to MMP-7 4 Figure 4 Structure of the 1400 MMP inhibitors 6 Figure 5 Click synthesis of Matrix Metalloprotease (MMP) inhibitors 8 Figure 6 Labeling mechanism of activity-based MMP probes 11 Figure 7 Proposed structure of site-specific affinity-based MMP probe and the 12 labeling mechanism Figure 8 Structures of MMPI warhead used for solid phase synthesis 14 Figure 9 Three broad-spectrum potent MMP inhibitors in clinical trials 14 Figure 10 Structures of 4 MMPI warheads which bear functional groups 16 Figure 11 Structure of 400-member hydroxamate inhibitors. Diversity was 17 generated at P2’ and P3’ positions with 20 natural amino acids. Figure 12 (a) Microarray image of the 400-member library screened against 21 thermolysin. Samples were spotted in duplicate. Spots of selected inhibitors (labeled by their P2’–P39 sequence) with IC50 (in brackets) were boxed. (b) Image in (a) shown as dendrogram before (left panel) or after Cluster Analysis (right panel) based on inhibition potency. Figure 13 Structure of (top) general hydroxamate inhibitors and (bottom) 24 “click chemistry” inhibitors reported herein against MMPs. Figure 14 (a) Inhibitor fingerprints of I) MMP-7, II) thermolysin and III) viii 27 collagenase - represented as “barcodes”. Black: min inhibition; Red: max inhibition. Figure 15 Screening of “clicked” inhibitors against MMP-7. Heat map 30 obtained using TreeView displays the inhibition fingerprint obtained, with most potent inhibitors indicated in bright red. Figure 16 Structure of second generation MMP probes 32 Figure 17 Fingerprints of 12 probes against 6 metalloenzymes. Strongest 33 relative labeling is visualized in red according to the scale shown. Figure 18 Protein microarray of various metalloenzymes screened by the Leu probe. 34 Figure 19 Structure of proposed activity-based MMP probes 53 Figure 20 LC-MS profiles of representative samples (Crude) from the 96 member 64 MMPI library Figure 21 Two isomers of the second generation MMP probes ix 70 LIST OF SCHEMES Scheme 1 Procedure for the synthesis of 1400-member MMP inhibitors 18 on solid-phase Scheme 2 Nanodroplet SMM strategy for high-throughput profiling of 19 potential MMP inhibitors Scheme 3 Synthetic route of constructing non-peptide based MMPI library 25 using click chemistry Scheme 4 Mechanism of acetals as latent electrophiles that interact with 34 catalytic nucleophile at the active site of matrix metalloproteases Scheme 5 Synthesis route of hydrophobic MMPI warheads. 36 Scheme 6 Synthesis route of warhead 6I and 6J 43 Scheme 7 Synthesis route of warhead 6K 46 Scheme 8 Synthesis route of MMPI warhead 6L 48 Scheme 9 Procedure for the synthesis of 400-member MMP inhibitors 50 on solid-phase Scheme 10 Synthesis route of Fmoc-Lys(Biotin)-OH 51 Scheme 11 Procedure for the synthesis of 400-member MMP inhibitors 55 on solid-phase Scheme 12 Synthesis route of alkyne building block 56 Scheme 13 Side reaction in the last TFA cleavage step 59 Scheme 14 Structure and synthesis of 12 Azide-containing blocks 60 Scheme 15 Assembling of MMPI library using “Click Chemistry” 61 x Scheme 16 Synthesis route of Azide 32 65 Scheme 17 Construction of MMP probes library (A-H) using “Click Chemistry” 67 Scheme 18 Construction of MMP probes library (I-L) using “Click Chemistry” 68 xi INDEX OF ABBREVIATIONS Ala Alanine Boc t-Butoxy carbonyl br Broad dd Doublet of doublets δ Chemical shift DCC N,N′-Dicyclohexylcarbodiimide DCM Dichloromethane DIEA Diisopropylethylamine 2D-GE 2-dimentional gel electrophoresis DMAP 4-Dimethylaminopyridine DMF Dimethylformamide EA Ethyl acetate Et Ethyl ESI Electron spray ionization Fmoc 9-Fluorenylmethoxycarbonyl Glu Glutamic acid Gly Glycine H Histidine HATU O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HOBt 1-Hydroxybenzotriazole HBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate xii HRMS High resolution mass spectrometry Hz Hertz hrs Hours Leu Leucine M Molar MeOH Methanol m Multiplet min Minute mmol Millimole MMP Matrix metalloproteases MMPI Matrix metalloproteases inhibitor MS Mass spectrometry NaHMDS Sodium bis(trimethylsilyl)amide NMR Nuclear magnetic resonance nM Nanomolar PDC Pyridinium dichromate Ph Phenyl Phe Phenylalanine ppm Parts per million q Quartet RBF Round-bottomed flask SMM Small molecule microarray s Singlet xiii sat. Saturated t Triplet tBuOK Potassium tert-butoxide TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography UV Ultraviolet xiv PUBLICATIONS Wang, J.; Uttamchandani, M.; Sun, L.P.; Yao, S.Q.* “Activity-Based High-Throughput Profiling of Metalloprotease Inhibitors Using Small Molecule Microarrays”, Chem. Commun. 2006, 717-719 Wang, J.; Uttamchandani, M.; Li, J.; Hu, M.; Yao, S.Q.* “Rapid Assembly of Matrix Metalloprotease (MMP) Inhibitors Using Click Chemistry” Org. Lett., 2006, 8, 38213824. Wang, J.; Uttamchandani, M.; Li, J.; Hu, M.; Yao, S.Q.* “ “Click” Synthesis of Small Molecule Probes for Activity-Based Fingerprinting of Matrix Metalloproteases” Chem.Commun. 2006, 3783-3785. Wang, J.; Uttamchandani, M.; Sun, H.; Yao, S.Q.* “Application of Microarrays with special tagged libraries”, QSAR Comb. Sci. 2006, 11, 1009-1019. Uttamchandani, M.; Wang, J.; Yao, S.Q.* “Protein and Small Molecule Microarrays: Powerful Tools for High-Throughput Proteomics”, Mol. BioSyst. 2006, 2, 58-68. Sun, H.; Chattopadhaya, S.; Wang, J.; Yao, S.Q.* “Recent development in microarraybased enzyme assays: from functional annotation to substrate/inhibitor fingerprinting”, Anal. Bioanal. Chem. 2006, 386, 416-426. xv ABSTRACT Matrix Metalloproteases (MMP) inhibition is currently brought to the focus of medicinal chemistry research due to their essential roles in many human diseases. To elucidate the functions of each individual MMP, it is imperative to synthesise small molecule/probes which can selectively target a particular MMP instead of those which exhibit broad spectrum inhibition against the whole family. In this project, we aim at profiling MMPs substrate specificities in a high-throughput manner using small molecule microarrays as well as elucidating their biological functions through activity-based protein labeling. Herein we introduce synthetic routes toward different P1’ substituted MMPI warheads and their use in the MMPI library synthesis on solid phase. In addition, by taking the advantages of the relative ease and convenience of “Click Chemistry” in constructing focused chemical libraries, we have been able to apply this strategy to synthesise a 96-membered library of metalloprotease inhibitors followed by in situ screening. Moreover by using the same strategy, we successfully demonstrate that the facile synthesis of various affinity-based hydroxamate probes that enables the generation of activity-based fingerprints of a variety of metalloproteases, including matrix metalloproteases (MMPs), in proteomics experiments. xvi Chapter 1 Introduction 1.1 Metalloproteases Metalloproteases, together with serine, cysteine and aspartic proteases, represent the four major classes of proteolytic enzymes which mediate the hydrolysis of the amide bond. These proteases have been found to be involved in a variety of cell functions such as DNA replication, cell-cycle progression, cell proliferation, differentiation, migration. Deregulations of protease activities are known to cause many diseases such as cancer, HIV, malaria, Alzheimer’s diseases. Overall proteases represent 5-10% of the potential drug targets. 1 Matrix metalloproteinases (MMPs) belong to a family of homologous zinc endopeptidases that are capable of hydrolyzing all known constituents of the extracellular matrix (ECM). 2 There are currently at least 23 members of human MMPs that have been identified so far. All MMPs are expressed as proMMPs which are activated by proteolytic cleavage between the pro and catalytic domains or chemical modification of the cysteine side chain followed by dissociation of the pro domain. 3 MMPs are characterized by a highly conserved zinc binding sequence HEXXHXXGXXH (X is any amino acid) at the active site followed by a conserved methionine residue located beneath the active site zinc. The catalytic zinc ion is coordinated to three histidine residues and is responsible for the activation of the water molecule at the active side. The pKa of the water molecule is lowered by coordination to the zinc ion and hydrogen bonding to the glutamic acid residue at the catalytic site and is thus activated towards nucleophilic attack of the electron-deficient carbon centre of the scissile peptide bond. As shown in Figure 1, during the hydrolysis, the zinc ion stabilizes the negative charge formed in the tetrahedral 1 intermediate, while the Glu and Ala residues aid in the proton transfer from the water molecule to the nitrogen atom, making it a good leaving group. The ammonium group then leaves and the substrate is cleaved. 4 Figure 1 Catalytic mechanism of MMPs Despite their well-documented pro-tumorigenic actions, only three MMPs - MMP1, 2 and 7 - have been experimentally validated as potential cancer targets. Another three (MMP-3, 8 and 9) have recently been classified as antitargets due to the key role they play in normal tissue homeostasis. With the precise biological functions of other human MMPs remaining largely unknown, the development of novel chemical and biological methods capable of high-throughput identification and characterization of MMPs has become increasingly urgent.2a 1.2 Matrix Metalloprotease inhibition Two general methods have been applied to the identification of matrix metalloprotease inhibitors: one method is the substrate-based design of pseudopeptide 2 derivatives; another approach is the random screening of nature compound libraries as well as synthetic small molecule libraries. 5 However such random screening of large libraries yields minimal results, with few compounds found to be potent and selective MMP inhibitors. 6 Thus most of the research efforts are still devoted to the design of peptide based inhibitors. The simplest form of an effective MMP inhibitor is a zincbinding group (ZBG) conjugated with a peptide sequence which mimics the natural substrate of MMPs. 7 Based on the selection of the ZBG; the corresponding peptide sequence could be positioned either on the left-hand side (LHS) or the right-hand side (RHS), or on both sides of the cleavage site. Among all the ZBGs developed so far, the hydroxamic acid has been found to be one of the most potent warheads against MMPs. 8 It is noted while some novel heterocyclic zinc-binding groups which are more potent and selective than acetohydroxamic acid (AHA) have been reported by Cohen et al, 9 no full inhibitor based on the those ZBGs has been synthesized so far. The design and development of inhibitors employing these promising ZBGs could potentially be another interesting research project. With the hydroxamic acid as the ZBG, inhibitors that are designed to target the enzyme subpockets on the right hand side of the active side exhibit particularly potent inhibition. The standard nomenclature used to designate substrate / inhibitor residues that bind to corresponding enzyme subsites is adopted here 10 as shown in Figure 2. 3 Figure 2 Standard nomenclature for substrate residues and their corresponding binding Another factor critical to achieving potent inhibition is the length between the carbon bearing P1’ substitution and the hydroxamic acid. It was shown by Johnson and co-workers that succinyl hydroxamic acid derivatives are more potent inhibitors of MMP-1 than either the corresponding malonyl or glutaryl derivatives. 11 For other ZBGs like the thiol, formylhydroxylamine, and phosphonate groups, the insertion of a single methylene spacer between the ZBG and the carbon bearing the P1’ substituent also resulted an improvement in activity. Thus peptide-based succinyl hydroxamates has became one of the most widely exploited scaffolds of MMP inhibitors. Inhibitors containing the hydroxamate ZBG typically exhibit broad-spectrum inhibition towards most metalloproteases, rather than exclusively towards MMPs. As shown in the following Figure 3, the P1’ position had the greatest interaction with the S1’ subpocket of MMP-7 when it is placed in the position β to the carbonyl group of the hydroxamate. 12 Glu269 O His His Zn2+ His O O OH O NH O P1' S3' O NH NH R O NH Ala162 Tyr30 Pro238 P2' N H S2' Leu181 S1' Figure 3 Binding of a hydroxamate inhibitor to MMP-7 The greatest challenge in constructing MMP inhibitors is achieving selectivity targeting specifically one particular member of MMP while having little or no effect on 4 other MMP members. This is of particular importance, especially considering the fact that some MMPs play essential roles in the normal cellular function and are defined as anti-targets, inhibition of which will counterbalance the benefits of target inhibition.2a Selective inhibition could be achieved by simultaneously targeting multiple different MMP binding pockets. Among all the substrate binding pockets of MMPs, the S1’ pocket has been identified as the major determinant of substrate specificity. According to the structural studies utilizing X-ray crystallography, NMR and computer modeling techniques, the MMPs can be divided into two structural classes depending on the depth of the S1' pocket. 13 This selectivity pocket is relatively deep for the majority of the enzymes like gelatinase A (MMP-2), stomelysin-1 (MMP-3) or collagenase-3 (MMP-13), but in the case of human fibroblast collagenase (MMP-1) or matrilysin (MMP-7) it is partially or completely occluded due to an increase in size of the side-chain of amino acid residues that form the pocket. As a result, selective inhibition of the deep pocket enzymes over the short pocket enzymes is easy to achieve by incorporating of an extended P1' group in MMP inhibitors, whereas the presence of smaller P1' groups generally leads to broad-spectrum inhibition. However, it has also been found that the S1’ pocket can undergo conformational changes in order to accommodate substrates or inhibitors that can form favorable interactions with the amino acid residues in the S1’ pocket. 14 One aim of our project is to achieve both potent and selective inhibition aganist MMPs. Our design focused on the co-operative effects across P1’, P2’ and P3’ positions that contributes to overall inhibition potency and selectivity. Moreover, biotin tag was 5 incorporated into the inhibitor design as a latent tag which could be applied to the future microarray profiling. (Figure 4) Natural Amino Acid side chain R O HO H N N H O O P2' P3' N H H N O N H O H N O O NH2 HN O H H N NH H S O Warhead R= O Hydrophobic NH2 OH O O OH Basic O Acidic Hydrophilic S O Sulfone Figure 4 Structure of the 1400 MMP inhibitors 1.3 Fragment-based discovery of metalloprotease inhibitors Fragment-based drug discovery has recently been brought to the attention of current research in medicinal chemistry, enabling us to profile the N2 possibilities with N+N combinations. 15 Compared to traditional high throughput screening and structure based drug design, fragment-based drug discovery offers two unique advantages: First, a library of small fragments represents a much higher proportion of the available ‘chemical space’ for low molecular weight compounds than a large library of drug-sized molecules does for higher molecular weight compounds, because the number of possible molecules 6 rises exponentially as molecular weight increases; Secondly, it decreases the synthetic effort, since only the selected fragments need to be assembled and tested with the target protein(s). 16 Fragment-based drug discovery typically involves a two-step process: optimal fragments are first identified by means of functional or direct binding assays at high concentrations, or by other techniques such as NMR-Based Screening, MassSpectrometry-based methods and Crystallography-based approaches etc. This is followed by linking the individual fragments using chemoselective ligation reactions like sulfurdisulfide exchange reaction, click chemistry. 20 17 oximine formation, 18 amide bond reaction 19 as well as In additional to this traditional process of fragment based drug discovery, there is an emerging research direction in this field which relies on the enzyme as the template to assemble its own inhibitors. 21 This is in a way similar to dynamic combinatorial chemistry, with the ligation reaction occurring only between the building blocks that are amplified in the presence of enzymes template in which they occupy. Fragment-based drug discovery not only allows us to identify enzyme inhibitors with novel structures, it also offers us a straightforward way of modifying existing lead drugs. We previously constructed a 400-member MMPI library and identified a potent inhibitor against thermolysin with IC50 =9.9 nM and Ki = 2.4 nM inhibition. 22 However, despite its promising results in vitro, peptide-based compounds have no in vivo activity due to rapid degradation by endogenous proteases. In developing non-peptide based small molecule inhibitors, fragment-based synthesis offers a convenient way to modify existing peptide-based inhibitors to construct inhibitors with non-amide bond linking two different components of the inhibitor. By simply splitting the model compound into two fragments, 7 each targeting a different binding pocket of the enzyme, we can easily generate a library of analogues for each fragment, with a reactive group appropriate for the ligation reaction appended to each fragment. Herein, we successfully applied this strategy employing the [1,3] dipolar cycloaddition of alkynes and azides as the ligation reaction for the rapid assembly and in situ screening of MMP inhibitors as shown in Figure 5. HO O H N N H O O P1' + O N H N N N H N n O Pn' "Click Chemistry" H N N3 n HO O H N 96 MMP inhibitors Pn' Figure 5 “Click” Synthesis of Matrix Metalloprotease (MMP) inhibitors 1.4 Inhibitor Fingerprinting In the post genomic era, research efforts have been shifted from enzyme identification to enzyme catalytic activity characterization. 23 The latter is important as it reveals not only the catalytic mechanism of the enzyme, but also generates a unique pattern of “inhibitor fingerprinting” against a known set of inhibitors. 24 In contrast to the traditional small molecule microarray (SMM) screening methods which simply rely on non-covalent ligand-protein interactions, “inhibitor fingerprinting” generates a unique inhibition pattern based specifically on the catalytic activity of the enzyme, thus eliminating false positives which result from inconsequential affinity between the ligand and non-targeted regions of the protein. The first “inhibitor fingerprinting” experiment was demonstrated by Diamond and Gosalia who developed a novel nanoliter-scale screening assay by utilizing nanoliter droplets as microreactors for testing small molecule 8 inhibitors against three different human caspases. A total of 352 small molecule inhibitors were printed as individual spots of glycerol on a glass slide. Subsequently an aerosolized mixture of caspase and a fluorogenic substrate were applied to assemble multi-component reactions at each reaction center. 25 The strength of the fluorescence signal generated reciprocally correlates with inhibitor potency. This strategy revealed a caspase inhibitor that showed high potency against all three of the caspase isoforms screened. However, the use of the viscous mediums such as glycerol and DMSO limits general applicability of this strategy in scenarios requiring predominantly aqueous environments. We have developed an alternative “nanodroplet” method for screening of inhibitors. In our earlier work, 37 enzymes belonging in different classes were spotted in spatially addressable, segregated droplets on a glass slide coated with suitable fluorogenic substrates (either small molecule- or protein-based). Upon incubation, nanodroplets containing active enzymes showed up on the microarray as discrete fluorescent spots whose intensities directly correlated with the relative activity of the spotted enzymes. 26 The feasibility of our strategy for high-throughput identification of enzyme inhibitors was demonstrated by screening a 400 member peptide-based small molecule library against thermolysin. Each member of the library had a hydroxamic acid “warhead” and an invariant isobutyl moiety at the P1’ position while diversity was generated by substituting P2’ and P3’ positions with combinations of all the 20 natural amino acids.22 By printing pre-incubated nanodroplets of enzyme-inhibitor mixes onto a protease-sensitive glass surface, we obtained the inhibitor fingerprint profiles for thermolysin in the terms of fluorescence intensity of the spots. Overall this strategy offers not only a rapid method 9 for inhibitor profiling and discovery, but also a viable method for the chemical screening of huge combinatorial libraries against virtually any enzyme class. 1.5 Activity-based metalloprotease profiling With the completion of human genome project, there is an urgent need to identify, characterize and assign biological functions of all proteins expressed by the genome, 27 because it is the expressed proteins, not the genome, which are eventually responsible for the different biological functions inside the cell, like cell signaling, differentiation, proliferation and translocation. 28 However the traditional methods of high throughput proteome profiling like two-dimensional gel electrophoresis (2D-GE) only allow us to detect the abundance of the protein, while revealing no information of the activity of the protein. 29 The activity-based proteome profiling approach which was initially developed by Cravett et al 30 uses active site-directed, small molecule probes that chemically react with certain classes of enzymes in a complex proteome, and can therefore report unique profiles of enzymes on the basis of their catalytic activities. The probe normally consists of two components: the reactive “warhead” which is responsible for the selective covalent modifying the active site of a particular enzyme family based on the unique enzyme catalytic mechanism; a reporter group like a fluorescence or biotin tag that is also necessary for the facile detection or purification purpose.29b Presently, this strategy has been successfully applied to profile all the four major classes of protease, such as aspartic, 31a serine, 30 cysteine proteases 32b, 32c and metalloproteases.32d, 32e It is necessary to mention that in the case of profiling enzymes whose hydrolytic mechanism does not involve any covalent intermediates, such as metalloproteases, the affinity-based strategy is applied. For affinity based protein profiling as shown in Figure 6, the reactive 10 “warhead” delivers the probe to the target protein and forms only non-covalent interaction with the active site. Immobilization of the probe to the protein results from UV irradiation, activating the photo-labile group and forming a highly reactive radical which reacts with the protein to form a robust covalent bond. In addition to these two units, a reporter group (fluorescence or biotin tag) is also necessary. Our group has pioneered in this field by introducing and demonstrating this strategy for the profiling of metalloproteases and aspartic proteases. 32a, 32f Herein, we expand the same strategy by taking a step further in the aim of designing specific metalloprotease inhibitors which selectively target a particular metalloprotease in the presence of other family members, in contrast to the previous probes which only discriminates between different enzyme families. The selectivity was primarily achieved by introducing different P1’ substitutions in the warhead, as this substitution is the major determinant of selectivity and activity of right-hand based matrix metalloprotease inhibitors. Our probe design takes advantage of the click chemistry reaction to quickly assemble the two fragments together. Figure 6 Labeling mechanism of activity-based MMP probes. Abbreviations: WH = warhead; BP = benzophenone; UV = ultraviolet; TER = tetraethylrhodamine. Comprehensive knowledge of the location of proteins within cellular microenvironments is critical for understanding their functions and interactions. Ideally, localization information indicates not only where a protein is found, but also temporal and spatial movements. 32 Our project aims at visualization of a particular matrix 11 metalloprotease within specific organelles. The results from this study will help us better understanding the relationship between the disease stages and the proteolytic activity of matrix metalloproteases. Specific targeting of affinity-based MMP probes to a predefined organelle could be achieved by highly orthogonal binding pairs, such as the DHFR (dihydrofolate reductase ) and MTX (Methotrexate) binding in this case. 33 O OH O Fluorenscence group COOH Warhead H N O HO N H O O HN O O NH O N N N H N N N O N Receptor tag N N H2N Receptor tag(DHFR) Receptor tag(DHFR) Tetra-functional probe NH2 = H N O HOOC O Photolabile Group MTX Protein at specific organelle Target metalloprotease Figure 7 Proposed structure of site-specific affinity-based MMP probe and the labeling mechanism 12 Chapter 2 Developing Solid Phase Synthesis Strategy of Succinyl Hydroxamate-based Matrix Metalloprotease Inhibitors (MMPIs) 2.1 Design of synthesis route of warheads bearing hydrophobic P1’ substitutions Although several MMPIs have successfully entered phase III clinical trials, the results have turned out to be disappointing. 34 There are still no MMPIs approved for cancer treatment so far. While it is debatable that the reason for the clinical failure of MMPs is due to their broad-based, non-specific inhibitory activity across the MMP family, the fundamental issue is that the specific role that each individual MMP in maintaining normal cellular function has not been properly elucidated. Defining these roles clearly will be necessary to identify potential treatment strategies. To address this problem, what is needed is the selective inhibition or labeling of only one particular MMP, while leaving other MMPs unaffected. It is thus desirable to develop highly efficient synthetic and screening strategies that allow rapid generation and screening of small molecule inhibitors/probes possessing not only high potency but more importantly good selectivity towards MMPs. All previous succinyl hydroxamate based MMPIs have been synthesized in solution phase via multiple transformations. 35 However in order to develop a facile synthesis method which allows us to generate large small molecule libraries mimicking MMP’s natural substrate, we have to rely on solid phase synthesis, as it is one of the cheapest 13 and most convenient way to construct libraries. 36 We have been able to design and synthesize trityl protected MMPI warheads with the structure CPh3ONH-Suc(2-P1’)COOH, which were shown to be compatible with standard Fmoc peptide chemistry/TFA cleavage procedures (Figure 8). Our warhead design was based on known molecular templates of Marimastat, Batimastat, and GM6001, three broad-spectrum hydroxamate inhibitors of matrix metalloproteases (Figure 9). It is noted that Overkleeft and coworkers independently reported a similar method based on the synthesis of N-Boc-OTBS-hydroxamates which have also been used for the solid phase synthesis of succinyl hydroxamates. 37 P1' O OH Ph3COHN O Figure 8 Chemical structure of MMPI warhead used for solid phase synthesis O HO N H H N O O OH O Marimastat HO N H N H H N S O O S Batimastat O N H HO N H H N O O N H NH GM6001 Figure 9 Three broad-spectrum potent MMP inhibitors in clinical trials 2.2 Design synthesis route of warheads with P1’ substitution mimicking nature amino acid side chains Five major interactions have been involved in the formation of protein 3-D structure, namely hydrogen bonding, hydrophobic interaction, π-π interaction, electrostatic 14 interaction and Van der Waals interaction. It is useful to design an inhibitor which can selectively form electro-static interaction with the target protein, which is the strongest interaction among them, thus simultaneously achieving both selectivity and potency. 38 Our previously developed solution phase synthesis route of MMPI warheads only allows us to synthesis those warheads which bear hydrophobic side chains (6A-6H). A new synthetic strategy is needed to synthesis succinyl hydroxamate warheads which contain all natural amino acid side chains, if possible. This is reasonable as MMPs specifically and efficiently hydrolyses the endogenous substrate with natural amino acid side chain in the P1’ position. In order to mimic the strong binding interactions between MMP and its natural substrate, we aimed to expand our warhead library size to include those containing acidic, basic and hydrophilic side chains. In this project, four hydroxamates (6I-6L) introduces side chains possessing acidic and basic groups, as well as hydrophilic groups of hydrogen-bonding property, at the P1’ position, thus are synthesized and expected to be able to form favorable electrostatic interactions with different S1’ binding pockets of MMPs. The acid-labile protecting groups in 6I-6K were chosen such that the warheads are compatible with standard solid-phase Fmoc chemistry, allowing them to be used in future for large-scale synthesis of probe libraries. Detailed synthesis of the above four warheads will be described in the experimental section. There are several considerations in designing the synthesis route: it should be general, enabling the facile incorporation of a wide variety of P1’ side chains and, when necessary, precise stereospecific control can be exerted over the chiral center located in the warhead (except 6L). The synthesis was accomplished by standard enolate chemistry coupled with Evan’s oxazolidinone auxillary, with different side chains being introduced from their 15 corresponding alkyl bromides (except sulfone warhead). The hydroxamate was protected with a trityl group, ensuring its compatibility with standard Fmoc peptide chemistry/TFA cleavage procedures. O OCPh3 NHBoc O O Ph3CO OH N H O 6I Ph3CO N H O OH Ph3CO O 6J COOtBu OO S O OH N H O Ph3CO OH N H 6K O 6L Figure 10 Structures of 4 MMPI warheads which bear functional groups 2.3 Spilt and pool synthesis of small molecule library using IRORI We first synthesized a 400-member small molecule library with the scaffold HONH-Suc(2-iBu)–P2’–P3’–Gly–Gly–Lys(biotin)–CONH2, as shown in Figure 11. Each inhibitor in the library comprises a succinic hydroxamate ‘‘warhead’’ (a highly potent zinc-binding group against metalloproteases), in which the P1’ residue was maintained as an isobutyl group throughout. The design was based on the structures of Marimastat, Batimastat, and GM6001, three broad-spectrum potent hydroxamate inhibitors of MMPs (Figure 2.1.2). With variations across P2’ and P3’ positions in the library, we aimed to profile both the potency and selectivity of individual members against different metalloproteases, in particular MMPs. A flexible linker and biotin were incorporated into each inhibitor for future proteomic applications. The construction of 400 members MMP inhibitors library was achieved by using standard Fmoc solid phase peptide synthesis, IRORI split and pool directed sorting technology (Scheme 2.3). 39 Final product was released from the solid phase by standard TFA cleavage protocol. The average 16 concentration of individual inhibitors in DMSO stock solution was measured to around 230µM (estimated using ACC dye conjugation as 1% additive in the final coupling step). O HO N H O HN O P3' HN P2' O H N O O N H HN O NH2 NH H HN H O H N S O Figure 11 Structure of 400-member hydroxamate inhibitors. Diversity was generated at P2’ and P3’ positions with 20 natural amino acids. Following the same synthesis protocol, we next expanded the library size to 1000 member by incorporating 10 different warheads (6A, 6C, 6D, 6E, 6F, 6G, 6I, 6J, 6K, 6L) in the P1’ position and 10 variations across P2’ and P3’ positions. A minor change has been made in the last step solid phase coupling reaction: instead of using HOBt/HBTU/DIEA as coupling reagent; we chose HATU/DIEA which had been shown to be more effective for this step’s transformation and improve the purity of the final products. 17 P3' O O H N H2N O N H O O O H2N O HN N H NH H HN H H N O d O HN N H N H O S S O Ph3CO O OH Ph3CO N H O O OH Ph3CO N H O A P3' P2' O H N N H HN O Ph3CO O N H Ph3CO O N H O Ph3CO OH O O O P1' O HN P3' P2' Ph3CO O O O J Ph3CO OH N H OO S O Ph3CO O K OH N H O L O HN N H O O OH N H I H N HN O OH N H f NHBoc COOtBu O O OH N H G OCPh3 Ph3CO O O D F S N H OH N H C E O Ph3CO O O OH N H NH H HN H H N O OH Ph3CO N H B O O O NH H HN H H N O HN a H2N O e O N H S O H2N O H2N NH H HN H H N b c O O N H NH H HN H H N S O g O HO N H P1' O HN O P3' HN P2' O H N O O N H HN O NH2 NH H HN H O H N S O Scheme 1 Procedure for the synthesis of 1400-member MMP inhibitors on solid-phase. Reagents and conditions: (a) i:Fmoc-Lys(Biotin)-OH, HOBt, HBTU, DIEA, DMF, 12 hrs; ii: 20% piperidine/DMF, 2hrs; (b)i:Fmoc-Gly-OH, HOBt, HBTU, DIEA, 6 hrs; ii:20% piperidine/DMF, 2hrs; (c) i:Fmoc-Gly-OH, HOBt, HBTU, DIEA, 6 hrs; ii: 20% piperidine/DMF, 2hrs; (d) i:Fmoc-AA3-OH, HOBt, HBTU, DIEA, 8 hrs; ii: 20% piperidine/DMF, 2hrs; (e) i:Fmoc-AA2-OH, HOBt, HBTU, DIEA, 8 hrs; ii: 20% piperidine/DMF, 2hrs; (f) i:CPh3ONH-Suc(2-P1’)-COOH, 1% Fmoc-ACC-COOH, HATU, 2,4,6-collidine or (HOBt, HBTU, DIEA), 12 hrs; ii: 20% piperidine/DMF, 2hrs; (g) 95% TFA/ 5% TIS, 2hrs. 2.4 Activity-Based High-Throughput Profiling of Metalloprotease Inhibitors Using Small Molecule Microarrays Small molecule microarrays have surfaced as important tools for screening large chemical libraries against a variety of protein targets for the rapid discovery of bioactive compounds. 40 , 24 This method has generally involves the immobilization of libraries of compounds in addressable grids on glass slides, onto which fluorescently tagged proteins are applied in attempts to isolate the strongest binders. The major limitation of such high- 18 throughput screening systems lies not so much in hit identification as in hit validation. Screening merely based on simple ligand binding potency invariably introduces false positives; arising as a result of inconsequential affinity to non-targeted regions of the protein. Without time-consuming validation, it remains unconfirmed whether any of the initial “hits” detected on traditional array platforms is relevant to the desired biological context. In order to address these existing limitations of small molecule microarrays in chemical screening, we have developed an alternative approach that allows small molecule modulators of protein function to be directly evaluated on the microarray, in an activity-based manner. By pre-coating slides with fluorogenic enzyme substrates, followed by programmed application of premixed enzyme and libraries of putative small molecule modulators (Scheme 2), we had been able to take advantage of the highthoughput, miniaturized microarray platform and at the same time directly isolate specific small molecules with high inhibition potency. Furthermore our strategy does not require tagging of the enzyme with a fluorophore, or secondary detection using antibodies, allowing proteins to be evaluated in their native form, and in real-time. + Enzyme with potent inhibitor Coat fluorogenic substrates Low fluorescence Enzyme with weak inhibitor Bodipy FL casein + Fluorescent!! Print mixtures of enzyme + inhibitor Scan to detect fluorescence Scheme 2 Nanodroplet SMM strategy for high-throughput profiling of potential MMP inhibitors. 19 We next used the nanodroplet strategy to screen the 400 hydroxamates against thermolysin and collagenase, as they exhibit similarity to many vertebrate metallopeptidases, in particular to those of the MMP family. 41 , 32e To validate our results, separate experiments were performed in standard microplate format. Advantages of the approach were immediately evident. First, the entire 400-member library (in duplicate) was readily accommodated on a single slide, effectively allowing >800 assays to be performed with merely 6 ml of bodipy FL casein (Figure 12 a). With few exceptions, results obtained from SMM and microplate formats were in good agreement, giving a relatively high Pearson correlation coefficient (r = 0.852). Second, the relative potency of each inhibitor was immediately revealed by the fluorescence intensity generated from its corresponding nanodroplet (small boxes in Figure 12 a) with more potent inhibitors giving weaker fluorescence signals, thus avoiding tedious hit validation. This was unambiguously confirmed by enzyme kinetic experiments carried out in microplates on selected inhibitors. Notably, the nanodroplet SMM strategy was able to discern slight differences in inhibitor potency. Finally, because the enzyme ‘‘inhibitor fingerprint’’ was generated in a single experiment under uniform conditions, the results could be used directly for further SAR analysis to address not only potency, but more importantly selectivity, of any given inhibitor (Figure 12 b): the dendrograms, before (left) and after (right) cluster analysis, show the relative potency of each inhibitor against thermolysin with regards to its P2’ and P3’ substitutions. Our results indicate that Cys, Glu and Asp were disfavored at both P2’ and P3’ positions. Potent thermolysin inhibitors appear to be those containing aromatic (i.e. Trp/Tyr), small (i.e. Ala), hydrophobic (i.e. Leu/Ile), basic (i.e. Lys/ Arg) and polar (i.e. Gln/Asn) residues in a variety of P2’/ P3’ combinations, with 20 considerable variations across rows and columns, indicating cooperativity from both P2’ and P3’ residues is critical to achieve maximum inhibition. Interestingly, screening results obtained with collagenase were distinctly different from those with thermolysin, with potent inhibitors comprising predominantly aromatic (i.e. Tyr/Trp/Phe) and hydrophobic residues (i.e. Leu/Ile) at the P2’ position, and Trp at the P3’ position. This underlines the potential of our platform in detecting subtle substrate preferences amongst different MMPs. One of the most potent inhibitors identified from our screen was HONH–Suc(2iBu)–Tyr–Lys–Gly–Gly–Lys(Biotin)–CONH2 (IC50 =9.9 nM; Ki = 2.4 nM), consisting of Tyr and Lys at its P2’ and P3’ sites, respectively, and was 10-fold more potent than GM6001.This finding, to our knowledge, provides the first direct evidence of P2’/ P3’ selectivity in thermolysin inhibitors. It is further supported by inspection of the active site structure of thermolysin, showing predominantly hydrophobic S2’ and solvent-accessible S2’ pockets. (a) Leu-Phe (144.5 nM) GM6001 (23.9 nM) Tyr-Gln (38.5 nM) Tyr-Lys (9.9 nM) No Inhibitor Ser-Ser (176.7 nM) Ser-Tyr (107.3 nM) Tyr-Asn (33.1 nM) (b) P3’ A R N D C Q E G H I L K M F P S T W Y V P2’ 21 0% 100% Inhibition C N T G H M I L K V R W A Q F Y S P E D N L H E S T F R K M G A W Q V Y C D P I A R N D C Q E G H I L K M F P S T W Y V Figure 12 (a) Microarray image of the 400-member library screened against thermolysin. Samples were spotted in duplicate. Spots of selected inhibitors (labeled by their P2’–P3’ sequence) with IC50 (in brackets) were boxed. (b) Image in (a) shown as dendrogram before (left panel) or after Cluster Analysis (right panel) based on inhibition potency. See Supporting Information for details. In conclusion, we have developed a nanodroplet SMM strategy for high-throughput profiling of inhibitors against metalloproteases, potentially extendable to other enzymes. It enables potent and highly selective inhibitors to be directly identified without the need of time-consuming hit validation. Our strategy thus provides a new tool in the ever expanding SMM technologies for the inhibitor fingerprinting of enzymes. Notwithstanding, a key issue remains to be addressed before the technique can be applied for routine high-throughput screening of enzyme inhibitors; with the current method, inhibitor/enzyme mixtures are individually prepared before spotting, and the microarray is processed immediately post-spotting. This inevitably limits the throughput of the screening, especially with multiple enzymes. We are currently investigating possible solutions to this and will report our findings in due course. 22 Chapter 3 Fragment Based Discovery of Non-peptide Based Metalloprotease Inhibitors 3.1 MMPI library design using click chemistry “Click Chemistry” is a concept originally introduced by Sharpless et al which refers to several classes of chemical transformations that share a number of important properties including very high reaction efficiency (in both conversion and selectivity) under mild conditions, and a simple workup. 42 The Cu(I)-catalyzed 1,3-cycloaddition between an azide and an alkyne is one such reaction that has recently been explored in the discovery of drugs and materials. This strategy has been successfully applied to the synthesis and identification of inhibitors against a number of biological targets, including HIV protease, 43 a, 44b sulfotransferases,44c fucosyltransferases,44d protein tyrosine phosphatases,44e acetylcholinesterase 44f and others.44g Herein, we describe, for the first time, a “click chemistry” approach for the rapid synthesis/assembly of a small molecule library based on different succinyl hydroxamates and its subsequent in situ screening for identification of candidate hits which possess moderate inhibitory activity against MMPs over other metalloproteases. Our approach thus lays the foundation for future exploration of more potent and selective MMP inhibitors, in high throughput, using “click chemistry”. Our library design was based on the general structure of hydroxamate-based MMP inhibitors (Figure 13), which were previously shown that (1) hydrophobic P1’ residues are in general preferred, (2) a variety of substitutions are tolerated at P2’ and P3’ positions, (3) hydrophobic P4’ residues are preferred and sometimes could confer a good degree of specificity amongst different MMPs. 23 His His His 2+ Zn O O N H P 1' H N O O HO N H P 3' O H N N H O P2' H N N N N H N n O P4' O Figure 13 Structure of (top) general hydroxamate inhibitors and (bottom) “click chemistry” inhibitors reported herein against MMPs. 3.2 Rapid assembly and in situ screening of metalloprotease inhibitors Starting from the eight hydrophobic warheads (6A-6H) which we previously synthesized, a total of eight succinyl hydroxamates (AlkyneA-AlkyneH) bearing a variety of alkyl, cycloalkyl and aromatic side chains were synthesized as shown in Scheme 3.2. A common alkyne handle was introduced to each warhead, facilitating the subsequent assembly with twelve different azides (Azide1-Azide12) using “click chemistry” (Scheme 3). The azides which bearing a hydrophobic moiety connected via a linker with a varying alkyl length (n = 2-4) were synthesized via a highly efficient two step procedures as described. 44e By changing the linker length of the azides, our aim is to project the hydrophobic moiety into the P4’ or even P5’ binding pocket of a targeted MMP, thus improving both potency and specificity of the resulting inhibitors. With both the alkyene and azide building blocks available, a 96-member inhibitor library was assembled using “click chemistry” in a 96-deep well block. Each of the eight alkyne warheads was mixed with each of the twelve azides (in slight excess) in a tBuOH/H2O solution, followed by addition of catalytic amounts of sodium ascorbate and CuSO4. The 24 “click chemistry” proceeded with high efficiency at room temperature for > 12 hours before analysis by LC-MS; results indicated, in almost all cases, the complete consumption of the alkynes and quantitative formation of the desired triazole products, thus ensuring that they may be used directly for subsequent in situ enzymatic screening without any further purification. Screens of the bidentate compounds against representative metalloproteases provided discerning inhibition fingerprints, revealing compounds with low micromolar potency against MMP-7. The relative ease and convenience of the strategy in constructing focused chemical libraries for rapid in situ screening of MMPs is thereby demonstrated. O P1' OH Ph3COHN O propagyl amine DMF P1' H N O HATU, DIEA Ph3COHN O HO DCM P1' H N O TFA N H O AlkyeneA-AlkyeneH 6A-6H + O O acid chloride RNH2 R Br N H CuSO4, Sodium ascorbate tBuOH/H20 HO n n = 2, 3 or 4 NaN3/DMF 50-60oC R N H N3 O N H P1' H N N N N O 96 member MMPI H N n O R n Azide1-Azide12 Scheme 3 Synthetic route of constructing non-peptide based MMPI library using click chemistry MMP-7 is one of the few MMPs that is secreted by cancer cells, and contributes to proliferation of intestinal adenomas as well as pancreatic cancer. 44 It was chosen for this study, together with collagenase and thermolysin that have roles in the progression bacterial corneal keratitis and the metabolism of Bacillus spp., respectively. 45 It was screened in a high-throughput, automated fashion against the 96-member library panel using standard fluorescence assays in microplates. The inhibitor potency was evaluated from the reduction in fluorescence output when introduced in standard enzymatic assays with quenched substrates. The resulting inhibition fingerprints obtained are displayed in 25 Figure 14 a, demonstrating how such enzymes may be easily discerned through their unique inhibition “barcodes”, which differ from previous fingerprint profiles generated by other methods 46 in that our current method is able to directly reflect an enzyme’s inhibition profiles (potency and selectivity). By taking advantage of such inhibition fingerprints, it would become possible to characterize and group proteins in an activitydependent manner. Furthermore, such analysis may prove useful in distinguishing not only inhibitors that are potent against specific enzyme classes, but also those that provide good discriminatory potential, thereby minimizing off-target effects of potential drug candidates. A broader evaluation of the inhibition profile obtained against MMP-7 (as shown in Figure 14 b) revealed a unique and consistent trend. Hydroxamate warheads containing alkyl and cycloalkyl side chains at the P1’ position (i.e. isobutyl, cyclohexyl and cyclopentyl in AlkyneB, AlkyneF and AlkyneG, repesctively) contributed highly to potency of inhibitors against MMP-7. The strongest inhibition was observed for scaffolds containing the latter two unnatural cyclic analogues. This correlates well with generic inhibitors against metalloproteases such as GM6001 and batimastat that are designed ’ with a small hydrophobic residue (Leu), i.e. isobutyl in the P1 pocket. 47 Our results thus highlight the potential of such pharmacophores in the design novel inhibitors against MMP-7 and possibly other MMPs. Another observation is that, azides having a 4-canbon linker (n = 4; F8-11, G8-11 in Figure 3b) appeared to contribute negatively to the inhibitor potency, which indicates the linker might be too long to properly accommodate ’ the hydrophobic moiety of the azides into P4 pocket of MMP-7. 26 (b) 1 2 3 4 5 6 7 8 9 10 11 12 Azide Alkyne (a) A1-12 B1-12 C1-12 D1-12 E1-12 F1-12 G1-12 H1-12 A B C D E F G H 0 I II 100% III Figure 14 (a) Inhibitor fingerprints of I) MMP-7, II) thermolysin and III) collagenase represented as “barcodes”. Black: min inhibition; Red: max inhibition. Black: min inhibition; Red: max inhibition. (b) Screening of “clicked” inhibitors against MMP-7. Heat map obtained using TreeView displays the inhibition fingerprint, with most potent inhibitors indicated in bright red. To unambiguously confirm the potency of these scaffolds from the preliminary screen, two compounds, F5 and G6, representing each of the cyclohexyl and cyclopentyl warheads were selected for detailed evaluations. These molecules were purified and fully characterized by NMR and LC-MS, before being evaluated against the panel of metalloproteases to elucidate the relevant inhibition constants. As shown in Table 3.2, F5 and G6 indeed inhibited MMP-7 strongly with a Ki of 1.4 μM and 3.8 μM respectively. More importantly, these inhibitors were 10 to 35 times more potent towards MMP-7 than the bacterial metalloproteases tested, demonstrating the potential of the strategy in elucidating inhibitors with both good potency and high selectivity against MMPs. 27 Table 1 IC50 (in μM) and Ki (in μM) of selected inhibitors MMP-7 thermolysin collagenase Ki IC50 IC50 IC50 F5 3.8 12.5 >50 >50 G6 1.4 6.5 >50 >50 H N O HO N H H N N N N O N H O F5 O O OH O HO N H H N N N N O 28 O N H O O G6 Chapter 4 Activity-based Metalloprotease Profiling 4.1 Designing affinity-based matrix metalloprotease probes With the precise biological functions of most human MMPs remaining largely unknown, the development of novel chemical and biological methods capable of highthroughput identification and characterization of MMPs has become increasingly urgent. Activity-based profiling, originally developed by Cravatt et al, 30 is one such technique that has recently been adapted for the study of metalloproteases including MMPs. 48 ABP works by selectively targeting enzymes of choice from a crude proteome, in an activitybased manner, using the so-called activity- and affinity-based small molecule probes. In the case of MMPs, small molecule probes possessing a) a hydroxamic acid recognition moiety known to chelate to the actives-site zinc of MMPs (as well as other zinccontaining proteins such as Thermolysin and Collagenase), b) a photolabile group (usually benzophenone or diazirine) capable of covalent crosslinking to the target enzyme, and c) a fluorescent/affinity tag for easy visualization/isolation of the cross-linked enzyme, have been successfully documented.32d, 42b Furthermore, in cases where a repertoire of probes having different recognition elements is available, one can obtain the “activity-based fingerprint” of the target enzyme, which reveals not only affinity, but more importantly specificity, of the enzyme/probe complex. We previously reported firstgeneration MMP probes which were peptides containing a C-terminal hydroxamic acid.42b Despite simplicity in their chemical synthesis, these so-called “left-handed” probes suffer a major drawback in that they bind to MMPs with relatively low affinity, making them less suitable for sensitive detection of MMPs from a complex proteome. In this project, we demonstrate 1) the synthesis of second-generation “right-handed” probes 29 aided by “click chemistry” (Figure 15) 2) their application in gel-based activity-based fingerprinting of numerous metalloproteases (including MMPs), and 3) the related preliminary microarray-based experiments, which, for the first time, demonstrate affinitybased probes are indeed compatible with protein microarrays for potential enzyme profiling experiments. P1'= probeA probeB probeC probeD O O N+ probeE O HO N H P1' O H N N N N O H N O N H probeF probeG probeH O H N O OH NH2 O O OH O " Click Chemistry" O O S O N probeI Second generation of MMP probeA-probeL probeJ probeK probeL Figure 15 Structures of second generation MMP probes 4.2 Activity based fingerprinting of metalloprotease Protein fingerprinting, by its name suggests, is a distinctive pattern generated against a panel of focused small molecule probes, which reflects the protein’s catalytic activity or binding property. Since our probes were designed based on known inhibitors of metalloproteases, the resulting inhibitor-based fingerprint not only offers invaluable information for decoding the enzymes’ physiological roles, but also facilitates the discovery of potent and selective inhibitors as potential drugs. A diverse group of different classes of metalloproteases were chosen in our experiment, such that they highlight the potential of the strategy not only in distinguishing between both close and distant members, but also in potential identification and characterization of various disease-related enzymes, i.e. MMPs and anthrax lethal factor. In additon, carbonic 30 anhydrase, a well-known zinc-binding protein (but not a metalloprotease) was also tested. Figure 16 displays the results obtained using gel-based fingerprinting with the probe library. Notably, we were able to produce distinct and reproducible fingerprints for each of these proteins, thus providing a unique capability of identifying and classifying these proteins according to their labeling profiles. Generally the Lys and Ile probes showed the greatest degree of labeling and seemed to strongly label nearly all the enzymes tested. The other probes were however more discerning in their labeling patterns. It was observed that the strongest labeling for MMP-3 was that of Long-OH and Lys probes. This agrees well with the known long hydrophobic pocket of MMP-3 that has been previously reported to bind designed inhibitors with such long hydrophilic scaffolds. MT1 MMP also shares a similar long pocket as MMP-3 and is observed to possess greater affinity to the Long-Phe as well as the Lys probe relative to the other probe scaffolds. The short S1’ pocket of MMP-7 was shown to accommodate both the Val and Phe probes. The Asp and Sulfone probes showed the weakest labeling with most of the enzymes, indicating these moieties are generally unfavoured for most of the metalloproteases tested. Overall the fingerprints enabled different enzymes to be classified according to their similarity. MMP-3 gave a distinct profile compared to the other enzymes screened. The labeling pattern of carbonic anhydrase was similar to that of MMP-7. Both Anthrax Lethal Factor and MT-1 MMP show strong selective labeling with one of the probe library set, namely Ile and Lys respectively. Importantly the panel of probes we have designed enables sufficient coverage for one enzyme to be distinguished from the next. 31 0% 100% Figure 16 Fingerprints of 12 probes against 7 metalloenzymes. Strongest relative labeling is visualized in red according to the scale shown. We next tested the feasibility of these probes to be used in a protein microarray for potential high-throughput discovery of metalloproteases. Previously, only activity-based, and not affinity-based, probes have been shown to detect enzymes immobilized in a protein microarray.47c, 49 Five different enzymes, of which three metalloproteases (i.e. Collagenase, Thermolysin and Anthrax LF), one serine protease (i.e. β-Chymotrypsin) and carbonic anhydrase, were spotted in triplicate on a glass slide, and subsequently screened with the Leu probe (Figure 17). Results indicate that the probe was in general able to distinguish metalloenzyme activity over other non-metalloenzyme activities, in most cases generating positive fluorescence signals only with metalloproteases (e.g. Collagenase and Thermolysin), as well as carbonic anhydrase (a zinc-binding enzyme), but not with β-Chymotrypsin. Despite several attempts, we were unable to detect the fluorescence labeling of Anthrax LF, as well as several MMPs (data not shown), on the microarray. As these proteins were only available from commercial sources in very low stock concentrations, we attibuted our failure to the less-than-optimal immobilization of the proteins. 32 1 1 1 2 2 2 3 4 3 4 3 4 5 5 5 Figure 17 Protein microarray of various metalloenzymes screened by the Leu probe. Five different proteins were spotted in triplicate: 1. Carbonic anhydrase (300 μg/ml); 2. Collagenase (300 μg/ml); 3. Thermolysin (300 μg/ml); 4. Anthrax LF (6 μg/ml); 5. βChymotrypsin (300 μg/ml). In conclusion, we have used “click chemistry” to successfully synthesize a secondgeneration library of metalloprotease probes containing succinyl warheads with a variety of P1’ functionalities. With these probes, we have been able to generate unique activitybased fingerprints against various metalloproteases including MMPs and other therapeutically important enzymes such as anthrax LF. Such fingerprinting strategies may lead to future identification and characterization of new MMPs, and the development of potential potent and selective inhibitors. We have also for the first time shown that affinity-based probes may be equally amenable for high-throughput screening of metalloprotease activities in a protein microarray. 4.3 Perspective-Activity based metalloprotease probes Despite the success of utilizing photo-labile group (benzophenone or diazarine) for covalent linking the probe with target metalloprotease, the labeling mechanism is still affinity-based, which means their interaction is not based on the enzyme’s catalytic mechanism. The major drawback of affinity-based probe is non-specific labeling contributing to interfering background signals. Presently, all the MMP probes designed 33 exhibit their inhibitory activity by chelation to the catalytic zinc ion. MMPs differ from cysteine and serine proteases in that the nucleophile of MMP hydrolysis is a zinc bounded water molecule, which is not part of the enzyme and thus cannot be utilized for reaction with inhibitors or probes. However upon closer examination, it is found that there is a glutamic acid residue at the catalytic site which is involved in hydrogen bonding to the nucleophilic water molecule. The glutamic acid, like the –SH and –OH groups in cysteine and serine proteases respectively, can serve as a nucleophile for covalent modification. Several irreversible metalloprotease inhibitors based on modifying the active site glutamic acid have been reported, (Scheme 4) 50 which could be used as a guideline for designing activity-based metalloprotease probes. (Figure 18) Protein protein Zn2+ O protein Protein Zn2+ O S O O S S O O O - protein S- O protein O O Glu-404 Glu-404 Scheme 4 Mechanism of acetals as latent electrophiles that interact with catalytic nucleophile at the active site of matrix metalloproteases. Firstly, the biphenyl moiety is projected into the deep hydrophobic pocket in the active sites of gelatinases, and this process brings the thiirane moiety close to the active-site zinc ion. Finally, the active site glutamate nucleophilic attacks the activated thiirane moiety resulting in irreversible inhibition. P1' H N S n* O H N TMR O Figure 18 Structure of proposed activity-based MMP probes 34 Chapter 5 Experimental Section 5.1 General Information Starting materials and reagents were purchased commercially and used without further purification, unless otherwise stated. All moisture-sensitive reactions were performed under a positive pressure of nitrogen. 1HNMR spectra were recorded on a 300 MHz Bruker ACF300 or DPX300 NMR spectrometer, Chemical shifts are reported in parts per million referenced to internal standard ((CH3)4Si = 0.00 ppm). ESI mass spectra were acquired in the positive mode using a Finnigan/Mat TSQ7000 spectrometer. Analytical RP-HPLC separations were performed on Phenomex C18 (150 x 3.0 mm) column, using a Shimadzu Prominence HPLC system equipped with a Shimadzu SPD20A detector. Eluents A (0.1 % TFA/acetonitrile) and B (0.1 % TFA/water) were used as the mobile phases. Thermolysin (E.C. 3.4.24.27) and GM6001 were purchased from Calbiochem (USA) and Bacterial collagenase (E.C. 3.4.24.3) was purchased from SigmaAldrich (Milwauki, USA) at the highest grade available and used without further purification. 5.2 Synthesis of succinyl hydroxamate-based warhead bearing hydrophobic side chains Synthesis of mono-substituted succinyl hydroxamate involved five steps (Scheme 5). First the acid was condensed with 2-oxazolidone in the presence of DCC/DMAP. This step was quite efficient and gave ~90% high yield. Subsequently, reaction of the oxazolidinone 2 with sodium hexamethyldisilazide and then tert-butyl bromoacetate 35 produced the racemic alkylated product 3 in ~80% yield. Deprotection of the tert-butyl ester followed by coupling with trityl hydroxylamine generated the succinyl hydroxamate 5 in ~70% yield over two steps. Removal of the oxazolidone protection group with lithium hydroxide/hydrogen peroxide produced the desired racemic monosubstituted succinyl hydroxamic acid 6 in ~80% yield. O HN O P1' O O O P1' OH N DCC, DMAP Br O O O O O P1' 3 2 1 O N O NaHMDS O TFA/DCM ~100% O O P1' LiOH OH Ph3COHN 6 O H2O2 O P1' O O N Ph3COHN DCC, DCM O O CPh3ONH2 N 6B 4 6C O HO 5 6A P1' O 6D O 6E 6F 6G 6H Scheme 5 Synthesis route of hydrophobic MMPI warheads. General procedure for the coupling reaction of acids 1 with 2-oxazolidinone: 51 To a suspension of 2-oxazolidinone (5.30 g, 60 mmol), DMAP (0.96 g, 7.8 mmol) and 1 (78 mmol) in CH2Cl2 (100 mL) at 0 °C, under a nitrogen atmosphere, was added DCC in one portion (16.1 g, 78 mmol). After 10 min the temperature was raised to RT and stirring was continued overnight. The dicyclohexylurea formed was filtered and the precipitate washed with CH2Cl2 (20 ml). The filtrate was washed with sat. NaHCO3 (100 ml), dried with Na2SO4 and concentrated at reduced pressure to furnish the crude product, which was purified by silica gel chromatography (20%-30% EtOAc in hexanes) to afford 2 as a colorless oil. 36 General procedure for the alkylation of oxazolidinone 2 with tert-butyl 2-bromoacetate: 52 To a flame-dried 500 ml three-necked RBF was added 9.26 g (50 mmol) of oxazoline 2 in 100 ml of dry THF. This mixture was cooled to -78oC, and 1.0 M sodium bis(trimethylsilyl)amide in THF (65 ml, 65 mmol) was added dropwise. The internal o reaction temperature was kept below -71 C. The solution was then stirred at -78 oC for 1 hour, then neat tert-butyl bromoacetate (11.1 ml, 75 mmol) was added via a syringe over a 10-minute period. The mixture was stirred at -78 oC for 2 hours then warmed to 0oC and quenched with Et2O and aqueous 2 M NH4Cl. The aqueous layer was separated and extracted with diethyl ether. The combined organic layers were washed with brine, dried, filtered, and concentrated to give a brown oil. The oil was purified by silica gel chromatography (15%-20% EA in hexane) to isolate desired product 3 as a colorless oil. General procedure for the cleavage of the tert-butyl group in 3: Compound 3 (40 mmol) was dissolved in 320 ml of DCM, then TFA (30.6 ml, 400 mmol) was added, the mixture was vigorously stirred at room temperature for 4 hours. After removal of the solvent, the product was diluted with ethyl acetate (100 ml), then washed with water and brine, followed by drying in Na2SO4. The solution was concentrated to give the corresponding carboxylic acid 5 as a brownish oil, which was dried in vacuo for 4 hours to completely remove TFA. General procedure for the coupling of carboxylic acids 4 with trityloxyamine: Carboxylic acid 4 (50 mmol) in DCM (150 ml) was cooled down to 0oC, before addition of DCC (12.4 g, 60 mmol). Ten minutes later, the trityl-protected hydroxyamine (14.6 g, 60 mmol) was subsequently added to the reaction mixture, and the mixture was allowed to warm up to room temperature and stirred for 3 hours. The dicyclohexylurea formed was filtered and the filtrate was washed with saturated NaHCO3, brine, dried over Na2SO4, then concentrated to give a brown oil, which was purified by silica gel chromatography (20%-30% EA in hexane) to isolate the desired product 5 as a white solid. General procedure for the hydrolytic cleavage of the 2-oxazolidinone moiety in 5: To imide 5 (35 mmol) in 4/1 THF/H2O (600 ml) was added 30% aqueous H2O2 (14.3 ml, 136 mmol) at 0oC over 5 minutes. The internal temperature was kept at or below 5oC. Lithium hydroxide (2.8 g, 116.7 mmol) in 20 ml of water was then added over 5 minutes to ensure the internal temperature was kept below 4oC. After addition, the mixture was allowed to warm to room temperature. After two hours, the reaction mixture was cooled to 0oC and sodium nitrite (7.2 g, 103.7 mmol.) was added. The mixture was allowed to warm to room temperature and concentrated under reduced pressure. The resulting liquid was extracted with CHCl3. The combined organic layers were washed with aqueous 0.1 M NaOH. The combined aqueous layers were acidified with aqueous 1 M HCl to pH = 7, then extracted with ethyl acetate. The combined organic layers were dried, filtered, and concentrated to give 6 as an off-white solid. 37 3-(3-methylbutanoyl)oxazolidin-2-one (2A): Yield = 88%. 1H-NMR (300 MHz, CDCl3) δ 4.41 (t, J = 8.34 Hz, 2H), 4.05 (t, J = 8.37 Hz, 2H), 2.81 (d, J = 6.96 Hz, 2H), 2.30 – 2.08 (m, 1H), 0.98 (d, J = 6.60 Hz, 6H); ESI-MS: m/z [M+Na]+ = 194.0. 3-(4-methylpentanoyl)oxazolidin-2-one (2B): Yield = 93%. 1H-NMR (300 MHz, CDCl3) δ 4.26 (t, J = 16.1 Hz, 2H), 3.85 (t, J = 16.1 Hz, 2H), 2.73 (t, J = 15.2 Hz, 2H), 1.50-1.33 (m, 3H), 0.77 (d, J = 7.2 Hz, 6H); ESIMS cald for C9H15NO3Na [M+Na]+ =208.2, found 208.0. 3-(3-methylpentanoyl)oxazolidin-2-one (2C): Yield = 76 %. 1H -NMR (300 MHz, CDCl3) δ 4.37 (t, J = 8.01 Hz, 2H), 3.98 (t, J = 8.03 Hz, 2H), 2.91 – 2.84 (dd, J = 16.02 Hz, J = 5.58 Hz, 1H), 2.74 – 2.66 (dd, J = 16.00 Hz, J = 7.98 Hz, 1H), 2.00 – 1.87 (m, 1H), 1.46 – 1.32 (m, 1H), 1.28 – 1.12 (m, 1H), 0.96 – 0.82 (m, 6H); ESI-MS: m/z [M+Na]+ = 208.2. 3-(3-phenylpropanoyl)oxazolidin-2-one (2D): Yield = 91%. 1H-NMR (300 MHz, CDCl3) δ 7.34 – 7.19 (m, 5H), 4.38 (t, J = 8.01 Hz, 2H), 4.00 (t, J = 8.01 Hz, 2H), 3.26 (t, J = 7.49 Hz, 2H), 2.99 (t, J = 7.50 Hz, 2H); ESI-MS: m/z [M+Na]+= 242.2. 3-(5-phenylpentanoyl)oxazolidin-2-one (2E): Yield = 82%. 1H-NMR (300 MHz, CDCl3) δ 7.42 – 7.04 (m, 5H), 4.38 (t, J = 8.01 Hz, 2H), 3.99 (t, J = 8.01 Hz, 2H), 2.95 (m, 2H), 2.65 (t, J = 6.96 Hz, 2H), 1.73 – 1.68 (m, 4H); ESI-MS: m/z [M+Na]+= 270.3. 3-(2-cyclohexylacetyl)oxazolidin-2-one (2F): Yield = 95%. 1H-NMR (300 MHz, CDCl3) δ 4.39 (t, J = 8.19 Hz, 2H), 4.01 (t, J = 8.19 Hz, 2H), 2.80 (d, J = 6.96 Hz, 2H), 2.01 – 1.79 (m, 1H), 1.79 – 1.54 (m, 5H), 1.38 – 0.86 (m, 5H); ESI-MS: m/z [M+Na]+ = 234.3. 3-(3-cyclopentylpropanoyl)oxazolidin-2-one (2G): Yield = 70%. 1H-NMR (300 MHz, CDCl3) δ 4.38 (t, J = 8.01 Hz, 2H), 3.99 (t, J = 8.19 Hz, 2H), 2.89 (t, J = 7.67 Hz, 2H) 1.77 – 1.64 (m, 3H), 1.62 – 1.47 (m, 6H), 1.11 – 1.09 (m, 2H); ESI-MS: m/z [M+Na]+ = 234.2. 3-(4-(benzyloxy)butanoyl)oxazolidin-2-one (2H): Yield = 82%. 1H-NMR (300 MHz, CDCl3) δ 7.38-7.26 (m, 5H), 4.47 (s, 2H), 4.29 (t, J = 8.37 Hz, 2H), 3.99 (t, J = 8.37 Hz, 2H), 3.55 (t, J = 6.27 Hz, 2H), 3.03 (t, J = 7.32 Hz, 2H), 1.98 (t, J = 6.63 Hz, 2H). ESI-MS: m/z [M+Na]+ = 286.2. tert-butyl 4-methyl-3-(2-oxooxazolidine-3-carbonyl)pentanoate (3A): Yield = 66%. 1H-NMR (300 MHz, CDCl3) δ 4.39 (t, J = 7.32 Hz, 2H), 4.09 – 3.98 (m, 3H), 2.82 – 2.78 (m, 1H), 2.41 – 2.35 (dd, J = 16.87 Hz, J = 3.15 Hz, 1H), 2.03 – 38 1.94 (m, 1H), 1.40, (s, 9H), 0.99 – 0.97 (m, 3H), 0.87 (d, J = 6.96 Hz); ESI-MS: m/z [M+Na]+ = 308.2. tert-butyl 5-methyl-3-(2-oxooxazolidine-3-carbonyl)hexanoate (3B): Yield = 83%. 1H-NMR (300 MHz, CDCl ) δ 4.37 (t, J = 17.2 Hz, 2H), 4.20-4.10 3 (m, 1H), 3.97 (t, J = 17.2 Hz, 2H), 2.63 (q, 1H), 2.35 (q, 1H), 1.52-1.48 (m, 2H); 1.35 (s, 9H), 1.17-1.24 (m, 1H), 0.84 (q, 4H). ESI-MS cald for C15H25NO5Na [M+Na]+ =322.3, found 322.1. tert-butyl 4-methyl-3-(2-oxooxazolidine-3-carbonyl)hexanoate (3C): Yield = 88%. 1H-NMR (300 MHz, CDCl3) δ 4.40 – 4.34 (m, 2H), 4.12 – 3.94 (m, 3H), 2.86 – 2.72 (m, 1H), 2.38 – 2.25 (m, 1H), 1.47 – 1.32 (m, 1H), 1.37 (s, 9H), 1.28 – 1.14 (m, 2H), 0.92 – 0.77 (m, 6H); ESI-MS: m/z [M+Na]+ = 322.1. tert-butyl 3-benzyl-4-oxo-4-(2-oxooxazolidin-3-yl)butanoate (3D): Yield = 83%. 1H-NMR (300 MHz, CDCl3) δ 7.34 – 7.17 (m, 5H), 4.44 – 4.28 (m, 3H), 4.08 – 3.88 (m, 2H), 3.08 – 3.02 (dd, J = 12.87 Hz , J = 5.58 Hz, 1H), 2.83 – 2.74 (dd, J = 17.05, J = 11.16 Hz, 1H), 2.60 – 2.52 (dd, J = 13.06 Hz, J = 9.75 Hz, 1H), 2.33 – 2.26 (dd, J = 17.07 Hz, J = 3.84 Hz, 1H), 1.37 (s, 9H); ESI-MS: m/z [M+Na]+ = 356.4. tert-butyl 3-(2-oxooxazolidine-3-carbonyl)-6-phenylhexanoate (3E): Yield = 85%. 1H-NMR (300 MHz, CDCl3) δ 7.31 – 7.11 (m, 5H), 4.41 – 4.32 (m, 2H), 4.17 – 3.93 (m, 3H), 2.80 – 2.71 (dd, J = 16.71 Hz, J = 10.80 Hz, 1H), 2.66 – 2.51 (m, 2H), 2.42 – 2.35 (dd, J = 16.71 Hz, J = 4.17 Hz, 1H), 1.78 – 1.58 (m, 4H), 1.35 (s, 9H); ESI-MS: m/z [M+Na]+ = 384.4. tert-butyl 3-cyclohexyl-4-oxo-4-(2-oxooxazolidin-3-yl)butanoate (3F): Yield = 67%. 1H-NMR (300 MHz, CDCl3) δ 4.52 – 4.36 (m, 2H), 4.15 – 4.01 (m, 3H), 2.86 – 2.77 (m, 1H), 2.50 – 2.43 (m, 1H), 1.74 – 1.63 (m, 6H), 1.43 (s, 9H) 1.39 – 0.94 (m, 5H); ESI-MS: m/z [M+H]+ = 326.4. tert-butyl 3-(cyclopentylmethyl)-4-oxo-4-(2-oxooxazolidin-3-yl)butanoate (3G): Yield = 74%. 1H-NMR (300 MHz, CDCl3) δ 4.43 – 4.38 (m, 2H), 4.24 – 4.15 (m, 1H), 4.07 – 3.98 (m, 2H), 2.78 – 2.69 (dd, J = 16.53 Hz, J = 10.80 Hz, 1H), 2.49 – 2.42 (dd, J = 16.53 Hz, J = 4.17 Hz, 1H), 1.80 – 1.66 (m, 3H), 1.66 – 1.47 (m, 6H), 1.40 (s, 9H), 1.18 – 1.00 (m, 2H); ESI-MS: m/z [M+Na]+ = 348.4. tert-butyl 5-(benzyloxy)-3-(2-oxooxazolidine-3-carbonyl)pentanoate (3H): Yield = 78%. 1H-NMR (300 MHz, CDCl3) δ 7.35 – 7.20 (m, 5H), 4.50 – 4.30 (m, 2H), 4.30 – 4.20 (m, 2H), 4.17 – 3.81 (m, 2H), 3.69 – 3.52 (m, 3H), 2.87 – 2.76 (dd, J = 16.5 Hz, J = 10.44 Hz, 1H), 2.85 – 2.76 (dd, J = 16.71 Hz, J = 4.53Hz, 1H), 2.09 – 1.98 (m, 1H), 1.35 (s, 9H); ESI-MS: m/z [M+Na]+= 400.3. 4-methyl-3-(2-oxooxazolidine-3-carbonyl)pentanoic acid (4A): 39 Yield = 98%. 1H-NMR (300 MHz, CDCl3) δ 4.50 – 4.39 (m, 2H), 4.12 – 3.98 (m, 3H), 2.98 – 2.80 (m, 1H), 2.56 – 2.50 (dd, J = 17.60 Hz, J = 3.32 Hz, 1H), 2.16 – 2.03 (m, 1H), 1.02 – 0.87 (m, 6H); ESI-MS: m/z [M-H]- = 228.3. 5-methyl-3-(2-oxooxazolidine-3-carbonyl)hexanoic acid (4B): Yield = 99%. 1H-NMR (300 MHz, CDCl3) δ 4.44-4.37 (m, 2H), 4.28-4.12 (m, 1H), 4.05-3.95 (m, 2H), 2.81 (q, 1H), 2.52 (q, 1H); 1.57-1.51 (m, 2H), 1.35-1.23 ( m, 1H), + 0.91(q, 4H); ESI-MS cald for C11H17NO5Na [M+H] 244.2, found 244.0. 4-methyl-3-(2-oxooxazolidine-3-carbonyl)hexanoic acid (4C): Yield = 99%. 1H-NMR (300 MHz, CDCl3) δ 8.87 (br s,1H), 4.43 – 4.37 (m, 2H), 4.14 – 3.92 (m, 3H), 2.95 – 2.69 (m, 1H), 2.54 – 2.37 (m, 1H), 2.20 – 1.68 (m, 1H), 1.45 – 1.20 (m, 2H), 1.00 – 0.78 (m, 6H); ESI-MS: m/z [M-H]- = 242.4. 3-benzyl-4-oxo-4-(2-oxooxazolidin-3-yl)butanoic acid (4D): Yield = 98%. 1H-NMR (300 MHz, CDCl3) δ 7.35 – 7.17 (m, 5H), 4.45 – 4.27 (m, 3H), 4.08 – 3.87 (m, 2H), 3.12 – 3.06 (dd, J = 13.23 Hz, J = 5.22 Hz, 1H), 2.94 – 2.84 (dd, J = 17.58 Hz, J = 10.80 Hz, 1H), 2.60 – 2.53 (dd, J = 13.23 Hz, J = 9.75 Hz, 1H), 2.43 – 2.36 (dd, J = 17.42 Hz, J = 3.84 Hz, 1H); ESI-MS: m/z [M-H]- = 276.3. 3-(2-oxooxazolidine-3-carbonyl)-6-phenylhexanoic acid (4E): Yield = 99%. 1H-NMR (300 MHz, CDCl3) δ 8.26 (br s, 1H), 7.42 – 7.15 (m, 5H), 4.45 – 4.37 (m, 2H), 4.35 – 4.15 (m, 1H), 4.04 – 3.93 (m, 2H), 2.94 – 2.85 (dd, J = 17.42 Hz, J = 10.44 Hz, 1H), 2.64 – 2.48 (m, 3H), 1.76 – 1.58 (m, 5H); ESI-MS: m/z [M-H]- = 304.3. 3-cyclohexyl-4-oxo-4-(2-oxooxazolidin-3-yl)butanoic acid (4F): Yield = 99%. 1H-NMR (300 MHz, CDCl3) δ 8.60 (br s, 1H), 4.45 – 4.38 (m, 2H), 4.14 – 3.94 (m, 3H), 2.99 – 2.76 (m, 1H), 2.57 – 2.50 (m, 1H), 1.74 – 1.62 (m, 6H), 1.31 – 0.82 (m, 5H); ESI-MS: m/z [M-H]- = 268.2. 3-(cyclopentylmethyl)-4-oxo-4-(2-oxooxazolidin-3-yl)butanoic acid (4G): Yield = 97%. 1H-NMR (300 MHz, CDCl3) δ 8.15 (br s, 1H), 4.46 – 4.38 (m, 2H), 4.27 – 4.12 (m, 1H), 4.00 (m, 2H), 2.91 – 2.82 (dd, J = 17.25 Hz, J = 10.80 Hz, 1H), 2.61 – 2.55 (dd, J = 17.24 Hz, J = 4.17 Hz, 1H), 1.79 – 1.54 (m, 9H), 1.25 – 1.08 (m, 2H); ESI-MS: m/z [M-H]- = 268.4. 5-(benzyloxy)-3-(2-oxooxazolidine-3-carbonyl)pentanoic acid (4H): Yield = 99%. 1H-NMR (300 MHz, CDCl3) δ 7.72 (br s, 1H), 7.46-7.28 (m, 5H), 4.58-4.42 (m, 2H), 4.38 – 4.20 (m, 2H), 4.05-3.85 (m, 2H), 3.72-3.52 (m, 3H), 2.98 – 2.85 (dd, J = 17.25 Hz, J = 10.80 Hz, 1H), 2.61 – 2.55 (dd, J = 17.24 Hz, J = 4.17 Hz, 1H), 2.08-1.79 (m, 2H); δ ESI-MS: m/z [M-H]- = 320.3. 4-methyl-3-(2-oxooxazolidine-3-carbonyl)-N-(trityloxy)pentanamide (5A): 40 Yield = 74%. 1H-NMR (300 MHz, CDCl3) δ 7.30 – 7.15 (m, 16H), 4.34 (t, J = 8.0 Hz, 2H), 4.09 – 3.90 (m, 3H), 2.41 – 2.32 (m, 1H), 1.88 – 1.70, (1H, m), 1.46 – 1.27 (m, 1H), 0.71 – 0.60 (m, 6H); ESI-MS: m/z [M+Na]+ = 509.2. 5-methyl-3-(2-oxooxazolidine-3-carbonyl)-N-(trityloxy)hexanamide (5B): Yield = 70%. 1H-NMR (300 MHz, CDCl3) δ 7.66 (br s, 1H), 7.35 (s, 15H), 4.35 (t, J = 16.4Hz, 2H), 4.13-3.89 (m, 3H), 2.33 (q, 1H); 1.66 (br s, 1H), 1.36-1.07 ( m, 3H), 0.82(m, 6H); ESI-MS cald for C30H32N2O5Na [M+Na]+ 523.2, found 523.1. 4-methyl-3-(2-oxooxazolidine-3-carbonyl)-N-(trityloxy)hexanamide (5C): Yield = 78%. 1H-NMR (300 MHz, CDCl3) δ 7.66 (br s, 1H), 7.42 – 7.28 (m, 15H), 4.33 (t, J = 7.83 Hz, 2H), 4.01 – 3.70 (m, 3H), 2.46 – 2.33 (m, 1H), 2.04 – 1.86 (m, 1H), 1.74 – 1.58 (m, 1H), 1.41 – 0.62 (m, 8H); ESI-MS: m/z [M+Na]+ = 523.6. 3-benzyl-4-oxo-4-(2-oxooxazolidin-3-yl)-N-(trityloxy)butanamide (5D): Yield = 82%. 1H-NMR (300 MHz, CDCl3) δ 7.60 (br s, 1H), 7.38 – 7.19 (m, 20H), 4.38 – 4.20 (m, 3H), 3.98 – 3.78 (m, 2H), 3.10 – 2.76 (m, 2H), 2.41 – 2.21 (m, 2H); ESIMS: m/z [M+Na]+ = 557.5. 3-(2-oxooxazolidine-3-carbonyl)-6-phenyl-N-(trityloxy)hexanamide (5E): Yield = 81%. 1H-NMR (300 MHz, CDCl3) δ 7.71 (br s, 1H), 7.44 – 7.11 (m, 20H), 4.34 – 4.29 (t, J = 8.37 Hz, 2H), 4.00 – 3.65, (m, 3H), 2.61 – 2.28 (m, 4H), 1.55 – 1.25 (m, 4H); ESI-MS: m/z [M+Na]+ = 585.3. 3-cyclohexyl-4-oxo-4-(2-oxooxazolidin-3-yl)-N-(trityloxy)butanamide (5F): Yield = 74%. 1H-NMR (300 MHz, CDCl3) δ 7.60 (br s, 1H), 7.49 – 7.28 (m, 15H), 4.45 – 4.23 (m, 2H), 4.04 – 3.59 (m, 3H), 2.41 – 2.32 (m, 1H), 1.75 – 1.30 (m, 7H), 1.19 – 0.78 (m, 5H); ESI-MS: m/z [M+Na]+ = 549.6. 3-(cyclopentylmethyl)-4-oxo-4-(2-oxooxazolidin-3-yl)-N-(trityloxy)butanamide(5G): Yield = 54%. 1H-NMR (300 MHz, CDCl3) δ 7.45 – 7.30 (m, 15H), 4.33 (t, J = 8.01 Hz, 2H), 4.05 – 3.88 (m, 3H), 2.46 – 2.34 (m, 1H), 1.73 – 1.46 (m, 10H), 1.15 – 0.87 (m, 2H); ESI-MS: m/z [M+Na]+ = 549.5. 5-(benzyloxy)-3-(2-oxooxazolidine-3-carbonyl)-N-(trityloxy)pentanamide (5H): Yield = 72%. 1H-NMR (300 MHz, CDCl3) δ 7.48-7.30 (m, 20H), 4.48-4.30 (m, 2H), 4.25 – 4.13 (m, 2H), 3.98-3.82 (m, 2H), 3.52-3.38 (m, 3H), 2.47-2.25 (m, 1H), 2.12-1.98 (m, 1H), 1.79-1.65 (m, 2H); ESI-MS: m/z [M+Na]+ = 601.5. 2-isopropyl-4-oxo-4-(trityloxyamino)butanoic acid (6A): Yield = 76%. 1H-NMR (300 MHz, CDCl3) δ 7.80 (br s, 1H), 7.49 – 7.26, (m, 15H), 2.74 – 2.59 (m, 1H), 2.45 – 2.15 (m, 2H), 1.82 – 1.68 (m, 1H), 1.06 – 0.66 (m, 6H); 13C-NMR (75 MHz, CDCl3) δ 174.4, 171.9, 146.8, 129.3 – 126.5 (m), 82.2, 42.9, 28.7, 27.7, 19.5, 17.4; ESI-MS: m/z [M+Na]+ = 440.3. 4-methyl-2-(2-oxo-2-(trityloxyamino)ethyl)pentanoic acid (6B): 41 Yield = 80%. 1H-NMR (300 MHz, CDCl3) δ 7.46 (br s, 1H), δ 7.32 (br s, 15H), 4.10 (q, 1H), 2.59-2.53 (m, 1H), 2.32-2.20 (m, 1H), 1.45-1.32 (m, 3H); 0.84(m, 6H); ESIMS cald for C27H29NO4Na [M+Na]+ 454.2, found 454.0. 3-methyl-2-(2-oxo-2-(trityloxyamino)ethyl)pentanoic acid (6C): Yield = 70%. 1H-NMR (300 MHz, CDCl3) δ 7.43 – 7.28 (m, 16H), 2.95 – 2.86 (m, 1H), 2.75 – 2.56 (m, 1H), 2.44 – 2.37 (m, 1H), 2.21 – 2.06 (m, 1H), 1.53 – 1.05 (m, 2H), 0.98 – 0.69 (m, 6H); 13C-NMR (75 MHz, CDCl3) δ 175.1, 172.2, 146.8, 130.1-126.5 (m), 82.1, 41.1, 34.6, 26.9, 26.8, 14.0, 11.7; ESI-MS: m/z [M+Na]+ = 454.2. 2-benzyl-4-oxo-4-(trityloxyamino)butanoic acid (6D): Yield = 79%. 1H-NMR (300 MHz, CDCl3) δ 7.49 – 6.96 (m, 20H), 3.03 – 2.86 (m, 1H), 2.59 – 2.42, (m, 2H), 2.09 – 1.92 (m, 1H), 1.69 – 1.52 (m, 1H); 13C-NMR (75 MHz, CDCl3) δ 174.5, 171.8, 146.8, 136.5, 129.3 – 127.2 (m), 82.1, 38.4, 36.1, 30.6; ESI-MS: m/z [M+Na]+ = 488.3. 2-(2-oxo-2-(trityloxyamino)ethyl)-5-phenylpentanoic acid (6E): Yield = 65%. 1H-NMR (300 MHz, CDCl3) δ 7.38 – 7.22 (m, 21H), 2.59 – 2.43 (m, 4H), 2.31 – 2.15 (m, 1H), 1.54 – 1.26 (m, 4H); 13C-NMR (75 MHz, CDCl3) δ 178.2, 174.9, 146.8, 141.7, 130.0 – 125.5 (m), 82.2, 36.9, 35.4, 31.6, 30.6, 28.2; ESI-MS: m/z [M+Na]+ = 516.6. 2-cyclohexyl-4-oxo-4-(trityloxyamino)butanoic acid (6F): Yield = 64%. 1H-NMR (300MHz, CDCl3) δ 7.33 – 7.18 (m, 15H), 3.16 – 2.91 (m, 1H), 2.79 – 2.71 (m, 1H), 2.46 – 2.42 (m, 1H), 2.50 – 1.52 (m, 6H), 1.24 – 1.09 (m, 5H); 13 C-NMR (75 MHz, CDCl3) δ174.4, 171.9, 146.8, 130.0 – 126.5 (m), 82.0, 42.5, 38.6, 30.2, 28.3, 27.7, 25.8; ESI-MS: m/z [M+Na]+ = 480.6 2-(cyclopentylmethyl)-4-oxo-4-(trityloxyamino)butanoic acid (6G): Yield = 83%. 1H-NMR (300 MHz, CDCl3) δ 7.42 – 7.19 (m, 15H), 2.20 – 2.05 (m, 3H), 1.41 – 1.26 (m, 9H), 1.14 – 0.68 (m, 2H); 13C-NMR (75 MHz, CDCl3) δ 176.2, 173.0, 146.8, 128.5 – 127.0 (m), 92.8, 45.7, 37.9 – 36.6 (m), 32.4 – 31.9 (m), 25.0; ESIMS: m/z [M+Na]+ = 480.6. 2-(2-(benzyloxy)ethyl)-4-oxo-4-(trityloxyamino)butanoic acid (6H): Yield = 73%. 1H-NMR (300 MHz, CDCl3) δ 7.48 – 7.20 (m, 20H), 4.55 - 4.34 (m, 2H), 3.62-3.36 (m, 2H), 2.61-2.57 (m, 1H), 2.41-2.30 (m, 1H), 2.22-2.15 (m, 1H), 1.821.62 (m, 2H); 13C-NMR (75 MHz, CDCl3) δ 176.2, 173.0, 146.8, 128.5 – 127.0 (m), 92.8, 45.7, 37.9 – 36.6 (m), 32.4 – 31.9 (m), 25.0; ESI-MS: m/z [M+Na]+ = 532.6. 5.3 Synthesis of succinyl hydroxamate-based warheads with P1’ substitution mimicking nature amino acid sides 5.3.1 Synthesis of warheads 6I and 6J Synthesis of hydrophilic and acidic P1’ substituted warheads involves 7 steps. Both of them started with mono-protection of the diol 7. This was followed by PDC oxidation 42 of alcohol 8 to give carboxylic acid 9. Upon installing oxazolidinone, enolate chemistry was carried out to introduce the succinyl template, giving rise to 11 and 12, respectively. To synthesize warhead 6I, trityl-protected hydroxylamine was first coupled to 11 using standard DCC procedures, followed by selective removal of the benzyl group on the alcohol using Pd/C with H2 to give 13. Subsequently, the free alcohol was protected with the acid-labile trityl group, followed by base hydrolysis of oxazolidinone to give the final warhead 6I. To synthesize warhead 6J, the alcohol moiety in 12 was oxidized to the corresponding acid, followed by coupling with trityl-protected hydroxylamine, giving 14. Finally, base hydrolysis of oxazolidinone furnished the final warhead 6J. O HO Ag2O OH n O PDC OH PhCH2Br 9 8 (1) CPh3Cl DMAP, TEA OH Ph3COHN (2) LiOH, H2O2 O O N Ph3COHN OH H2O2 N Ph3CHNO HO (1)NaHMDS O (1) PDC, DMF N O (2) CPh ONH , DCC HO 3 2 Br O O O O 14 O O O 11 O O O O LiOH N HO O O Ph3COHN O O O O (2) Pd/C, H2 N n O 10 (2) TFA/DCM O (1) CPh3ONH2, DCC 13 O O DCC,DMAP O O 6I O n O OH OCPh3 O OH n 7 6J O 2-oxazolidone O (3) Pd/C, H2 O 12 OH OH HO 7a 7b Scheme 6 Synthesis route of warhead 6I and 6J General procedure for the conversion of 7a-b to 8a-b: 53 To a solution of the diol 7 (a or b; 120 mmol) in DCM (300 ml) was added Ag O 2 (41.7 g, 180 mmol) and benzyl bromide (15.7 ml, 132 mmol) at room temperature under a nitrogen atmosphere. The reaction was stirred further for 4 hrs before the solid was removed by filtration. The resulting filtrate was concentrated in vacuo and purified by flash chromatography (10 – 30% EA/hexane) to give 8 as a colorless oil. 5-(benzyloxy)pentan-1-ol (8a): Yield = 84%. 1H-NMR (300 MHz, CDCl ) δ 7.40-7.25 (m, 5H), 4.50 (s, 2H), 3.58 3 (t, J = 12.2 Hz, 2H), 3.48 (t, J = 12.9 Hz, 1H), 2.28 (s, 1H), 1.67-1.54 (m, 4H), 1.51-1.40 (m, 2H); ESI-MS: m/z [M+Na]+ = 217.3. 4-(benzyloxy)butan-1-ol (8b): Yield = 80%. 1H-NMR (300 MHz, CDCl ) δ7.48 – 7.21, (m, 5H), 4.49, (s, 2H), 3 3.57 (t, J = 6.48 Hz, 2H), 3.48 (t, J = 5.93 Hz, 2H), 2.91 (br s, 1H), 1.70 – 1.57 (m, 4H); ESI-MS: m/z [M+Na]+= 203.2 43 General procedure for oxidation of 8a-b to 9a-b: 54 To a solution of 8 (a or b; 96 mmol) in DMF (250 ml) at 0 ºC under a nitrogen atmosphere was added pyridinium dichromate (170.6 g, 453 mmol). The mixture was gradually raised to room temperature and stirred overnight. Water was subsequently added and the resulting mixture was extracted with ether. The combined ether layers were washed with brine, dried over Na2SO4, filtered and concentrated. The oil obtained was purified by flash chromatography (10 – 20% EA/hexane) to give 9. 5-(benzyloxy)pentanoic acid (9a): Yield = 50%.1H-NMR (300 MHz, CDCl3) δ 9.96 (br s, 1H), 7.42-7.25 (m, 5H), 4.51 (s, 2H), 3.50 (t, J = 10.8 Hz, 2H), 2.38 (t, J = 12.6 Hz, 1H), 2.71 (m, 4H); ESI-MS: m/z [M-H]-= 207.2. 4-(benzyloxy)butanoic acid (9b): Yield = 64%.1H-NMR (300 MHz, CDCl3) δ 10.10 (br s, 1H), 7.42-7.30 (m, 5H), 4.54 (s, 2H), 3.56 (t, J = 5.94 Hz, 2H), 2.51 (t, J = 6.96 Hz, 2H), 1.98 (t, J = 6.63 Hz, 2H). ESI-MS: m/z [M-H]-= 193.1. Procedures for conversion of 9a-b to 10a-b, then to 11 & 12: The synthesis of 11 and 12 from 9a-b was accomplished using procedures described previously. 3-(5-(benzyloxy)pentanoyl)oxazolidin-2-one (10a): Yield = 80%. 1H-NMR (300 MHz, CDCl3) δ 7.33-7.22 (m, 5H), 4.60 (s, 2H), 4.35 (t, J = 16.2 Hz, 2H), 3.90 (t, J = 15.9 Hz, 2H), 3.50 (t, J = 12.9Hz, 2H), 2.96 (t, J = 12.9 Hz, 2H), 1.82-1.67 (m, 4H); ESI-MS: m/z [M+Na]+ = 300.2. 3-(4-(benzyloxy)butanoyl)oxazolidin-2-one (10b): Yield = 82%. 1H-NMR (300 MHz, CDCl3) δ 7.38-7.26 (m, 5H), 4.47 (s, 2H), 3.56 (t, J = 5.94Hz, 2H), 2.51 (t, J = 6.96 Hz, 2H), 1.98 (t, J = 6.63 Hz, 2H). ESI-MS: m/z [M+Na]+ = 286.2. 6-(benzyloxy)-3-(2-oxooxazolidine-3-carbonyl)hexanoic acid (11): The oxazolidine precursor of 11 was synthesized from 10a accordingly (Yield = 71%), and characterized by NMR and MS. 1H-NMR (300 MHz, CDCl3) δ 7.38-7.26 (m, 5H), 4.47 (s, 2H), 4.38-4.30 (m, 2H), 4.25-4.16 (m, 1H), 4.08-3.92 (m, 2H), 3.46 (m, 2H), 2.83-2.74 (dd, J = 17.97 Hz, J = 10.68 Hz, 1H), 2.45-2.38 ( dd, J = 16.70 Hz, J = 4.11 Hz, 1H), 1.78-1.56 ( m, 4H); ESI-MS: m/z [M+Na]+= 414.2. The above intermediate (13.3 g, 34 mmol) was treated with TFA (26.2 ml, 340 mmol) in DCM (170 ml), based on a previously published procedure, to give 11 (11.3 g, 33.7 mmol) in 99% yield. 1H-NMR (300 MHz, CDCl3) δ 7.38-7.26 (m, 5H), 4.38 (s, 2H), 4.42-4.28 (m, 2H), 4.24-4.12 (m, 1H), 4.08-3.92 (m, 2H), 3.50 (m, 2H), 2.83-2.74 ( dd, J = 17.26 Hz, J = 10.18 Hz, 1H), 2.56-2.42 (dd, J = 16.70 Hz, J = 4.11 Hz, 1H), 1.78-1.56 ( m, 4H); ESI-MS: m/z [M-H]- = 334.3. 44 tert-butyl 5-hydroxy-3-(2-oxooxazolidine-3-carbonyl)pentanoate (12): The oxazolidine precursor of 12 was synthesized from 10b accordingly (Yield = 78%), and characterized by NMR and MS. 1H-NMR (300 MHz, CDCl3) δ7.35 – 7.20 (m, 5H), 4.50 – 4.30 (m, 2H), 4.30 – 4.20 (m, 2H), 4.17 – 3.81 (m, 2H), 3.69 – 3.52 (m, 3H), 2.87 – 2.76 (dd, J = 16.5 Hz, J = 10.44 Hz, 1H), 2.85 – 2.76 (dd, J = 16.71 Hz, J = 4.53Hz, 1H), 2.09 – 1.98 (m, 1H), 1.35 (s, 9H); ESI-MS: m/z [M+Na]+ = 414.3. Hydrogenolysis of above intermediate (13.2 g, 35 mmol) was accomplished in THF (175 ml) with Pd/C (1.32 g) by stirring the mixture at room temperature under a hydrogen atmosphere for 1.5 hrs. The mixture was filtered, and the filtrate concentrated and purified by flash chromatography (80-100% EA/hexane) to yield 12 (7.96 g, 27.7 mmol, 79%). 1H-NMR (300 MHz, CDCl3) δ 4.43 – 4.35 (m, 2H), 4.24 – 4.14 (m, 2H), 4.05 – 3.96 (m, 2H), 3.65 – 3.60 (t, J = 5.94 Hz, 2H), 2.83 – 2.74 (dd, J = 16.89 Hz, J = 10.47 Hz, 1H), 2.46 – 2.39 (dd, J = 16.89 Hz, J = 4.20 Hz, 1H), 1.92 – 1.65 (m, 2H), 1.35 (s, 9H); ESI-MS: m/z [M+Na]+ = 310.2. 6-hydroxy-3-(2-oxooxazolidine-3-carbonyl)-N-(trityloxy)hexanamide (13): Coupling of acid 11 (11.4 g, 34.0 mmol) with trityl-protected hydroxylamine (11.2 g, 40.8 mmol) and DCC (8.42 g, 40.8 mmol) in DCM (120 ml), following previously published procedures, yielded the corresponding intermediate (80%) after purification by flash chromatography (40-60% EA/hexane). 1H-NMR (300 MHz, CDCl3) δ 7.68 (br s, 1H), 7.38-7.26 (m, 20H), 4.45 (s, 2H), 4.38-4.22 (m, 2H), 4.04-3.82 (m, 3H), 3.37 (m, 2H), 2.40-2.37 (m, 1H), 2.12-2.01 ( m, 1H), 1.58-1.32 (m, 4H); ESI-MS: m/z [M+Na]+ = 615.1. The above intermediate (16.1 g, 27.2 mmol) in THF (130 ml) and Pd/C (1.61 g) was stirred at room temperature under a hydrogen atmosphere for 1.5 hrs. The mixture was filtered, and the filtrate concentrated and purified by column chromatography (80100% EA/hexane) to afford 13 (10.8g, 21.5 mmol, 75%).1H-NMR (300 MHz, CDCl3) δ 7.68 (br s, 1H), 7.38-7.26 (m, 15H), 4.47-4.34 ( m, 2H), 4.06-3.85 (m, 3H), 3.56-3.42 (m, 2H), 2.43-2.34 (m, 1H), 2.02-1.88 (m, 1H), 1.48-1.20 (m, 4H); ESI-MS: m/z [M+Na]+ = 525.1. 2-(2-oxo-2-(trityloxyamino)ethyl)-5-(trityloxy)pentanoic acid (6I): To a solution of 13 (10.8 g, 21.5 mmol), DMAP (0.26 g, 2.15 mmol) and TEA (5.36 ml, 38.7 mmol) in DCM (240 ml) was added CPh3Cl (6.00 g, 21.5 mmol) and the reaction was stirred at room temperature overnight. DCM was removed in vacuo to yield a yellow semi-solid. Subsequently, 20% EA/hexane was added to the residue. The white solid obtained was filtered off and washed again with 20% EA/hexane to afford the pure intermediate (15.6 g, 21.0 mmol, 98%).1H-NMR (300 MHz, CDCl3) δ 7.18-7.56 (m, 31H), 4.22 (t, J = 7.83 Hz, 2H), 3.61 (t, J = 8.73 Hz, 2H), 3.14-3.06 (q, 2H), 2.99-2.97 (m, 1H), 2.38-2.22 (m, 1H), 1.96-1.78 (m, 1H), 1.48-1.20 (m, 4H); ESI-MS: m/z [M+Na]+= 767.8. The above intermediate (15.6 g, 21.0 mmol) was treated with LiOH (1.51 g, 63.0 mmol) and 30 % H2O2 (11.8 ml, 113 mmol) using previously published procedures, followed by purification with flash chromatography (5-10% MeOH) to give 6I (9.66 g, 14.3 mmol, 68%).1H-NMR (300 MHz, CDCl3) δ 7.25-7.16 (m, 31H), 3.18-2.82 (m, 2H), 2.71-2.55 (m, 1H), 2.48-2.25 (m, 1H), 2.18-2.02 (m, 1H), 1.72-1.25 (m, 4H);13C-NMR 45 (75 MHz, CDCl3) δ 176.0, 174.5, 146.8, 129.2-126.4 (m), 88.9, 82.0, 68.6, 36.2, 35.4, 24.7, 22.1; ESI-MS: m/z [M-H]-= 674.3. tert-butyl 5-oxo-3-(2-oxooxazolidine-3-carbonyl)-5-(trityloxyamino)pentanoate (14): Oxidation of alcohol 12 (7.96 g, 27.7 mmol) was accomplished by following the procedure described for the conversion of 8a-b to 9a-b, using PDC (31.3 g, 83.1 mmol) in DMF (70 ml), to furnish the corresponding intermediate (6.51 g, 21.6 mmol, 78%).1HNMR (300 MHz, CDCl3) δ 4.48 – 4.34 (m, 3H), 4.08 – 3.93 (m, 2H), 2.84 – 2.69 (m, 2H), 2.54 – 2.38 (m, 2H), 1.40 (br s, 9H); ESI-MS: m/z [M-H]- = 300.3. Above intermediate (65.1 g, 21.6 mmol) was coupled with trityl-protected hydoxylamine (7.13 g, 25.9 mmol) and DCC (5.34 g, 25.9 mmol) in DCM (65 ml), following previously published procedures. Upon purification by flash chromatography (50-70% EA/hexane), the desired product 14 was isolated (7.37 g, 13.2 mmol, 61%).1HNMR (300 MHz, CDCl3) δ 7.47 – 7.32 (m, 16H), 4.37 (t, J = 8.01 Hz, 2H), 4.12 (q, J = 7.32 Hz, 1H), 3.96 (t, J = 7.32 Hz, 2H), 2.39 – 1.85 (m, 4H), 1.40 (s, 9H); ESI-MS: m/z [M+Na]+ = 581.5. 2-(2-tert-butoxy-2-oxoethyl)-4-oxo-4-(trityloxyamino)butanoic acid (6J): Hydrolytic cleavage of the oxazolidinone moiety in 14 (7.37 g, 13.2 mmol) was accomplished by LiOH (0.95 g, 39.6 mmol) and 30 % H2O2 (6.36 ml, 60.7 mmol) using previously published procedures, which upon purification by flash chromatography (7080% EA/hexane) yielded 6J (4.72 g, 9.64 mmol, 73%).1H-NMR (300 MHz, CDCl3) δ 7.43 – 7.29 (m, 16H), 3.02-2.87 (m, 1H), 2.79-2.63 (m, 1H), 2.48-2.25 (m, 2H), 2.18-2.02 (m, 1H), 1.43 (s, 9H);13C-NMR (75 MHz, CDCl3) δ 177.0, 172.2, 171.9, 141.8, 140.8, 81.0, 65.0, 40.6, 36.7, 29.8, 27.9; ESI-MS: m/z [M+Na]+ = 512.4. 5.3.2 Synthesis of warhead 6K O H2N O (1) (Boc)2O, NaOH OH BocHN O N (2) 2-oxazolidone DCC, DMAP O 16 15 (1)NaHMDS (2) Pd/C, H2 O O Br NHBoc NHBoc (1)CPh3ONH2, DCC O OH Ph3COHN 6K (2) LiOH, H2O2 O O N HO O O 17 O Scheme 7 Synthesis route of warhead 6K tert-butyl 6-oxo-6-(2-oxooxazolidin-3-yl)hexylcarbamate (16) To a solution of 6-aminohexanoic acid 15 (7.87 g, 60 mmol) and NaOH (2 M solution, 50ml) in dioxane/water (2:1; 180 ml) at 0 °C was added (Boc)2O (15.7 g, 72 mmol). The reaction was allowed to proceed at room temperature overnight. 46 Subsequently, dioxane was removed under reduced pressure. The resulting mixture was acidified to pH 2 with 1 M HCl, then extracted with EA (3 x 80 ml). The combined organic extracts were dried over Na2SO4, filtered and concentrated to furnish the desired product as colorless oil (13.8 g, 99%), which was further reacted with 2-oxazolidinone (4.35 g, 50 mmol), DMAP (0.92 g, 7.5 mmol) and DCC (15.5g, 75 mmol) based on the general procedures described for the synthesis of 10a-b. Upon column purification (20 40% EA/hexane), the desired product 16 was isolated as white solid (15.6 g, 52.0 mmol, 65%).1H-NMR (300 MHz, CDCl3) δ 4.55 (br s, 1H), δ 4.39 (t, J = 8.01 Hz, 2H), 3.99 (t, J = 8.19 Hz, 2H), 3.19 – 3.02 (m, 2H), 2.89 (t, J = 7.49 Hz, 2H), 1.72 – 1.58 (m, 2H), 1.57 – 1.35 (m, 4H), 1.41 (s, 9H); ESI-MS: m/z [M+Na]+ = 323.2. 7-(tert-butoxycarbonyl)-3-(2-oxooxazolidine-3-carbonyl)heptanoic acid (17) Compound 16 (15.6 g, 52.0 mmol) was alkylated with benzyl 2-bromoacetate (15.5 g, 67.6 mmol) using NaHMDS (67.6 ml, 67.6 mmol) in 180 ml THF following previously described procedures. Upon column purification (20 – 35% EA/hexane), the product/intermediate was isolated as colorless oil (16.6 g, 36.9 mmol, 71%).1H-NMR (300 MHz, CDCl3) δ 7.30 (m, 5H), δ 5.11 – 4.97 (m, 2H), 4.56 (br s, 1H), 4.42-4.28 (m, 2H), 4.26-4.16 (m, 1H), 4.02-3.78 (m, 2H), 3.07-3.05 (m, 2H), 2.98-2.82 (dd, J = 17.07Hz, J = 6.27Hz , 1H), 2.55-2.48 (dd, J = 16.91Hz, J = 3.84Hz , 1H), 1.65 (m, 2H), 1.41 (m, 11H), 1.26 (m, 2H). ESI-MS; m/z [M+Na]+ = 437.3. Hydrogenolysis of the above intermediate (16.6 g, 36.9 mmol) in THF (130 ml) with Pd/C (1.66 g) was accomplished using procedures described earlier for the synthesis of 13. The desired product 17 was obtained without column purification (13.2 g, 36.9 mmol, 99%).1H -NMR (300 MHz, CDCl3) δ 4.63 (br s, 1H), 4.39 (t, J = 8.01 Hz, 2H), 4.18-4.10 (m, 1H), 3.98 (t, J = 8.15 Hz, 2H), 3.07-3.05 (m, 2H), 2.91-2.80 (dd, J = 17.07Hz, J = 6.27 Hz , 1H), 2.53-2.45 (dd, J = 16.91 Hz, J = 3.84Hz , 1H), 1.68-1.63 (m, 2H), 1.43-1.38 (m, 13H); ESI-MS: m/z [M-H]- = 357.3. 6-(tert-butoxycarbonyl)-2-(2-oxo-2-(trityloxyamino)ethyl)hexanoic acid (6K) Acid 17 (13.2 g, 36.9 mmol) was coupled with trityl-protected hydroxylamine (13.2 g, 48.0 mmol) and DCC (9.90 g, 48.0 mmol) in DCM (180 ml) using procedures previously described. Upon column purification (30 – 50% EA/hexane), the intermediate was obtained as a white solid (18.2 g, 29.5 mmol, 80%). 1H -NMR (300 MHz, CDCl3) δ 7.72 (br s, 1H), 7.34 ( m, 15H), 4.52 (br s, 1H), 4.33 (t, J = 16.38Hz, 2H), 4.04-3.88 (m, 3H), 3.04-3.052 (m, 2H), 2.40-2.30 (m, 1H), 2.08-2.02 (m, 1H), 1.48-1.26 (m, 15H); ESIMS: m/z [M-H]- = 615.2. Hydrolysis of above intermediate was achieved by the treatment with LiOH (2.12 g, 88.5 mmol), H2O2 (12.4 ml, 118 mmol) in THF/H2O (3/1; 360 ml) using procedures previously described, and purified to give the desired product 6K as a while solid (10.9 g, 20.0 mmol, 68%). 1H -NMR (300 MHz, CDCl3) δ 8.30 (br s, 1H), 7.33 (m, 15H), 4.59 (br s, 1H), 3.02-2.95 (m, 2H), 2.52-2.40 (m, 1H), 2.27-2.16 (m, 1H), 1.50-1.26 (m, 15H);13C-NMR (75 MHz, CDCl3) δ 174.9, 171.8, 146.8, 144.5, 130.0 – 126.5 (m), 82.2, 79.6, 40.4, 36.9, 31.5, 30.5, 28.4, 23.2; ESI-MS: m/z [M+Na]+= 569.1. 5.3.3 Synthesis of warhead 6L 47 O phenyl 2-bromoacetate m-CPBA O NaHMDS S O O O O O O O 20 t-BuOK tert-butyl 2-bromoacetate S SH 18 O O O O m-CPBA 19 O S O NaHMDS O O TFA/DCM OS O O O O CPh3ONH2 DCC piperidine O O S O Ph3COHN O 6L O OH Ph3COHN OS O O O O 26 HO OH O 23 DCC,DMAP DCM, FmocOH O 21 O OS O O 22 phenyl 2-bromoacetate O O O Pd/C O OS O O O 25 O OS O O O 24 Scheme 8 Synthesis route of MMPI warhead 6L tert-butyl 2-(4-methoxyphenylthio)acetate (19): To a stirred solution of 18 (8.61 ml, 70 mmol) in THF (110 ml) under a nitrogen atmosphere at 0 ºC was added t-BuOK over 5 min. Tert-butyl bromoacetate was then added dropwise. The reaction mixture was stirred for another 3 hrs, after which the white solid formed was filtered away. The filtrate was concentrated and purified by column chromatography (100% hexane – 5% DCM/hexane) to furnish 19 as colorless oil (15.0 g, 58.8 mmol, 84%).1H-NMR (300 MHz, CDCl3) δ 7.40 (d, J = 9.06 Hz, 2H), 6.82 (d, J = 9.06 Hz, 2H), 3.76 (s, 3H), 3.41 (s, 2H), 1.38 (s, 9H); ESI-MS: m/z [M+Na]+ = 277.2. 4-benzyl 1-tert-butyl 2-(4-methoxyphenylthio)succinate (20): Compound 19 (7.12 g, 28.0 mmol) was alkylated with benzyl 2-bromoacetate (12.8 g, 56.0 mmol) using NaHMDS (56.0 ml, 56.0 mmol) in 180 ml THF following general procedures described previously. Upon column purification (2 – 7% EA/hexane), the desired product 20 was obtained as colorless oil (8.69 g, 21.6 mmol, 77%).1H-NMR (300 MHz, CDCl3) δ 7.43 (d, J = 8.70 Hz, 2H), 7.34 (s, 5H), 6.84 (d, J = 8.70 Hz, 2H), 5.12 (s, 2H), 3.79 (m, 4H), 2.94-2.85 (dd, J = 16.89 Hz, J = 9.75 Hz , 1H), 2.74-2.67 (dd, J =17.07 Hz, J = 5.55 Hz , 1H), 1.39 (s, 9H); ESI-MS: m/z [M+Na]+= 425.0. tert-butyl 2-(4-methoxyphenylsulfonyl)acetate (21): To a solution of 19 (7.12 g, 28.0 mmol) in DCM (140 ml) under a nitrogen atmosphere at 0 ºC was added m-CPBA (12.1 g, 70.0 mmol) in small portions. The reaction mixture was stirred overnight. The white solid formed was filtered away, and the filtrate was concentrated and purified by column chromatography (15 – 40% DCM/hexane), to furnish 21 (5.61 g, 19.6 mmol, 70%).1H-NMR (300 MHz, CDCl3) δ 7.79 (d, J = 8.71 Hz, 2H), 7.00 (d, J = 8.72 Hz, 2H), 3.99 (s, 2H), 3.87 (s, 3H), 1.37 (s, 9H); ESI-MS: m/z [M+Na]+= 308.9. 4-benzyl 1-tert-butyl 2-(4-methoxyphenylsulfonyl)succinate (22): Compound 20 (8.69 g, 21.6 mmol) was oxidized with m-CPBA (9.32 g, 54.0 mol) using procedures described for the conversion of 19 to 21. Upon column purification (60 48 – 80% EA/hexane), the desired product 22 was isolated as colorless oil (7.13 g, 16.4 mmol, 76%) Sulfone 21 (5.61 g, 19.6 mmol) was alkylated with benzyl 2-bromoacetate (8.98 g, 39.2 mmol) using NaHMDS (39.2 ml, 39.2 mmol) in 100 ml THF following procedures described earlier. Upon column purification (60-80% EA/hexane), the desired product 22 was isolated (6.39 g, 14.7 mmol, 75%).1H-NMR (300 MHz, CDCl3) δ 7.79 (d, J = 9.06 Hz, 2H), 7.34 (s, 5H), 7.01 (d, J = 9.06 Hz, 2H), 5.11 (s, 2H), 4.33 (t, J = 14.97Hz, 1H), 3.88 (s, 3H), 3.10 (d, J = 7.32 Hz, 2H), 1.34 (s, 9H); ESI-MS: m/z [M+Na]+ = 457.0. 4-(benzyloxy)-2-(4-methoxyphenylsulfonyl)-4-oxobutanoic acid (23): Cleavage of the tert-butyl group in 22 (13.0 g, 30 mmol) with TFA (23.1 ml, 300 mmol) in DCM (150 ml) was accomplished using procedures described previously to give 23 (11.1 g, 29.4 mmol, 98%).1H-NMR (300 MHz, CDCl3) δ 7.78 (d, J = 8.73 Hz, 2H), 7.33 (s, 5H), 7.01 (d, J = 8.73 Hz, 2H), 5.64 (br s, 1H), 5.10 (q, 2H), 4.46 (t, J = 14.61 Hz, 1H), 3.87 (s, 3H), 3.11 (d, J = 7.68 Hz, 2H); ESI-MS: m/z [M+Na]+ = 401.1. 1-(9H-fluoren-9-yl)methyl 4-benzyl 2-(4-methoxyphenylsulfonyl)succinate (24): Acid 23 (11.1 g, 29.4 mmol) was coupled with Fmoc-OH (4.35 g, 29.4 mmol), using DCC (7.28 g, 35.3 mmol) and DMAP (0.43 g, 3.53 mmol) following procedures described previously. Upon column purification (10 - 25% EA/hexane), the desired product 24 was isolated (15.5 g, 27.9 mmol, 95%).1H-NMR (300 MHz, CDCl3) δ 7.77 (q, 4H), 7.60 (d, J = 7.28 Hz, 2H), 7.52 (d, J = 7.28 Hz, 2H), 7.42 ( t, J = 12.28 Hz, 2H), 7.30 (m, 7H), 6.96 (d, J = 8.73 Hz, 2H), 5.11 (q, 2H), 4.56 (t, J = 14.97 Hz, 1H), 4.38 (q, 1H), 4.23 (q, 1H), 4.10 (q, 1H), 3.81 (s, 3H), 3.19 (d, J = 7.32 Hz, 2H); ESI-MS: m/z [M+Na]+= 579.2. 4-((9H-fluoren-9-yl)methoxy)-3-(4-methoxyphenylsulfonyl)-4-oxobutanoic acid (25): Hydrogenolysis of 24 (15.5 g, 27.9 mmol) in THF (140 ml) with Pd/C (1.55 g) was accomplished using procedures described for the synthesis of 13. Upon column purification (5-10% DCM/hexane), the desired product 25 was isolated (12.2 g, 26.2 mmol, 94%).1H-NMR (300 MHz, CDCl3) δ 7.78 (q, 4H), 7.60 (d, J = 7.28 Hz, 1H), 7.52 (d, J = 7.28 Hz, 1H), 7.42 (t, J = 12.28 Hz, 2H), 7.30 (m, 2H), 6.96 (d, J = 8.73 Hz, 2H), 4.50 (m, 2H), 4.25 (t, J = 17.76 Hz, 1H), 3.81 (s, 3H), 3.15 (d, 1H), 3.81 (s, J = 9.09 Hz, 2H); ESI-MS: m/z [M+Na]+ = 489.1. (9H-fluoren-9-yl)methyl 2-(4-methoxyphenylsulfonyl)-4-(trityloxyamino)butanoate (26): Acid 25 (12.2 g, 26.2 mmol) was coupled with trityl-protected hydroxylamine (8.65 g, 31.4 mmol) and DCC (6.48 g, 31.4 mmol) in DCM (130 ml) using procedure described previously. After column purification (20 – 30% EA/hexane), 26 (13.8 g, 19.1 mmol, 73%) was isolated as a white solid.1H-NMR (300 MHz, CDCl3) δ 7.78 (q, 4H), 7.60 (d, J = 7.28 Hz, 1H), 7.52 (d, J = 7.28 Hz, 1H), 7.35 (m, 20H), 6.93 (d, J = 9.06 Hz, 2H), 4.48-4.35 (m, 1H), 4.30-4.08 ( m, 3H), 3.83 (s, 3H), 2.50 (m, 1H), 2.08 (m, 1H); ESI-MS: m/z [M+Na]+ = 746.2. 2-(4-methoxyphenylsulfonyl)-4-oxo-4-(trityloxyamino)butanoic acid (6L): 49 26 (13.8 g, 19.1 mmol) was stirred in 20% piperidine (in DMF; 100ml) at room temperature for 15 min. The reaction mixture was concentrated in vacuo and purified by column chromatography (5-10% MeOH/DCM) to furnish 6L as an off-white solid (10.1 g, 18.5 mmol, 97%).1H-NMR (300 MHz, CDCl3) δ 7.72 (d, J = 8.34 Hz, 2H), 7.35-7.24 (m, 16H), 4.10-4.02 (m, 1H), 3.87 (s, 3H), 2.58-2.49 (m, 1H), 2.47-2.32 (m, 1H);13CNMR (75 MHz, CDCl3) δ 164.2, 146.8, 140.6, 131.4, 129.0 – 127.5 (m), 114.3, 65.7, 55.7, 45.8, 29.7. ESI-MS: m/z [M+Na]+ = 568.2. 5.4 Solid phase synthesis of 400 member library containing 20 amino acids in the P2’ and P3’ positions The construction of the 400-members MMP inhibitors library was achieved by standard Fmoc solid phase peptide synthesis, IRORI split-and-pool directed sorting technology (Scheme 5. 3). The synthesis involved the use of only 20 reaction bottles in three rounds of synthesis and sorting. Final products were released from the solid phase by standard TFA cleavage protocol. The average concentration of individual inhibitors in DMSO stock solution was adjusted to a uniform 230 μM (estimated using ACC dye conjugation as 1% additive in the final coupling step). O H2N H2N O O N H a NH H HN H H N H2N c b O O HN N H N H O H H N S NH H HN O S O d O H2N P3' HN P2' H N O O HN N H O O N H O HN H H N P3' e NH H H2N O H N O N H O HN O O HN N H O P3' O H N HN P2' O N H O HN O N H O NH H HN H H N S g O O HO N H O HN O P3' HN P2' H O H N O O N H HN O NH2 HN O H H N S O 50 NH H NH H S O f O HN H N S O Ph3CO O N H Scheme 9 Procedure for the synthesis of 400-member MMP inhibitors on solid-phase. Reagents and conditions: (a) i: Fmoc-Lys(Biotin)-OH, HOBt, HBTU, DIEA, 12 hrs; ii: 20% piperidine/DMF, 2 hrs (b) i: Fmoc-Gly-OH, HOBt, HBTU, DIEA, 6 hrs; ii: 20% piperidine/DMF, 2 hrs (c) i: Fmoc-Gly-OH, HOBt, HBTU, DIEA, 6 hrs; ii: 20% piperidine/DMF, 2 hrs (d) i: Fmoc-AA3-OH, HOBt, HBTU, DIEA, 8 hrs; ii: 20% piperidine/DMF, 2 hrs (e) i: Fmoc-AA2-OH, HOBt, HBTU, DIEA, 8 hrs; ii: 20% piperidine/DMF, 2 hrs (f) i: CPh3ONH-Suc(2-iBu)-OH, HOBt, HBTU, DIEA, 12 hrs; ii: 20% piperidine/DMF, 2 hrs (g) 95% TFA/ 5% TIS, 2 hrs 5.4.1. Synthesis of Fmoc-Lys(Biotin)-OH EDC, NHS S S DMF 85% HO O O HN H NH H HN H NH H HN H O O O O NH H Fmoc-Lys(NH2)-OH PH=8, Dioxane/water O S N H 82% N O FmocHN COOH O Scheme 10 Synthesis route of Fmoc-Lys(Biotin)-OH Biotin-NHS. To a solution of D-biotin (24 g, 100 mmol) in DMF was added Nhydroxysuccinimide (14 g, 120 mmol) and EDC (23 g, 120 mmol). The reaction was allowed to proceed overnight. The resulting mixture was dried in vacuo to remove DMF. The gel-like residue was recrystallized from EtOH/Acetic acid/H2O(95:1:4) to afford biotin-NHS as a white solid (29 g; 85% yield):1H-NMR (300 MHz, CD3OD) δ 6.40 (br s, 1H), 6.35 (br s, 1H), 4.33-4.29 (m, 1H), 4.17-4.13 (m, 1H), 3.12-3.08 (m, 1H), 2.86-2.81 (m, 5H) including 2.81 (s, 4H), 2.67 (t, J = 7.2 Hz, 2H), 2.61-2.59 (m, 1H), 1.70-1.24 (m, 6H); ESI-MS cald for C14H20N3O5S [M+H]+ 342.1, found 342.1. Fmoc-Lys(Biotin)-OH. To a solution of Fmoc-Lys(Boc)-OH (39.8 g, 85 mmol) in DCM (0.4 ml) was added TFA (80 ml). The reaction was stirred at room temperature for 1 hour and concentrated in vacuo. The oil residue was dissolved in 280 ml of 1:1 Dioxane/water co-solvent, and the pH of the solution was adjusted to 8~8.5 by using a 4 M NaOH solution at 0oC. Biotin-NHS (29 g, 85 mmol) and was subsequently added, and the reaction mixture was stirred at room temperature overnight. The resulting gelatinous solid formed was added ether, stirred for 5 minutes. The supernatant ether was decanted followed by addition of acetone and adjusting the pH of the resulting solution to 3 using 2 M HCl at 0oC. Finally, the solid was filtered, washed several times with MeOH to afford the pure Fmoc-K(Biotin)-OH as a white solid (41.5 g, 82%):1H-NMR (300 MHz, CD3OD) δ 7.89 (d, J = 7.6 Hz, 2H), 7.72 (d, J = 7.7 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H), 7.33 (t, J = 7.3 Hz, 2H), 6.41 (br s, 1H), 6.35 (br s, 1H), 4.31-4.22 (m, 4H), 4.13-4.09 (m, 1H), 3.91-3.84 (m, 1H), 3.10.-3.00 (m, 3H), 2.80 (dd, J = 4.9 Hz, J = 12.2 Hz, 1H), 2.56 51 (d, J = 12.5 Hz, 1H), 2.03 (t, J = 7.3 Hz, 2H), 1.61-1.29 (m, 12H); ESI-MS cald for C31H39N4O6S [M+H]+ 595.3, found 595.2. 5.4.2 Experimental details of the solid phase synthesis Synthesis of Fmoc-Lys (biotin)-OH resin. To a 1-litre glass bottle was added a solution of 20% piperidine in DMF and 12 g of Rink Amide AM resin (loading: 0.5 mmol/g). The mixture was shaken for 2 hours. The resin was filtered and washed with DMF (3 x), DCM (3 x) and DMF (3 x). The preactivated solution containing Fmoc-Lys(biotin)-OH (4.0 eq), HOBt (4.0 eq), DIC (4.0 eq), DIEA (4.0 eq) in DMF (400 ml) was added. The reaction was shaken overnight at room temperature. The resin was then washed with DMF (3 x), DCM (3 x) and DMF (3 x). Subsequently, Fmoc was removed following 20% piperidine treatment as described below. Loading GG linker. To a 1-litre bottle was added biotin-linked resin and a preactivated solution of Fmoc-Gly-OH (4.0 eq), HOBt (4.0 eq), HBTU (4.0 eq) and DIEA (8.0 eq) in 500 ml DMF. The reaction mixture was shaken for 6 hours and the resin was then washed with DMF, DCM, and DMF. Subsequently, Fmoc was deprotected by 20% piperidine in DMF for 2 hours. The procedures were repeated to load the second Fmoc-Gly-OH coupling. Loading of P2’, P3’ residues. To a 1-litre glass bottle was added a solution of 20% piperidine in DMF and 400 microreactors (each containing 30 mg of Lysin(biotin)-GGFmoc resin and an Rf tag). The mixture was shaken for 2 hours and then the microreactors were washed with DMF (3 x), DCM (3 x) and DMF (3 x). The microreactors were then distributed into twenty bottles and each bottle was subjected to a solution of a unique Fmoc-protected amino acid (4.0 eq), and HOBt (4.0 eq), HBTU (4.0 eq) and DIEA (8.0 eq) in DMF (50 ml). After shaken for 8 hours, the microreactors were washed with DMF (3 x 400 ml), DCM (3 x 400 ml) and DMF (3 x 400 ml). The Fmoc group was then deprotected by 20% piperidine, and the microreactors were washed with ’ DMF. To load the residue of P , repeat the above procedure. 2 Loading P1’ residue: succinic acid derivative (w/ 1% Fmoc-ACC). The 400 microreactors were added a preactived solution of succinic acid derivative (4.0 eq), Fmoc-ACC (0.04 eq), HOBt (4.0 eq), HBTU (4.0 eq) and DIEA (8.0 eq) in DMF. The reaction mixture was shaken overnight and then the microreactors were washed with DMF, DCM and DMF. The Fmoc group was removed and the microreactors were washed with DMF, MeOH and dried under vacuum. Cleavage from the solid support. Each dried microreactor was treated with an 1-ml solution containing TFA (95%), TIS (5%) and the mixture was shaken for 4 hours at rt. The cleavage solution was then transferred to 96-well plates and then concentrated in vacuo. Precipitation. To each vessel was added ether and the plate was kept in a -20oC freezer overnight. Removal of the ether layer after centrifugation for 10 minutes gave 400 peptide products. Then each peptide was dissolved in DMSO (0.5 ml). 52 Quantification. The final coupling step incorporated 1% coumarin to each of the small molecule inhibitors in order to facilitate a fluorescence-based approach for concentration determination. 10 μl of the peptide solutions were diluted into PBS buffer (pH 7.4) to a final volume of 200 μl. The solutions were transfer to 96-well microtitre plates scanned for fluorescence under the coumarin channel λex= 360 nm and λem = 455 nm using a TM SpectraMAX Gemini XS fluorescence plate reader (Molecular Devices, USA). A linear concentation gradient was also using unconjugated aminomethylcarboxycoumarin (ACC) and this standard was used to estimate concentrations of each library member. 5.4.3 Library characterization The purity of the final products was determined by Shimadzu automated RP-HPLC, with single major peaks obtained in most cases (214 nm). ESI-MS spectrometry was taken for selected samples to further confirm successful synthesis (Figure 5.3.3). Six samples were selected for Ki and IC50 determination as shown in Table 5.3.3. Figure 19 LC-MS profiles of the representative samples of the 400 MMPI library. 53 P2' - P3' IC50 (nM) Ki (nM) ESI-MS found (calculated) Leu-Phe 144.5 NDa 916.5 (917.1) Ser-Ser 176.7 104 830.2 (830.9) Ser-Tyr 107.3 ND 906.2 (907.0) Tyr-Gln 38.5 ND 947.3 (948.1) Tyr-Asn 33.1 ND 933.3 (934.1) Tyr-Lys 9.9 2.4 947.3 (948.1) GM6001 23.9 25.2 - a ND = Not determined Table 2 Ki/IC50 values of 6 selected inhibitors from the library together with commercial inhibitor GM6001 is tabulated together with results obtained from large-scale microarray and microplate screens. ESI-MS results are also displayed. 5.5 Solid phase synthesis of 1000 member library containing 10 amino acids in the P2’ and P3’ positions and 10 variations in the P1’ position The construction of 1000 member MMPI library was done by following the protocol as described in section 5.3, except minor change made in the final coupling step. Instead of using HOBt/HBTU/DIEA as coupling reagent, HATU/DIEA had been used to improve coupling efficiency and the purity of final product. 54 P3' H N H2N O O O N H O O O H2N O HN N H NH H HN H H N O d O HN N H N H O N H NH H HN H H N O H2N b c S H2N H N S S O a NH H HN H O O O Ph3CO e O OH Ph3CO N H O O OH Ph3CO N H 6A O OH N H O 6C 6D OCPh3 O O O H2N P3' HN P2' O H N O O O N H HN O Ph3CO O N H OH N H O 6E 6F S NHBoc O OH N H P1' O HN O P3' H N HN P2' Ph3CO O O 6I O O OO S O Ph3CO 6K OH N H O 6L O O HN N H O COOtBu OH N H 6J f O OH N H 6G O Ph3CO N H O O O Ph3CO N H O OH Ph3CO O NH H HN H H N Ph3CO OH N H Ph3CO N H O NH H HN H H N S O g O HO N H P1' O HN O P3' HN P2' O H N O O N H HN O NH2 NH H HN H O H N S O Scheme 11 Procedure for the synthesis of 400-member MMP inhibitors on solid-phase. Reagents and conditions: (a) i: Fmoc-Lys(Biotin)-OH, HOBt, HBTU, DIEA, 12 hrs; ii: 20% piperidine/DMF, 2 hrs (b) i: Fmoc-Gly-OH, HOBt, HBTU, DIEA, 6 hrs; ii: 20% piperidine/DMF, 2 hrs (c) i: Fmoc-Gly-OH, HOBt, HBTU, DIEA, 6 hrs; ii: 20% piperidine/DMF, 2 hrs (d) i: Fmoc-AA3-OH, HOBt, HBTU, DIEA, 8 hrs; ii: 20% piperidine/DMF, 2 hrs (e) i: Fmoc-AA2-OH, HOBt, HBTU, DIEA, 8 hrs; ii: 20% piperidine/DMF, 2 hrs (f) i: CPh3ONH-Suc(2-P1’)-OH, HATU, DIEA, 12 hrs; ii: 20% piperidine/DMF, 2 hrs (g) 95% TFA/ 5% TIS, 2 hrs 5.6 Rapid assembly of metalloprotease inhibitor using click chemistry Alkyne (A-H) synthesis 55 P1' O OH Ph3COHN O 6A-6H O HO N H H N propagyl amine DMF P1' H N O HATU, DIEA Ph3COHN O N H O AlkyneA-AlkyneG 27A-27H O HO O AlkyneA N H H N O HO O AlkyneB P1' H N O TFA/DCM HO N H H N O HO N H O AlkyneC H N O AlkyneD O O HO N H H N O AlkyneE O HO N H H N O HO O AlkyneF N H H N O HO O AlkyneG N H H N O AlkyneH Scheme 12 Synthesis route of alkyne building block General procedure for the coupling of 6A-6H with propargyl amine: To a solution of 6 (A to H) (5 mmol), HATU (6 mmol) and DIEA (6 mmol) in DMF (10 ml) was added propargyl amine (6 mmol) at room temperature under a nitrogen atmosphere. The mixture was stirred for 2-4 hrs. The solvent was removed in vacuo and the resulting oil residue was diluted with DCM and extracted with water. The combined DCM layers were dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (80% DCM/hexane – 10% MeOH/DCM) to afford 27 (A to H) in 80 – 91% yield, typically as a white or off-white solid. 2-isopropyl-N1-(prop-2-ynyl)-N4-(trityloxy)succinamide (27A): Yield = 88%.1H-NMR (300 MHz, DMSO-d6) δ 7.39 – 7.18 (m, 15H), 3.89 – 3.63 (m, 2H), 2.50 (s, 1H), 2.38 – 1.72 (m, 3H), 1.53 – 1.43 (m, 1H), 0.63 – 0.49 (m, 6H); ESI-MS: m/z [M+Na]+ = 477.3. 2-isobutyl-N1-(prop-2-ynyl)-N4-(trityloxy)succinamide (27B): Yield = 80%.1H-NMR (300 MHz, CDCl3) δ 7.43 – 7.38 (m, 15H), 3.85 – 3.69 (m, 2H), 2.80 (s, 1H), 2.45 – 2.31 (m, 1H), 2.23 – 2.08 (m, 2H), 1.42 – 1.28 (m, 3H), 0.84 – 0.68 (m, 6H); ESI-MS: m/z [M+Na]+ = 491.6. 2-sec-butyl-N1-(prop-2-ynyl)-N4-(trityloxy)succinamide (27C): Yield = 91%. 1H-NMR (300 MHz, CDCl3) δ 7.44 – 7.26 (m, 15H), 3.94 – 3.78 (m, 2H), 2.50 – 2.34 (m, 1H), 2.28 – 2.13 (m, 2H), 1.70 (s, 1H), 1.35 – 1.19 (m, 3H), 0.85 – 0.69 (m, 6H); ESI-MS: m/z [M+Na]+ = 491.5. 2-benzyl-N1-(prop-2-ynyl)-N4-(trityloxy)succinamide (27D): 56 Yield = 82%. 1H-NMR (300 MHz, CDCl3) δ 7.65 – 6.93 (m, 20H), 4.02 – 3.57 (m, 2H), 2.80 (s, 1H), 2.68 – 2.30 (m, 3H), 2.26 – 2.11 (m, 2H); ESI-MS: m/z [M+Na]+ = 525.4. 2-(3-phenylpropyl)-N1-(prop-2-ynyl)-N4-(trityloxy)succinamide (27E): Yield = 82%.1H-NMR (300 MHz, CDCl3) δ 7.45 – 6.99 (m, 20H), 3.90 – 3.73 (m, 2H), 2.79 (s, 1H), 2.52 – 2.36 (m, 5H), 1.49 – 1.32 (m, 4H); ESI-MS: m/z [M+Na]+ = 553.6. 2-cyclohexyl-N1-(prop-2-ynyl)-N4-(trityloxy)succinamide (27F): Yield = 83%. 1H-NMR (300 MHz, MeOD) δ 7.48 – 7.22 (m, 15H), 4.06 – 3.81 (m, 2H), 2.81 (s, 1H), 2.57 – 2.54 (m, 1H), 2.30 – 2.15 (m, 2H), 1.69 – 0.89 (m, 11H); ESI-MS: m/z [M+Na]+ = 517.4. 2-(cyclopentylmethyl)-N1-(prop-2-ynyl)-N4-(trityloxy)succinamide (27G): Yield = 85%.1H-NMR (300 MHz, CDCl3) δ 7.48 – 7.26 (m, 15H), 4.03 – 3.81 (m, 2H), 2.41 – 2.38 (m, 2H), 2.19 – 2.16 (m, 2H), 1.63 – 1.26 (m, 8H), 1.09 – 0.88 (m, 3H); ESI-MS: m/z [M+Na]+= 517.5. 2-(2-(benzyloxy)ethyl)-N1-(prop-2-ynyl)-N4-(trityloxy)succinamide (27H): Yield = 80%. 1H-NMR (300 MHz, CDCl3) δ 7.44 – 7.20 (m, 20H), 4.48 – 4.39 (m, 1H), 4.35 – 4.21 (m, 1H), 4.02 – 3.62 (m, 2H), 3.48 – 3.22 (m, 2H), 2.75-2.52 (m, 1H), 2.48-2.22 (m, 1H), 2.20 (s, 1H), 1.95-1.80 (m, 1H), 1.73-1.51 (m, 3H); ESI-MS: m/z [M+23]+ = 569.4. General procedure for the cleavage of trityl moiety in AlkyneA-AlkyneH To a solution of 27 (4 mmol) in DCM (8 ml) was added TFA (40 mmol) and TIS (8 μl). The mixture was stirred for 1 hr, following which the solvent was removed in vacuo. The crude product was purified by column chromatography (5% MeOH/DCM–10% MeOH/DCM). AlkyneA-AlkyneH was isolated in 40 – 55% yield. N4-hydroxy-2-isopropyl-N1-(prop-2-ynyl)succinamide (AlkyneA): Yield = 52%.1H-NMR (300 MHz, MeOD) δ 4.08 – 4.00 (m, 1H), 3.90 – 3.83 (m, 1H), 2.65 – 2.56 (m, 1H), 2.56 – 2.46 (m, 1H), 2.45 – 2.36 (m, 1H), 2.34 – 2.26 (m, 1H), 1.87 – 1.83 (m, 1H), 1.04 – 0.94 (m, 6H);13C-NMR (75 MHz, CDCl3) δ 177.2, 172.2, 81.3, 72.9, 37.3, 34.3, 32.9, 30.1, 21.5; HRMS cald for C10H16O3N2Na [M + Na]+ : 235.1053, found 235.1057. N4-hydroxy-2-isobutyl-N1-(prop-2-ynyl)succinamide (AlkyneB): Yield = 43%. 1H-NMR (800 MHz, MeOD) δ 4.06 – 3.96 (m, 1H), 3.93 – 3.87 (m, 1H), 2.86 – 2.76 (m, 1H), 2.64 – 2.53 (m, 1H), 2.39 – 2.29 (m, 1H), 2.23 – 2.13 (m, 1H), 1.61 – 1.56 (m, 2H), 1.26 – 1.16 (m, 1H), 0.98 – 0.89 (m, 6H); 13C-NMR (75 MHz, CDCl3) δ 177.9, 171.5, 81.3, 73.0, 43.4, 40.3, 30.3, 27.8, 24.4, 23.2; HRMS cald for C11H18O3N2Na [M + Na]+: 249.1220, found 249.1215. 57 2-sec-butyl-N4-hydroxy-N1-(prop-2-ynyl)succinamide (AlkyneC): Yield = 45%. 1H-NMR (800 MHz, MeOD) δ 4.07 – 3.98 (m, 1H), 3.97 – 3.82 (m, 1H), 2.72 – 2.62 (m, 1H), 2.61 – 2.53 (m, 1H), 2.45 – 2.35 (m, 1H), 2.31 – 2.21 (m, 1H), 1.71 – 1.61 (m, 1H), 1.52 – 1.43 (m, 1H), 1.24 – 1.14 (m, 1H), 0.99 – 0.89 (m, 6H); 13CNMR (75 MHz, CDCl3) δ 177.4, 172.4, 81.5, 72.9, 39.4, 36.4, 34.4, 30.2, 28.9, 17.0, 12.3; HRMS cald for C11H18O3N2Na [M + Na]+ : 249.1220, found 249.1221. 2-benzyl-N4-hydroxy-N1-(prop-2-ynyl)succinamide (AlkyneD): Yield = 48%. 1H-NMR (800 MHz, MeOD) δ 7.39 – 7.11 (m, 5H), 3.97 – 3.82 (m, 2H), 3.01 – 2.99 (m, 1H), 2.99 – 2.86 (m, 1H), 2.74 – 2.72 (m, 1H), 2.59 – 2.53 (m, 1H), 2.43 – 2.35 (m, 1H), 2.23 – 2.20 (m, 1H); 13C-NMR (75 MHz, CDCl3) δ 176.8, 171.5, 140.9, 131.0, 130.3, 128.4, 81.0, 73.0, 46.8, 40.3, 36.6, 30.2; HRMS cald for C14H16O3N2Na [M + Na]+ 283.1053, found 283.1061. N4-hydroxy-2-(3-phenylpropyl)-N1-(prop-2-ynyl)succinamide (AlkyneE): Yield = 41%. 1H-NMR (800 MHz, MeOD) δ 7.37 – 7.18 (m, 5H), 4.06 – 4.04 (m, 1H), 3.98 – 3.82 (m, 1H), 2.76 – 2.63 (m, 1H), 2.61 – 2.60 (m, 1H), 2.59 (s, 1H), 2.49 – 2.47, 2.19 – 2.16 (m, 1H), 2.40 – 2.31 (m, 1H), 1.66 – 1.51 (m, 3H), 1.50 – 1.47 (m, 1H); 13C-NMR (75 MHz, CDCl3) δ 177.6, 171.6, 144.2, 130.2, 127.6, 81.4, 73.0, 42.0, 40.0, 37.5, 33.7, 31.0, 30.3; HRMS cald for C16H19O3N2 [M-1]- : 287.1390, found 287.1389. 2-cyclohexyl-N4-hydroxy-N1-(prop-2-ynyl)succinamide (AlkyneF): Yield = 48%. 1H-NMR (800 MHz, MeOD) δ 4.08 – 3.97 (m, 1H), 3.86 – 3.80 (m, 1H), 2.56 (br s, 1H), 2.56 – 2.38 (m, 2H), 2.35 – 2.27 (m, 1H), 1.77 – 1.73 (m, 3H), 1.69 – 1.49 (m, 2H), 1.54 – 1.44 (m, 1H), 1.30 – 1.21 (m, 2H), 1.20 – 1.11 (m, 1H), 1.09 – 0.91 (m, 2H); 13C-NMR (75 MHz, CDCl3) δ 177.2, 172.2, 81.4, 72.9, 42.6, 37.2, 34.4, 32.6, 30.5, 28.3; HRMS cald for C13H20O3N2Na [M + Na]+ : 275.1366, found 275.1371. 2-(cyclopentylmethyl)-N4-hydroxy-N1-(prop-2-ynyl)succinamide (AlkyneG): Yield = 55%. 1H-NMR (800 MHz, MeOD) δ 4.03 – 4.01 (dd, J = 17.6 Hz, J = 2.4 Hz, 1H), 3.97 – 3.84 (m, 1H), 2.77 – 2.70, 2.59 – 2.56 (m, 1H), 2.56 (s, 1H), 2.44 – 2.42, 2.18 – 2.15 (m, 1H), 2.32 – 2.28 (m, 1H), 1.86 – 1.84 (m, 1H), 1.77 – 1.74 (m, 2H), 1.71 – 1.63 (m, 1H), 1.62 – 1.61 (m, 2H), 1.54 – 1.51 (m, 2H), 1.35 – 1.32 (m, 1H), 1.12 – 1.07 (m, 2H); 13C-NMR (75 MHz, CDCl3) δ 177.9, 174.1, 81.4, 72.9, 41.5, 40.4, 40.0, 34.9, 34.1, 30.3, 26.9; HRMS cald for C13H20O3N2Na [M + Na]+ : 275.1366, found 275.1362. 2-(2-(benzyloxy)ethyl)-N4-hydroxy-N1-(prop-2-ynyl)succinamide (AlkyneH): Yield = 52%. 1H-NMR (300 MHz, MeOD) δ 7.40 – 7.23 (m, 5H), 4.46 (s, 2H), 3.92 – 3.74 (m, 2H), 3.48 (br s, 1H), 3.02 – 2.84 (m, 1H), 2.57 (s, 1H), 2.40 – 2.25 (m, 2H), 1.38 – 1.30 (m, 2H); 13C-NMR (75 MHz, CDCl3) δ 177.3, 171.5, 140.5, 130.2, 129.8, 129.5, 81.4, 74.9, 73.2, 69.6, 41.9, 34.5, 30.4, 26.9; HRMS cald for C16H20O4N2Na [M + Na]+ : 327.1315, found 327.1316. 58 Characterizations of Side Reaction during TFA deprotection of warheads: A prominent side product was consistently observed in the last TFA cleavage step. Three of these side products were isolated, further characterized by NMR and confirmed to be the cyclic adducts, SP1, SP2 & SP3 (Figure 5.5). They are presumably generated from the acid-catalyzed cyclization reaction of the warheads as proposed in Figure 2. Nevertheless, moderate yields (40-55%) were routinely obtained in this step. O P1' O HO N H H2+ N TFA HO N O SP1 OOCF3C+H3N HO N O O - O HO N HO N + O O O P1' O SP2 SP3 Scheme 13 Side reaction in the last TFA cleavage step 3-cyclohexyl-1-hydroxypyrrolidine-2,5-dione (SP1): 1 H-NMR (300 MHz, CDCl3) δ 2.79-2.72 (m, 1H), 2.71-2.63 (m, 2H), 2.51-2.44 (dd, J=17.33 Hz, 3.20Hz, 1H), 1.97-1.85 (m, 1H), 1.83-1.62 (m, 4H), 1.55-1.45 (m, 1H), 1.351.02 (m, 5H); 3-benzyl-1-hydroxypyrrolidine-2,5-dione (SP2): 1 H-NMR (300 MHz, CDCl3) δ 7.33 – 7.21 (m, 3H), 7.17 – 7.15 (m, 2H), 3.26-3.18 (m, 1H), 3.17-3.10 (m, 1H), 2.87 – 2.79 (m, 1H), 2.71-2.62 (dd, J= 18.15 Hz, 8.46 Hz, 1H), 2.70 (s, 1H), 2.46-2.38 (dd, J= 18.17 Hz, 4.04Hz, 1H). 1-hydroxy-3-(3-phenylpropyl)pyrrolidine-2,5-dione (SP3): 1 H-NMR (300 MHz, CDCl3) δ 7.29-7.24 (m, 2H), 7.19-7.14 (m, 3H), 2.80-2.75 (m, 2H), 2.66-2.60 (m, 3H), 2.32-2.22 (m, 1H), 1.92-1.88 (m, 1H), 1.75-1.63 (m, 2H), 1.61-1.48 (m, 1H). Azide (Azide1 -Azide12) Synthesis Azide1-Azide7 were synthesized from the corresponding acid chlorides and amines via the bromides based on a recently reported procedure.44e Azide8-Azide12 were previously reported. 59 O acid chloride R RNH2 O NaN3/DMF Br N H R 50-60oC n O N3 O N3 N H N H O N3 O O H N O N3 O O NH H N H N N3 N3 O O NH F OH Azide4 O O N3 N3 Azide7 Azide6 Azide5 O HN N3 Azide3 Azide2 Azide1 O n O N H H N N3 Azide1-Azide12 n = 2, 3 or 4 O N H HN N3 HN N3 O O HN S N3 NH N O HO O HO O Azide8 Azide9 Azide10 O O Azide11 Azide12 Scheme 14 Structure and synthesis of 12 Azide-containing blocks 3-azido-N-(9H-fluoren-2-yl)propanamide (Azide1): Yield = 69%. 1H-NMR (300 MHz, CDCl3) δ 7.86 (br s, 1H), 7.70 (t, J = 7.40 Hz, 2H), 7.53 – 7.49 (m, 1H), 7.35 – 7.26 (m, 4H), 3.86 (s, 2H), 3.73 (t, J = 6.24 Hz, 2H), 2.61 (t, J = 6.33 Hz, 2H); ESI-MS: m/z [M+Na]+ = 301.3. 4-azido-N-(9H-fluoren-2-yl)butanamide (Azide2): Yield = 82%. 1H-NMR (300 MHz, CDCl3) δ 7.87 (br s, 1H), 7.73 – 7.68 (t, J = 6.72 Hz, 2H), 7.51 (d, J = 7.23 Hz, 1H), 7.38 – 7.25 (m, 4H), 3.87 (s, 2H), 3.44 (t, J = 6.42 Hz, 2H), 2.49 (t, J = 7.08 Hz, 2H), 2.06-2.01 (m, 2H); ESI-MS: m/z [M+Na]+ = 315.3. 5-azido-N-(9H-fluoren-2-yl)pentanamide (Azide3): Yield = 73%. 1H-NMR (300 MHz, CDCl3) δ 7.87 (br s, 1H), 7.73 – 7.68 (t, J = 7.23 Hz, 2H), 7.51 (d, J = 7.23 Hz, 1H), 7.38 – 7.27 (m, 4H), 3.87 (s, 2H), 3.34 (t, J = 6.59 Hz, 2H), 2.42 (t, J = 7.23 Hz), 1.89 – 1.80 (m, 2H), 1.75 – 1.68 (m, 2H); ESI-MS: m/z [M+23]+ = 329.3. methyl 2-(3-azidopropanamido)-3-(1H-indol-3-yl)propanoate (Azide4): 60 Yield = 75%. 1H-NMR (300 MHz, CDCl3) δ 8.27 (br s, 1H), 7.52 (d, J = 7.71 Hz, 1H), 7.34 (d, J = 7.74 Hz), 7.25 – 7.09 (m, 2H), 6.98 (s, 1H), 6.18 (br d, 1H), 4.99- 4.93 (m, 1H), 3.70 (s, 3H), 3.61 – 3.51 (m, 2H), 3.33 (t, J = 3.30 Hz, 2H), 2.37 – 2.32 (m, 2H); ESI-MS: m/z [M+1]+ = 316.3. methyl 2-(4-azidobutanamido)-3-(1H-indol-3-yl)propanoate (Azide5): Yield = 72%. 1H-NMR (300 MHz, CDCl3) δ 8.25 (br s, 1H), 7.53 (d, J=7.74 Hz, 1H), 7.36 (d, J=7.89 Hz, 1H), 7.23-7.10 (m, 2H), 6.97 (d, J=2.31 Hz, 1H), 4.98 – 4.92 (m, 1H), 3.71 (s, 3H), 3.32 (t, J = 5.51 Hz, 2H), 3.26 (t, J = 6.66 Hz, 2H), 2.21 (t, J = 7.23 Hz, 2H), 1.86 (quintet, J = 6.90 Hz); ESI-MS: m/z [M+1]+ = 330.1. methyl 2-(4-azidobutanamido)-3-(4-hydroxyphenyl)propanoate (Azide6): Yield = 75%. 1H-NMR (300 MHz, CDCl3) δ 6.96 – 6.93 (m, 2H), 6.76 – 6.71 (m, 2H), 6.06 (br s, 1H), 4.90 – 4.83 (m, 1H), 3.75 (s, 2H), 3.30 – 3.25 (m, 2H), 3.13 – 2.94 (m, 2H), 2.27 (t, J = 7.41 Hz, 2H), 1.87 (quintet, J = 6.86 Hz, 2H); ESI-MS: m/z [M+23]+ = 329.1. N-(4-fluorobenzyl)-3-azidopropanamide (Azide7): Yield = 75%.1H-NMR (300 MHz, CDCl3) δ 7.25 – 7.20 (m, 2H), 7.02 – 6.97 (t, J = 8.63 Hz, 2H), 6.39 (br s, 1H), 4.37 (d, J = 5.91 Hz, 2H), 3.60 (t, J = 6.41 Hz, 2H), 2.43 (t, J = 6.33 Hz, 2H); ESI-MS: m/z [M-1]- = 221.5. Procedure for Libary Assembly Using “Click Chemistry” O HO N H P1' H N O AlkyeneA-AlkyeneH O + R N H N3 CuSO4, Sodium ascorbate tBuOH/H20 n Azide1-Azide12 O HO N H P1' H N N N N O 96 member MMPI H N n O R Scheme 15 Assembling of MMPI library using “Click Chemistry” The 96-membered library of MMP inhibitors were assembled in situ in a 96-deep well microplate. Both the alkynes (5 mmol, 1 eq) and azides (1.2 eq) were dissolved in a minimal amount of DMSO before loading into each well in proportions. The azides were added to the reaction in slight excess so as to ensure that all alkynes were consumed completely.4 Subsequently, 1 ml of a mixture of tBuOH/H2O (1:1 volume ratio) was applied to each well. The microplate was shaken for a few minutes, followed by sequential addition of catalytic amounts of sodium ascorbate (~10%) and CuSO4 (~1%) into each well to initiate the “Click Chemistry” assembly, which was continued at room temperature for another 8-12 hrs with shaking. The assembled products were used directly for subsequent LC-MS analysis and enzymatic assays without any further purification. LC-MS analysis of all 96 in situ-assembled products indicated the complete consumption of the alkynes and quantitative formation of the correct triazole products in almost every case. The LC-MS profiles of representative examples were shown below. HPLC conditions: 5% A to 95% A in 20 min gradients. 61 m AU(x1,000) 254nm ,4nm (1.00) 2.5 O 2.0 HO 1.5 N N N H N N H O H N C25H33N7O6 Exact Mass: 527.25 ESI-MS:[M+1]+ = 528 O O O A4 1.0 N H 0.5 0.0 0.0 2.5 5.0 7.5 1 0.0 12.5 15.0 17.5 m AU(x100) 5.0 254nm ,4nm (1.00) O 4.0 HO 3.0 N N N H N N H H N O O C22H32N6O5 Exact Mass: 460.24 ESI-MS:[M+1]+ = 461 O 2.0 A10 1.0 0.0 0.0 2.5 5.0 7.5 1 0.0 12.5 15 .0 17.5 15.0 17.5 m AU(x1,000) 2 54nm ,4nm (1 .00) 1.5 O HO 1.0 N N N H N N H C19H26N6O4 Exact Mass: 402.2 ESI-MS:[M+1]+ = 403 O O 0.5 H N A12 0.0 0.0 2.5 5.0 7.5 10.0 12.5 m AU(x100) 254nm ,4nm (1.00) O 5.0 HO H N N H N N N H N C26H38N6O7 Exact Mass: 546.28 ESI-MS:[M+1]+ = 547 O O O 2.5 O C8 OH 0.0 0.0 2.5 5.0 7.5 1 0.0 12.5 15 .0 17.5 15.0 1 7.5 m AU(x100) 254nm ,4n m (1.00) 5.0 O 4.0 HO 3.0 2.0 N H H N O N N N H N O C9 N S C19H29N7O4S Exact Mass: 451.2 ESI-MS:[M+1]+ = 452 1.0 0.0 0.0 2.5 5.0 7.5 10.0 62 12.5 m AU(x100) 254nm ,4nm (1 .00) H N 7.5 O 5.0 HO 2.5 N H C30H35N7O6 Exact Mass: 589.26 ESI-MS:[M+1]+ = 590 O N N N H N Stereoisomers (identical MS) N H O O O D5 0.0 0.0 2.5 5.0 7.5 1 0.0 12.5 15.0 17.5 m AU(x1,000) 254nm ,4n m (1.00) 1.5 O 1.0 HO 0.5 N H O C33H36N6O4 Exact Mass: 580.28 ESI-MS:[M+1]+ = 581 O N N N H N N H E2 0.0 0.0 2.5 5.0 7.5 1 0.0 12.5 15.0 17.5 15.0 17.5 12.5 15.0 17.5 12 .5 15.0 17.5 m AU(x100) 254nm ,4nm (1 .00) 4.0 C25H29FN6O4 Exact Mass: 496.22 ESI-MS:[M+1]+ = 497 3.0 O 2.0 HO 1.0 N N N H N N H O H N O E7 F 0.0 0.0 2.5 5.0 7.5 10.0 m AU(x10 0) 2.0 254nm ,4nm (1.00) OH 1.5 O HO 1.0 12.5 N H O N H O 0.5 C27H38N6O7 Exact Mass: 558.28 ESI-MS:[M+1]+ = 559 O N N N H N O F6 0.0 0.0 2 .5 5.0 7.5 10.0 m AU(x100) 6.0 254nm ,4nm (1 .00) 5.0 O 4.0 HO 3.0 N H H N N N N H N O 2.0 O O F11 O C27H38N6O7 Exact Mass: 558.28 Mol. Wt.: 558.63 OH 1.0 0.0 0.0 2.5 5.0 7.5 10.0 63 10.0 m AU(x100) 254nm ,4nm (1.00) H N 7.5 O 5.0 HO 2.5 H N N H N N N O C29H39N7O6 Exact Mass: 581.3 ESI-MS:[M+1]+ = 582 O O N H O G5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 15 .0 17.5 15.0 17.5 15 .0 17.5 m AU(x100) 254nm ,4nm (1 .00) 7.5 O O 5.0 HO H N N H 2.5 N N N C32H39N7O7 Exact Mass: 633.29 ESI-MS:[M+1]+ = 634 O N H O H2 0.0 0.0 7.5 2 .5 5.0 7.5 10.0 12.5 m AU(x100) 254nm ,4n m (1.00) O 5.0 O HO 2.5 N N N H N N H C34H38N6O5 Exact Mass: 610.29 ESI-MS:[M+1]+ = 611 H N O O H3 0.0 0.0 2.5 5.0 7.5 10.0 12.5 m AU(x100) 2 54nm ,4nm (1.00) 4.0 O 3.0 O HO 2.0 1.0 N H H N O F N N N H N C26H31FN6O5 Exact Mass: 526.23 ESI-MS:[M+1]+ = 527 O H7 0.0 0.0 2.5 5.0 7.5 10.0 12.5 Figure 20 LC-MS profiles of representative samples (Crude) from the 96 member MMPI library 64 5.7 Rapid Assembly of Metalloprotease Probes Using Click Chemistry 5.7.1 Synthesis of azide 32 O O O O H2N O 1. (Boc)2O, NaOH OH BocHN H2 N O 2. EDC, DIEA, NHS 28 N (Boc)2O H2N NH2 O BocHN BocHN 33 TFA/DCM BocHN NH2 OOCF3C+H3N N3 N3 (2) TFA/DCM 36 (1) HBTU, DIEA 35 34 O 30 - NaOH OH N H DIEA O 29 OH O O O N O O O O TMR O H N 32 N H H N HBTU, DIEA O - OOCF3C+H3N N3 O N H H N N3 O 31 N+ Scheme 16 Synthesis route of Azide 32 NHS ester (29): To a solution of 6-aminohexanoic acid 28 (7.87 g, 60 mmol) and NaOH (2 M, 50 ml) in dioxane/water (2/1; 180 ml) at 0 °C was added (Boc)2O (15.7 g, 72 mmol). The reaction was allowed to proceed at room temperature overnight. Subsequently, dioxane was removed under reduced pressure and the resulting mixture was acidified to pH 2 with 1 M HCl, followed by extraction with EA (3 x 80 ml). The combined organic layers were dried over Na2SO4, filtered and concentrated to furnish colorless oil, which was subsequently reacted with NHS (8.06 g, 70 mmol) and EDC (13.42 g, 70 mmol) in DMF at room temperature overnight. Upon column purification (20-40% EA/hexane), the desired product 29 was obtained as colorless oil (13.8 g, 42.0 mmol, 70%). 1H-NMR (300 MHz, CDCl3) δ 4.60 (br s, 1H), 3.16 – 3.05 (m, 2H), 2.82 (s, 4H), 2.60 (t, J = 7.49 Hz, 2H), 1.81 – 1.70 (m, 2H), 1.51 – 1.40 (m, 13H); ESI-MS: m/z [M+Na]+= 351.3. 3-(4-benzoylphenyl)-2-(6-(tert-butoxycarbonyl)hexanamido)propanoic acid (30): 29 (1.64 g, 5 mmol) dissolved in 15ml DMF was added H-p-Bz-Phe-OH (1.35 g, 5 mmol) and DIEA (1.05 ml, 6 mmol). The reaction mixture was stirred under N2 overnight. After that, DMF was removed in vacuo. The residue was taken into EA and washed with 1 M HCl. The EA layer was dried over Na2SO4, filtered and concentrated. Upon column chromatography (5-10% MeOH/DCM), the final product 30 was obtained as colorless oil (2.05 g, 4.25 mmol, 85%). 1H-NMR (300 MHz, CDCl3) δ 8.08 (s, 1H), 7.74 – 7.66 (m, 4H), 7.63 – 7.56 (m, 1H), 7.49 – 7.44 (m, 2H), 7.31 – 7.26 (m, 2H), 4.83 (br s, 1H), 3.41 – 2.96 (m, 2H), 2.21 – 2.05 (m, 2H), 1.61 – 1.47 (m, 2H), 1.47 – 1.32 (m, 11H), 1.30 – 1.14 (m, 2H); ESI-MS: m/z [M-1]- = 481.5. tert-butyl 2-aminoethylcarbamate (34): 65 A solution of di-tert-butyl dicarbonate (6.1 g, 28 mmol) in DCM (400 ml) was added dropwise over 6 h to a solution of ethylenediamine (11.2 ml, 166.7 mmol) predissolved in DCM (50 ml) while maintaining vigorous stirring. The reaction was continued with stirring for another 24 h at room temperature. Upon concentration, the resulting oil was taken into aqueous sodium carbonate (600 ml) and extracted with dichloromethane (2 x 300 ml). The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to yield 5 as colourless oil (4.47 g, 98%). 1H-NMR (300 MHz, CDCl3) δ 5.07 (br s, 1H), 3.17 – 3.06 (m, 2H), 2.74 (t, J = 5.91 Hz, 2H), 1.39 (s, 9H); ESI-MS: m/z [M+1]+= 161.2. tert-butyl 2-azidoethylcarbamate (35): Sodium azide (2.25 g, 34.7 mmol) was dissolved in a mixture of H2O (5.7 ml) and CH2Cl2 (9.5 ml) at 0oC. Triflyl anhydride (1.18 ml, 7.04 mmol) was subsequently added dropwise to the solution. The reaction was continued for 2 h. The CH2Cl2 layer was removed and the aqueous portion was extracted with CH2Cl2 (2 x 4.75 ml). The combined organic fractions, which contain triflyl azide, were washed once with saturated Na2CO3 and directly added to a solution containing 34 (564 mg, 3.52 mmol), K2CO3 (731 mg, 5.3 mmol), CuSO4 pentahydrate (8.8 mg, 35.2 μmol), distilled H2O (11.4 ml) and CH3OH (22.7 ml). The resulting mixture was stirred at ambient temperature overnight. Subsequently, the organic solvents were removed under reduced pressure and the aqueous slurry was diluted with H2O (75 ml) and acidified to pH 2 with 6 N HCl. Following extraction with DCM (3 × 50 ml), the combined organic layers were washed with H2O, dried over MgSO4, and concentrated in vacuo. Upon column purification, the desired product 35 was isolated as colorless oil (590 mg, 3.17 mmol, 90%). 1H-NMR (300 MHz, CDCl3) δ 5.14 (br s, 1H), 3.22 – 3.11 (m, 2H), 2.79 (t, J = 5.91 Hz, 2H), 1.45 (s, 9H); ESI-MS: m/z [M+1]+= 187.1. 2-azidoethanamine (36): 35 was deprotected by TFA/DCM as described above to give 36 (97% yield). 1HNMR (300 MHz, MeOD) δ 3.67 (t, J = 5.75 Hz, 2H), 3.07 (t, J = 5.58 Hz, 2H); ESI-MS: m/z [M+1]+= 87.1. 6-amino-N-(1-(2-azidoethylamino)-3-(4-benzoylphenyl)-1-oxopropan-2-yl) hexanamide (31): To compound 30 (1.93 g, 4 mmol) dissolved in DMF was added HBTU (1.90 g, 5 mmol) and DIEA (0.87 ml, 5 mmol) at 0oC. The mixture was stirred for 10 min before addition of 36 (0.43 g, 5 mmol). The mixture was further agitated for another 12 h at room temperature, after which DMF was removed in vacuo and the residue was taken into ethyl acetate (50 ml). The organic layer was washed with saturated NaHCO3 (2 x 30 ml), 1 M HCl (2 x 30ml), brine (2 x 30 ml), dried over anhydrous Na2SO4, and concentrated under reduced pressure to afford a yellow oily product. Purification of this compound by flash chromatography (silica gel, ethyl acetate/hexane = 3:1) furnished the intermediate as a white solid (1.65g, 3mmol, 75%). 1H-NMR (300 MHz, CDCl3) δ 7.81 – 7.65 (m, 4H), 7.53 (t, J = 7.32 Hz, 1H), 7.41 (t, J = 7.50 Hz, 2H), 7.28 (m, 2H), 4.88 – 66 4.72 (m, 1H), 3.55 – 3.36 (m, 4H), 3.36 – 3.04 (m, 4H), 2.14 (t, J = 7.49 Hz, 2H), 1.62 – 1.48 (m, 2H), 1.47 – 1.31 (m, 11H), 1.27 – 1.22 (m, 2H); ESI-MS: m/z [M+23]+= 573.4. Deprotection of above intermediate (1.65 g, 3 mmol) with TFA (10 eq) in DCM gave 31 as yellow oil (1.35 g, 3 mmol, ~100%). 1H-NMR (300 MHz, MeOD) δ 7.91 – 7.75 (m, 4H), 7.66 (t, J = 7.32 Hz, 1H) 7.54 (t, J = 7.49 Hz, 2H), 7.44 (d, J = 8.37 Hz, 2H), 4.73 – 4.68 (m, 1H), 3.41 – 3.31 (m, 4H), 3.28 – 3.21 (m, 1H), 3.04 – 2.97 (m, 1H), 2.91 – 2.85 (m, 2H), 2.25 – 2.20 (m, 2H), 1.66 – 1.50 (m, 4H), 1.36 – 1.23 (m, 2H); ESIMS: m/z [M+1]+ = 451.4. Azide (32): Coupling of 31 with Rhodamine followed the same procedures as conversion 30 to 31. Column purification (5%-10%-15% MeOH/DCM) was carried out to isolate the pure product (50% yield). 1H-NMR (300 MHz, MeOD) δ 8.60 – 8.42 (m, 1H), 8.26 – 8.08 (m, 1H), 7.82 (s, 1H), 7.71 – 7.37 (m, 10H), 7.15 – 6.97 (m, 6H), 4.70 – 4.65 (m, 1H), 3.78 – 3.61 (m, 8H), 3.35 – 3.32 (m, 2H), 3.31 – 3.19, (m, 1H), 2.99 – 2.89 (m, 1H), 2.19 (t, J = 7.49 Hz, 2H), 1.63 – 1.46 (m, 4H), 1.39 – 1.20 (m, 14H); ESI-MS: m/z [M+1]+ = 920.2. 5.7.2 Construction of MMP probes (A-H) using “Click Chemistry” P1'= O 32 + HO P1' probeA H N probeB probeC N H probeD O O AlkyneA-AlkyneH CuSO4, Sodium ascorbate tBuOH/H20 probeE probeG probeF probeH O N+ O HO N H P1' O H N N N N O H N O H N N H O O O O N Second generation of MMP probeA-probeH Scheme 17 Construction of MMP probes library (A-H) using “Click Chemistry” Synthesis of the above 8 hydrophobic alkyne-containing warheads followed procedures similar to ones previously, and details will be reported elsewhere. As shown in Figure 5.7.2, the alkyne (24 μmol, 1.2 eq) and the azide 32 (20 μmol, 1 eq) were dissolved in a minimal amount of DMSO. A mixture of tBuOH/H2O solution (1:1; 1 ml) 67 was subsequently added and the reaction was shaken for a few minutes to obtain a clear solution. The “click chemistry” was initiated by sequential addition of catalytic amounts of sodium ascorbate (0.1 eq) and CuSO4 (0.01 eq). The reaction was continued with shaking at room temperature for another 12 hrs. Upon further dilution with DMSO (1 ml), the reaction product was directly injected on LC-MS; results indicated the complete consumption of the azide and quantitative formation of the triazole final product in all cases. The final probes (probeA-probeH) were subsequently purified by semi-prep reverse phase HPLC and characterized by MS. Semi-prep HPLC conditions: 30% to 80% A in 60 min gradients. 5.7.3 Synthesis of probes (I-L): The synthesis of probes (probeI-probeL) following scheme shown in Figure 5.7.2 was problematic, due to the polar nature of the unprotected side chains which rendered the warheads difficult to purify before the “Click Chemistry” step. Consequently, the scheme below (Figure 5.7.3) was used to synthesize the probes instead. Briefly, warheads 6I-6L were converted to the corresponding alkynes, 6i-6l. Subsequently, “Click Chemistry” was carried out with the protected warheads, followed by direct TFA treatment to remove the protecting groups. Upon HPLC purification, the final probes (probeI-probeL) were obtained in pure form and characterized by LC-MS. O O Ph3CO N+ O Ph3CO P1'PG H N N H N N N H N N H O O HATU, DIEA DMF 32 O H N O CuSO4, Sodium ascorbate tBuOH/H20 O O P1' N H 6I-6L propagyl amine O Ph3CO O P1' N H N OH O H N O 6i-6l TFA/TIS/DCM OH NH2 P1'= O O HO N H P1' O H N N N N O H N O N H N+ probeI O O probeJ O H N O OH O O S O O N probeK Second generation of MMP probeI-probeL probeL Scheme 18 Construction of MMP probes library (I-L) using “Click Chemistry” General procedure for the coupling of 6I-6L with propargyl amine: To a solution of acid (6I to 6L) (5 mmol), HATU (6 mmol) and DIEA (6 mmol) in DMF (10 ml) was added propargyl amine (6 mmol) at room temperature under a nitrogen atmosphere. The mixture was stirred for 2-4 hrs. The solvent was removed in vacuo and the resulting oil residue was diluted with DCM and extracted with water. The combined DCM layers were dried over Na2SO4, filtered and concentrated. The crude product was 68 purified by column chromatography (80% DCM/hexane – 10% MeOH/DCM) to afford the alkyne (6i to 6l) in 75 – 90% yield, typically as a white or off-white solid. 1 4 N -(prop-2-ynyl)-N -(trityloxy)-2-(3-(trityloxy)propyl)succinamide (6i) Yield = 85%. 1H-NMR (300 MHz, MeOD) δ 7.42-7.27 (m, 30H), 3.99-3.78 (m, 2H), 3.12-2.95 (m, 2H), 2.89 (s, 1H), 2.62-2.52 (m, 1H), 2.50-2.38 (m, 1H), 2.25-2.10 (m, 1H), 1.50-1.21 (m, 4H); ESI-MS: m/z [M+23]+= 736.1. tert-butyl 7-oxo-5-(prop-2-ynylcarbamoyl)-7-(trityloxyamino) heptylcarbamate (6j) Yield = 80%. 1H-NMR (300 MHz, MeOD) δ 7.46-7.33 (m, 15H), 4.00-3.75 (m, 2H), 3.09-2.92 (m, 2H), 2.62-2.58 (m, 1H), 2.52-2.45 (m, 1H), 2.25-2.11 (m, 1H), 2.092.02 (m, 1H), 1.50 (s, 9H), 1.48-1.25 (m, 4H), 1.25-1.15 (m, 4H); ESI-MS: m/z [M+23]+ = 606.3. tert-butyl 5-oxo-3-(prop-2-ynylcarbamoyl)-5-(trityloxyamino) pentanoate (6k) Yield = 90%. 1H-NMR (300 MHz, MeOD) δ 7.43-7.26 (m, 15H), 3.99-3.57 (m, 4H), 2.80 (s, 1H), 2.48-2.32 (m, 1H), 2.30-2.15 (m, 2H), 1.42 (s, 9H); ESI-MS: m/z [M+23]+= 549.3. 1 4 2-(4-methoxyphenylsulfonyl)-N -(prop-2-ynyl)-N -(trityloxy)succinamide (6l) Yield = 75%. 1H-NMR (300 MHz, MeOD) δ 7.42-7.22 (m, 19H), 4.04-3.62 (m, 4H), 3.05 (s, 3H), 2.35-2.22 (m, 1H), 2.18 (s, 1H), 2.12-1.90 (m, 2H); ESI-MS: m/z [M+23]+= 605.2. “Click Chemistry” followed by TFA deprotection The alkyne 6i-6l (24 μmol, 1.2 eq) and the azide 32 (20 μmol, 1 eq) were dissolved in a minimal amount of DMSO. A mixture of tBuOH/H2O solution (1:1; 1 ml) was subsequently added and the reaction was shaken for a few minutes to obtain a clear solution. The “click chemistry” was initiated by sequential addition of catalytic amounts of sodium ascorbate (0.1 eq) and CuSO4 (0.01 eq). The reaction was continued with shaking at room temperature for another 12 hrs. Upon further dilution with DMSO (1 ml), the reaction product was directly injected on LC-MS; results indicated the complete consumption of the azide and quantitative formation of the triazole final product in all cases. Subsequently, TFA (4 ml) and TIS (200 μl) were added to the reaction mixtures. The mixture was stirred for 1 hr, following which the solvent was removed in vacuo. The crude product was directly purified by semi-prep RP-HPLC (conditions: 30% to 100% A in 60 min). 5.7.4. LC-MS characterization of the final probes (A-L): Due to the presence of two isomeric forms of the Rhodamine dye used (e.g. 5-and 6-Tetraethylrhodamine) in the synthesis, both isomers of all 12 final probes were produced as a result (Figure 20). They were unambiguously confirmed by LC-MS profiles which indicate, in every final probe, two peaks with equal intensity and identical molecular weight, but differing slightly in the retention time, were produced. We were able to isolate both isomeric forms in all but one case (probeA-probeL). 69 O 5- or 6- isomer O HO N H P1' H N N N N O H N O O H N N H N+ O O O O N Figure 21 Two isomers of the second generation MMP probes ProbeA: LC conditions: 30%-80% A in 20 min mAU (x100) 254nm,4nm (1.00) 1.25 1.00 P1'= 0.75 0.50 Isomer 1 C63H75N10O10Exact Mass: 1131.57 ESI-MS: [M+1]+ = 1132.3 0.25 0.00 -0.25 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 12.5 15.0 17.5 min mAU (x100) 254nm,4nm (1.00) 2.0 1.5 1.0 C63H75N10O10Exact Mass: 1131.57 ESI-MS: [M+1]+ = 1132.3 P1'= Isomer 2 0.5 0.0 0.0 2.5 5.0 7.5 10.0 ProbeB: LC conditions: 30%-80% A in 20 min 70 mAU (x1,000) 254nm,4nm (1.00) C64H77N10O10Exact Mass: 1145.58 ESI-MS: [M+1]+ = 1146.6 3.0 P1'= 2.0 Isomer 1 1.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min mAU (x1,000) 254nm,4nm (1.00) 3.0 C64H77N10O10Exact Mass: 1145.58 ESI-MS: [M+1]+ = 1146.6 2.0 P1'= 1.0 Isomer 2 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min ProbeC: LC conditions: 30%-80% A in 20 min mAU (x1,000) 254nm,4nm (1.00) 1.5 C64H77N10O10Exact Mass: 1145.58 ESI-MS: [M+1]+ = 1146.6 P1'= 1.0 Isomer 1 0.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min mAU (x1,000) 254nm,4nm (1.00) 1.5 1.0 C64H77N10O10Exact Mass: 1145.58 ESI-MS: [M+1]+ = 1146.6 P1'= 0.5 Isomer 2 0.0 0.0 2.5 5.0 7.5 10.0 71 12.5 15.0 17.5 min ProbeD: LC conditions: 30%-80% A in 20 min mAU (x100) 2.0 254nm,4nm (1.00) 1.5 C67H74N10O10+ Exact Mass: 1178.56 ESI-MS: [M+1]+ = 1179 P1'= 1.0 Isomer 1 0.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 12.5 15.0 17.5 min 12.5 15.0 17.5 min mAU (x100) 254nm,4nm (1.00) 4.0 C67H74N10O10+ Exact Mass: 1178.56 ESI-MS: [M+1]+ = 1179 3.0 P1'= 2.0 Isomer 2 1.0 0.0 0.0 2.5 5.0 7.5 10.0 ProbeE: LC conditions: 30%-80% A in 20 min mAU (x100) 254nm,4nm (1.00) 1.25 1.00 C69H78N10O10+ Exact Mass: 1206.59 ESI-MS: [M+1]+ = 1207 0.75 P1'= 0.50 0.25 Isomer 1 0.00 -0.25 0.0 2.5 5.0 7.5 10.0 72 mAU (x100) 254nm,4nm (1.00) 2.5 2.0 C69H78N10O10+ Exact Mass: 1206.59 ESI-MS: [M+1]+ = 1207 1.5 1.0 0.5 P1'= Isomer 2 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 12.5 15.0 17.5 min 12.5 15.0 17.5 min ProbeF: LC conditions: 30%-80% A in 20 min mAU (x100) 2.5 254nm,4nm (1.00) 2.0 1.5 1.0 C66H78N10O10+ Exact Mass: 1170.59 ESI-MS: [M+1]+ = 1172 P1'= Isomer 1 0.5 0.0 0.0 2.5 5.0 7.5 10.0 mAU (x100) 4.0 254nm,4nm (1.00) 3.0 C66H78N10O10+ Exact Mass: 1170.59 ESI-MS: [M+1]+ = 1172 P1'= 2.0 Isomer 2 1.0 0.0 0.0 2.5 5.0 7.5 10.0 ProbeG: LC conditions: 30%-80% A in 20 min 73 mAU (x1,000) 254nm,4nm (1.00) 1.5 1.0 C66H79N10O10Exact Mass: 1171.6 ESI-MS: [M+1]+ = 1172.6 P1'= Isomer 1 0.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min mAU (x1,000) 254nm,4nm (1.00) 3.0 2.0 C66H79N10O10Exact Mass: 1171.6 ESI-MS: [M+1]+ = 1172.6 P1'= 1.0 Isomer 2 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min ProbeH: LC conditions: 30%-0% A in 20 min mAU (x1,000) 254nm,4nm (1.00) 3.0 C69H79N10O11Exact Mass: 1223.59 ESI-MS: [M+1]+ = 1224 Isomer 1 2.0 O 1.0 P1'= 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min mAU (x1,000) 254nm,4nm (1.00) 1.5 C69H79N10O11Exact Mass: 1223.59 ESI-MS: [M+1]+ = 1224 1.0 O Isomer 2 0.5 P1'= 0.0 0.0 2.5 5.0 7.5 10.0 ProbeI: 74 12.5 15.0 17.5 min LC conditions: 30%-100% A in 20 min mAU (x1,000) 254nm,4nm (1.00) 3.0 OH 2.0 Isomer 1 Isomer 2 C63H75N10O11Exact Mass: 1147.56 ESI-MS: [M+1]+ = 1148 P1'= 1.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min *We did not manage to isolate the two isomeric forms of probe I by semi-prep HPLC, as shown above. ProbeJ: LC conditions: 30%-100% A in 20 min mAU (x100) 254nm,4nm (1.00) 7.5 NH2 5.0 C64H78N11O10Exact Mass: 1160.59 ESI-MS: [M+1]+ = 1161 P1'= Isomer 1 2.5 0.0 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 14.0 15.0 16.0 17.0 18.0 19.0 min mAU (x1,000) 254nm,4nm (1.00) 1.5 NH2 1.0 C64H78N11O10Exact Mass: 1160.59 ESI-MS: [M+1]+ = 1161 P1'= 0.5 Isomer 2 0.0 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 14.0 15.0 16.0 17.0 18.0 19.0 min ProbeK: 75 LC conditions: 30%-100% A in 20 min mAU (x1,000) 1.5254nm,4nm (1.00) O C62H71N10O12Exact Mass: 1147.53 ESI-MS: [M+1]+ = 1148 1.0 OH P1'= 0.5 Isomer 1 0.0 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 14.0 15.0 16.0 17.0 18.0 19.0 min mAU (x1,000) 254nm,4nm (1.00) 1.5 O C62H71N10O12Exact Mass: 1147.53 ESI-MS: [M+1]+ = 1148 1.0 OH P1'= 0.5 Isomer 2 0.0 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 14.0 15.0 16.0 17.0 18.0 19.0 min ProbeL: LC conditions: 30%-100% A in 20 min mAU (x1,000) 254nm,4nm (1.00) 3.0 O 2.5 2.0 C67H75N10O13SExact Mass: 1259.52 ESI-MS: [M+1]+ = 1260 P1'= O 1.5 S O 1.0 Isomer 1 0.5 0.0 -0.5 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 14.0 15.0 16.0 17.0 18.0 19.0 min 76 mAU (x1,000) 254nm,4nm (1.00) 3.0 O 2.0 C67H75N10O13SExact Mass: 1259.52 ESI-MS: [M+1]+ = 1260 P1'= O S O 1.0 Isomer 2 0.0 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 14.0 15.0 16.0 17.0 18.0 19.0 min 77 Chapter 6 References 1 . 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Chem. 1995, 38, 2570-2581. 83 Chapter 7 Appendices 84 85 86 87 88 89 90 91 92 93 94 95 [...]... route of constructing non-peptide based MMPI library 25 using click chemistry Scheme 4 Mechanism of acetals as latent electrophiles that interact with 34 catalytic nucleophile at the active site of matrix metalloproteases Scheme 5 Synthesis route of hydrophobic MMPI warheads 36 Scheme 6 Synthesis route of warhead 6I and 6J 43 Scheme 7 Synthesis route of warhead 6K 46 Scheme 8 Synthesis route of MMPI... fingerprints of a variety of metalloproteases, including matrix metalloproteases (MMPs), in proteomics experiments xvi Chapter 1 Introduction 1.1 Metalloproteases Metalloproteases, together with serine, cysteine and aspartic proteases, represent the four major classes of proteolytic enzymes which mediate the hydrolysis of the amide bond These proteases have been found to be involved in a variety of cell functions... Figure 19 Structure of proposed activity-based MMP probes 53 Figure 20 LC-MS profiles of representative samples (Crude) from the 96 member 64 MMPI library Figure 21 Two isomers of the second generation MMP probes ix 70 LIST OF SCHEMES Scheme 1 Procedure for the synthesis of 1400-member MMP inhibitors 18 on solid-phase Scheme 2 Nanodroplet SMM strategy for high-throughput profiling of 19 potential MMP... Deregulations of protease activities are known to cause many diseases such as cancer, HIV, malaria, Alzheimer’s diseases Overall proteases represent 5-10% of the potential drug targets 1 Matrix metalloproteinases (MMPs) belong to a family of homologous zinc endopeptidases that are capable of hydrolyzing all known constituents of the extracellular matrix (ECM) 2 There are currently at least 23 members of human... of novel chemical and biological methods capable of high-throughput identification and characterization of MMPs has become increasingly urgent.2a 1.2 Matrix Metalloprotease inhibition Two general methods have been applied to the identification of matrix metalloprotease inhibitors: one method is the substrate-based design of pseudopeptide 2 derivatives; another approach is the random screening of nature... combinations of all the 20 natural amino acids.22 By printing pre-incubated nanodroplets of enzyme-inhibitor mixes onto a protease-sensitive glass surface, we obtained the inhibitor fingerprint profiles for thermolysin in the terms of fluorescence intensity of the spots Overall this strategy offers not only a rapid method 9 for inhibitor profiling and discovery, but also a viable method for the chemical. .. methods of high throughput proteome profiling like two-dimensional gel electrophoresis (2D-GE) only allow us to detect the abundance of the protein, while revealing no information of the activity of the protein 29 The activity-based proteome profiling approach which was initially developed by Cravett et al 30 uses active site-directed, small molecule probes that chemically react with certain classes of. .. successfully applied to profile all the four major classes of protease, such as aspartic, 31a serine, 30 cysteine proteases 32b, 32c and metalloproteases. 32d, 32e It is necessary to mention that in the case of profiling enzymes whose hydrolytic mechanism does not involve any covalent intermediates, such as metalloproteases, the affinity-based strategy is applied For affinity based protein profiling as shown... visualization of a particular matrix 11 metalloprotease within specific organelles The results from this study will help us better understanding the relationship between the disease stages and the proteolytic activity of matrix metalloproteases Specific targeting of affinity-based MMP probes to a predefined organelle could be achieved by highly orthogonal binding pairs, such as the DHFR (dihydrofolate reductase... GM6001, three broad-spectrum hydroxamate inhibitors of matrix metalloproteases (Figure 9) It is noted that Overkleeft and coworkers independently reported a similar method based on the synthesis of N-Boc-OTBS-hydroxamates which have also been used for the solid phase synthesis of succinyl hydroxamates 37 P1' O OH Ph3COHN O Figure 8 Chemical structure of MMPI warhead used for solid phase synthesis O HO .. .CHEMICAL BIOLOGY OF MATRIX METALLOPROTEASES Wang Jun Under the supervision of Associate Professor Yao Shao Qin A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY... the facile synthesis of various affinity-based hydroxamate probes that enables the generation of activity-based fingerprints of a variety of metalloproteases, including matrix metalloproteases (MMPs),... HNMR and CNMR of Alkyne A 86 7.4 HNMR and CNMR of Alkyne B 87 7.4 HNMR and CNMR of Alkyne C 88 7.4 HNMR and CNMR of Alkyne D 89 v 7.4 HNMR and CNMR of Alkyne E 90 7.4 HNMR and CNMR of Alkyne F