ABSTRACT Title of dissertation: THE SYNTHESIS OF A DIVERSE LIBRARY OF AI-2 ANALOGS TO INVESTIGATE BACTERIAL QUORUM SENSING Jacqueline A.I. Smith, Doctor of Philosophy, 2011 Directed by: Assistant Professor Herman O. Sintim, Department of Chemistry & Biochemistry Bacteria have evolved several mechanisms to promote their survival, which sometimes come at the cost of human health. They use toxins known as virulence factors to cause the symptoms associated with infections. They also form communities called biofilm, which allow them to thrive and resist attacks by the host?s immune system. Conventional antibiotics fail to penetrate the biofilm matrix. The expression of virulence factors and formation of biofilm are both regulated by a phenomenon known as quorum sensing. Quorum sensing is a form of cell-to-cell communication, which allows bacteria to coordinate gene expression via the secretion of signaling molecules, known as autoinducers, and the subsequent detection of these molecules. The ultimate goal of this dissertation was to identify new small molecules that would be used to disrupt quorum sensing in bacteria. AI-2, which is a universal quorum sensing autoinducer, found in over 60 bacterial species, was targeted. In this study a new facile synthesis of AI-2 was achieved and this new methodology was adapted to the synthesis of a library of analogs. These analogs were screened for their ability to modulate AI-2 mediated quorum sensing in Vibrio harveyi, Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. It was found that AI-2 analogs were able to cause synergistic agonism of bioluminescence in V. harveyi. Furthermore, several analogs were able to repress quorum sensing in E. coli yet very few analogs were active in the homologous quorum sensing system of S. typhimurium. These analogs were processed by the AI-2 processing enzymes in E. coli. Finally some AI-2 analogs were found to inhibit quorum sensing in P. aeruginosa in pure culture as well as in mixed cultures. These findings will provide the framework for the development of new small molecules which are able to modulate quorum sensing and thus act as tools in the inhibition of bacterial virulence and biofilm formation. THE SYNTHESIS OF A DIVERSE LIBRARY OF AI-2 ANALOGS TO INVESTIGATE BACTERIAL QUORUM SENSING By Jacqueline A.I. Smith Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2011 Advisory Committee: Assistant Professor Herman O. Sintim Professor Philip DeShong Professor Daniel Falvey Assistant Professor Nicole LeBlanc-LaRonde Copyright by Jacqueline A.I. Smith 2011 ii Dedication This dissertation is dedicated to my daughter Jasmine, my mom Phyllis and the entire Smith and Watkins families. In loving memory of my grandmother Mary Alice Watkins iii Acknowledgements I would like to thank God for blessing me with the ability to pursue this degree and for giving me strength and peace as I went through graduate school. Thank you Jesus! I would like to thank my advisor Dr. Herman Sintim for guiding my research and giving me insight into the scientific profession. I would also like to thank my family for supporting me; and all my friends, especially Kathy and Rey, for keeping me grounded as I went through this journey. I would like to thank my church Light of the World Family Ministries for their support and prayers. I thank the Meyerhoff Scholarship Program and my friends from UMBC for encouraging me to pursue my PhD and staying in contact with me over the years. I would like to acknowledge my collaborators Dr. William E. Bentley (and the Bentley Lab), and Dr. Vincent Lee (and the Lee Lab). Special thanks goes to Varnika Roy for conducting the ??galactosidase screening, in vitro phosphorylation TLC and synthetic ecosystem experiments shown in Chapter 3. Also thanks to my labmate Jingxin Wang for help with characterization of the quinoxaline derivatives and Sonja Gamby for help with analogs in Chapter 2. I would like to acknowledge my dissertation committee members Dr. Philip DeShong, Dr. Nicole LaRonde-LeBlanc, Dr. Daniel Falvey, Dr. Surgei Sukarahev. I?d like to thank the NFS Bridge to the Doctorate Fellowship (2006- 2008), GAANN Fellowship (2009-2010) and the Ann E. Wiley Dissertation Fellowship (Spring 2011) for financial support. Finally I would like to thank the entire staff of the University of Maryland Department of Chemistry and Biochemistry for their support. iv Table of Contents Dedication?????????????????????...??????.???ii Acknowledgements????????????????..?..?????????iii Table of Content??????????????????.?...????????.iv List of Tables????????????????????...???????..?.vi List of Figures????????????????????????..????.vii List of Schemes??????????????????..????????...?.xi List of Abbreviations??????????????????????..???...xii Chapter One: Introduction????????????????????...???..1 1.1 New approachs to anti-infective chemotherapy???????????.?1 1.2 Quorum sensing in gram-negative and gram-positive bacteria???.???4 1.3 Quorum sensing inhibitors?????????????????...??..6 1.4 Autoinducer-2 mediated quorum sensing??????????????13 1.5 AI-2 inhibitors???????????????????????..?22 1.6 Objective, hypothesis and specific aims???????????...??...25 1.7 Dissertation outline??????????????????????..26 Chapter Two: Facile synthesis of AI-2 and a diverse library of analogs????...?..27 2.1 Introduction: Discovering the chemical identity of AI-2????????27 2.2 Previous syntheses of AI-2?????????????????...?...28 2.3 Previous syntheses of AI-2 analogs??????????????.??31 2.4 Results: New facile synthesis of AI-2???????????...???...32 2.5 Synthesis of C1 analogs of AI-2???????????...?????...37 2.6 Synthesis of C4 and C5 analogs of AI-2 ??????????????.39 2.7 Discussion??????????????????????????41 2.8 Conclusion?????????????????????????...42 Chapter Three: Biological evaluation of analogs in V. harveyi, E. coli, S. typhimurium and P. aeruginosa?????????????????????????.??.43 3.1 Bioluminescence???????????????????????..43 3.2 Synergist agonism in V. harveyi???????????????...?...47 3.3 Discussion- V. harveyi.?????????????????????50 3.4 Quorum sensing in enteric bacteria???????????????.?.51 3.5 Inhibition and processing in enteric bacteria???????????.?..52 3.6 Discussion- E. coli and S. typhimurium??????????????...59 3.7 Pseudomonas aeruginosa????????????????????62 3.8 P. aeruginosa pyocyanin production modulation??????...???.....63 3.9 Discussion- P. aeruginosa???????????????????...68 3.10 Ester-protected AI-2 and analogs???????????????.?..69 Chapter Four: Conclusions, Broader Impact and Future Work?????....??..?76 4.1 Conclusion?????????????????????????...76 4.2 Broader Impact???????????????????????.?78 4.3 Future Work?????????????????????????.79 Chapter Five: Experimental, supplementary figures and references?????.?......83 5.1 Methods of synthesis??????????????????...?...?.83 5.2 Method of biological evaluation???????????..?????....85 5.3 Supplementary figures?????????????????.???....87 v 5.4 NMR characterizations????..????????????????..98 5.5 Spectra???????????????????????????126 5.6 References?????????????????????????..277 vi List of Tables Table 1.1: Organisms which have the LuxS/AI-2???????????????18 Table 3.1: V. harveyi strains and genotypes ????..??????????...??47 Table 3.2: Enteric bacteria strains and genotypes ????..?..???????...?.51 Table 3.3: P. aeruginosa strains and genotypes ???.??...???????..?.....63 Table 4.1: Inhibitory concentrations of select C1-analogs???????????...77 vii List of Figures Figure 1.1: Autoinducers used in quorum sensing????????????.?.?..4 Figure 1.2: TraR bound to DNA and AHLs???????????????...??5 Figure 1.3: Natural quorum sensing inhibitors and their synthetic derivatives????..6 Figure 1.4: Methylthioadenosine nuclease (MTAN) inhibitors???????.....?.....8 Figure 1.5: AHL analogs??????????????????????..??..9 Figure 1.6: Analogs of AIP1 and AIP2????????????????...??..11 Figure 1.7: Compounds identified by high throughput screening????.??...??13 Figure 1.8: Crystal structure of S-THMF-borate bound to LuxPQ and R-THMF bound to LsrB????????????????????????????..????..14 Figure 1.9: Quorum sensing in enteric bacteria??????????.???...??15 Figure 1.10: Quorum sensing in V. harveyi???????????.????...?..16 Figure 1.11: LuxS inhibitors?????????????????????...?..22 Figure 1.12: Structural analogs of AI-2?????????????.????...?23 Figure 1.13: Structurally unrelated inhibitors of AI-2???????..????...?.24 Figure 2.1: H1 NMR of the equilibrium mixture of compounds derived from DPD?....35 Figure 2.2: H1 NMR of the quinoxaline derivatives of AI-2???????????36 Figure 2.3: Library of diverse C1-analogs of AI-2????????????...??37 Figure 3.1: Crystal structure of LuxPQ dimer??????????????.??.45 Figure 3.2: Bioluminescence induction in V. harveyi MM32 (at 8hrs) by addition of a mixture of 2?M C1-analogs, 12nM AI-2 and 100?M boric acid and 50?M C1-analogs and 100?M boric acid?????????????????????????....49 viii Figure 3.3: AI-2 dependent ?- galactosidase production in E. coli ZK126 pLW11 and S. typhimurium MET708 (both luxS+) in response to linear, branched and deoxy-analogs..54 Figure 3.4: Phosphorylation of DPD by LsrK in the presence of radio-label ATP and representative TLC analysis of the LsrK mediated phosphorylation. ATP alone, AI-2, butyl-DPD, isobutyl-DPD and deoxyl-isobutyl-DPD treated with LsrK for 2hrs???55 Figure 3.5: AI-2 dependent ?- galactosidase production in E. coli SH3 (LsrK-, LuxS-) and E. coli LW7 (LuxS-) in response to ethyl-DPD??????????????.56 Figure 3.6: AI-2 dependent ?- galactosidase in E. coli ZK126 (LsrR+) and E. coli LW8 (LsrR-) in response to methyl-DPD (AI-2), butyl-DPD, isobutyl-DPD and deoxy isobutyl-DPD?????????????????????????????.57 Figure 3.7: AI-2 dependent ?- galactosidase production in S. typhimurium MET708; AI- 2 dependent bioluminescence production in V. harveyi BB170 and AI-2 dependent GFP induction in E. coli W3110 pCT6 (all strains are luxS+) in response to isobutyl-DPD and isopropyl-DPD????????????????????????????...58 Figure 3.8: Predicted tertiary structure of S. typhimurium and E. coli LsrR proteins?..61 Figure 3.9: P. aeruginsosa virulence factor, pyocyanin ?????????..??....63 Figure 3:10: Pyocyanin production in P. aeruginosa PAO1 in response to methyl-DPD, ethyl-DPD, heptyl-DPD, isobutyl-DPD, cyclopentyl-DPD and phenyl-DPD ????????????????????????????????...??.64 Figure 3.11: AI-2 dependent ?- galactosidase production in E. coli LW7 and S. typhimurium MET715 (both luxS-) in response to a mixture of a) 40 ?M synthetic DPD and cyclic and b) synthetic DPD and aromatic analogs???????.??????65 ix Figure 3.12: AI-2 dependent ?- galactosidase production in S. typhimurium MET708; Pyocyanin production in P. aeruginosa PAO1; AI-2 dependent RFP induction in E. coli W3110 pCT6 dsRed (all strains are luxS+) in response to isobutyl-DPD, phenyl-DPD and a cocktail of isobutyl-DPD and phenyl-DPD?????????????????67 Figure 3.13: Bioluminescence induction in V. harveyi MM32 in response to ester protected AI-2 and hexyl-DPD both in the absence and presence of exogenous AI- 2??????????????????????????????..???..70 Figure 3.14: Bioluminescence induction in V. harveyi BB170 in response to ester protected AI-2 over time?????????????????????????????????71 Figure 3.15: ?- galactosidase production E. coli LW7 in response to isobutyl-DPD, diacetate isobutyl, hexyl-DPD and diacetate hexyl in the presence of exogenous AI- 2????????????????????????????????...?..72 Figure 3.16: Model pathway of ester protected AI-2 and analogs in V. harveyi and E. coli????????????????????????????????.?73 Figure S1: ?- galactosidase production in E. coli LW7 in response to AI-2, hexyl-DPD, isobutyl-DPD and their diacetate derivatives after 4 weeks of incubation at various temperatures?????????????????????????????...87 Figure S2: Bioluminescence induction in V. harveyi BB886 (LuxQ-) in response to select C1-analogs?????????????????????????????.....88 Figure S3: Bioluminescence induction in V. harveyi BB721 (LuxO-) in response to C1- analogs???????????????????????????????...89 x Figure S4: ?- galactosidase production in E. coli LW7 and S. typhimurium MET715 (both luxS-) in response to linear, branched and deoxy analogs??????????90 Figure S5: ?- galactosidase production in E. coli LW9 (LsrB-) in response to linear, branched and deoxy analogs of AI-2??...?????????????????..91 Figure S6: Pyocyanin production in response to linear, branched, cyclic and aromatic C1-analogs????????????????????????????.??92 Figure S7: ?- galactosidase production in E. coli LW7 and S. typhimurium MET715 (both are luxS-) in response to cyclic and aromatic analogs???????????..93 Figure S8: Effect of ester-protected AI-2 and analogs on V. harveyi growth??.??.94 Figure S9: ?- galactosidase production in E. coli LW7 in response to AI-2 and diacetate AI-2....................................................................................................................................95 Figure S10: Dose-response curves of C1 analogs in the presence of AI-2 measured by ?- galactosidase in E. coli LW7 and S. typhimurium MET715(both are luxS-)???.........96 Figure S11: Phosphorelay used for signal transduction in the AI-2 mediated quorum sensing pathway of V. harveyi?????????????????????????..97 Figure S12: Synergistic agonsism of V. harveyi MM32 (LuxS-) in the prescence of various concentrations of AI-2 and Hexyl-DPD????????????????97 xi List of Schemes Scheme 1.1: Biosynthesis of AHLs?????????????????????8 Scheme 1.2: Equilibrium mixture of AI-2 compounds????????????.....13 Scheme 1.3: Retrosynthetic analysis of AI-2??????????...????...?..26 Scheme 2.1: Biosynthesis of 4,5-dihydroxyl-2,3-pentadiene (DPD)...???????.27 Scheme 2.2: Janda?s synthesis of DPD????????????????...??..29 Scheme 2.3: Semmelhack?s synthesis of DPD????????????......???29 Scheme 2.4: Vanderleyden?s synthesis of DPD????????????????30 Scheme 2.5: Doutheau?s synthesis of DPD???????????????..??.31 Scheme 2.6: Synthesis of Trifluoromethyl-DPD????????????..?..?..32 Scheme 2.7: A new synthesis of AI-2????????????????...???33 Scheme 2.8: Resonance states of acyldiazo??????????????......??34 Scheme 2.9: Condesation of DPD with 1,2-phenylenediamine to form quinoxaline?...36 Scheme 2.10: New synthesis of diacetate AI-2 and diacetate analogs???????..39 Scheme 2.11: Synthesis of deoxy-AI-2 analogs????????????????40 Scheme 3.1: Mechanism of bioluminescence production with riboflavin as the luciferin???????????????????????????????..44 xii List of Abbreviations AB= Autoinducer Bioassay Ac= Acetate AcOH= Acetic Acid ACP= Acyl-carrier protein AHL= Acyl homoserine lactone AIP= Autoinducing peptide AI-2= Autoinducer 2 ATP= Adenosine triphosphate CAI-1= Cholerae Autoinducer-1 DABCO= 1,4-Diazabicyclo[2.2.2]octane DBU= 1,8-Diazobicyclo[5.4.0]undec-7-ene DCM= Dichloromethane DMSO= Dimethyl sulfoxide DMS= Dimethyl sulfide DPD= 4,5-Dihydroxyl-2,3-pentadione EtOH= Ethanol GFP= Green fluorescent protein HAI-1= Harveyi Autoinducer-1 HSL= Homoserine lactone LDA= Lithium diisopropylamide LM= Luria Marine MeCN= Methyl acetonitrile xiii MeI= Methyliodide MeOH= Methanol MRSA= Methicillin-resistant Staphylococcus aureus MTA= Methylthioadenosine MTAN= Methylthioadenosine nuclease MTR= Methylthioribose nBuLi= (Normal) Butyl Lithium ONPG= O-nitrophenyl-galactoside PQS= Pseudmonas quinolone signal R-DHMF= R-dihydroxymethyl furanone RLU= Relative light units R-THMF= R-tetrahydroxymethyl furanone SAH= S-adenosylhomocysteine SAM= S-adenosylmethionine S-DHMF= S-dihydroxymethyl furanone SRH= S-ribosehomocysteine S-THMF= S-tetrahydroxymethyl furanone TBAF= Tetrabutylammoniafluoride TBS= Tertbutyldimethylsilyl THF= Tetrahydrofuran VRE= Vancomycin-resistant Entereococcus VRSA= Vancomycin-resistant Staphyloccocus aureus 1 Chapter One Introduction 1.1 New approaches to anti-infective chemotherapy Over the last two decades the treatment of bacterial infections has become non- trivial due to the rapid development of resistance and the emergence of multi-drug resistant organisms.1 By 2003, 50% of all hospital infections were caused by methicillin- resistant Staphylococcus aureus (MRSA).1 In 2007, 70% of all hospital-acquired infections were resistant to at least one or more antibiotic.2 Today, community-acquired MRSA as well as vancomycin-resistant S. aureus (VRSA) and vancomycin-resistant Enterococci (VRE) are less susceptible to newer drugs such as daptomycin and linozelid.1 Since antibiotics are designed to be lethal to bacteria (bactericidal) or inhibit their growth (bacteriostatic), evolutionary pressure is placed on the organism to develop mechanisms of resistance.2-3 Such mechanisms include alteration of the drug target, degradation of the drug molecule or rapid expulsion of the drug out of the cell.2-3 Progress has been made in developing anti-infective agents which have novel targets including riboswitches, fatty acid synthesis, and programmed cell death.3 However, since these processes are vital for the survival of bacteria it is inevitable that resistance will soon develop.3 Bacteria communicate through the secretion and detection of small molecules in a process known as quorum sensing.3a, 4 Once a critical population is reached bacteria coordinate the expressions of genes required for processes such as virulence, biofilm formation, and bioluminescence.4 Virulence factor expression is responsible for the symptoms associated with bacterial infections whereas biofilm formation accounts for the 2 persistence of infections as well as difficulties encountered when trying to kill bacteria with antibiotics.2-3 Virulence is accomplished through the secretion of factors such as toxins and proteases, which directly affect host cell function.2 Bacteria only express these factors when the population is large enough to be effective.2 It has been shown that quorum sensing regulates the expression of these virulence genes.5 As bacteriocidal drugs put enormous pressure on bacteria to develop resistance, it has been suggested that strategies such as quorum sensing inhibition, which attenuate bacterial virulence but do not kill bacteria, might lead to less resistance development.2-3 Biofilm is a community of bacteria encapsulated in a polysaccharide matrix; this matrix can form on surfaces such as living tissues or medical devices.6 Once incorporated in a biofilm matrix, bacteria are resistant to traditional antibiotics and are rarely cleared by the host immune system.6-7 Also the biofilm environment increases the probability of antibiotic-resistant plasmid being transferred between bacteria.6b Biofilm is involved in over 60% of bacterial infections.3a, 6a However there is currently no anti-biofilm drug in clinical use.3a, 6a Since quorum sensing has been shown to be involved in biofilm formation, anti-quorum sensing agents may provide a mean to clear biofilm infections. Although not proven clinically there are several reasons why attenuating virulence and biofilm formation by interfering with quorum sensing is less likely to cause resistance. Firstly mutation of quorum sensing proteins in order to overcome the action of anti-quorum sensing agents would cause the organism to be unresponsive to the natural signaling molecules. Therefore it would not be able to detect when a threshold concentration of bacteria is present. Secondly, since quorum sensing is a community- 3 dependent behavior, mutating a quorum sensing protein will cause the organism to be ?out of sync? with the other bacteria. Although the mutated organism will be able to overcome the action of the anti-quorum sensing agents its neighbors will not. Thus the mutated organism will turn quorum sensing ?on? independently. Ultimately its efforts to conduct processes such as biofilm formation and virulence expression will be inadequate and the host immune system can easily clear these lone mutants. Finally there is no growth advantage in developing anti-quorum sensing-resistance like that of antibiotic- resistance where mutation allows the organism to thrive and replicate. If a mutation of quorum sensing proteins does occur, the forementioned discussions suggest that this mutant may be at a growth disadvantage. Therefore targeting quorum sensing as a new means of anti-infective treatment is less likely to cause rapid resistance. 4 1.2 Quorum sensing of gram-negative and gram-positive organism Figure 1.1: Autoinducers used in quorum sensing3a Quorum sensing involves the release of signaling molecules called autoinducers. Generally gram-negative bacteria use acylhomoserine lactones (AHLs) with varying acyl-chain lengths (3-7; Figure 1.1) whereas gram-positive species use oligopeptides, which may be post-translationally modified (1-2; Figure 1.1) for intra-species communication.4a,8 In gram-negative bacteria, such as Vibrio fischeri and Pseudomonas aeruginosa, AHLs are produced by a LuxI-type synthase proteins and detected by a LuxR-type cytoplasmic receptor proteins, which bind to DNA in order to activate or repress genes (See Figure 1.2).3a, 4a, 9 5 Figure 1.2: a) crystal structure of TraR bound to DNA b) AHL in binding site of TraR The bioluminescent marine bacterium, V. fischeri, uses 3-oxo-hexanoyl- homoserine lactone (3OC6-HSL; 7) and N-octanoyl-homoserine lactone (C8-HSL; 5) for intraspecies communication.3a, 10 The opportunistic organism P. aeruginosa uses two AHLS: N-butyryl-homoserine lactone (C4-HSL; 3) and N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL; 6) in a dual quorum sensing system.3a, 4a, 10 3OC12-HSL is produced by LasI and detected by LasR while C4-HSL is produced by RhlI and is detected by RhlR.3a, 4a The las system controls the rhl system as well as several virulence factors.3a, 4a P. aeruginosa also secrete other signaling molecules including 2-heptyl-3- hydroxyl-4-quinolone (Pseudomonas quinolone signal; (PQS), 8) and piperazines (9).3a, 4a Other Vibrios such as V. cholerae and V. harveyi use parallel quorum sensing signals.11 In V. cholorae, the signaling molecules are S-3 hydroxydodecan-4-one (CAI-1; 10) and AI-2 (11-13).3a, 4a, 11 CAI-1 and AI-2 are synthesized by CqsA and LuxS, respectively and then detected by the membrane bound proteins CqsS and LuxPQ, respectively. 3a, 4a, 11 In V. harveyi, a third signal HAI-1 (3OHC4-HSL; 3) is synthesized by LuxM and detected a) b) 6 by LuxN. 3a, 11 Enterohaemorrhagic E. coli (EHEC) use an unidentified compound known as AI-3 for signaling along with the hormones epinephrine and norepinephrine.4b, c, 12 AI- 3 binds the membrane bound protein QseC, which initiates a phospho-relay and triggers the expression of genes responsible for attaching and effacing lesions.4b, c, 12 The quorum sensing systems of the bacteria described above are well understood and attempts to target these organisms for anti-infective chemotherapy have been pursued.10, 13 1.3 Quorum sensing inhibitors Researchers have found that in nature bacteria target the quorum sensing communication system of other organisms in competition for resources. This is accomplished through quorum quenching enzymes (i.e. lactonases and acylases)14 which degrade the signaling molecules before they are able to initiate quorum sensing. Unfortunately anti-quorum sensing chemotherapies are unlikely to use proteins due to their potential to cause an immune system response. Instead small molecules are better candidates for anti-quorum sensing studies. Figure 1.3: Natural quorum sensing inhibitors and their synthetic derivatives 7 Nature provides several examples of small molecules that interfere with quorum sensing (Figure 1.3). For example, patulin (14) and penicillic acid (15), present in the broth of Penicillium, were able to down regulate gene expression in P. aeruginosa.15 A component of garlic extract, GC-7 (16), was able to inhibit V. fischeri signaling systems.3a The food additive cinnamaldehyde (17) was found to be a potent inhibitor of AI-2 mediated quorum sensing in several Vibrios.16 Derivatives of cinnamaldehyde were also screened however only 4-NO2-cinnamaldehyde was more active than the parent compound, but it suffered from toxicity.16 The mechanism of inhibition of cinnamaldehyde was found to be due to binding to the master regulator LuxR found in V. harveyi. 16 The most well known natural inhibitors of quorum sensing are the brominated furanones, which are produced by red algae.17 Brominated furanones have been found to inhibit biofilm formation in S. typhimurium and P. aeruginosa.18 Synthetic derivatives of these brominated furanones (18 and 19) have demonstrated inhibition of P. aeruginosa biofilm formation in vitro as well as in vivo on mice models.17-18 Both natural (20) and synthetic brominated furanones (21) are able to reduce biofilm formation in S. typhimurium.18b Though the synthetic analog (21) was slightly less active, it had a lower toxicity.18b Initial studies found that brominated furanones inhibit quorum sensing by targeting LuxR.17, 19 However, a recent study found that these compounds can covalently modify LuxS.20 These examples confirm that small molecules can indeed effect quorum sensing in vivo and in vitro. 22, 18b 8 Taking lead from nature, synthetic molecules have been developed to block the synthesis, binding or other downstream signaling events caused by autoinducers using structural analogs or compounds identified by high throughput screening.13 Scheme 1.1: Biosynthesis of AHLs3a (ACP= acyl-carrier proteins) In the biosynthesis of AHLs, LuxI-type enzymes catalyze the reaction of the fatty acid portion of acyl carrier proteins with S-adenosylmethionine (SAM (22) to form AHLs and methylthioadenosine (MTA, 23; see Scheme 1.1).3a, 10, 21 The nucleosidase, Pfs then converts MTA into methylthioribose (MTR; 24), which forms methionine (25).3a, 10, 21 Figure 1.4: Methylthioadenosine nuclease (MTAN) inhibitors 9 Recent work by Schramm involved the synthesis of a library of transition state analogs of MTA as inhibitors of the methylthioadenosine nuclease (MTAN, (Pfs); See Figure 1.4).22 Originally, early stage transition state analogs were designed and p- chlorophenylthio-ImmA (pClPhT-ImmA; 26) was found to be the tightest binder of Pfs.22 A newer generation of analogs were designed to mimic late stage transition states; in this series p-chlorophenylthio-DADMe-ImmA (27) was also the best binder.22 Additionally But-DADMe-ImmA (28) was able to effect biofilm formation in V. cholerae and E. coli O157:H7.23 Although this approach has given promising results, MTANs are vital for polyamine synthesis, methyl transfer and methionine synthesis in human cells as well as in bacteria. 23 Therefore these molecules are likely to be toxic due to their potential to interfere with important metabolic process in human cells. Figure 1.5: AHL analogs 10 Structural analogs of AHLs are the most widely explored set of small molecule modulators of quorum sensing (Figure 1.5).10 As previously described, AHLs of different chain lengths are used for signaling between specific species of bacteria.4a, 10 Early studies showed that alteration of the structure of the AHL often resulted in reduced agonism or in some cases antagonism via competition with the natural ligand for binding to LuxR-type proteins.24 Initial results showed that C9-AHL (29), which is only one carbon longer than the natural AHL, was a potent inhibitor of LuxR mediated bioluminescence in V. fischeri.24 Following the introduction of a new synthetic route, over 90 analogs with non-native AHLs have been synthesized and evaluated in A. tumefaciens, P. aeruginosa and V. fischeri by Blackwell.25 Out of this library several analogs were found, including iodophenyl HSL, 30 and compound 31, to be antagonists against all three bacteria .25 Additionally, bromophenyl HSL (33) and indole-HSL (32) were found to reduce biofilm formation of P. aeruginosa.26 Interestingly 34 acted as a potent agonist in V. fischeri although most other aryl analogs acted as antagonists.25 Despite these successes AHLs are susceptible to hydrolysis by lactonases.14a Therefore analogs that lack the lactone moiety are more desirable. Suga has synthesized analogs which have the lactone ring replaced with other cyclic structures.27 The cyclohexanone (36), cyclopentanol (35) and phenol (37) derivatives of 3-oxo-C12-HSL all acted as antagonist while cyclohexanol (38) was found to be an agonist of P. aeruginosa.27b Other structural alterations, for instance replacing the amide functionality with an amino- sulfonyl group or sulfide, afforded antagonists 40 and 39.28 11 Figure 1.6: Analogs of AIP1 and AIP2 Analogs of quorum sensing in gram-positive bacteria have primarily targeted S. aureus. In this system oligopeptides are modified, cyclized and transported out of the cell. These autoinducing peptides (known as AIPs) then bind the membrane bound protein AgrC which induces a phosphorelay mechanism and controls virulence expression. In S. aureus, four groups of AIPs are produced by different strains and are detected by specific AgrCs.29 It has been shown that AIPs of different groups bind and inhibit the response of the natural AIP.29 This cross-inhibition stimulated the evaluation of structural analogs of AIPs across groups (i.e. AIP-II analogs were tested on AgrC-1; Figure 1.6).29a Initial replacement of aspartate with alanine (AIP-1 D5A; 45) resulted in a potent inhibitor of all 4 AgrCs.30 Other variations such as 44 gave an inhibitor of AgrC-2 and AgrC-4 only.30 Truncation of AIP-1 D5A (trAIP-1 D2A; 46) provided an equally potent inhibitor of AgrC-1-4.30 Likewise truncated AIP-II (41) was found to be a potent inhibitor across all four groups.30 Structure activity relationship (SAR) studies of this compound uncovered two additional derivatives, 42 and 43, which are more potent against AgrC-1 and AgrC-2.31 12 Figure 1.7: Compounds identified by high throughput screening Other modulators of quorum sensing have been found by high throughput screenings (Figure 1.7). PD12 (47) and TP-5 (49) are inhibitors of AHL signaling in P. aeruginosa.32 TP-5 was the only antagonists amongst a series of structurally similar compounds all of which acted as agonists.32b LED209 (48) was found through high throughput screenings to inhibit Enterohaemorrhagic E. coli through binding to the membrane bound receptor QseC (for further details see p.6 and the indicated references).33 Competition with AHLs for binding to the membrane bound receptor protein, LuxN, in V. harveyi has recently been investigated by Bassler.34 Out of the 30,000 compounds screened, 15 non-toxic candidates were identified most of which were structurally unrelated to AHLs (50 and 51).34 In a subsequent study, Bassler screened these LuxN inhibitors for activity against LuxR-type proteins owing to the fact the AHLs 13 have the capacity to bind both membrane bound and cytoplasmic proteins.34 Chlorothiolactone (52) was identified as an inhibitor of a human pathogen Chromobacterium violaceum, which uses the CviR as the LuxR-type cytoplasmic receptor.35 Further studies on chlorothiolactones, 53 and chlorolactone, 54, revealed that inhibition can occur through binding to CviR and inhibiting transcription or by preventing binding of CviR to DNA which also inhibits transcrition.35 Though it has been shown that anti-quorum sensing chemotherapy is possible, the challenge to find a common target among many bacteria still remains. 1.4 Autoinducer-2 mediate quorum sensing Most autoinducers are species-specific but there exists a universal autoinducer, AI-2, which has been detected in several species of bacteria.36 Scheme 1.2: Equilibrium mixture of AI-2 compounds AI-2 is not a single compound but a collection of inter-converting compounds 55- 60 (Scheme 1.2).37 Once formed, DPD undergoes spontaneous rearrangements to give a mixture of compounds; (2S, 4S)-2,4-dihydroxy-2-methyldihydroxyfuran-3-one (S- 14 DHMF; 56), (2R, 4S)-2,4-dihydroxy-2-methyldihydroxyfuran-3-one (R-DHMF; 59), (2S, 4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (S-THMF; 57) and (2R, 4S)-2-methyl- 2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF; 60).38 In the presence of borate salts, S- THMF can form S-THMF-borate (58).37 It has been shown that different species of bacteria recognize different forms of DPD (See Figure 1.8); S. typhimurium detects R- THMF (60)38 whereas V. harveyi, which is found in marine environments detects S- THMF-borate (59).37 Figure 1.8: Crystal structure of a) S-THMF-borate bound to LuxPQ and b) R-THMF bound to LsrB 15 Figure 1.9: Quorum sensing in enteric bacteria3a In enteric bacteria, AI-2 is internalized through the LsrABC transporter and phosphorylated by LsrK (Figure 1.9).39 Phosphorylated AI-2 (P-AI-2) acts as a substrate of the LsrR protein.39 In a non-quorum sensing state, LsrR represses the lsr operon.40 When P-AI-2 binds to LsrR, the lsr operon is derepressed and the LsrR-regulated genes are expressed.39 LsrG, an enzyme encoded by the lsr operon, catalyzes the degradation of P-AI-2 into phosphoglycolic acid (PG) and an unknown C3 compound.41 It has been shown that the internalization of AI-2 by enteric bacteria interferes with the quorum sensing process of other bacteria.42 Similar internalization of AI-2 is executed in A. actinomycetemcomitans by the ribose binding protein, RbsB.43 In this organism phosphorylated AI-2 interacts with a two-component system consisting of QseBC, which controls biofilm formation and iron uptake.43 S. meliloti also internalizes AI-2 through a LsrB homolog although this organism does not have LuxS.44 16 Figure 1.10: Quorum sensing in V. harveyi3a As previously mentioned, V. harveyi, a marine bacterium also uses AI-2 for signaling.45 V. harveyi uses quorum sensing to encode genes, which control bioluminescence and virulence.46 Under normal conditions (low cell density), LuxPQ acts as a histidine-kinase and initiates the phosphorylation of LuxU which subsequently phosphorylates LuxO (Figure 1.10).47 LuxO along with ?54 activates five small regulatory RNAs (sRNAs).11 48 These sRNAs along with the Hfq chaperone destabilize the transcriptional regulator, LuxR preventing its production.49 LuxR regulates the expression of genes responsible for bioluminescence and virulence.50 Therefore under normal conditions, LuxR is not present to induce the expression of genes responsible for bioluminescence and virulence factor production.50 In the quorum sensing state (high cell density), when AI-2 binds, LuxPQ acts as phosphatase and dephosphorylates LuxU and LuxO.11 This results in the inactivation of the sRNAs and ultimately allows LuxR to 17 activate bioluminescence and virulence genes.49 A similar phospho-relay is initiated upon AI-2 binding in V. cholerae but HapR, the homologue of LuxR, represses biofilm formation.51 4, 5-Dihydroxy-2, 3-pentanedione (DPD; 55), the linear precursor of AI-2, is synthesized by the LuxS protein, which is highly conserved in both gram-positive and gram-negative bacteria.36, 45 Therefore it has been suggested that AI-2 functions as an interspecies signal unlike the intraspecies AHLs and oligopeptides.4a, 8 The luxS gene is present in over 60 species of bacteria and AI-2 production has been reported in many of these organisms.52, 3 Table 1.1 outlines a subset of these organisms and LuxS/AI?s role. Therefore drugs which target the LuxS/AI-2 quorum sensing system could have the potential to have broad-spectrum anti-quorum sensing activity. 18 Table 1.1: Organisms which have the LuxS/AI-2 system Bacteria Observed phenotype in LuxS mutants Complementation mode Ref Oral pathogens Streptococcus oralis Reduced mutualistic biofilm growth Synthetic DPD; Plasmid containing luxS gene 53 Streptococcus gordinii Downregulation genes required for carbohydrate metabolism; Reduced mixed biofilm formation Plasmid containing luxS gene 54 Aggregatibacter actinomycetemcomitans Reduced biofilm growth Partially purified DPD; Plasmid containing luxS gene 43, 55 Porphyromonas gingivalis Reduced mixed biofilm growth; Synthetic DPD; Plasmid containing luxS gene 54 Streptococcus mutans Attenuated biofilm formation AI-2 producing bacterial strains 56 Actinomyces naeslundii Reduced mutualistic biofilm growth Synthetic DPD; Plasmid containing luxS gene 53 Food-borne pathogens Salmonella typhimurium Inactive AI-2 internalization In vitro synthesized AI-2; Plasmid containing luxS gene 57 Clostridium perfringens Reduced toxin production Culture supernatant 58 Campylobacter jejuni No differentiation of genes (via microarray analysis) In vitro synthesized AI-2 59 Bacillus cereus Normal biofilm formation In vitro synthesized AI-2* 60 Listeria monocytogenes Increased biofilm formation In vitro synthesized AI-2** 61 Vibrio chlorae Increased biofilm formation Synthetic DPD* 62 Vibrio angullarium Pigmentation Synthetic DPD 63 Vibrio ichthyoenter No change in biofilm formation or virulence N/A 64 Vibrio vulnificus Decreased protease and increased haemolysin production Plasmid containing luxS gene 65 Edwardsiella tarda Reduced biofilm formation, type III secretion system gene expression and virulence Culture supernatant containing AI-2 66 Vibrio harveyi Reduced bioluminescence Synthetic DPD 46a Opportunistic pathogens Staphylococcus aureus Increased virulence factor synthesis Synthetic DPD 67 Staphyloccous epidermidis Increased biofilm and enhanced virulence Plasmid containing luxS gene; Culture supernatant containing AI-2* 68, 69 Streptococcus intermedius Increased antibiotic susceptibility Synthetic DPD 70 Streptococcus anginosus Increased antibiotic susceptibility Synthetic DPD 71 Symbiotic bacteria Escherichia coli Reduced biofilm formation In vitro synthesized AI-2 66 Lactobacillus reuteri Increase biofilm thickness Purified AI-2** 72 Lactobacillus rhamnosus Decreased metabolism and biofilm formation Culture supernatant; Synthetic DPD** 73 Human pathogens Neisseria meningitidis Metabolic byproduct Culture supernatent 74 Helicobacter pylori Lost of motility Plasmid containing luxS gene; In 75 19 vitro synthesized AI-2 Proteus mirabilis No effect on virulence or motility N/A 76 Borrelia burgdorferi No effect N/A 77 *addition of in vitro synthesized AI-2 caused a reduction in biofilm; **Failed to effect biofilm formation Despite the ubiquitous nature of AI-2 there is still some debate as to whether it is indeed used for signaling in all organisms which posses LuxS or if it simply acts as a metabolic byproduct of the methyl cycle.78, 79 In addition to DPD, LuxS also produces homocysteine, which is converted to cysteine and used in the biosynthesis of the essential amino acid, methionine. 80 Studies using LuxS mutants often show alterations in biofilm formation/architecture and/or virulence factor expression but due to the role of LuxS in metabolism it is often difficult to determine whether these alterations are the result of interfering with quorum sensing or due to the disruption of the active methyl cycle. 78 79 Previous studies have relied on complementation of LuxS mutant strains with, plasmids containing luxS, culture supernatant containing AI-2, or in vitro synthesized DPD, partially puriefied AI-2 or synthetic DPD in attempts to decipher the role of the LuxS/AI- 2 system in bacteria. 78, 79 In some organisms the role of AI-2 in pathogenesis is now clear.79 A luxS mutation in Vibrio vulnificus, an organism responsible for septicemia and wound infections, caused aberrant expression of virulence factors.65 Upon addition of culture supernatant containing AI-2, these virulence factors were restored to basal levels.65 In luxS mutants of Clostridium perfringens, the gram-positive pathogen responsible for gangrene, toxin production was restored to wild-type levels when culture supernatant containing AI-2 was added.58 However, this complementation strategy can be misleading since other components in culture supernatant cannot be ruled out as effecting virulence expression. Helicobacter pylori, the gram-negative organism responsible for peptidic 20 ulceration, gastric cancer and some types of gastric lymphoma, is controlled by AI-2 mediated quorum sensing.75 Complementation with synthetic DPD in a luxS mutant restored motility through flagellar transcription whereas complementation with cysteine did not restore this phenotype. 75 In addition to phenotypes, transcriptome analysis has also been used to identify genes which are modulated in the presence of AI-2. Transcriptome analysis in Staphylococci reveals AI-2?s importance in metabolism and virulence.67, 69, 68 Transcriptome analysis in S. aureus revealed that deletion of the luxS gene affected several metabolic enzymes as well as genes responsible for the production of capsular polysaccharide (CP), a virulence factor on bacteria cell walls which allow cells to evade phagocytes of the host immune system.67 Similarly transcriptome analysis of S. epidermidis luxS mutants showed metabolic genes being modulated but also modulation of pro-inflammatory and immune evasion factors.69, 68 In addition to virulence factor expression, AI-2 has also been shown to control biofilm formation in several bacteria.74 A luxS mutant strain of Streptococcus oralis was unable to produce biofilm and grow mutualistically with Actinomyces naeslundii.53 This malfunction was restored by the addition of synthetic AI-2. Another oral pathogen responsible periodontal disease, Aggregatibacter actinomycetemcomitans, also lacked the ability to form biofilm and cause virulence in luxS mutant strains; partially purified AI-2 was able to restore this activity.55 Finally biofilm formation and architecture was restored by in vitro synthesized AI-2 in Escherichia coli.66 In some organisms such as Vibrio cholerae, a human pathogen responsible for the gastrointestinal disease cholera62 and B. cereus, AI-2 signaling results in the repression of biofilm formation.60 Other phenotypes, which are modulated by AI-2 include antibiotic susceptibility and production. For 21 example, antibiotic susceptibility was restored in a luxS mutant of Streptococcus anginosus71 and Streptococcus intermedius 70 after addition of synthetic DPD. A few examples point to AI-2 as a by-product of metabolism.81, 78, 79 Studies with the probiotic, Lactobacillus rhamnosus, reveal that neither growth nor biofilm formation was restored to wild type levels in luxS mutants with culture supernatant containing AI-2 or with chemically synthesized DPD.73 Though cysteine was successfully able to significantly restore growth and biofilm formation.73 Additionally, purified AI-2 was unable to restore biofilm in luxS mutants of Lactobacillus reuteri67 and Listeria monocytogenes.61 Transcriptome analysis in a luxS mutant of the human pathogen, Campylobacter jejuni, revealed that the differentiation of genes associated with metabolism, rather than quorum sensing, was responsible for this organism?s loss of motility.59 Also the addition of in vitro synthesized AI-2 had no effect on gene expression in this analysis, indicating that motility is affected by metabolic disturbance rather than quorum sensing.57 Similar transcriptional analysis using DNA microarrays in Neisseria meningitides indicated that the attenuation of virulence observed in luxS mutant was not due to quorum sensing as addition of culture supernatant containing AI-2 did not affect gene expression.81 Finally, the role of AI-2 in S. typhimurium has been questioned owing to the fact that complementation with chemically synthesized DPD was not able to restore biofilm formation.82 Cysteine and the other components of the active methyl cycle were also ineffective at restoring biofilm.82 Therefore it is unclear what caused biofilm perturbation in S. typhimurium.82 The relevance of quorum sensing in S. typhimurium has also been questioned because of the similarities in AI-2 processing with sugar metabolism (i.e. binding, internalization and phosphorylation) and the lack of function of 22 AI-2 in S. typhimurium other than internalization.79 Other organisms for example Borrelia burgdorferi77 and Proteus mirabilis76 exhibit no difference in wild type versus luxS mutant strains indicating that LuxS may be present only for its role in the active methyl cycle. The aforementioned examples reveal that not all organisms, which contain LuxS and produce AI-2 use it for quorum sensing. Though LuxS is important for metabolism and in some cases AI-2 has been shown to be a by-product of metabolism, it is clear that AI-2 can modulate virulence factor expression, biofilm formation and antibiotic susceptibility in several bacteria.74, 53, 71 Additionally, some organisms which do not have LuxS have been able to respond to exogenously added AI-2.44, 83 This could suggest that over time AI-2 as a by-product of metabolism became an indicator for some bacteria that other bacteria were present in their surroundings. Furthermore, AI-2 has been shown to form independently of LuxS from ribulose-5-phosphate.84 Therefore the designation of AI-2 as an interspecies signaling molecule is still valid as no other single molecule has been shown to be relevant to as many organisms as AI-2.8 1.5 AI-2 inhibitors Figure 1.11: LuxS Inhibitors85 23 AI-2 is ubiquitous and chemotherapies which target this molecule could provide a broad-spectrum anti-infective agent. Though inhibitors of the AI-2 mediated quorum sensing systems have not been rigorously pursued until recently. Initial attempts to inhibit AI-2 synthesis have involved the construction of analogs of the natural substrate of LuxS S-ribosylhomocysteine (Figure 1.11). 78, 84 Studies by Zhou revealed two active analogs: S-anhydroribosylhomocysteine, 61 and S-homoribosylcysteine, 62.85a These analogs were found to inhibit AI-2 synthesis but no other biological tests were conducted.84 Other acyclic analogs of SRH (i.e., 63) were found to bind differently to the LuxS of different organisms including B. subtilis, V. harveyi and E. coli, although to varying extents.85b LuxS inhibition would not be a useful target for anti-quorum sensing therapies though due to the fact that other metabolic processes are controlled by LuxS80 and the biological response of inhibitors could be the result of interfering with these processes rather than quorum sensing. 78, 79 Figure 1.12: Structural analogs of AI-279, 86, 87, 88 The inhibition of AI-2 via competition for receptors or processing enzymes has been pursued through the synthesis of structural analogs of AI-2 (Figure 1.12). 79, 86, 87, 88 24 Initial studies looked at the natural compounds laurencione (64) and MHF (65) due to the structural similarities with the linear and cyclic forms of AI-2, respectively.86 Although both showed some activity they were not as effective at causing bioluminescence induction as AI-2 in V. harveyi.86 Synthetic efforts by Janda found that the enantiomer (4R)-DPD (66) was also less active than the natural molecule.86 Eventually new synthetic routes were published allowing for variations on the hydroxyl moieties of AI-2 analogs (68).87 Unfortunately, no biological tests have been reported for this analog although similar acetylated analogs were found to act identical to AI-2.89 An analog which replaced the hydrogens in the C1 methyl group of DPD with fluorines (trifluoromethyl- DPD; 67) was found to be more active than the natural compounds (64, 65) or the enantiomer of DPD (66) in inducing bioluminescence in V. harveyi.88 With these initial results it became clear that AI-2 analogs often acted as less potent agonist of AI-2 mediated processes rather as antagonists. Subsequent studies by Janda identified butyl- DPD (69) as a good inhibitor of ?-galactosidase transcription in S. typhimurium although the mechanism of this inhibition was not revealed.90 Although butyl-DPD as well as propyl-DPD acted as antagonist in S. typhimurium they were also found to cause synergist agonism in V. harveyi.90 25 Figure 1.13: Structurally unrelated inhibitors of AI-2 QS 91, 92, 63 Other compounds have been screened which are structurally unrelated to AI-2 (Figure 1.13). 91, 92, 63 The commercial compound pyrogallol (70) and its derivative (71) have been identified as a potent inhibitor of quorum sensing controlled- bioluminescence.91 Likewise various boronic acids (72 and 73)92 inhibit bioluminescence at low concentrations although the toxicity of both the pyrogallol and boronic acid will likely prevent clinical use. A new structural class of phenothiazine derivatives also have been found to inhibit AI-2 quorum sensing.93 Recently, a new set of compounds were screened for activity against V. harveyi and other vibrios. LMC-21 (74)63 was found to be a potent inhibitor of biofilm formation in V. anguillarium and V. vulnificus as well as block V. harveyi infection of Artemia shrimp.63 Although structurally similar to SAM and SAH, the mode of inhibition of this nucleoside was found be through blocking the LuxPQ receptor.63 26 From the forementioned discussion of AI-2 inhibitors it is evident that there is a need for a more extensive development of structural analogs of AI-2 as these molecules are more likely to modulate quorum sensing in a diverse set array of bacteria. 1.6 Objective, hypothesis and specific Aims The objective of this dissertation is to create a superior synthesis of AI-2 which facilitates the design of a large library of analogs. Our hypothesis is that structural analogs will allow for probing into the promiscuity or specificity of quorum sensing proteins. Also since AI-2 is a universal signaling molecule we hypothesize that analogs of AI-2 will have broad range applicability as modulators of quorum sensing or probes to identify new AI-2 receptors. The specific aims are as follows: 1. To develop a new synthesis of AI-2 and construct analogs. Scheme 1.3: Retrosynthetic Analysis of AI-2 2. To compare analog activities to AI-2 activity in several organisms by monitoring: a. Bioluminescence induction/inhibition in V. harveyi b. ?-galactosidase production/inhibiton in E. coli and S. typhimurium c. Pyocyanin production/inhibition in P. aeruginosa 3. To monitor the effect of AI-2 and analogs in mixed bacteria cultures which mimic real life scenarios where bacteria co-habit in an ecosystem with other organisms. 27 1.7 Dissertation Outline Chapter 2 describes the newly developed synthesis of AI-2. This chapter will demonstrate the ability of this new synthesis to obtain a library of diverse C1 analogs as well as C4 and C5 analogs. Chapter 3 describes the evaluation of the newly developed analogs on V. harveyi, E. coli, S. typhimurium and P. aeruginosa. This chapter also describes the evaluation of AI-2 and analogs on mixed cell cultures. Chapter 4 will outline the conclusions, broader impact and future direction of this research Chapter 5 provides the experimental procedure, spectroscopic characterization and biological protocols used. 28 Chapter Two Facile synthesis of AI-2 and a diverse library of analogs 2.1 Introduction: Discovering the chemical identity of AI-2 AI-2 is now considered a universal quorum sensing molecule. Initial genetic studies found that E. coli, S. typhimurium, V. cholerae, V. harveyi and E. faecium all contained the luxS gene which encodes for LuxS, the synthase enzyme for AI-2.46 The luxS gene is located near metK and Pfs, genes encoding proteins known to be involved in the active methyl cycle.36 Scheme 2.1: Biosynthesis of 4,5-dihydroxyl-2,3-pentadiene (DPD) The active methyl cycle is an important S-adenosylmethionine (SAM; 22) metabolic pathway which produces DPD and cysteine (Scheme 2.1).80 A methyltransferase removes a methyl group from the methionine moiety of SAM to form S- adenosylhomocysteine (SAH; 75).36 Next, the Pfs enzyme removes the adenine group producing S-ribosylhomocysteine (SRH; 76).36 SRH is the substrate which is subsequently degraded by LuxS to produce 4,5-dihydroxyl-2,3-pentadione (DPD; 55) and 29 homocysteine.36 As homocysteine is known to be recycled to become cysteine and then methionine, it was proposed that DPD was an interspecies autoinducer responsible for light production in many organisms in addition to V. harveyi whereas HAI-1 (see Figure 1.10; p. 4) is a species-specific signaling molecule.46 AI-2 can be synthesized in vitro using purifed proteins (Pfs and LuxS) and the required substrate (SAH; which is converted to SRH by Pfs).36 It can also be isolated as partial purified AI-2 using chloroform-methanol extraction or a boron affinity column.94 Despite these efforts a chemical synthesis of DPD, the precursor to AI-2, was needed to provide spectroscopic confirmation of the structure of AI-2.36 Ultimately a chemical synthesis of AI-2 would also provide the means to make analogs of AI-2, which could be used to perturb AI-2 signaling in bacteria. 2.2 Previous syntheses of AI-2 Several groups were interested in chemical synthesis of AI-2/DPD80, 87, 92 yet despite its fairly simple framework, DPD is highly functionalized, prone to hydration and polymerization and is unstable to column chromatography.95 Therefore the chemical synthesis of this molecule is non-trivial. 30 Scheme 2.2: Janda's Synthesis of DPD Reagents and conditions: a) oxalyl chloride, DMSO, CH2Cl2; then Et3N; b) CBr4, Ph3P,CH2Cl2; c) tBuLi, MeI, THF; d) 60% AcOH; e) CH(OMe)3(neat), H2SO4(cat.); f) KMnO4, acetone, buffer(aq); g) H2O, pH 6.5 (K2HPO4/KH2PO4 (0.1 M), NaCl (0.15M)), 24 hr. 95 The first synthesis of AI-2, published by Janda in 2004, required seven steps (Scheme 2.2).95 The key step of Janda?s synthesis is a Corey-Fuch reaction to prepare alkyne 78.95 It was later observed that the acetal protecting group was difficult to deblock therefore compound 78 was converted to compound 79 and a subsequent potassium permanganate oxidation of alkyne 79 afforded diketone 80 (Scheme 2.2).95 Acidic cleavage of 80 was monitored by NMR and confirmed for the first time that AI-2 indeed existed as a mixture of the linear, DPD and cyclic products.95 The Mallaird reaction of the dione moiety of DPD and 1,2-phenylene diamine gave one single compound confirming that linear and cyclic species were in equilibrium with each other.95 31 Scheme 2.3: Semmelhack's Synthesis of DPD; Reagents and conditions: a) KIO4, K2CO3, H2O/CH2Cl2 (76% yield) b) Ph3P, CBr4 (67%) c) i.nBuLi ii. H2O (79%) d) i. nBuLi ii. CH3I (64%/99%) e) cat. RuCl2 , NaIO4 (70%) f) pH 1.5 (100%). 96 In 2005, Semmelhack reported a synthesis of AI-2, which was similar to what Janda had reported.96 However Semmelhack utilized a cyclohexylidene protecting group for the diol instead of an acetal group that was used by Janda (Scheme 2.3).96 Also, in Semmelhack?s synthesis, rhodium acetate/sodium periodate was used to oxidize the alkyne moiety into the diketone functionality instead of the harsher potassium permanganate reagent used by Janda.96 The major drawbacks of both Semmelhack?s and Janda?s syntheses are two-fold: 1) They both require several chromatographic separation steps which would not facilitate the rapid generation of analogs; 2) because the diol remains protected throughout their syntheses, the synthesis chemical probes of AI-2 whereby one or both of the alcohol groups are functionalized is not possible by these methods. 32 Scheme 2.4: Vanderleyen Synthesis of DPD a) NH(CH3)2, EtOH; b) CH2=C(CH3)MgBr, Et2O, THF; c) DOWEX 50X8-100, MeOH; d) O3, MeOH, Me2S. 82 Two additional syntheses of AI-2 were reported in 2005.82 The synthesis reported by Vanderleyden required five steps (Scheme 2.4).82 Key steps of Vanderleyden?s synthesis are the nucleophilic addition of a Grignard reagent to amide 88 and a subsequent ozonolysis to access the dicarbonyl functionality of DPD (Scheme 2.4).82 Cleavage of the acetal was performed using DOWEX resin.82 Scheme 2.5: Doutheau Synthesis. Reagents and conditions: (a) THF, DABCO (0.25 equiv), 0oC (b) TBAF (1 equiv), THF, rt. (c) O3, MeOH, -78 oC, then DMS, -78oC to rt.89 Lastly, Doutheau reported a three step synthesis of AI-2 using a Baylis-Hilman reaction and ozonolysis as key steps to access AI-2.89 Although the Doutheau synthesis is shorter than the previously reports, for AI-2 analogs whereby the starting material enone is not commercially available several steps are required to make the starting material, vide vida. 33 2.3 Previous syntheses of AI-2 analogs Scheme 2.6: Synthesis of Trifluromethyl-DPD.88 Despite the obvious need for AI-2 analogs for biological testing, only a handful of AI-2 analogs had been synthesized and investigated for biological activities prior to this work.88 In 2006 Doutheau synthesized trifluoromethyl-DPD in six steps.88 In this work, the key Baylis-Hillman reaction was shown not to be widely applicable, as it resulted in an inseparable mixture of the desired product 96 and the aldehyde dimer 97 (Scheme 2.6).88 Doutheau has also reported that the bis-(O)-acetylated AI-2 derivative is a stable analog of AI-2.87 In biological media the ester groups of this analogue are cleaved to release active AI-2.87 Additionally an AI-2 analog with a tertbutyl ester group was synthesized as well as ethyl-DPD and a 4,5-dihyroxyl-2,3-hexandione compound but these analogs were not tested for biological activity.89 From the foregoing discussions about AI-2?s synthesis and adaptation of the reported methodologies toward the synthesis of AI-2 analogs, it was evident that there was the need for a much simpler synthesis of AI-2 that will be amenable to the rapid synthesis of a large library of diverse AI-2 analogs. 34 2.4 Results: New facile synthesis of AI-2 Scheme 2.7: A new synthesis of AI-2.97 We have developed a very simple synthesis of AI-2, which is amenable to analog synthesis.97 Our synthesis of AI-2 (Scheme 2.7) begins with the condensation of acyldiazomethane (102) with commercially available 2-(tert-butyl dimethylsilyloxy)- acetaldehyde (91).97 The acyldiazomethane was formed via the reaction of acetyl chlorides with diazomethane.98 Nucleophilic addition of diazo compounds to aldehydes has previously been achieved through deprotonation of the diazo functionality by a strong base such as LDA or NaOH.99 Our initial attempts using LDA to deprotonate acyldiazomethane proved problematic, presumably due to the decomposition of the lithiated diazo intermediate. Purification of the product was also challenging because several side products were formed. In search of a milder method, we employed the DBU-catalyzed condensation of diazo compounds and aldehydes, first reported by Wang.100 This facile conversion was conducted at room temperature and gave the TBS- protected hydroxydiazo: 5-(tert-butyldimethylsilyloxy)-3-diazo-4-hydroxypentan-2-one in 58% yield (not shown). 35 Scheme 2.8: Resonance states of acyldiazo Subsequently a TBAF deprotection of the silyl group was conducted to give the diazodiol 103.97 Analysis by 13C NMR indicated that this diol remained in the linear form; there was an absence of signal between 100 and 120 ppm, which is indicative of lactols and a ketone peak at 192 ppm (See Experimental Section). This is most likely due to the fact that the diazo carbonyl is less electrophilic because of resonance contributor 106 (Scheme 2.8). Also an IR stretch of 2135 cm-1 indicated that the diazo functionality remained intact after treatment with TBAF. During the course of our research we learned that we could conduct the TBAF deprotection without purification of the nucleophilic addition product. We therefore performed the nucleophilic addition and deprotection successively and obtained a yield of 50% over two steps.97 The formation of compound 103 set the stage for a facile oxidation to DPD. Due to its instability to column chromatography, in any successful synthesis of AI-2, the last step must involve the use of reagents that are readily removed. For the oxidation of diazodiol 103 to DPD, we strategically chose the highly reactive dioxirane because it is volatile and easily removed.97 Dioxirane was prepared as a concentrated solution in acetone and added to the diazodiol 103.97 Upon disappearance of starting material, the excess reagent and acetone were evaporated.97 1H NMR showed an 36 equilibrium mixture of compounds, as expected for the interconverting isomers of AI-2 (See Figure 2.1).97 Our NMR data for synthetic AI-2 is identical to literature data.80, 87, 92 Figure 2.1: H1 NMR of equilibrium mixture of compounds derived from DPD *(indistinguishable mixture of Hs) a d e bc 37 Scheme 2.9: Condensation of DPD with 1,2 phenylenediamine to form quinoxaline Finally the reaction of our synthetic AI-2 with 1, 2-phenylenediamine gave quinoxaline 107 as one species in 1H NMR (See Figure 2.2).97 Figure 2.2: H1 NMR of quinoxaline derivative of AI-2 Our concise synthesis differs from all other published approaches due to the clean final step.97 DPD synthesized using this method was stable at room temperature for at least 4 weeks and stable upon refrigeration for several months. e d c b a 38 2.5 Synthesis of C1 analogs of AI-2 Figure 2.3: Library of diverse C1- analogs of AI-2.97, 101 With this facile synthesis in hand, we proceeded to synthesize 22 AI-2 analogs with branched, cyclic and aromatic as well as linear alkyl groups at the C1 position (108- 129; Figure 2.3).97 Janda?s AI-2 synthesis is not amenable to the synthesis of analogs with branched alkyl groups due to the difficulty of alkylation with secondary or tertiary groups.90 AI-2 analogs with branched and cyclic alkyl groups could provide important insights into the constraints of the active site in AI-2 receptor proteins such as LuxP and LsrB as well as other known processing enzymes (LsrK and LsrR).41 Since there are 39 several commercially available acid chlorides, we were able to prepare diazo carbonyls with various alkyl groups via the reaction of diazomethane and acid chlorides.98 Desired alkyl groups whose acid chlorides were not commercially available were obtained from the carboxylic acid compounds. After the diazo carbonyl was acquired, synthesis of this diverse set of analogs followed the previously described method.97, 101 Our new synthesis of AI-2 provided the route needed to access C1 analogs of AI-2 (Compounds 108-129, see Figure 2.3). These were synthesized using various commercially available acid chlorides without difficulty and without the need for any alterations in our synthetic strategy. The one-pot condensation-deprotection step was accomplished with moderate yields for analogs (108-129). NMR analysis of these analogs (108-129) showed an equilibrium mixture of linear and cyclic analogs with exceptions being observed with neopentyl-DPD (118) and isobutyl-DPD (116). The proton NMR of both neopentyl-DPD (118) and isobutyl-DPD (116) in D2O indicated that these analogs existed predominantly as linear forms. Also a single species was observed in H1 NMR of DPD analogs in chloroform. Differences in the equilibrium ratios of DPD and cyclic forms of different analogs may have the potential to affect the biological profile of analogs. 40 2.6 Synthesis of C4 and C5 analogs of AI-2 Scheme 2.10: New synthesis of diacetate AI-2 and analogs Although our synthesis of AI-2 is mild, due to the chemical reactivity of AI-2, purification cannot be achieved. Therefore we endeavored to synthesize ester-protected AI-2 and analog derivatives, since this modification would allow purification to be preformed on silica gel. Using our new methodology, we are able to synthesize the diacetate analog of AI-2 by treating diazodiol 130 with acetic anhydride to give 131 (Scheme 2.10). Dioxirane oxidation of this compound gave diacetate DPD analog (132a) which remained in the linear confirmation. Acetate protection was also conducted using select analogs (hexyl (132b) and isobutyl (132c)). These analogs would be used to observe the effect of protecting the diol on biological activity (See Section 3.10). Also protection of AI-2 (methyl-DPD; 55) and hexyl-DPD (112) with different ester groups were also conducted (see Supplementary S8). Biological activity of these variants was also investigated (Section 3.10). NMR analysis of ester-protected analogs was much cleaner than free DPD analogs. As expected blocking the C4 and C5 hydroxyl groups prevents cyclization, therefore only one species was observed in both H1 and C13 NMRs spectra of the compounds. 41 Since AI-2 is known to dimerize at high concentrations, these ester-protected analogs could also provide a way to store AI-2 and analogs for prolonged periods without dimerization. To test this hypothesis, ester analogs of AI-2 were stored at different temperatures for up to four weeks and biological testing was conducted on analogs to determine their stability under these conditions. Results showed that both free and ester- protected analogs maintained their biological profile after 4 weeks. However, these acetate analogs were found to be slightly less active than free analogs (See Supplementary Figure S1). Therefore the instability of AI-2 may be the result of harsh preparation methods by different synthetic routes or concentration. Scheme 2.11: Synthesis of deoxy-AI-2 analogs.101 Additional variations of the aldehyde used with our method provided 5-deoxy- analogs of DPD (Scheme 2.11).101 Briefly diazocarbonyls were reacted with acetaldehyde to give 133.101 Dioxirane oxidation of 133 gave 134 as deoxy-analogs of DPD.101 Although deoxy-analogs are essential ?locked? in the linear form, H1 NMR revealed that 42 the two carbonyl groups are readily hydrated. Synthesis of deoxy-AI-2 as well as other select analogs allowed the biological probing of the importance of the C5 hydroxyl group to be conducted (see Section 3.5).101 2.7 Discussion Six syntheses of AI-2 have been presented since 2004, yet several years lapsed before this research lead to the introduction of a library of analogs of AI-2. This is evidence that published syntheses are too lengthy and do not provide easy access to AI-2 or analogs. Elaboration of the C1 position of AI-2 using either the Janda or Semmelhack protocol requires an alkylation step via an SN2 displacement reaction. 90 This places some constraints on the type of C1 analogs that can be readily obtained via these methods. Furthermore both Janda and Semmelhack?s methods protect the diol unit in AI-2 with an acetal protection and this does not allow easy variation at the C4 or C5 position. Additionally as evident in Janda?s synthesis the acidic cleavage of the diol moiety is not always straightforward.95 In Semmelhack?s synthesis although the cyclohexanone by- product is shown not to effect cell growth; it remains to be seen if this by-product effects bioluminescence or can be internalized and/or processed by quorum sensing proteins.96 Finally Doutheau?s synthesis though concise and keeps the diols free, requires starting enones that are not readily available.89 Moreover the Baylis-Hillman reaction can be problematic when attempting to synthesize analogs.88 Other recently published syntheses require several chromatographic separation steps (>3) and have not be demonstrated to produce analogs.89 43 2.8 Conclusion In conclusion, our new synthesis of AI-2 is short and the most amenable to analog synthesis.101 Access to analogs of various shapes and sizes will allow the specificity or promiscuity of quorum sensing proteins to be deduced. Not only does our synthesis allow for the rapid development of a large library of C1 analogs, but is also capable of constructing analogs with variations at the C4 and C5 position. Furthermore this synthesis is shown to make stable AI-2 and analogs have been shown to remain active at various temperatures. (See Supplemental Figure S1) This new synthesis has and will continue to aid in the discovery of new protein targets for anti-quorum sensing chemotherapies. 101 44 Chapter Three: Biological evaluation of analogs in V. harveyi, E. coli, S. typhimurium and P. aeruginosa 3.1 Bioluminescence Quorum sensing was first discovered in the marine bacterium V. fischeri, which produces light when in a symbiotic association with the Hawaiian Bobtail squid.4a It was observed that light production, known as bioluminescence, was highly dependent on cell density and researchers soon discovered that bacteria could coordinate gene expression through the detection of signaling molecules. This phenomenon was termed quorum sensing.102 Bioluminescence is caused by an enzyme-catalyzed reaction, which is controlled by the lux (luciferase) gene operon.46b Light is produced by the oxidation of a reduced flavin mononucleotide (FMNH2) reacting with molecular oxygen and a fatty aldehyde. 46b This reaction results in the emission of blue-green light at 490 nm.46b Bioluminescence occurs in several other species of the Vibrio genera as well as the Xenorhabdus, Photobacterium, and Shewanella generas.46b Different substrates and proteins control light production in each bioluminescent organism with the only similarity being the use of molecular oxygen.46b The lux operon is usually made up of luxCDABE genes required for the synthesis of the luciferase (luxAB) and aldehyde substrate.46b The aldehyde substrate is formed via a fatty acid reductase encoded by luxCDE required to convert polypeptides to the fatty aldehyde.46b The lux operon of V. fischeri consists of luxRICDABEG with the additional genes: luxR, luxI and luxG.46b luxG has been proposed to encode the flavin reductase responsible for synthesizing the reduced flavin 45 mononucleotide.46b We now know that luxI and luxR encode the synthase (LuxI) and receptor (LuxR) of the AHL autoinducer.9 After quorum sensing was first discovered in V. fischeri, researchers sought to understand the quorum sensing process in other organisms. Scheme 3.1: Mechanism of bioluminescence production with riboflavin as the luciferin V. harveyi, another marine organism which uses quorum sensing to regulate bioluminescence, uses a quorum sensing system that greatly differs from that of V. fischeri and other gram-negative bacteria. In V. harveyi the lux operon consists of luxCDABEGH, with the additional gene luxH encoding for the synthesis of the riboflavin precursor, 3,4-dihydroxy-2-butanone-4-phosphate.46b This indicates that riboflavin is the luciferin (luciferase substrate) in V. harveyi.46b Interestingly, the lux operon of V. harveyi does not contain genes encoding for LuxI and LuxR.11 This is due to the fact that V. harveyi does not use the LuxI-LuxR system for quorum sensing like V. fischeri.11 Instead a region separate from the luxCDABEGH operon has been identified in V. harveyi, which also controls the luminescence phenotype. This region contains the regulatory gene luxR (which has no homology to the luxR of V. fischeri),47b which is a key transcriptional 46 factor required for expression of the luxCDABEGH operon.46b LuxR acts as both an activator and repressor of quorum sensing-controlled processes; it activates bioluminescence, while repressing type III secretion factors. 11, 45a LuxR is controlled by a phosphorelay mechanisms involving a series of proteins which are switched on at high cell density (See Section 1.4).11 Figure 3.1: Dimer of 2 LuxPQ complexes bound to 2 molecules of AI-2 (LuxP (green) bound to AI-2 and periplasmic domain of LuxQ (red); LuxP?(cyan) bound to AI-2 and periplasmic domain of LuxQ? (magenta)) As previously described, the S-THMF-borate isomer is the active AI-2 found bound to LuxP in V. harveyi.37 The presence of boron is likely due to the marine environment in which V. harveyi resides, where boron concentrations can reach up to 0.4 mM.11 The AI-2 pathway of V. harveyi is quite unique because its receptor exists as a dimer (LuxPQ).47a Crystallography and mutagenesis of this LuxPQ complex have been studied and much insight is now known about the role of this interesting pair in controlling quorum sensing. It is known that LuxQ is in a complex with LuxP even at 47 low cell density.47a Also certain contacts intrinsically exist between LuxQ and LuxP, which inhibits the conversion of LuxQ from kinase to phosphatase.47a These contacts are disrupted upon binding of AI-2.47a Additionally, it has been revealed that two LuxPQ dimers merge in the presence of AI-2 and exist at a 140 degree angle to each other.103 The formation of this tetramer results in the release of LuxQ, switching it from kinase to phosphatase, and therefore turning quorum sensing ?on?.103 Since these conformational changes are vital for AI-2 to mediate quorum sensing, it may be possible to design small molecules which interfere with these changes and perturb the signaling pathway. LuxP and analogous phosphorelay mechanisms are found in most Vibrios11 Also LuxR homologs have been discovered in V. cholera (HapR)104, V. angullarium (VanT)105, V. parahaemolyticus (OpaR)106, V. vulnificus (SmcR) 107 and V. fischeri (LitR)108. It is likely that homologous proteins exist in organisms in which quorum sensing circuits have not yet been defined. Therefore investigations into how small molecules modulate the bioluminescence production of V. harveyi by targeting LuxR and other proteins could have applications in other human and fish pathogens. 48 3.2 Synergistic agonism in V. harveyi Table 3.1: V. harveyi strains and genotypes 47b,102 V. harveyi strains Relevant genotype and/or property BB120 Wild type BB170 Wild type luxN::Tn5 (sensor 1-, sensor 2+); AI- 1+, AI-2+ MM32 luxN::Tn5; luxS::Tn5 (sensor 1-, sensor 2+); AI-1+, AI-2- BB886 Wild type luxPQ::Tn5 Kan BB721 Wild type luxO::Tn5 V. harveyi bioluminescence induction is often used as a reporter of AI-2 signaling activity.102 After the development of our facile synthesis, we sought to investigate how C1 analogs of different sizes and shapes modulate the quorum sensing circuit of V. harveyi through the monitoring of bioluminescence induction.97 Initial screenings for bioluminescence induction in V. harveyi were conducted on a diverse subset of the entire C1 analog library created.97 Since the LuxN/HAI-1 pathway is the dominant signaling pathway in V. harveyi109 it is important to suppress detection of HAI-1 by LuxN. Also in order to control the amount of AI-2 in this model system, AI-2?s synthase, LuxS, must be inactivated. Therefore a LuxN, LuxS double mutant strain (designated MM32) was used for testing. At high concentrations (50?M) only ethyl-DPD (108; Figure 2.3) and cyclopropyl-DPD (119; Figure 2.3) were able to induce bioluminescence after 8 hours albeit to a 10-fold lower degree than AI-2 (Figure 3.1a).97 The other analogs tested were not able to induce luminescence even at this high concentration.97 At lower 49 concentrations (2?M) none of the analogs significantly induced bioluminescence (Figure 3.1b).97 Ironically in the presence of 12nM AI-2, analogs were found to synergize the action of AI-2.97 Ethyl-DPD (108; Figure 2.3) and cyclohexyl-DPD (122; Figure 2.3) gave the most pronounced synergistic agonism causing 4.3-fold and 9.1-fold activation, respectively.97 The remaining analogs gave moderate enhancement: propyl-DPD (109; 2.6 fold), butyl-DPD (110; 2.7 fold), isopropyl-DPD (114; 2.4 fold), tert-butyl-DPD (115; 2.9 fold) and cyclopropyl (119; 3.1 fold) (See Figure 2.3 for structures).97 Other researchers90, 110 have reported similar synergistic agonism however the observation of synergism in analogs of a range of shapes and sizes suggest promiscuity in the quorum sensing proteins responsible for bioluminescence induction in V. harveyi.97 50 Figure 3.2: a) Bioluminescence induction in V. harveyi MM32 (at 8hrs) by addition of 2?M analog, 12nM AI-2 and 100?M boric acid b) Bioluminescence induction in V. harveyi MM32 by addition of 50?M analogs and 100?M boric acid.97 In order to shed light on the mechanism of synergistic agonism by AI-2 analogs, bioluminescent strains with deletions in select proteins involved in signal transduction in V. harveyi have been tested (For a detailed scheme of the AI-2 signaling pathway in V. harveyi see Supplementary Figure S11, p121). V. harveyi BB886 is a LuxQ mutant strain which cannot respond to AI-2.102 Therefore any modulation observed in this strain would be the result of analogs acting on the AI-1 pathway. Since analogs were not able to induce bioluminescence in this strain, it is probable that synergistic agonism is derived in the AI-2 pathway. (See Supplemental Figure S2) Another possible target of synergistic bioluminescence is the transcriptional regulator LuxR. Small molecules have been shown to bind LuxR and destabilize its interaction with the lux operon.16 Therefore it is plausible to suggest that C1 analogs could also bind LuxR, and enhance stabilization, 51 resulting in synergism with AI-2. V. harveyi BB721, is a LuxO mutant strain and since LuxO indirectly represses luminescence through LuxR this strain is always bright.47b If C1 analogs target proteins downstream of LuxO, incubation with V. harveyi BB721 should give the same synergistic response in this mutant (BB721). No synergism was observed with C1 analogs in this strain (See Supplemental Figure S3). Although these observations suggest that the target of C1 analogs is upstream of LuxO, more experimentation is needed to definitively determine the mechanism of synergistic agonism. 3.3 Discussion-V. harveyi Synergistic agonism by C1 analogs of AI-2 provides further insight into the well known quorum sensing circuit of V. harveyi. Mutant bioluminescent strains showed that deletion of LuxQ abolished the observed synergisms. Also a lack of response following the mutation of the response regulator, LuxO, suggests that analogs do not bind LuxR. These findings point to the action of C1 analogs being at the LuxPQ level. Analogs are unable to induce bioluminescence on their own. This may lead to the assumption that these molecules do not bind LuxPQ effectively. However it is possible that analogs bind LuxPQ allosterically and sensitize LuxP to AI-2. Mutagenesis at the LuxP:LuxQ interface resulted in sensitizing LuxP to lower AI-2 concentrations.47a Therefore, it is possible that C1 analogs similarly bind to one active site of the LuxPQ dimer while AI-2 binds to the other active site. The conformational change that results from this hetero- ligand binding probably leads to a more efficient kinase activity by LuxQ. Future investigations are needed to determine the actual origin of synergistic agonism. 52 3.4 Quorum sensing in enteric bacteria Table 3.2: Enteric bacteria strains and genotypes 101,39 E. coli strains Relevant genotype and/or property W3110 Wild type LW7 W3110 ?lacU160-tna2 ?luxS::Kan ZK126 W3110 ?lacU169-tna2 LW8 ZK126_lsrR::Kan LW9 ZK126 ?(lsrACDBFG)::Kan SH3 W3110 ?lacU160-tna2 ?luxS ?lsrK ::Kan; Cm S. typhimurium strains MET708 rpsl putRA :: Kan-lsr-lacZYA luxS::T-POP MET715 rpsl putRA :: Kan-lsr-lacZYA In addition to bioluminescence induction in Vibrios, the AI-2 signaling pathways of enteric bacteria (i.e. E. coli and S. typhimurium) are well characterized.41 The lsrACDBFGE operon controls the expression of the proteins involved in the transport and processing of AI-2 in these organisms.39 The lsr operon is AI-2-dependent hence the designation LuxS-regulated (lsr).39 In addition to transport proteins, the lsr operon divergently transcribes genes which encode LsrK, the AI-2 kinase and LsrR, the transcriptional repressor.39 LsrR plays a vital role in the biofilm architecture of E. coli.108 It was reported that a LsrR mutant strain of E. coli produced biofilm which was structurally different from biofilm produced in the wild-type strain.74, 108 Investigations 53 into quorum sensing controlled expression of the lsr operon could contribute to the development of anti-biofilm treatments through small molecules which target LsrR. 3.5 Inhibition and processing in enteric bacteria In addition to V. harveyi bioluminescence, we investigated the effect of diverse linear and branched C1 analogs on the uptake and processing of AI-2 in enteric bacteria.101 Since lsr is under AI-2 mediated- quorum sensing control, we used lsr-lacZ reporter strains to observe modulation of ?-galactosidase production by AI-2 analogs. The lacZ gene expresses the enzyme ?-galactosidase, which in nature cleaves lactose into glucose and galactose. lacZ is often fused to gene operons where an analog of lactose, called o-nitrophenyl-?-galactoside (ONPG), is used as a colometric indicator of operon activity.111 Once the target operon is activated and lacZ is expressed, ?-galactosidase is produced.111 If ONPG is introduced into the system, it is cleaved and generates o- nitrophenol.111 O-nitrophenol is yellow and has a UV absorbance at 420 nm.111 Therefore the intensity of o-nitrophenol detected is proportional to the amount of ?-galactosidase produced. Since the lsr operon is under quorum sensing control, bacterial strains containing the lsr-lacZ fusion, produces ?-galactosidase in response to AI-2. Conversely small molecules which interrupt AI-2 mediate lsr expression will not produce ?- galactosidase. Initial results using LuxS mutant strains in both E. coli and S. typhimurium showed that only ethyl-DPD (110; Figure 2.3) was able to activate transcription of lsr on its own101 (See Supplementary Figure S5). Next we tested our analogs for their ability to antagonize the AI-2 signaling.101 In bacterial strains which produced their own AI-2 54 (LuxS+) several analogs were able to compete with AI-2 in E. coli including all linear analogs with chain-length greater than 2-carbons (propyl-DPD (109), butyl-DPD (110), pentyl-DPD (111), hexyl-DPD (112), and heptyl-DPD (113); see Figure 2.3 for structures) and several branched analogs (isopropyl-DPD (114), isobutyl-DPD (116), secbutyl-DPD (117), neopentyl-DPD (118)).101 Ironically in the LuxS+ S. typhimurium reporter strain, fewer linear analogs (butyl-DPD; 110 and to a lesser extent propyl-DPD; 109) and a single branched analog (isobutyl-DPD; 116) were able to antagonize AI-2 signaling.101 Therefore isobutyl-DPD (116)was identified as a potent inhibitor of both E. coli and S. typhimurium quorum sensing.101 Identical results emerged when exogenous AI-2 was added to LuxS mutant strains in the presence of the C1 analogs.101 55 Figure 3.3: AI-2 dependent ?-galactoside production in E. coli ZK126 pLW11 and S. typhimurium MET708 (both luxS + ) in response to a) linear analogs and b) branched and deoxy analogs.101 To further understand the mechanism by which analogs cause inhibition of quorum sensing, we tested their ability to be processed by the AI-2 internalization and phosphorylation proteins, LsrB and LsrK, respectively.101 A lacZ reporter strain lacking the transporter, LsrB, was tested for activity in the presence of AI-2 and analogs in E. coli.101 Ironically, both agonists (methyl-DPD; 55 and ethyl-DPD; 108) and antagonists (C3 and greater linear; branched analogs) remained effective in modulating quorum sensing in the absence of the AI-2 transporter (See Supplementary Figure S6).101 It is possible that analogs are able to freely diffuse into the cell or use alternative transporters. Also in vitro phosphorylation of AI-2 and analogs using radio-labeled ATP were 56 conducted to test whether analogs were able to be phosphorylated by LsrK.101 TLC shows that all DPD-analogs are phosphorylated whereas deoxy-analogs, which lack a hydroxyl group at the C5 position (See Section 2.6 for structure of deoxy-analogs) are not phosphorylated (Figure 3.4).101 This confirms Bassler?s assignment of phospho-AI-2 as having the phosphate group on the C5 hydroxyl rather than the C4 hydroxyl.41 Interestingly, even analogs which do not antagonize AI-2 signaling are able to be phosphorylated by LsrK.101 Therefore phosphorylation must not be the only determinant which controls inhibition of the lsr operon. Figure 3.4: a) Phosphorylation of DPD by LsrK in the presence of ATP b) Representative radioactive TLC analysis of LsrK mediated phosphorylation. ATP, AI-2, Butyl-DPD, Isobutyl-DPD and deoxy-Isobutyl treated with LsrK for 2hrs. 101 57 Binding of phospho-AI-2 to the transcriptional repressor, LsrR is key to the expression of genes in the quorum sensing system of enteric bacteria.41, 110 Phospho-AI-2 has been shown to bind LsrR and subsequently destabilize its interaction with the lsr operon promoter region therefore de-repressing the operon.112 We predict that it is the phosphorylated form of ethyl-DPD (108), which causes agonism through destabilization of the LsrR-operon complex. To support this notion, reporter strain lacking LsrK did not show agonism (Figure 3.5).101 Figure 3.5: AI-2 dependent ?-galactoside production in E. coli SH3 (LsrK-, LuxS-) and E. coli LW7 (LuxS-) in response to ethyl-DPD.101 58 . Figure 3.6: AI-2 dependent ?-galactoside production in E. coli ZK126 (LsrR+) and E. coli LW8 (LsrR-) in response to methyl-DPD (AI-2), butyl-DPD, isobutyl-DPD and deoxy-isobutyl-DPD.101 Likewise, phosphorylated forms of antagonists probably bind LsrR and stabilize its interaction with the lsr operon, sustaining its repression of quorum sensing-controlled genes. Screening of the most potent inhibitors (butyl-DPD; 110 and isobutyl-DPD; 116) in strains lacking the LsrR repressor revealed that inhibition was not observed (Figure 3.6).101 This indicates that inhibition of the lsr operon occurs through LsrR. 59 Figure 3.7: a) AI-2 dependent ?-galactoside production in S. typhimurium MET708 b) AI-2 dependent bioluminescence production in V. harveyi BB170 and c) Flow cytometry analysis of AI-2 dependent GFP induction in E. coli W3110 pCT6 (all strains are luxS + ) in response to isobutyl-DPD and isopropyl-DPD.101 It is well known that AI-2 signaling in one species can be detected and interfere with the quorum sensing network of another unrelated species.42 Thus we postulated that inhibitors of AI-2 signaling would be able to perturb several quorum sensing systems simultaneously. To test this hypothesis, we examined our most potent analog isobutyl- DPD (116), in a tri-species synthetic ecosystem.101 This synthetic ecosystem was strategically designed so that the response of each organism to AI-2 was uniquely identifiable and could be quantified separately.101 The following responses were utilized: 60 ?-galactoside production in S. typhimurium MET708, GFP induction in E. coli W3110 pCT6, and bioluminescence induction in V. harveyi BB170.101 As anticipated isobutyl- DPD (116) was a potent inhibitor of E. coli GFP induction and S. typhimurium ?- galactosidase production in the synthetic ecosystem (Figure 3.7a and 3.7c).101 Surprisingly isobutyl-DPD (116) was also able to inhibit V. harveyi bioluminescence (Figure 3.7b).101 Although C1 analogs of AI-2 are known to cause synergistic agonism of bioluminescence in AB media97, it has been shown that once a quorum is already formed they act as antagonists.101 This antagonism is only observed in LM media.101 Since bioluminescence is highly dependent on environment, we assume that components of the media may change the observed effect of analogs. Therefore isobutyl-DPD (116) has emerged as a broad-spectrum inhibitor of quorum sensing. While isobutyl-DPD (116) showed broad inhibition, isopropyl-DPD (114) was found to be a selective inhibitor of E. coli which did not effect S. typhimurium (Figure 3.7a and 3.7c).101 Selectivity is of great benefit when pathogenic bacteria exist in a niche where symbiotic organisms exist. Similar to isobutyl-DPD, isopropyl-DPD was also found to inhibit V. harveyi bioluminescence (Figure 3.7b).101 Therefore we have found both a broad and a selective inhibitor which were effective in a mixed culture environment.101 3.6 Discussion- E. coli and S. typhimurium Three proteins have been identified as key AI-2 signaling in enteric bacteria, LsrB, LsrK and LsrR and may act as checkpoints when AI-2-like molecules are introduced into the system.101 For the first checkpoint, we?ve shown through an LsrB 61 mutant that analogs are able to enter the bacterial cell through diffusion or alternative transporters.101 AI-2 has been shown to be transported by the ribose binding protein RbsB in other organisms.55 Therefore it is plausible that AI-2 analogs also use this apparatus as an alternative entry into the cell or perhaps they can freely diffuse into the cell.101 For the second checkpoint, in vitro phosphorylation was shown in all analogs.101 Moreover deoxy-analogs were not phosphorylated indicating that the hydroxyl group on the C5 position is necessary for phosphorylation.101 Also we?ve confirmed that phosphorylation is required for gene expression, as the LsrK mutant was unable to activate the lsr operon. 101 From this observation we can assume that it is the phosphorylated form of analogs which cause agonism or antagonism of the lsr operon. 101 Finally lack of inhibition in LsrR mutants indicate that LsrR is the target of C1 analogs and the various biological profiles of the analogs are likely due to varying binding affinities for LsrR. We predict that the C1 alkyl chains interact with LsrR and cause the protein to bind to the DNA promoter region of the lsr operon to different extents. 101 Since a minimum 3-carbon chain length is required for antagonism we can assume the side chain binds to a hydrophobic region and causes a stronger LsrR-DNA complex to form. 101 In contrast to AI-2 (methyl-DPD; 55) and ethyl-DPD (108) cause LsrR not to bind and therefore de- represses the operon. 101 The differences observed in quorum sensing modulation in S. typhimurium and E. coli indicate that subtle differences may exists which make the S. typhimurium system more robust. Interestingly several linear and branched analogs were able to repress lsr transcription in E. coli while only butyl- and isobutyl-DPD were effective in S. typhimurium.101 These findings were unexpected since the two quorum sensing systems 62 are homologous.101 Alignment studies have shown that LsrK and LsrR proteins in these organisms share 82% and 77% homology, respectively.101 Also the E. coli LsrR binding site showed 83% homology to the S. typhimurium promoter region.101 In addition predicted secondary and tertiary structures show similar folds for these organisms (Figure 3.8).101 Further studies are needed to determine why these systems show divergent response to C1-analogs. Figure 3.8: Predicted tertiary structures of the LsrR proteins of S. typhimurium (green) and E. coli (cyan) provided by ESyPred3D 101 The observation that analogs of AI-2 can be either broad- or selective-inhibitors in mixed cultures is medically relevant since bacteria rarely exist in isolation.113 Therefore quorum sensing targets which remain effective in mixed cultures that most resembles natural environments are valuable. Overall, linear and branched analogs gave much insight into the ability of AI-2 like molecules to be processed by the AI-2 machinery of enteric bacteria. 63 3.7 Pseudomonas aeruginosa P. aeruginosa is an opportunistic pathogen which causes biofilm-related lung infections in cystic fibrosis patients.114 P. aeruginosa quorum sensing uses the typical LuxI-LuxR-type system found most often in gram-negative bacteria.7 Quorum sensing has been shown to control biofilm in this organism as mutant strains with alterations in the quorum sensing pathway are deficient in biofilm formation.114 Biofilm formation was restored upon addition of the signaling molecule, N-3-oxo-dodecanoyl-homoserine lactone (See Figure 1.1; 6).114 This observation suggests that N-3-oxo-dodecanoyl- homoserine lactone (6) is vital for biofilm formation and that small molecules which can compete with this signaling molecule may be able to prevent biofilm formation.114 Several small molecules analogs of AHLs have been identified which modulate Pseudomonas biofilm formation yet they face solubility and stability issues.25-26 Therefore there is a need for structurally diverse small molecules which target quorum sensing and are able to clear P. aeruginosa associated biofilm infections. AI-2 is not synthesized in P. aeruginosa nor have any AI-2 receptors been identified in this organism.83 Genetic analysis has shown that AI-2 up-regulates some genes required for Pseudomonas pathogenesis.83 This is an interesting phenomenon as AI-2 has been suggested to be a universal signaling molecule and therefore able to be sensed by an array of bacteria. C1 analogs of AI-2 contain side chains which resemble the side chains found in AHLs. It has been suggested that these analogs freely diffuse through the cell in a manner similar to how AHLs diffuse into the cells and directly bind the LuxR-type transcription proteins required for quorum sensing in gram-negative bacteria. If AI-2 64 analogs are AI-1-like and therefore able to affect the AI-1 pathway of gram-negative bacteria in addition to the AI-2 pathway of enteric bacteria, this dual action is another means of universally affecting quorum sensing. 3.8 P. aeruginosa pyocyanin production modulation Table 3.3: P. aeruginosa strains, genotypes and references P. aeruginosa strains Relevant genotype and/or property PAO1 Wild type AHL analogs of various shapes and sizes have been found to modulate quorum sensing and biofilm formation in P. aeruginosa.10 Likewise we decided to screen our entire library of C1 analogs including cyclic and aromatic (See Figure 2.3; 108-129) on P. aeruginosa. Figure 3.9: P. aeruginosa virulence factor pyocyanin 65 Figure 3.10: Pyocyanin production in P. aeruginosa PAO1 in response to methyl-DPD, ethyl- DPD, heptyl DPD, isobutyl-DPD, cyclopentyl-DPD and phenyl-DPD The effect of analogs on the production of a virulence factor, pyocyanin, is an interesting method to evaluate the effect of small molecules on this quorum sensing- controlled process. Pyocyanin is an aromatic compound which absorbs at 540 nm and produces a blue-green pigment (Figure 3.9).115 In the wild-type strain PAO1, only a few C1 analogs were found to reduce pyocyanin production. Several linear analogs moderately inhibit pyocyanin production while some cyclic and aromatic analogs are more effective. A panel of analogs are shown in Figure 3.10. Heptyl-DPD (112), cyclopentyl-DPD (121) and phenyl-DPD (125) (see Figure 2.3 for analog structures) were the most effective modulators of pyocyanin production (See Supplementary Figure S6 for biological profile of analogs in P. aeruginosa). In addition the cyclic and aromatic analog subsets were screened in E. coli and S. typhimurium for their ability to effect the lsr operon. Initial agonism assays showed that 0 20 40 60 80 100 120 140 160 C el ls o nl y M et hy l E th yl H ep ty l Is ob ut yl C yc lo pe nt yl P he ny l % Py ocy an in p ro du ct io n 66 none of the cyclic or aromatic analogs were able to induce gene expression on their own (See Supplementary Figure S7). Next the analogs were screened in the presence of synthetic DPD in order to determine if any analogs act as antagonists. 0 20 40 60 80 100 120 140 160 C el ls + M et hy l-D P D O nl y Lu xS- C el ls Is ob ut yl C yc lo pr op yl C yc lo bu ty l C yc lo pe nt yl C yc lo he xy l C H 2- C yc lo he xy l C yc lo he pt yl % R es po ns e (B et a- ga la ct os id as e Ac tiv ity) E. coli S. typhimurium a) 67 Figure 3.11: AI-2 dependent ?-galactoside production in E. coli LW7 and S. typhimurium MET715 (both luxS - ) in response to 40 ?M synthetic DPD and a) cyclic analogs and b) aromatic analogs. Of the cyclic analogs only cyclopentyl-DPD (121) significantly antagonized AI-2 activity while cyclobutyl-DPD (120) gave minimal inhibition in E. coli (see Figure 3.11). Larger cyclic analogs (cyclohexyl-DPD (122), CH2-cyclohexyl-DPD (124) and cycloheptyl- DPD (123); see Figure 2.3) all did not give significant knockdown. Therefore AI-2 processing enzymes may be unable to accommodate large groups at the C1 position greater than cyclopentyl-DPD (121). Also since 5-membered rings are more flexible than 3- and 4- membered rings, a desired conformation may be required for inhibition that is inaccessible to the more strained cyclopropyl-DPD (119) and cyclobutyl-DPD (120) analogs. None of the aromatic analogs were able to modulate quorum sensing in E. coli 0 20 40 60 80 100 120 140 C el ls + M et hy l-D P D O nl y Lu xS- C el ls Fu ra no sy l P he ny l M et ho xy ph en yl Fl ur op he ny l N itr op he ny l % R es po ns e (B et a- ga la ct os id as e Ac tiv ity) E. coli S. typhimurium b) 68 or S. typhimurium including the 5-membered aromatic analog, furanoyl-DPD. It is likely that some degree of flexibility is required at the C1 position for analogs to be processed, which the flat aromatic compounds lack. Cyclopentyl-DPD (121) was not effective at inhibiting S. typhimurium lsr expression. This is consistent with our previous finding that the two enteric bacteria show differing levels of susceptibility.101 Further studies and molecular modeling will be conducted to determine if cyclic and aromatic groups are incompatible with AI-2 processing enzymes due to their bulkiness and rigidity. Figure 3. 12: a) AI-2 dependent ?-galactoside production in S. typhimurium MET708 b) Pyocyanin production in P. aeruginosa PAO1 and c) Flow cytometry analysis of AI-2 dependent RFP induction in E. coli W3110 pCT6 dsRed (all strains are luxS + ) in response to isobutyl-DPD, phenyl-DPD and a cocktail containing both isobutyl-DPD and phenyl-DPD. 69 A new synthetic ecosystem was constructed using E. coli, S. typhimurium and P. aeruginosa. As previously discussed, in order to decipher the response of each bacterium different reporters were used: AI-2 induced ?-galactoside production in S. typhimurium MET708, pyocyanin production in P. aeruginosa PAO1 and AI-2 dependent RFP induction in E. coli W3110 pCT6 dsRed. Previously isobutyl-DPD (116) was found to be an inhibitor of E. coli, S. typhimurium and V. harveyi yet it is not able to inhibit pyocyanin production. Phenyl-DPD (125) is a potent inhibitor of pyocyanin production, therefore we developed a cocktail containing these two complementary analogs. This cocktail was able to knockdown quorum sensing in all three organisms of this new synthetic ecosystem. The analog cocktail approach is a new avenue to simultaneously perturbing the quorum sensing system of several organisms to be further explored. 3.9 Discussion- P. aeruginosa These results show that expanding the diversity of groups at the C1 position of AI-2 to include cyclic and aromatic groups allow for diverse bacteria to be targeted. In addition to the 3-carbon length minimum previously identify for E. coli inhibition, cyclic analogs reveal that a five-membered ring (cyclopentyl-DPD; 121) may be the optimal ring size able to effect AI-2 processing enzymes. Also smaller rings and aromatic analogs suggest that some degree of flexibility is required for inhibition. Although P. aerguinosa is a part of the human microflora, lung infections caused by these pathogens are difficult to treat due to robust biofilm.115 AHL analogs which have been shown to perturb quorum sensing regulated biofilm formation are known to have solubility issues and are susceptible to quenching by acylases, lactonases as well as other 70 native defense enzymes.14b, 116 AI-2 analogs, which are able to interfere with the AI-2 pathway in enteric bacteria as well as the AI-1 signaling pathway in organisms such as P. aeruginosa, would be a valuable addition to the arsenal of quorum sensing small molecules currently available. 3.10 Ester-protected AI-2 and analogs Although ester-protection may be desired for the stability and purification of AI-2 and analogs, it remains to be seen if these derivative will behave similarly in biological systems. Acetate-protected AI-2 has been shown to induce bioluminescence in a manner similar to free AI-2.87 It is therefore hypothesized that analogs of AI-2 with ester protecting groups would also act in a manner synomonous to the free DPD analog. Also different ester protecting groups may offer varying levels of bioactivity. Ester protected pro-drugs of AI-2 and hexyl-DPD were synthesized and first screened in V. harveyi MM32 (LuxN-, LuxS-). Also ester-protected analogs did not inhibit bacterial growth (see Supplementary Figure S8). We found that ester analogs of AI-2 were able to induced bioluminescence to a significant extent (Figure 3.13a; black bars) were as ester-protected hexyl analogs were not able to induce bioluminescence in their own (Figure 3.13b; white bars). This would be expected since C1 analogs do not induce bioluminescence on its own (with the exception of ethyl-DPD; 108). 71 Figure 3.13: Bioluminescence induction in V. harveyi MM32 (LuxS-) in response to various ester protections on AI-2 and hexyl-DPD Next, analogs were screened for synergistic agonism. Synergistic agonism was observed in V. harveyi MM32 (LuxS-) in response to ester protected hexyl analogs in the presence 0 2000000 4000000 6000000 8000000 10000000 12000000 Blank Diaceate AI-2 Dipropionate AI-2 Dibutyrate AI-2 Divalerate AI-2 R LU Analogs alone Analogs plus 500nM AI-2 72 of exogeneous 500nM AI-2 (Figure 3.13b; light gray bars). Surprisingly ester-protected analogs caused more synergism that free hexyl-DPD. Similarly AI-2 prodrugs were also tested in the presence of 500nM AI-2 (Figure 3.13a). Figure 3.14: Bioluminescence induction in V. harveyi BB170 (LuxS+) in response to various AI-2 pro- drugs over time Although ester protected AI-2 derivatives were active they did not cause bioluminescence to the degree to which free AI-2 induced (Figure 3.14a) in V. harveyi BB170 (LuxS+). It is possible that these analogs are limited by the time needed for cleavage of the ester groups. As Figure 3.14 shows the activity of ester protected AI-2s increased significantly from 3hrs to 6hrs. This suggests that these analogs may have a time-dependent response. 0 200000 400000 600000 800000 1000000 1200000 AI-2 Diacetate AI-2 Dipropionate AI-2 Dibutyrate AI-2 Divalerate AI-2 Blank R LU 3hrs 6hrs 73 Figure 3.15: AI-2 dependent ?-galactoside production in E. coli LW7 (LuxS-) in response to isobutyl, diaceate isobutyl, hexyl and diacetate hexyl in the presence of AI-2 (1:1 ratio) Finally ester protected analogs were screen for activity in E. coli. Since isobutyl- DPD was identified as the best inhibitor in E. coli, this analog and diacetate isobutyl (132c) was compared for antagonism of ?-galactosidase production in the presence of exogeneous AI-2. Hexyl-DPD (111) and diacetate hexyl (132b) was also tested. Results showed that the ester protected inhibitors were able to compete with AI-2 as well as the free analogs (Figure 3.15). Similarly diacetate AI-2 (132a) was able to induce ?- galactosidase production although to a lesser extent than AI-2 (Supplementary Figure S9). 0 20 40 60 80 100 120 140 160 LW7 LW7+AI-2 Isobutyl Diacetate Isobutyl Hexyl Diacetate Hexyl M ill er U ni ts 74 Figure 3.16: Models of biological activity of ester protected; a) Required entry and exit of ester analogs suggests time-dependent activity in V. harveyi; b) Required entry only suggests non-time-dependent activity in E. coli 75 In conclusion although ester protected AI-2 and analogs have similar bioactivity as the free DPDs there seem to be a dependence on time for bioluminescence induction. This would be expected since these analogs need to be cleaved before they can obtain the active cyclic forms of AI-2 (and presumably analogs). This time dependence is not observed in the case of ?-galactosidase production in E. coli. Figure 3.16 provides models of the proposed path of ester protected AI-2 in these two systems. It has been suggested that AI-2 and analogs can traverse cell membranes through passive diffusion. Therefore it is plausible that in the case of V. harveyi, ester protected analogs must first diffuse into the cell where it can be cleaved by esterases. Free analogs then must exit and cyclize into the active form (S-THMF-borate) to induce bioluminescence by binding LuxPQ. In E. coli ester protected analogs likely have a more direct path to initiating activity. Once inside the cell, ester protected analogs can be cleaved and get directly phosphorylated by LsrK. Since AI-2 binding proteins exist inside the cell in E. coli there is no need for the cleaved products to exit and re-enter through LsrB. Since there are several transport pathways in E. coli further studies are need to confirm this model. These models suggest that the V. harveyi system is more sensitive to the time required for ester analogs to become fully active. 76 Chapter Four Conclusions, Broader Impact and Future Work 4.1 Conclusion The development of a new synthesis of AI-2 has enabled the construction of a large library of analogs with diverse groups at the C1 position.93, 97 Linear, branched, cyclic and aromatic analogs of AI-2 have been synthesized which allow for probing into the length, bulk and electronic requirements of the C1 position.97, 101 In addition, variations at the C4 and C5 positions have been made which also give insight into the structural requirements of AI-2 for signaling and stability. Since AI-2 is a universal signaling molecule, access to this library of analogs has allowed for the perturbation of AI-2 signaling in several quorum-sensing systems.101 In V. harveyi AI-2 analogs of various shapes and sizes all caused synergistic agonism of AI-2 induced bioluminescence.97 In E. coli, linear analogs with chain lengths of three carbons or greater as well as branched analogs were shown to antagonize the lsr based AI-2 response.101 Ironically in S. typhimurium, which is known to have homologous quorum sensing proteins as E. coli, only butyl-DPD (110) and isobutyl-DPD (116) were found to be inhibitors. 101 Overall several potent inhibitors were identified many of which are active in more than one organism. Isobutyl-DPD (116) is the most potent inhibitor of enteric bacteria while butyl-DPD (110) and cyclopentyl-DPD (121) are also very potent inhibitors in E. coli (see Table 4.1). Additionally in P. aeruginosa, although AI-2 is not produced in this organism, C1 analogs moderately reduced pyocyanin production. 77 Table 4.1: Inhibitor Concentration (IC50) of the most potent C1-inhibitors Inhibitory Concentrations (IC50/?M) E. coli Standard Error (logIC50) S. typhimurium Standard Error (logIC50) Isobutyl-DPD 0.1073 0.1813 40 0.1164 Butyl-DPD 5.934 0.1927 >40 ND Cyclopentyl-DPD 4.885 0.1464 >40 ND Heptyl-DPD 31.87 0.244 >40 ND Phenyl-DPD >40 ND >40 ND Finally individual AI-2 analogs were found to be both broad- and selective- inhibitors of mixed culture synthetic ecosystems.101 Also a ?cocktail? of analogs including isobutyl-DPD (116) and phenyl-DPD (125) were found to simultaneously affect diverse organisms in a synthetic ecosystem. The versatility of this new synthesis is demonstrated not only the construction of C1 analogs but also C4 and C5 analogs. Deoxy-analogs lacking the hydroxyl group on C5 allowed for investigations into the importance of phosphorylation on inhibition in enteric bacteria. Also ester-protection at both the C4 and C5 position offer the advantage of silica purification. Moreover ester-protected antagonists , isobutyl diacetate (132c) and hexyl diacetate (132b), showed the same activity as free analogs in E. coli. Although agonist, methyl diacetate (132a), demonstrate slightly lesser activity. A slow-releasing mechanism involving cell entry and cleavage by the ester analogs has been proposed. This may allow sustained modulation of quorum sensing processes in V. harveyi or other organism. Overall the conciseness and versatility of this new synthesis has provided new insights in the quorum sensing system of several organisms. 78 4.2 Broader Impact The broader impact of this unique body of work is quite diverse. In addition to a new synthetic route to AI-2, which is amenable to several variations (i.e. C1 alkyl groups, C4 and C5 groups), the preliminary biological work presented opens the field for massive exploration. Not only has possible anti-quorum sensing therapeutic targets been identified, these findings have illuminated new aspects of the universal nature of AI-2 (i.e., P. aerugionsa inhibition, cocktail approach in mixed cultures, synergistic agonism). The ability of AI-2 to respond to an organism in which it is not produced is an interesting phenomenon.83 The flexibility of this new synthesis would allow analogs to be designed containing functional groups capable of ?tagging? proteins with which it intereacts. Using this strategy, new AI-2 receptor proteins can be identified in organisms where the AI-2 signaling pathway is not well understood. Although much research has been done in recent years to better understand how AI-2 effects pathogenesis, a detailed understanding only exists in a handful of organisms.42, 74 V. harveyi bioluminescence induction is a typical reporter of AI-2 activity and synergistic agonism by analogs have wide reaching implications. Recently it has been found that the quorum sensing regulator, LuxR, represses type III secretion in V. harveyi in addition to activating bioluminescence.46a Therefore synergistic agonists may enhance the suppression of type III secretion in this organism. Similarly in V. cholerae, AI-2 (and CAI-1) activated gene expression results in repression of biofilm formation and virulence.51 Thus, synergistic agonists may inhibit pathogenesis in this organism as well. Furthermore, as previously mentioned several other Vibrios, which are fish pathogens 79 have homologues quorum sensing systems as V. harveyi.44a, 60, 97, 111 It remains to be seen how C1 analogs will affect quorum sensing controlled processes in these organisms. Additionally the role of AI-2 mediated quorum sensing in biofilm formation is not fully understood in many organisms.78 The recent discovery that biofilm architecture in E. coli is affected by LsrR (and AI-2), is key to understanding how organisms control biofilm.103 Our finding that C1 analogs are processed by AI-2 enzymes in E. coli and likely bind LsrR will be useful in understanding what effect altering the C1 position of AI-2 has on biofilm formation and architecture in E. coli. The potential insights obtained from these studies could then be extended to understand biofilm formation on a more global platform. Moreover our finding that AI-2 analogs are effective in a variety of mixed culture environments is extremely relevant to oral microbes which often are involved in mixed culture biofilm formation.43, 53 The ability of our analogs to either selectively or broadly disturb these organisms? quorum sensing network could be useful in dental care. 4.3 Future Work This research has revealed and defined the structural requirements of AI-2 signaling pathways. Although synergistic agonism is observed among all analogs in V. harveyi, it is known that the cyclic form of AI-2 is the active form in V. harveyi. Therefore synthesis of ?locked? cyclic analogs may provide enhanced synergistic effects especially if side chains which showed more pronounce effects in this work are incorporated. Likewise in enteric bacteria our in vitro phosphorylation suggested that it is the phosphorylated forms which bind to LsrR in E. coli. The varying levels of 80 inhibition among C1 analogs observed in this organism could be due to some analogs more readily adapting the linear conformer and thus being more efficiently phosphorylated by LsrK than analogs which may prefer to exist in the cyclic conformer. Therefore synthetic design of stable, cell permeable-phospho-analogs may be more potent inhibitors than the unphosphorylated C1 analogs identified in this work. Since preferred C1 side chains have been found which effect E. coli and S. typhimurium, new analogs could be designed to be selective or broad-spectrum based on the side chain installed. In addition to structural insights, biological insights have also been gained. In V. harveyi the absence of synergistic agonism in mutant strains strongly suggest that this effect is caused by allosteric binding of analogs to LuxPQ. The complex interactions between LuxP and LuxQ have been proven to effect AI-2 sensitivity (see Section 3.1).47a However direct binding assays could be problematic since AI-2 most likely has a much higher binding affinity for LuxPQ than analogs. Therefore a fluorescence-based sensor may be a more effective method to monitor binding of analogs to LuxPQ. Pei has constructed a LuxPQ which contains a fluorophore and quencher at the LuxQ hinge of LuxP.117 Therefore monitoring the behavior of this LuxPQ-probe in the presence of C1 analogs may be more beneficial since fluorescence is highly sensitive. Any subtle changes which the analogs cause could be detected and analyzed to understand their synergistic effect. Other methods including orthogonal chemical genetics have also been used to explore the affect of small molecules on proteins such histidine kinases.118 The different responses observed in lsr expression in E. coli and S. typhimurium is quite intriguing. These systems have been assumed to be homologous based on the 81 presence of identical proteins involved in AI-2 processing in both organisms.40 Our C1 analogs have revealed that subtle variations exists which alter S. typhimurium?s susceptibility. C1 analogs in this manner could be used as actual probes to decipher where biological variations originate. For example E. coli transporter mutants were tested and it was found that alternative transport into the cell exists. This alternative pathway may not exist in S. typhimurium therefore testing our C1 inhibitors in a transporter mutant strain for this organism could show a difference. Another possible approach would be to compare the substrate specificity of LsrK in the two organisms. LsrK of S. typhimurium has been shown to be substrate specific not being able phosphorylating glucose or ribose.38 Thus the conclusions made from in vitro phosphorylation using E. coli LsrK may not directly coorelate to S. typhimurium. Finally binding assays of C1 analogs using purified LsrR from E. coli and S. typhimurium may show that the divergent biological effects of analogs is due to different binding affinities for LsrR. Ultimately the wide variety of C1 analogs available will allow the underlying dissimilarities of these enteric bacteria to be unraveled in the future. Additionally the biological relevance of AI-2 internalization and processing in enteric bacteria is thought to be a method of quenching the AI-2 signal of other bacteria in its niche.40, 106 To prove this theory it is necessary to conduct a more exhaustive study of synthetic ecosystems. A concentration-dependent effect of exogenous AI-2 and analogs on the response of an organism in the presence and absence of various other organisms could give valuable insights on the role of AI-2 in mixed culture environments. 82 The observation that AI-2 and analogs are able to perturb quorum sensing in P. aeruginosa is fascinating yet the idea that such effects are due to AI-1 likeness requires further investigation. The effect of analogs on other quorum sensing process in P. aeruginosa could be tested such as elastase B production. Also screening with mutant strains are needed to identify what pathway AI-2 and analogs are acting on to effect quorum sensing in P. aeruginosa (i.e., las, rhl, etc.). Additionally analogs should be screened in other AI-1 dominate pathways such as V. cholerae to see if similar effects are observed. Once a thorough understanding of AI-2/analogs role in effecting the quorum sensing circuitry of these unlikely organism, combination therapies maybe desired which incorporated AI-2 analogs with other known quorum sensing inhibitors. 83 Chapter Five Experimental, Supplemental Figures and References 5.1 General Methods of Synthesis Air and moisture sensitive reactions were carried out in oven-dried glasswares sealed with rubber septa under a positive pressure of dry argon or nitrogen, unless otherwise indicated. Reactions were stirred using Teflon-coated magnetic stir bars. Organic solutions were concentrated using a B?chi rotary evaporator with an aspirator pump. Dry tetrahydrofuran was obtained using PureSolvent? prior to use. Dry acetonitrile was distilled from CaH2 prior to use. Thin-layer chromatography (TLC) was performed on Merck Kieselgel 60 F254 plates with a 365 nm fluorescent indicator. The TLC was visualized by ultraviolet light and acidic p-anisaldehyde stain followed by gentle heating. The crude reaction mixtures were purified by flash chromatography on silica gel (230-400 mesh). NMR spectra were measured on Bruker AV-400, Bruker DRX-400 (1H at 400 MHz, 13C at 100MHz), Bruker DRX-500 (1H at 500 MHz, 13C at 125MHz) or Bruker AVIII-600 (1H at 600 MHz, 13C at 150MHz). Data for 1H -NMR spectra are reported as follows: chemical shift (ppm, relative to residual solvent peaks or indicated external standards; s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, td = triplet of doublets, m = multiplet), coupling constant (Hz), and integration. Data for 13C - NMR are reported in terms of chemical shift (ppm) relative to residual solvent peak. Mass spectra (MS) and high resolution mass spectra (HRMS) were recorded by JEOL AccuTOF-CS (ESI positive, needle voltage 1800~2400 eV). Infrared spectra (IR) were recorded by a ThermoNicolet IR200 Spectrometer. 84 Synthesis of Diazodiols: To a solution of the diazocarbonyl in anhydrous acetonitrile (0.2M) was added DBU (0.16-0.20 eq) and the requisite aldehyde (2-(tert- butyldimethylsilyloxy)) acetaldehyde or acetaldehyde) (1-1.5 eq). The reaction was stirred at room temperature under nitrogen for 4-8 hours and monitored by TLC. Upon disappearance of starting material, the reaction was quenched with sodium bicarbonate. The organic layer was extracted with dichloromethane (3 x 20 mL) and dried with magnesium sulfate. The solvent was evaporated under reduced pressure. To a solution of crude product in anhydrous tetrahydrofuran (0.2M) TBAF was added (1-2 eq) at 0o C. The solution was allowed to warm to room temperature and stirred for 1-3 hours under nitrogen. The solvent was evaporated and the crude product was purified by column chromatography. The product eluted as yellow oil with 1:3 to 3:2 ethyl acetate:hexane. Synthesis of DPDs: To a solution of diazodiol (1 eq) in acetone (1-2 mL) was added dioxirane (15-20 mL) in acetone dropwise. The reaction was allowed to stir at room temperature (1-2 hrs) until complete disappearance of starting material as indicated by TLC (loss of UV activity). Solvent and excess reagent was evaporated under reduced pressure. NMR was taken without further purification. Synthesis of Quinoxaline Derivatives: To a solution of DPD-analog was added 1, 2- phenylenediamine (1.5 eq). The reaction was stirred at room temperature for 10 minutes and then the reaction mixture was washed with (2M) HCl. The crude mixture was purified on silica. 85 5.2 Methods of Biological Evaluation Bacterial Strains and Growth Condition S. typhimurium and E. coli strains were cultured in Luria-Bertani medium (LB, Sigma) at either 30C or 37C with vigorous shaking (250 rpm) unless otherwise noted. The V. harveyi strains were grown in AB or LM medium. Antibiotics were used for the following strains: (60 or 100 ?g ml-1) kanamycin for S. typhimurium MET715, (50 ?g ml- 1), ampicillin for E. coli LW7 pLW11. (50 ?g ml-1) ampicillin and (50 ?g ml-1) kanamycin for E. coli MDAI-2 pCT6 and E. coli SH3 pLW11 along with (20?g ml-1) chloramphenicol for the latter and (20 ?g ml-1) kanamycin for V. harveyi BB170. Modulation of bioluminescence in V. harveyi The test compounds were evaluated for their (ant) agonistic activity in V. harveyi following reported protocol. Briefly, V. harveyi strain BB170 or MM32 was grown for 18 h at 30 ?C in AB (or LM) medium and then diluted 1:500 into fresh AB medium. Aliquots of analogs (and AI-2) were added to cells in a 96-well plate. Bioluminescence was taken at either 30 min or 1 h intervals. Measurement of ?-galactosidase production in E. coli and S. typhimurium. The QS response indicated by lsr gene expression was analyzed in pure culture studies by culturing E. coli LW7 pLW11, E. coli ZK126 pLW11 and S. typhimurium MET708, S. typhimurium MET715 overnight in LB medium supplemented with appropriate antibiotics as stated previously. These cells were then diluted into fresh LB medium (with 86 antibiotics) and grown to an OD600 of 0.8 - 1.0 at 30 ?C, 250 rpm. Cells were then collected by centrifugation at 10,000 xg for 10 minutes, and resuspended in 10 mM phosphate buffer. AI-2 (40 ?M) and the respective analog (40 ?M) were added to the E. coli or S. typhimurium suspension for 2 hours at 37 ?C. AI-2 dependent ?-galactosidase production was quantified by the Miller assay. 111 Measurement of pyocyanin production. Pyocyanin was extracted from culture supernatants of wild type PAO1, and measured as described by Essar et al.110 Briefly, 2 mL of chloroform was added to 2 mL of culture supernatant, taken from 19 h cultures grown in the presence of DPD analog. After extraction, 1 mL of the chloroform layer was transferred to a fresh tube and mixed with 180 ?L of 0.2 M HCl. After centrifugation, the aquaeous (top) layer was separated and its absorption measured at 520 nm. 87 Supplementary Figures a) b) 0 20 40 60 80 100 120 RT 4 (-20) ZK126 LW7 LW7 + AI-2 % R es po ns e (B et a- ga la ct os id as e ac tiv ity ) AI-2 Diacetate AI-2 0 20 40 60 80 100 120 RT 4 (-20) ZK126 LW7 LW7 + AI-2 % R es po ns e (B et a- ga la ct os id as e ac tiv ity ) Isobutyl-DPD Diacetate Isobutyl 88 c) Figure S1: ?-galactoside production in E. coli LW7 (LuxS-) in response to a) AI-2, b) hexyl-DPD, c) isobutyl-DPD and their diacetate derivatives in the presence of exogeneous AI-2 stored for 4- weeks at various temperatures Figure S2: Bioluminescence induction in V. harveyi BB886 (LuxQ-) in response to select C1 analogs 0 20 40 60 80 100 120 RT 4 (-20) ZK126 LW7 LW7 + AI-2 % R es po ns e (B et a- ga la ct os id as e ac tiv ity ) Hexyl-DPD Diacetate Hexyl 89 Figure S3: Bioluminescence induction in V. harveyi BB721 (LuxO-) in response to C1 analogs 90 Figure S4: ?-galactoside production in E. coli LW7 and S. typhimurium MET715 (both luxS-) in response to a) linear analogs and b) branched and deoxy analogs. 91 Figure S5: ?-galactoside production in E. coli LW9 (LsrB-) in response to a) linear analogs and b) branched and deoxy analogs101 92 0 20 40 60 80 100 120 140 160 180 Blank AI-2 Ethyl-DPD Propyl-DPD Butyl-DPD Pentyl-DPD Hexyl-DPD % P yo cy an in P ro du ct io n 93 Figure S6: Pyocanin production in response to a) linear b) branched c) cyclic and d) aromatic C1 analogs of AI-2 0 20 40 60 80 100 120 140 Cells only Cyclopropyl Cyclobutyl Cyclopentyl Cyclohexyl CH2- Cyclohexyl Cycloheptyl % P yo cy an in P ro du ct io n 94 Figure S7: ?-galactoside production in E. coli LW7 and S. typhimurium MET715 (both are LuxS-) in response to a) cyclic and b) aromatic analogs b) a) 95 Figure S8: Effect of ester-protected AI-2 and analogs growth in V. harveyi a) MM32 and b) BB170 a) b) 96 Figure S9: ?-galactosidase production in response to AI-2 and diacetate AI-2 in E. coli LW7 (LuxS-) a) 97 Figure S10: Dose-response curve of C1 inhibitors in the presence of AI-2 in a) E. coli and b) S. typhimurium Figure S11: Phosphorelay used for signal transduction in the AI-2 mediated quorum sensing pathway of V. harveyi b) 98 Figure S12: Synergistic agonsism of V. harveyi MM32 (LuxS-) in the prescence of various concentrations of AI-2 and Hexyl-DPD 5.2 NMR Characterizations 3-diazo-4, 5-dihydroxypentan-2-one (S1): 1H NMR (400 MHz, CDCl3) ? ppm 4.76 (1H, m), 3.85 (1H, dd, J = 11.4, 3.2 Hz), 3.75 (1H, dd, J = 11.4, 3.2 Hz), 3.46 (1H, s, br), 2.69 (1H, s, br), 2.26 (3H, s). 13C NMR (100 MHz, CDCl3) ? ppm 191.7, 66.3, 64.2, 25.6. IR: 3349, 2361, 2338, 2092, 1607 cm-1. Yield: 50% (over 2 steps) 99 4-diazo-5,6-dihydroxyhexan-3-one (S2): 1H NMR (400 MHz, CDCl3) ? ppm 4.75 (1H, m), 4.48 (1H, br), 4.21 (1H, br), 3.81-3.79 (1H, m), 3.72-3.70 (1H, m), 3.44 (1H, br), 2.50 (2H, q, J = 7.4 Hz), 1.13 (3H, t, J = 7.4 Hz); 13C NMR (100 MHz, CDCl3) ? ppm 195.2, 65.9, 64.1, 31.2, 8.2; IR: 3365, 2980, 2940, 2084, 1607 cm-1. Yield 33% (over 2 steps) 3-diazo-1,2-dihydroxyheptan-4-one (S3): 1H NMR (400 MHz, CDCl3) ? ppm 4.75 (1H, m), 4.08 (1H, br), 3.81 (1H, dd, J = 11.2, 3.4 Hz), 3.70 (1H, dd, J = 11.2, 5.4 Hz), 3.35 (1H, br), 2.45 (2H, t, J = 7.4 Hz), 1.68-1.63 (2H, m), 0.94 (3H, t, J = 7.4 Hz). 13C NMR (100 MHz, CDCl3) ? ppm 195.3, 66.6, 64.8, 40.5, 18.6, 14.1. IR: 3395, 2964, 2935, 2876, 2082, 1605 cm-1. Yield: 52% (over 2 steps) 3-diazo-1,2-dihydroxyoctan-4-one (S4): 1H NMR (400 MHz, CDCl3) 4.75 (1H, br), 3.83 (1H, dd, J = 3.9, 11.5 Hz), 3.72 (1H, dd, J = 5.3, 11.5 Hz), 3.02 (1H, s, br), 2.45- 2.49 (2H, m), 1.95 (1H, s, br), 1.57-1.65 (2H, m), 1.30-1.40 (2H, m), 0.91 (3H, t, J = 7.3 Hz) 13C NMR (100 MHz, CDCl3) ? ppm 194.9, 66.2, 64.1, 37.7, 26.5, 22.1, 13.6. IR: 3334, 2959, 2872, 2082, 1607 cm-1. Yield: 48% (over 2 steps) 100 3-diazo-1,2-dihydroxynonan-4-one (S5): 1H NMR (CDCl3, 400 MHz) ? 4.78 (1H, t, J= 4.4 Hz), 3.83-3.85 (1H, m), 3.73 (1H, dd, J= 16.8, 5.6 Hz), 2.49 (2H, t, J= 7.6 Hz), 1.65 (2H, t, J= 7.2 Hz), 1.32-1.33 (4H, m), 0.89-0.93 (3H, m); 13C NMR (CDCl3, 100 MHz) ? 195.9, 96.3, 67.1, 64.9, 39.1, 31.9, 24.9, 23.1, 14.5. IR: 3377, 2957, 2931, 2872, 2085, 1710, 1609 cm-1 Yield: 25% (over 2 steps) 3-diazo-1,2-dihydroxydecan-4-one (S6): 1H NMR (CDCl3, 400 MHz) ? 4.75 (1H, br), 3.77-3.79 (1H, m), 3.68 (1H, dd, J= 13.6, 4.8 Hz), 2.47 (2H, t, J= 6.4, 5.6 Hz), 1.58-1.64 (2H, m), 1.26-1.35 (6H, m), 0.87-0.91 (3H, m); 13C NMR (CDCl3, 100 MHz) ? 195.2, 70.5, 65.9, 64.5, 38.5, 31.7, 29.0, 24.8, 22.6, 14.2. IR: 3394, 2956, 2928, 2959, 2086, 1610 cm-1 Yield: 49% (over 2 steps) 3-diazo-1,2-dihydroxyundecan-4-one (S7): 1H NMR (CDCl3, 400 MHz) ? 4.78 (1H, br), 3.86-3.89 (1H, m), 3.77 (1H, dd, J= 15.2, 4.4 Hz), 3.54 (1H, br), 2.77 (1H, br), 2.50 (2H, t, J= 7.6 Hz), 1.66 (2H, q, J= 7.2 Hz), 1.27-1.32 (8H, m), 0.91 (3H, t, J= 6.8, 7.2 Hz); 13C NMR (CDCl3, 100 MHz) ? 195.8, 67.1, 64.8, 38.7, 32.0, 29.6, 29.4, 25.0, 23.0, 14.5. IR: 3393, 2926, 2857, 2084, 1610 cm-1 Yield: 61% (over 2 steps) 101 4-diazo-5,6-dihydroxy-2-methylhexan-3-one (S8): 1H NMR (400 MHz, CDCl3) ? ppm 4.74-4.76 (1H, m), 3.81 (1H, dd, J = 11.5, 3.9 Hz), 3.70 (1H, dd, J = 11.5, 5.6 Hz), 2.79- 2.86 (1H, m), 1.12 (6H, d, J = 6.8 Hz). 13C NMR (100 MHz, CDCl3) ? ppm 199.5, 66.7, 64.8, 36.5, 19.1. IR: 3357, 2971, 2929, 2362, 2084, 1738, 1609 cm-1. Yield: 25% (over 2 steps) 4-diazo-5,6-dihydroxy-2,2-dimethylhexan-3-one (S9): 1H NMR (400 MHz, CDCl3) ? ppm 4.78 (1H, t, J = 4.9 Hz), 3.83 (1H, dd, J = 4.2, 11.5 Hz), 3.74-3.70 (1H, m), 1.23 (9H, s). 13C NMR (100 MHz, CDCl3) ? ppm 200.6, 67.8, 64.0, 44.3, 26.6. IR: 3358, 2971, 2361, 2338, 2077, 1702, 1602 cm-1. Yield: 20% (over 2 steps) 3-diazo-1,2-dihydroxy-6-methylheptan-4-one (S10): 1H NMR (CDCl3, 400 MHz) ? 4.79 (1H, br), 4.18 (1H, br), 3.83 (1H, dd, J= 11.2, 3.6 Hz), 3.72 (1H, dd, J= 11.6, 5.6 Hz), 2.36 (2H, d, J= 6.8 Hz), 2.12-2.19 (1H, m), 0.97 (6h, d, J= 6.8 Hz); 13C NMR (CDCl3, 100 MHz) ? 195.1, 71.3, 66.6, 64.8, 47.5, 26.3, 22.9. IR: 3377, 2960, 2873, 2087, 1708, 1609 cm-1 Yield: 22% 102 3-diazo-1,2-dihydroxy-5-methylheptan-4-one (S11): 1H (CDCl3, 400MHz) ? 4.80 (1H, br), 3.83 (1H, dd, J= 15.6, 4.4 Hz), 3.73 (1H, dd, J= 16.4, 4.8 Hz), 2.65-2.70 (1H, m), 1.69-1.77 (1H, m), 1.44-1.51 (1H, m), 1.14-1.16 (3H, d, J= 6.8 Hz), 0.92-0.96 (3H, m); 13C (CDCl3, 100 MHz) ? 199.5, 67.2, 64.8, 43.6, 27.3, 17.1, 12.3. IR: 3405, 2967, 2935, 2878, 2084, 1755, 1605 cm-1 Yield: 54% (over 2 steps) 3-diazo-1,2-dihydroxy-6,6-dimethylheptan-4-one (S12): 1H NMR (CDCl3, 400 MHz) ? 4..77--4..79 ( (m, 1H), 3.82-3.85 (m, 1H), 3.72-3.75 (m, 1H) 2.36 (s, 2H), 1.05 (s, 9H); 13C NMR (CDCl3, 100 MHz) 194.8, 71.8, 66.9, 64.7, 50.9, 32.9, 30.0.IR: 3387, 2955, 2870, 2082, 1602, 1467cm-1 Yield: 53% (over 2 steps) 1-cyclopropyl-2-diazo-3,4-dihydroxybutan-1-one (S13): 1H NMR (400 MHz, CDCl3) ? ppm 4.81 (1H, s, br), 3.87-3.92 (1H, m), 3.79-3.83 (1H, m), 3.38 (1H, s, br), 2.62 (1H, s, br), 1.94-2.01 (1H, m), 1.13-1.17 (2H, m), 0.92-0.97 (2H, m); 13C NMR (100 MHz, CDCl3) ? ppm 193.5, 66.4, 63.7, 16.4, 9.5. IR: 3401, 2929, 2362, 2088, 1690, 1612 cm -1. Yield: 28% (over 2 steps) 103 1-cyclobutyl-2-diazo-3,4-dihydroxybutan-1-one (S14): H1 (CDCl3, 400MHz) ppm ? 4.75 (1H, br), 3.72-3.83 (2H, m), 2.32-2.34 (2H, m), 2.16-2.17 (2H, m), 1.97-2.00 (2H, m), 1.86-1.91 (2H, m); C13 (CDCl3, 100MHz) ppm ? 196.9, 67.0, 64.7, 42.5, 24.9, 18.3 IR: 3376, 2944, 2867, 2085, 1754, 1697, 1603 cm-1. Yield: 21% (over 2 steps) 1-cyclopentyl-2-diazo-3,4-dihydroxybutan-1-one (S15) : 1H NMR (400 MHz, CDCl3) ? ppm 4.75-4.77 (1H, m), 4.21 (1H, br), 3.77-3.81 (1H, m), 3.67-3.71 (1H, m), 3.50 (1H, br), 1.77-1.80 (4H, m), 1.86-1.89 (2H, m), 1.55-1.59 (2H, m) 13C NMR (100 MHz, CDCl3) ? ppm 198.4, 66.6, 64.9, 47.0, 29.8, 26.4 IR 3376, 2952, 2869, 2360, 2082, 1607 cm-1 Yield: 19% (over 2 steps) 1-cyclohexyl-2-diazo-3,4-dihydroxybutan-1-one (S16): 1H NMR (400 MHz, CDCl3) ? ppm 4.75 (1H, t, J = 4.6, 4.6 Hz), 3.85 (1H, dd, J = 4.1, 11.5 Hz), 3.75 (1H, dd, J = 4.9, 11.5 Hz), 3.41 (1H, s, br), 2.58 (1H, s, br), 2.49-2.56 (1H, m), 1.74-1.83 (3H, m), 1.68- 1.70 (1H, m), 1.42-1.51 (2H, m), 1.22-1.32 (3H, m). 13C NMR (100 MHz, CDCl3) ? ppm 198.4, 66.7, 64.3, 46.3, 28.8, 25.5. IR: 3399, 2975, 2932, 2362, 2085, 1616 cm-1. Yield: 36% (over 2 steps) 104 1-cyclohexyl-3-diazo-4,5-dihydroxypentan-2-one (S17): H1 (400MHz, CDCl3) ppm ?: 4.77 (1?, br s), 3.87-3.84 (1H, m), 3.75 (1H, dd, J= 10.8, 6.8 Hz), 2.35 (2H, d, J= 7.2 Hz), 1.66-1.79 (6H, m), 1.22-1.33 (4H, m), 0.99-1.05 (2H, m); C13 (100MHz, CDCl3) ppm ?: 195.8, 97.1, 64.7, 46.3, 35.6, 33.5, 26.4 1-cycloheptyl-2-diazo-3,4-dihydroxybutan-1-one (S18): 1H NMR (400 MHz, CDCl3) ? ppm 4.75-4.78 (1H, m), 3.82-3.89 (1H, m), 3.72-3.79 (1H, m), 2.90-2.97 (1H, br), 2.67- 2.75 (1H, m), 1.76-1.89 (5H, m), 1.63-1.72 (2H, m), 1.56-1.61 (4H, m), 1.43-1.51 (2H, m); 13C NMR (100 MHz, CDCl3) ? ppm 200.0, 66.9, 64.5, 48.1, 31.0, 28.6, 27.0; IR: 3401, 2924, 2857, 2086, 1615 cm-1 2-diazo-1-(furan-2-yl)-3,4-dihydroxybutan-1-one (S19): 1H NMR (400 MHz, CDCl3) ? ppm 7.50 (1H, dd, J=0.7, 1.7 Hz), 7.17 (1H, d, J= 0.7 Hz), 6.55 (1H, dd, J= 1.7, 3.6 Hz), 4.96 (1H, br), 3.95-3.91 (1H, m), 3.86-3.81 (1H, m), 3.58-3.55 (1H, m) 13C NMR (400 MHz, CDCl3) ? ppm 174.3, 151.3, 144.2, 118.0, 111.9, 66.9, 63.8 IR: 3246, 2359, 2341, 2105, 1571, 1543 cm-1 Yield: 20% (over 2 steps) 105 2-diazo-3,4-dihydroxy-1-phenylbutan-1-one (S20): 1H NMR (400 MHz, CDCl3) ? ppm 7.63-7.65 (2H, m), 7.54-7.56 (2H, m), 7.47-7.50 (1H, m), 4.97 (1H, br), 3.98-4.01 (2H, m); 13C NMR (400 MHz, CDCl3) ? ppm 190. 3, 137.4, 132.5, 129.2, 127.7, 127.6, 67.8, 64.7 IR: 3400, 2925, 2360, 2341, 1597, 1570 cm-1 Yield: 18% (over 2 steps) 2-diazo-3,4-dihydroxy-1-(4-methoxyphenyl)butan-1-one (S21): 1H NMR (400 MHz, CDCl3) ? ppm 7.61-7.62 (2H, m), 6.92-6.96 (2H, m), 4.93 (1H, t, J= 4.8, 4.8 Hz), 3.96 (1H, dd, J= 4.4, 11.6 Hz), 3.88 (3H, s), 3.85-3.89 (1H, m); 13C NMR (400 MHz, CDCl3) ? ppm 189.1, 163.1, 129.9, 114.3, 69.4, 64.7, 55.9 2-diazo-1-(4-fluorophenyl)-3,4-dihydroxybutan-1-one (S22): 1H NMR (400 MHz, CDCl3) ? ppm 7.61-7.64 (2H, m), 7.13-7.16 (2H, m), 4.93 (1H, t, J= 4.8, 4.4 Hz), 3.96 (1H, dd, J=4.4, 7.2 Hz), 3.87 (1H, dd, J= 4.8, 11.2 Hz) 13C NMR (400 MHz, CDCl3) ? ppm 188.6, 166.5, 163.9, 133.6, 130.2, 116.3, 67.7, 64.7; Yield: 9.5% (over 2 steps) 106 2-diazo-3,4-dihydroxy-1-(4-nitrophenyl)butan-1-one (S23): H1 (400MHz, d6- Acetone) ppm ? 8.37?8.40 (2?, m), 7.94-7.96 (2H, m), 4.85 (1H, br s), 3.81 (2H, d, J=4.8 Hz); C13 (100MHz, d6-Acetone) ppm ?: 149.7, 128.9, 124.2, 78.7, 66.6, 64.5; Yield: 16% (over 2 steps) 3-diazo-4-hydroxypentan-2-one (S24): 1H NMR (CDCl3, 400 MHz) ? 5.04 (d, J= 5.6 Hz, 1H), 3.22 (br, 1H), 2.28 (s, 3H), 1.77 (br, 1H), 1.40 (d, J= 6.4 Hz, 3H); 13C NMR (CDCl3, 100 MHz) ? 192.3, 62.4, 26.2, 19.5. IR: 3391, 2922, 2850, 2480, 2363, 2093, 1715, 1612 cm-1 Yield: 20% (over 2 steps) 3-diazo-2-hydroxy-6-methylheptan-4-one (S25): 1H NMR (CDCl3, 400 MHz) ? ppm 5.02 (d, J= 6.0 Hz, 1H), 3.53 (br, 1H), 2.35 (d, J= 7.2 Hz, 2H), 2.12-2.19 (m, 1H), 1.38 (d, J= 6.4 Hz, 3H), 0.95 (d, J= 6.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz) ? ppm 195.1, 72.9, 62.3, 47.6, 37.0, 26.2, 22.9, 19.6. IR: 3401, 2960, 2872, 2075, 1609 cm-1 Yield: 28% (over 2 steps) 107 1-cyclohexyl-2-diazo-3-hydroxybutan-1-one (S26): 1H NMR (CDCl3, 400 MHz) ? ppm 5.04 (1H, q, J= 6.4, 6.8, 6.4 Hz), 2.52-2.56 (1H, m), 1.76-1.79 (4H, m), 1.69-1.73 (2H, m), 1.39-1.54 (2H, m), 1.33 (3H, d, J= 5.2 Hz), 1.22-1.28 (2H, m); 13C NMR (CDCl3, 100 MHz) ? ppm 198.9, 62.4, 46.7, 29.1, 26.2, 21.7, 19.6, 14.6. IR: 2931, 2856, 2361, 2340, 2076, 1616 cm-1 DPD (4,5-dihydroxypentane-2,3-dione) and cyclic compounds (55): 1H NMR (400 MHz, D2O) ? ppm 4.24-4.28 (2H,m), 4.04-4.10 (6H, m), 3.93-3.95 (2H, m), 3.84-3.86 (2H, m), 3.67-3.72 (4H, m), 3.52-3.59 (3H, m), 3.44-3.48 (2H, m), 2.26 (3H, s), 1.30 (6H, s), 1.26 (6H, s). 13C NMR (100 MHz, D2O) ? ppm 103.9, 99.1, 74.3, 73.5, 71.2, 61.4, 24.8, 20.2, 19.6. Ethyl-DPD (1,2-dihydroxyhexane-3,4-dione) and cyclic compounds (108): 1H NMR (400 MHz, CDCl3) ? ppm 4.94 (1H, t, J = 3.1), 3.99-4.07 (2H, m), 2.91-3.01 (1H, m), 2.75-2.85 (1H, m), 1.80-1.88 (2H, m), 1.15 (3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3) ? ppm 200.2, 198.8, 75.2, 64.2, 30.9, 18.9, 6.9. Propyl-DPD (1,2-dihydroxyheptane-3,4-dione) and cyclic compounds (109): 1H NMR (400 MHz, CDCl3) ? ppm 4.91 (1H, t, J = 3.1 Hz), 3.96-4.04 (2H, m), 2.81-2.89 108 (1H, m), 2.70-2.78 (1H, m), 1.61-1.70 (3H, m), 0.88-0.99 (6H, m). 13C NMR (100 MHz, CDCl3) ? 199.3, 198.4, 74.7, 63.7, 38.7, 16.2, 13.5. Butyl-DPD (1,2-dihydroxyoctane-3,4-dione) and cyclic compounds (110): 1H NMR (400 MHz, CDCl3) ? ppm 4.94, (1H, t, J = 3.2 Hz), 3.98-4.06 (2H, m), 2.86-2.94 (1H, m), 2.74-2.83 (1H, m), 1.59-1.67 (2H, m), 1.34-1.43 (2H, m), 0.95 (3H, t, J = 7.3 Hz). 13C NMR (400 MHz, CDCl3) ? ppm 199.2, 198.2, 74.5, 63.5, 36.4, 24.4, 21.9, 13.5. Pentyl-DPD (1,2-dihydroxynonane-3,4-dione) and cyclic compounds (111): 1H NMR (D2O, 400 MHz) a: ? 3.85-3.87 (1H, m), 3.60-3.64 (1H, m), 3.48-3.43 (1H, m), 2.62-2.67 (2H, m), 1.51-1.61 (2H, m), 1.13-1.17 (2H, m), 0.70-0.73 (3H, m); b: ? 3.97-4.01 (1H, m), 3.73-3.80 (1H, m), 3.60-3.64 (1H, m), 1.40-1.43 (2H, m), 1.13-1.17 (6H, m), 0.70- 0.73 (3H, m); c: ? 3.75-3.80 (1H, m), 3.38-3.48 (1H, m), 3.34-3.38 (1H, m), 1.40-1.43 (2H, m), 1.13-1.17 (6H, m), 0.70-0.73 (3H, m); 13C NMR (CDCl3, 125 MHz) ? 212.9, 97.3, 74.6, 62.0, 37.3, 33.7, 32.3, 31.3, 23.3, 22.6, 14.2. 109 Hexyl-DPD (1,2-dihydroxydecane-3,4-dione) and cyclic compounds (112): 1H NMR (D2O, 600 MHz) a: ? 4.01-4.05 (1H, m), 3.64-3.68 (1H, m), 3.48-3.52 (1H, m), 2.59-2.70 (2H, m), 1.57-1.62 (2H, m), 1.16-1.18 (6H, m) 0.74-0.75 (3H, m); b: ? 4.25-4.27 (1H, m), 3.82-3.84 (1H, m), 3.64-3.68 (1H, m), 1.43-1.46 (2H, m), 1.16-1.18 (8H, m), 0.74- 0.75 (3H, m); c: ? 3.91-3.92 (1H, m), 3.48-3.52 (1H, m), 3.39-3.42 (1H, m), 1.43-1.46 (2H, m), 1.16-1.18 (8H, m), 0.74-0.75 (3H, m); 13C NMR (CDCl3, 150 MHz) ? 73.7, 61.1, 36.5, 30.7, 28.8, 27.8, 22.6, 21.7, 13.2. Heptyl-DPD (1,2-dihydroxyundecane-3,4-dione) and cyclic compounds (113): 1H NMR (D2O, 400 MHz) a: ? 4.04-4.05 (1H, m), 3.65-3.71 (1H, m), 3.49-3.54 (1H, m), 2.64-2.69 (2H, m), 1.58-1.63 (2H, m), 1.18-1.20 (8H, m), 0.74-0.76 (3H, m); b: ? 4.25- 4.27 (1H, m), 3.83-3.86 (1H, m), 3.65-3.71 (1H, m), 1.46-1.48 (2H, m), 1.18-1.20 (8H, m), 0.74-0.76 (3H, m); c: ? 3.91-3.94 (1H, m), 3.49-3.54 (1H, m), 3.40-3.44 (1H, m), 1.46-1.48 (2H, m), 1.18-1.20 (8H, m), 0.74-0.76 (3H, m); 13C NMR (CDCl3, 125 MHz) ? 216.5, 74.8, 69.5, 62.5, 37.5, 31.8, 29.0, 22.8, 14.2. 110 Isopropyl-DPD (1,2-dihydroxy-5-methylhexane-3,4-dione) and cyclic compounds (114): 1H NMR (400 MHz, CDCl3) ? ppm 4.92 (1H, t, J = 3.2 Hz), 3.98 (2H, d, J = 3.3 Hz), 3.72 (1H, q, J = 7.0 Hz), 3.34-3.41 (1H, m), 1.23-1.29 (3H, m), 1.19 (1H, dd, J = 6.9, 1.4 Hz) 1.15 (6H, dd, J = 6.9, 6.3 Hz), 1.03 (3H, dd, J = 6.9, 1.5 Hz), 0.92 (1H, d, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3) ? ppm 203.1, 199.5, 75.3, 63.9, 35.3, 34.1, 30.1, 17.7, 17.3. Tertbutyl-DPD (1,2-dihydroxy-5,5-dimethylhexane-3,4-dione) and cyclic compounds (115): 1H NMR (400 MHz, CDCl3) ? ppm 4.78 (1H, t, J = 3.5 Hz), 4.37-4.45 (2H, m), 3.91 (2H, d, J = 3.5 Hz), 3.69-3.74 (1H, m), 1.27 (9H, s), 1.23 (3H, s), 1.09 (1H, s), 1.06 (1H, s), 1.01 (6H, s). 13C NMR (100 MHz, CDCl3) ? ppm 213.8, 207.4, 201.4, 101.7, 75.5, 73.1, 66.8, 63.2, 42.9, 37.1, 26.6, 26.1, 24.6, 24.1. Isobutyl-DPD (1,2-dihydroxy-6-methylheptane-3,4-dione) (116): 1H NMR (D2O, 400 MHz) ? 3.85 (1H, dd, J=11.2, 3.6 Hz), 3.68 (1H, dd, J= 15.6, 3.6 Hz), 3.52 (1H, dd, J= 19.2, 7.6 Hz), 2.54-2.59 (2H, m), 1.97-2.02 (1H, m), 0.83-085 (6H, m); 13C NMR (CDCl3, 100 MHz) ? 211.7, 96.8, 73.9, 61.6, 45.7, 41.9, 23.8, 22.0. 111 Secbutyl-DPD (1,2-dihydroxy-5-methylheptane-3,4-dione) and cyclic compounds (117): 1H NMR (D2O, 400 MHz) a: ? 3.63-3.66 (1H, m), 3.46-3.51 (1H, m), 2.95-2.98 (1H, m), 1.46-1.58 (2H, m), 0.79-0.84 (3H, m), 0.68-0.77 (3H, m); b: ? 3.81-3.83 (1H, m), 3.70-3.74 (1H, m), 1.46-1.58 (2H, m), 1.19-1.28 (1H, m), 0.89-0.94 (3H, m), 0.68- 0.77 (3H, m); c: ? 3.81-3.83 (1H, m), 3.70-3.74 (1H, m), 1.46-1.58 (2H, m), 1.19-1.28 (1H, m), 0.89-0.94 (3H, m), 0.68-0.77 (3H, m); 13C NMR (CDCl3, 125 MHz) ? 217.2, 97.8, 75.2, 74.2, 72.4, 63.5, 62.3, 42.1, 40.9, 27.3, 26.9, 25.4, 24.2, 17.3, 16.9, 14.8, 12.1, 11.6, 11.3. Neopentyl-DPD (1,2-dihydroxy-6,6-dimethylheptane-3,4-dione) (118): 1H NMR (D2O, 400 MHz) ? 3.81-3.85 (1H, m), 3.65-3.69 (1H, m), 3.48-3.53 (1H, m), 2.60 (2H, dd, J= 44.0, 13.2 Hz), 0.91 (9H, s); 13C NMR (CDCl3, 100 MHz) ? 210.8, 202.1, 96.8, 74.3, 74.0, 63.2, 51.7, 48.3, 48.2, 31.2, 30.1, 29.3, 29.1. Cyclopropyl-DPD (1-cyclopropyl-3,4-dihydroxybutane-1,2-dione) and cyclic compounds (119): 1H NMR (400 MHz, CDCl3) ? ppm 4.90 (1H, t, J = 3.2 Hz), 4.00 112 (2H, d, J =3.2 Hz), 2.71-2.75 (1H, m), 1.09-1.24 (12H, m), 0.83-0.89 (4H, m).13C NMR (100 MHz, CDCl3) ? ppm 74.7, 63.7, 29.7, 16.3, 14.3. Cyclobutyl-DPD (1-cyclobutyl-3,4-dihydroxybutane-1,2-dione) and cyclic compounds (120): 1H NMR (400 MHz, D2O) ? ppm 4.78-4.81 (1H, m), 4.51-4.61 (1H, m), 4.39-4.42 (1H, m), 4.20-4.29 (2H, m), 4.11-4.16 (1H, m), 4.00-4.06 (1H, m), 3.87- 3.90 (1H, m), 3.75-3.79 (2H, m), 3.61-3.69 (2H, m), 3.42-3.49 (1H, m), 2.56-2.78 (2H, m), 2.81-2.89 (1H, m), 2.43-2.55 (2H, m), 1.97-2.13 (18H, m), 1.72-1.96 (24H, m); 13C NMR (100 MHz, D2O) 104.6, 75.5, 73.9, 71.7, 70.9, 69.4, 61.4, 54.2, 38.1, 30.6, 25.6, 25.5, 25.4, 23.0, 22.9, 22.5, 18.2, 17.8 Cyclopentyl-DPD (1-cyclopentyl-3,4-dihydroxybutane-1,2-dione) and cyclic compounds (121): 1H NMR (400 MHz, D2O) ? ppm 4.03-4.06 (1H, m), 3.88-3.90 (1H, m), 3.77-3.81 (1H, m), 3.65-3.69 (1H, m), 3.49-3.54 (1H, m), 3.31-3.35 (1H, m), 2.17- 2.26 (1H, m), 1.80-1.83 (2H, m), 1.39-1.67 (18H, m); 13C NMR (100 MHz, D2O) 218.1, 99.7, 76.4, 74.2, 69.4, 64.2, 47.9, 47.3, 34.5, 34.1 113 Cyclohexyl-DPD (1-cyclohexyl-3,4-dihydroxybutane-1,2-dione) and cyclic compounds (122): 1H NMR (400 MHz, CDCl3) ? ppm 4.93 (1H, br), 4.00 (2H, d, J = 2.8 Hz), 3.14-3.20 (1H, m), 1.71-1.92 (18 H, m), 1.23-1.48 (16H, m). 13C NMR (100 MHz, CDCl3) ? ppm 202.4, 199.6, 75.2, 63.9, 44.6, 28.1, 27.6, 26.1, 26.0, 25.9, 25.5. Cycloheptyl-DPD (1-cycloheptyl-3,4-dihydroxybutane-1,2-dione) (123): 1H NMR (400 MHz, D2O) ? ppm 3.78-3.82 (1H, m), 3.73-3.75 (1H, m), 3.64-3.66 (1H, m), 3.61- 3.63 (1H, m), 1.61-1.80 (5H,m), 1.50-1.60 (2H,m), 1.24-1.47 (4H,m) CH2-Cyclohexyl-DPD (1-cyclohexyl-4,5-dihydroxypentane-2,3-dione) (124): 1H NMR (400 MHz, D2O) ? ppm 3.65-3.69 (2H, m), 2.60-2.62 (1H, m), 1.79-1.83 (2H, m), 1.54- 1.60 (3H, m), 1.04-1.23 (6H, m), 0.82-0.94 (4H, m) 13C NMR (100 MHz, D2O) 199.5, 198.3, 75.2, 64.1, 44.7, 35.2, 33.6, 33.5, 33.4, 26.5, 26.4, 26.3 Phenyl-DPD (3,4-dihydroxy-1-phenylbutane-1,2-dione) (125): 1H NMR (400 MHz, D2O) ? ppm 8.06-8.09 (1H, m), 7.85-7.86 (1H, m), 7.66-7.71 (1H, m), 7.46-7.48 (8H, m), 114 7.32-7.34 (9H, m), 4.93-4.95 (1H, m), 4.46-4.49 (2H, m), 4.30-4.34 (3H, m), 4.23-4.27 (3H, m), 4.08-4.09 (1H, m), 3.98-4.00 (4H, m), 3.89-3.95 (4H, m), 3.66-3.74 (4H, m) 13C NMR (100 MHz, D2O) 137.8, 130.4, 129.6, 129.4, 129.1, 128.5, 128.4, 127.7, 127.4, 127.3, 100.5, 74.6, 73.7, 71.8, 69.1 Furanoyl-DPD (1-(furan-2-yl)-3,4-dihydroxybutane-1,2-dione) (126): 1H NMR (400 MHz, D2O) ? ppm 7.80 (1H, d, J= 0.5), 7.65 (1H, dd, J= 0.5, 3.7 Hz), 6.64 (1H, dd, J= 1.7, 3.7 Hz), 4.01 (1H, dd, J= 3.7, 7.6 Hz), 3.73 (1H, dd, J= 3.7, 11.8 Hz), 3.53 (1H, dd, J= 7.6, 11.8 Hz), 2.59-2.56 (1H, m); 13C NMR (100 MHz, D2O) 187.9, 149.9, 149.4, 125.2, 120.0, 113.5, 97.2, 75.6, 61.6 Fluorophenyl-DPD (1-(4-fluorophenyl)-3,4-dihydroxybutane-1,2-dione) (128): 1H NMR (400 MHz, D2O) ? ppm 8.13-8.16 (1H, m) 7.91-7.94 (2H, m), 7.70-7.74 (2H, m), 7.42-7.46 (4H, m), 7.13-7.19 (6H, m), 7.00-7.08 (4H, m), 4.55-4.58 (1H, m), 4.42-4.46 (1H, m), 4.27-4.30 (1H, m), 4.19-4.24 (1H, m), 4.00-4.03 (1H, m), 3.95-3.07 (1H, m), 3.87-3.91 (1H, m), 3.62-3.69 (2H, m) 115 Nitrophenyl-DPD (3,4-Dihydroxy-1-(4-nitro-phenyl)-butane-1,2-dione) (129): 1H NMR (D2O, 400 MHz) 8.17 (2H, dd, J= 0.8, 8.4 Hz), 7.68 (2H, dd, J= 2, 6.8 Hz), 4.47 (1H, t, J= 6.8, 7.2 Hz), 4.34-4.38 (1H, m), 4.25-4.28 (1H, m), 4.00-4.02 (1H, m), 3.95- 3.98 (1H, m) Deoxy-Methyl DPD (4-hydroxypentane-2,3-dione) (134a): 1H NMR (D2O, 400 MHz) ? 3.94 (1H, q, J= 6.5 Hz), 2.25 (3H, s), 1.08 (3H, d, J= 10.0 Hz); 13C NMR (CDCl3, 150 MHz) ? 211.3, 97.7, 69.8, 24.8, 15.4. Deoxy-Isobutyl DPD (134b): 1H NMR (D2O, 400 MHz) b: ? 4.23 (1H, q, J= 6.8, 6.8, 6.8 Hz), 2.82-2.86 (2H, m), 2.28 (2H, s), 1.36-1.38 (3H, m), 1.07-1.11 (6H, m); c: ? 3.84 (1H, q, J= 6.8, 6.8, 6.8 Hz), 2.76 (2H, d, J= 6.8 Hz), 2.41 (2H, s), 1.36-1.38 (3H, m), 1.07-1.11 (6H, m) 13C NMR (CDCl3, 125 MHz) ? 217.2, 97.8, 75.2, 74.2, 72.4, 63.5, 62.3, 42.1, 40.9, 27.3, 26.9, 25.4, 24.2, 17.3, 16.9, 14.8, 12.1, 11.6, 11.3. 1-(3-methylquinoxalin-2-yl)ethane-1,2-diol (S27): 1H NMR (500 MHz, CDCl3) ? ppm 8.03-8.06 (2H, m), 7.72-7.78 (2H, m), 5.11-5.16 (1H, m, br), 4.55 (1H, d, J = 7.6 Hz), 116 4.03-4.08 (1H, m), 3.86 (1H, dd, J = 11.4, 5.5 Hz), 2.83 (3H, s). 13C NMR (125 MHz, CDCl3) ? ppm 153.1, 152.2, 142.1, 139.6, 130.3, 129.8, 128.8, 128.6, 71.2, 66.2, 29.9, 22.2. HRMS (ESI+): Found 205.0978 Calc?d 205.0977 (M+H). 1-(3-ethylquinoxalin-2-yl)ethane-1,2-diol (S28): 1H NMR (500 MHz, CDCl3) ? ppm 8.10 (1H, dd, J = 8.4, 1.8 Hz), 8.02 (1H, d, J = 8.0 Hz), 7.71-7.78 (2H, m), 5.17 (1H, s, br), 4.56 (1H, d, J = 7.0 Hz), 4.01-4.06 (1H, m), 3.83 (1H, dd, J = 11.6, 5.7 Hz), 3.02- 3.16 (2H, m),1.46 (3H, t, J = 7.5 Hz). 13C NMR (125 MHz, CDCl3) ? ppm 156.4, 152.7, 142.3, 139.5, 130.2, 129.7, 128.9, 128.6, 70.9, 66.7, 29.6, 12.8. HRMS (ESI+): Found 219.1131 Calc?d 219.1134 (M+H). 1-(3-propylquinoxalin-2-yl)ethane-1,2-diol (S29): 1H NMR (500 MHz, CDCl3) ? ppm 8.06 (1H, dd, J = 8.3, 1.7 Hz), 8.02 (1H, d, J = 8.0 Hz), 7.70-7.77 (2H, m), 5.17 (1H, s, br), 4.56 (1H, d, J = 7.0Hz), 4.06-4.01 (1H, m), 3.83 (1H, dd, J = 11.7, 5.7 Hz), 3.02-3.16 (2H, m),1.46 (3H, t, J = 7.5Hz). 13C NMR (125 MHz, CDCl3) ? ppm 156.4, 152.7, 142.3, 139.5, 130.2, 129.7, 128.9, 128.6, 70.9, 66.7, 29.6, 12.8. HRMS (ESI+): Found 233.1331 Calc?d 233.1290 (M+H). 117 1-(3-butylquinoxalin-2-yl)ethane-1,2-diol (S30): 1H NMR (500 MHz, CDCl3) ? ppm 8.07 (1H, d, J = 8.0 Hz), 8.03 (1H, d, J = 7.5 Hz), 7.71-7.77 (2H, m), 5.17 (1H, s, br), 4.57 (1H, s, br), 4.04 (1H, dd, J = 11.6, 3.2 Hz), 3.81 (1H, dd, J = 11.6, 5.9 Hz), 2.99- 3.10 (2H, m), 1.79-1.94 (2H, m), 1.47-1.55 (2H, m), 1.00 (3H, t, J = 7.4 Hz). 13C NMR (125 MHz, CDCl3) ? ppm 155.8, 152.8, 142.3, 139.4, 130.2, 129.7, 128.9, 128.6, 70.9, 66.8, 34.3, 31.2, 23.1, 14.2. HRMS (ESI+): Found 247.1459 Calc?d 247.1447 (M+H). 1-(3-pentylquinoxalin-2-yl) ethane-1,2-diol (S31): 1H NMR (500 MHz, CDCl3) ? 8.02- 8.08 (2H, m), 7.72-7.76 (2H, m), 5.16-5.18 (1H, m), 4.02-4.05 (1H, m) 3.80-3.83 (1H, m), 3.01-3.06 (2H, m), 1.87-1.91 (2H, m), 1.44-1.46 (6H, m), 0.94 (3H, t, J= 7.0, 7.0 Hz); 13C NMR (125 MHz, CDCl3) ? 155.8, 152.8, 142.3, 139.4, 130.2, 129.7, 128.9, 128.6, 70.9, 66.8, 34.5, 32.1, 299, 28.7, 22.8, 14.2. HRMS (m/z): Found, 261.1612 Calc?d. 261.1603 (M+H). 1-(3-hexylquinoxalin-2-yl) ethane-1,2-diol (S32): 1H NMR (600 MHz, CDCl3) ? 8.03- 8.08 (2H, m), 7.73-7.77 (2H, m), 5.17 (1H, br), 4.56-4.57 (1H, m), 3.06 (1H, br), 3.81 (1H, m), 3.01-3.06 (2H, m), 1.86-1.89 (2H, m), 1.47-1.48 (2H, m), 1.35-1.37 (4H, m), 118 0.90-0.92 (3H, m); 13C NMR (150 MHz, CDCl3) ? 155.8, 152.8, 142.3, 139.5, 130.2, 129.7, 128.9, 128.6, 70.9, 66.8, 34.6, 31.9, 29.6, 29.0, 22.8, 14.3. HRMS (m/z): Found, 275.1753 Calc?d. 275.1760 (M+H). 1-(3-heptylquinoxalin-2-yl) ethane-1,2-diol (S33): 1H NMR (500 MHz, CDCl3) ? 8.02- 8.08 (2H, m), 7.72-7.76 (2H, m), 5.14 (1H, br), 4.55 (1H, br), 4.02-4.07 (1H, m), 3.79- 3.82 (1H, m), 3.01-3.06 (2H, m), 1.87-1.89 (2H, m), 1.46-1.49 (2H, m), 1.30-1.32 (4H, m), 0.90 (3H, t, J= 7.0 Hz); 13C NMR (125 MHz, CDCl3) ? 155.8, 152.8, 142.2, 139.4, 130.2, 129.7, 128.9, 128.6, 70.9, 66.8, 34.6, 31.9, 29.9, 29.4, 29.1, 22.8, 14.3. HRMS (m/z): Found, 289.1908 Calc?d. 289.1916 (M+H). 1-(3-isopropylquinoxalin-2-yl)ethane-1,2-diol (S34): 1H NMR (600 MHz, CDCl3) ? ppm 8.08 (1H, d, J = 7.8 Hz), 8.03 (1H, d, J = 7.8 Hz), 7.71-7.77 (2H, m), 5.22 (1H, s), 4.69 (1H, s), 4.03 (1H, dd, J = 11.4, 2.5 Hz), 3.77 (1H, dd, J = 11.6, 6.0 Hz), 3.43-3.47 (1H, m), 1.45 (3H, d, J = 6.7Hz), 1.39 (3H, d, J = 6.7 Hz). 13C NMR (150 MHz, CDCl3) ? ppm 160.4, 151.9, 142.5, 139.4, 130.0, 129.7, 129.1, 128.5, 70.7, 67.2, 31.2, 29.9, 22.9, 21.8. MS (ESI+): Found 233.13 Calc?d 233.12 (M+H). 119 1-(3-tert-butylquinoxalin-2-yl)ethane-1,2-diol (S35): 1H NMR (600 MHz, CDCl3) ? ppm 8.06 (1H, d, J = 8.4 Hz), 7.99 (1H, d, J = 7.8 Hz), 7.71-7.76 (2H, m), 5.42-5.44 (1H, m), 3.97 (1H, dd, J = 11.6, 2.9 Hz), 3.83 (1H, dd, J = 11.6, 6.3 Hz), 1.59 (9H, s). 13C NMR (100 MHz, CDCl3) ? ppm 161.1, 154.7, 141.1, 139.3, 130.2, 129.8, 129.5, 128.2, 71.6, 68.1, 39.1, 30.6. HRMS (ESI+): Found 247.1455 Calc?d 247.1447 (M+H). 1-(3-isobutylquinoxalin-2-yl) ethane-1,2-diol (S36): 1H NMR (400 MHz, CDCl3) ? 8.05-8.11 (2H, m), 7.76-7.81 (2H, m), 5.21 (1H, t, J= 3.2, 3.2 Hz), 4.05 (1H, dd, J= 11.6, 3.2 Hz), 3.79 (1H, dd, J= 11.6, 5.6 Hz), 2.95 (2H, d, J= 7.2 Hz), 2.44-2.47 (1H, m), 1.07 (3H, d, J= 4.0 Hz), 1.06 (3H, d, J= 3.6 Hz); 13C NMR (100 MHz, CDCl3) ? 155.3, 153.2, 142.4, 139.5, 130.4, 129.9, 129.2, 128.8, 71.0, 66.9, 43.2, 28.9, 23.2, 22.8. HRMS (m/z): Found, 247.1444 Calc?d. 247.1447 (M+H). 1-(3-sec-butylquinoxalin-2-yl) ethane-1,2-diol (S37): 1H NMR (400 MHz, CDCl3) ? 8.04-8.11 (2H, m), 7.73-7.79 (2H, m), 5.20-5.26 (1H, m), 4.03-4.08 (1H, m), 3.71-3.82 120 (1H, m), 3.18-3.22 (1H, m), 1.93-1.21 (1H, m), 1.75-1.85 (1H, m), 1.40 (3H, dd, J= 29.5, 6.5 Hz), 0.90 (3H, dt, J= 20.5, 7.5, 7.5 Hz); 13C NMR (100 MHz, CDCl3) ? 130.2, 129.9, 129.3, 128.7, 70.9, 67.3, 38.6, 38.3, 21.2, 20.2, 12.8, 12.6. HRMS (m/z): Found, 247.1446 Calc?d. 247.1447 (M+H). 1-(3-neopentylquinoxalin-2-yl) ethane-1,2-diol (S38): 1H NMR (400 MHz, CDCl3) ? 8.05-8.13 (2H, m), 7.76-7.79 (2H, m), 5.29-5.33 (1H, m), 4.01 (1H, dd, J= 4.5, 3.2 Hz), 3.73 (1H, dd, J= 6.0, 5.6 Hz), 3.02 (2H, q, J= 24.8,13.6, 13.6 Hz), 1.11 (9H, s); 13C NMR (100 MHz, CDCl3) ? 154.3, 153.7, 142.1, 130.4, 130.1, 129.4, 128.7, 71.4, 67.2, 46.2, 34.1, 30.3. HRMS (m/z): Found, 261.1604 Calc?d. 261.1603 (M+H). 1-(3-cyclopropylquinoxalin-2-yl)ethane-1,2-diol (S39): 1H NMR (500 MHz, CDCl3) ? ppm 8.01 (1H, dd, J = 8.0, 1.2 Hz), 7.96 (1H, dd, J = 8.3, 1.2 Hz), 7.65-7.72 (2H, m), 5.36-5.39 (1H, m), 4.85 (1H, d, J = 6.0Hz), 4.17 (1H, d, J = 11.2Hz), 3.84 (1H, dd, J = 6.0, 11.6Hz), 2.78 (1H, s, br), 2.27-2.32 (1H, m), 1.48-1.52 (1H, m), 1.23-1.27 (1H, m), 1.15-1.21 (2H, m). 13C NMR (125 MHz, CDCl3) ? ppm 156.2, 152.3, 142.3, 138.9, 129.9, 129.1, 128.8, 128.5, 71.1, 66.6, 13.8, 11.9, 10.6. HRMS (ESI+): Found 231.1133 Calc?d 231.1134 (M+H). 121 1-(3-cyclobutylquinoxalin-2-yl)ethane-1,2-diol (S40): 1H NMR (400 MHz, CDCl3) ? ppm 8.19-8.25 (2H, m), 7.80-7.90 (2H, m), 5.21-5.22 (1H, br), 4.04-4.11 (1H, m), 3.80- 3.83 (1H, m), 2.60-2.70 (2H, m), 2.33-2.47 (2H), 2.15-2.22 (2H, m), 1.93-2.07 (2H, m) 13C NMR (100 MHz, CDCl3) ? ppm 131.1, 131.0, 129.4, 126.9, 71.1, 66.7, 38.5, 28.1, 25.5, 18.4 MS (ESI+): Found 245.20 Calc?d 245.12 (M+H). 1-(3-cyclopentylquinoxalin-2-yl)ethane-1,2-diol (S41): 1H NMR (400 MHz, CDCl3) ? ppm 8.05-8.10 (2H, m), 7.73-7.77 (2H, m), 5.81 (1H, t, J= 3.2Hz), 4.07 (1H, dd, J= 3.2, 11.6 Hz), 3.78 (1H, dd, J= 6.0, 11.6 Hz), 3.53-3.56 (1H, m), 2.15-2.17 (2H, m), 1.97-2.00 (4H, m), 1.78-1.81 (2H, m); 13C NMR (100 MHz, CDCl3) ? ppm 159.5, 152.4, 142.7, 139.3, 130.2, 129.8, 129.3, 128.5, 71.1, 67.4, 42.9, 34.7, 33.7, 26.6, 26.5 HRMS (ESI+): Found 259.2224 Calc?d 259.1368 (M+H). 1-(3-cyclohexylquinoxalin-2-yl)ethane-1,2-diol (S42): 1H NMR (500 MHz, CDCl3) ? ppm 8.09 (1H, d, J = 8.0 Hz), 8.02 (1H, d, J = 8.0 Hz), 7.70-7.76 (2H, m), 5.21 (1H, s, 122 br), 4.66 (1H, s, br), 4.01-4.04 (1H, m), 3.75 (1H, dd, J = 11.5, 6.1 Hz), 3.01-3.07 (1H, m), 1.76-1.98 (6H, m), 1.44-1.49 (4H, m). 13C NMR (125 MHz, CDCl3) ? ppm 159.6, 152.0, 142.5, 139.4, 130.0, 129.6, 129.1, 128.5, 70.7, 67.3, 41.7, 33.2, 32.0, 26.8, 26.1. HRMS (ESI+): Found 273.1618 Calc?d 273.1603 (M+H). 1-(3-cycloheptylquinoxaline-2-yl) ethane-1,2-diol (S43): 1H NMR (400 MHz, CDCl3) ? ppm 8.03-8.12 (2H, m), 7.72-7.78 (2H, m), 5.20-5.27 (1H, m), 4.01-4.11 (1H, m), 3.73- 3.79 (1H, m), 3.19-3.28 (1H, m), 2.83-2.86 (1H, m), 2.14-2.22 (1H, m), 1.96-2.04 (4H, m), 1.75-1.83 (4H, m), 1.59-1.64 (4H, m) 13C NMR (100 MHz, CDCl3) ? 151.7, 130.2, 129.7, 129.3, 128.7, 70.9, 67.3, 35.6, 34.3, 28.5, 28.3, 27.6, 27.5. MS (ESI+): Found 287.27 Calc?d 287.17 (M+H). 1-(3-(cyclohexylmethyl)quinoxalin-2-yl)ethane-1,2-diol (S44): 1H NMR (500 MHz, CDCl3) 8.03-8.12 (2H, m), 7.75-7.79 (2H, m), 5.18-5.21 (1H, m), 4.03 (1H, dd, J= 3.3, 3.4 Hz), 3.73-3.79 (1H, m), 2.94 (2H, d, J= 7.0 Hz), 2.32-2.35 (1H, m), 1.12-1.17 (4H, m), 0.85-0.92 (6H, m) 13C NMR (100 MHz, CDCl3) ? 155.5, 153.6, 130.7, 130.3, 129.6, 129.1, 71.4, 67.4, 42.4, 38.9, 34.3, 33.9, 27.1, 26.9 MS (ESI+): Found 287.19 Calc?d 287.17 (M+H). 123 1-(3-phenyl-quinoxalin-2-yl)-ethane-1,2-diol (S45): 1H NMR (400 MHz, CDCl3) ? ppm 8.08-8.16 (2H, m), 7.78-7.82 (2H, m), 7.75 (1H, dd, J= 0.8, 2.4 Hz), 7.44 (1H, dd, J= 0.8, 2.8 Hz), 6.69-6.70 (1H, m), 5.74-5.76 (1H, m), 4.12 (1H, dd, J= 3.2, 11.6 Hz), 3.72 (1H, dd, J= 5.2, 11.6 Hz); 13C NMR (100 MHz, CDCl3) ? ppm 145.4, 131.0, 130.6, 129.5, 128.7, 114.6, 113.0, 71.6, 66.9. HRMS (ESI+): Found 257.1679 Calc?d 257.0848 (M+H). 1-(3-(furan-2-yl)quinoxalin-2-yl)ethane-1,2-diol (S46): 1H NMR (400 MHz, CDCl3) ? ppm 8.08-8.16 (2H, m), 7.78-7.82 (2H, m), 7.75 (1H, dd, J= 0.8, 2.4 Hz), 7.44 (1H, dd, J= 0.8, 2.8 Hz), 6.69-6.70 (1H, m), 5.74-5.76 (1H, m), 4.12 (1H, dd, J= 3.2, 11.6 Hz), 3.72 (1H, dd, J= 5.2, 11.6 Hz); 13C NMR (100 MHz, CDCl3) ? ppm 145.4, 131.0, 130.6, 129.5, 128.7, 114.6, 113.0, 71.6, 66.9. HRMS (ESI+): Found 257.1679 Calc?d 257.0848 (M+H). 124 1-(3-fluorophenyl-quinoxalin-2-yl)-ethane-1,2-diol (S47): 1H NMR (400 MHz, CDCl3) ? 8.51-8.59 (1H, m), 8.32-8.33 (1H, m), 7.98-8.00 (2H, m), 7.86-7.89 (2H, m), 7.74-7.78 (2H, m), 5.43 (1H, t, J= 4, 3.6 Hz), 4.02 (1H, dd, J= 3.2, 9.2 Hz), 3.71 (1H, dd, J= 4.4, 12 Hz) MS (m/z): Found 285.13 Calc?d. 285.15 (M+H). 1-(3-(4-nitrophenyl)quinoxalin-2-yl)ethane-1,2-diol (S48): 1H NMR (400 MHz, CDCl3) ? 8.44-8.47 (2H, m), 8.17-8.22 (2H, m), 7.99-8.01 (2H, m), 7.89-7.92 (2H, m), 5.19 (1H, t, J= 4.0, 4.4 Hz), 3.83 (1H, dd, J= 3.6, 8.0 Hz), 3.76 (1H, dd, J= 4.8, 6.8 Hz) MS (m/z): Found 312.12 Calc?d. 312.09 (M+H). 1-(3-methylquinoxalin-2-yl) ethanol (S49): 1H NMR (400 MHz, CDCl3) ? 8.03-8.06 (2H, m), 7.72-7.76 (2H, m), 5.19 (1H, q, J= 6.4 Hz), 2.77 (3H, s), 1.55 (3H, d, J= 6.4 Hz). 13C NMR (100 MHz, CDCl3) ? 157.4, 151.5, 142.0, 139.9, 130.1, 129.8, 128.8, 128.7, 67.0, 23.9, 22.3. HRMS (m/z): Found 189.1039 Calc?d. 189.1028 (M+H). 1-(3-isobutylquinoxalin-2-yl) ethanol (S50): 1H NMR (500 MHz, CDCl3) ? 8.06-8.10 (2H, m), 7.73-7.79 (2H, m), 5.25-5.30 (1H, m), 4.71 (1H, d, J= 7.6 Hz), 2. 88 (2H, d, J= 125 7.2 Hz), 2.42-2.49 (1H, m), 1.56 (3H, d, J= 6.4 Hz), 1.08 (3H, d, J= 6.8 Hz), 1.00 (3H, d, J= 7.6 Hz); 13C NMR (125 MHz, CDCl3) ? 158.2, 155.2, 142.8, 140.4, 130.2, 130.0, 129.5, 129.1, 67.4, 43.8, 29.3, 25.4, 23.5, 23.2. HRMS (m/z): Found 231.1505 Calc?d. 231.1497 (M+H). 1-(3-cyclohexylquinoxalin-2-yl) ethanol (S51): 1H NMR (400 MHz, CDCl3) ? 8.04-8.10 (2H, m), 7.72-7.76 (2H, m), 5.29-5.31 (1H, m), 4.85 (1H, d, J= 7.6 Hz). 2.92-3.01 (1H, m), 1.86-2.02 (6H, m), 1.61-1.63 (1H, m), 1.56 (3H, d, J= 6.4 Hz), 1.44-1.57 (3H, m); 13C NMR (100 MHz, CDCl3) ? 159.0, 156.4, 142.5, 139.7, 129.7, 129.6, 129.2, 128.7, 66.5, 41.8, 33.5, 31.8, 27.0, 26.8, 26.2, 25.4. MS (m/z): Found 257.24 Calc?d. 257.16 (M+H). 126 5.5. Spectra of AI-2 analogs 3-diazo-4, 5-dihydroxypentan-2-one (S1): 127 128 4-diazo-5,6-dihydroxyhexan-3-one (S2): 129 130 3-diazo-1,2-dihydroxyheptan-4-one (S3): 131 3-diazo-1,2-dihydroxyoctan-4-one (S4): 132 133 3-diazo-1,2-dihydroxynonan-4-one (S5): 134 3-diazo-1,2-dihydroxydecan-4-one (S6): 135 4-diazo-5,6-dihydroxy-2-methylhexan-3-one (S8): 136 137 4-diazo-5,6-dihydroxy-2,2-dimethylhexan-3-one (S9): 138 139 3-diazo-1,2-dihydroxy-6-methylheptan-4-one (S10): 140 141 142 3-diazo-1,2-dihydroxy-5-methylheptan-4-one (S11): 143 3-diazo-1,2-dihydroxy-6,6-dimethylheptan-4-one 144 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 1.01 8 1.04 2 1.04 6 2.05 2 2.35 7 3.71 9 3.73 4 3.74 8 3.82 4 3.84 2 3.85 2 4.77 2 4.78 4 4.79 5 7.27 4 10.0 2 2.0 5 1.3 0 1.2 5 1.0 0 PC 2.00 GB 0 LB 0.30 Hz SSB 0 WDW EM SF 399.7300000 MHz SI 32768 F2 ? Processing parameters SFO1 399.7324685 MHz PL1 ?4.00 dB P1 10.00 usec NUC1 1H ======== CHANNEL f1 ======== TD0 1 D1 1.50000000 sec TE 295.4 K DE 6.00 usec DW 66.000 usec RG 128 AQ 2.5999219 sec FIDRES 0.192317 Hz SWH 7575.758 Hz DS 2 NS 64 SOLVENT CDCl3 TD 39392 PULPROG zg30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 14.57 Date_ 20091021 F2 ? Acquisition Parameters PROCNO 1 EXPNO 10 NAME 33?smith?1021 Current Data Parameters 145 200 180 160 140 120 100 80 60 40 20 0 ppm 14.5 9 30.0 5 32.8 9 50.9 7 64.7 2 66.9 5 71.8 3 77.1 1 77.4 3 77.6 3 77.7 5 194.7 7 PC 1.40 GB 0 LB 1.00 Hz SSB 0 WDW EM SF 100.5121480 MHz SI 32768 F2 ? Processing parameters SFO2 399.7315989 MHz PL12 17.37 dB PL2 ?4.00 dB PCPD2 89.00 usec NUC2 1H CPDPRG2 waltz16 ======== CHANNEL f2 ======== SFO1 100.5242095 MHz PL1 0.00 dB P1 4.50 usec NUC1 13C ======== CHANNEL f1 ======== TD0 1 d11 0.03000000 sec D1 1.50000000 sec TE 296.1 K DE 6.00 usec DW 18.400 usec RG 362 AQ 1.2059124 sec FIDRES 0.414641 Hz SWH 27173.912 Hz DS 32 NS 1300 SOLVENT CDCl3 TD 65536 PULPROG zgdc30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 17.50 Date_ 20091021 F2 ? Acquisition Parameters PROCNO 1 EXPNO 20 NAME 33?smith?1021 Current Data Parameters 1-cyclopropyl-2-diazo-3,4-dihydroxybutan-1-one (S13): 146 147 1-cyclobutyl-2-diazo-3,4-dihydroxybutan-1-one (S14): 148 149 150 1-cyclopentyl-2-diazo-3,4-dihydroxybutan-1-one (S15): 151 152 1-cyclohexyl-2-diazo-3,4-dihydroxybutan-1-one (S16): 153 1-cyclohexyl-3-diazo-4,5-dihydroxypentan-2-one (S17): 154 155 156 1-cycloheptyl-2-diazo-3,4-dihydroxybutan-1-one (S18): 157 2-diazo-1-(furan-2-yl)-3,4-dihydroxybutan-1-one (S19): 158 159 2-diazo-3,4-dihydroxy-1-phenylbutan-1-one (S20): 160 2-diazo-3,4-dihydroxy-1-(4-nitrophenyl)butan-1-one (S23): 161 3-diazo-4-hydroxypentan-2-one (S24): 162 163 3-diazo-2-hydroxy-6-methylheptan-4-one (S25): 164 165 1-cyclohexyl-2-diazo-3-hydroxybutan-1-one (S26): 166 167 DPD (4,5-dihydroxypentane-2,3-dione) and cyclic compounds (55): 168 169 Ethyl-DPD (1,2-dihydroxyhexane-3,4-dione) and cyclic compounds (108): 170 171 Propyl-DPD (1,2-dihydroxyheptane-3,4-dione) and cyclic compounds (109): 172 173 Butyl-DPD (1,2-dihydroxyoctane-3,4-dione) and cyclic compounds (110): 174 175 Pentyl-DPD (1,2-dihydroxynonane-3,4-dione) and cyclic compounds (111): 176 177 178 Hexyl-DPD (1,2-dihydroxydecane-3,4-dione) 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 1.52 8 1.54 1 1.56 7 1.58 6 1.60 3 1.62 1 1.64 0 1.65 8 1.67 7 1.79 6 1.80 3 1.82 1 1.83 6 1.84 4 1.87 4 1.88 1 1.90 2 2.12 0 2.20 3 2.78 3 2.80 1 2.87 4 4.01 5 4.02 3 4.02 7 4.03 5 4.93 6 7.29 0 7.5 1 15.6 0 3.5 7 2.3 6 1.0 0 0.2 8 PC 2.00 GB 0 LB 0.30 Hz SSB 0 WDW EM SF 399.7300000 MHz SI 32768 F2 ? Processing parameters SFO1 399.7324685 MHz PL1 ?4.00 dB P1 10.00 usec NUC1 1H ======== CHANNEL f1 ======== TD0 1 D1 1.50000000 sec TE 294.9 K DE 6.00 usec DW 66.000 usec RG 256 AQ 2.5999219 sec FIDRES 0.192317 Hz SWH 7575.758 Hz DS 2 NS 64 SOLVENT CDCl3 TD 39392 PULPROG zg30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 13.00 Date_ 20090817 F2 ? Acquisition Parameters PROCNO 1 EXPNO 10 NAME 10?smith?0817 Current Data Parameters Sample Label hexyl_dpd Group sintim User Name smith 179 200 180 160 140 120 100 80 60 40 20 ppm 14.44 5 22.84 5 22.89 2 23.02 3 29.07 7 30.00 0 31.86 2 31.99 6 32.19 3 37.35 3 64.17 3 75.17 2 77.11 8 77.43 7 77.63 9 77.75 4 198.86 3 199.87 8 PC 1.40 GB 0 LB 1.00 Hz SSB 0 WDW EM SF 100.5121480 MHz SI 32768 SFO2 399.7315989 MHz PL12 17.37 dB PL2 ?4.00 dB PCPD2 89.00 usec NUC2 1H CPDPRG2 waltz16 ======== CHANNEL f2 ======== SFO1 100.5242095 MHz PL1 0.00 dB P1 4.50 usec NUC1 13C ======== CHANNEL f1 ======== TD0 1 d11 0.03000000 sec D1 1.50000000 sec TE 295.6 K DE 6.00 usec DW 18.400 usec RG 181 AQ 1.2059124 sec FIDRES 0.414641 Hz SWH 27173.912 Hz DS 16 NS 3800 SOLVENT CDCl3 TD 65536 PULPROG zgdc30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 23.08 Date_ 20090817 PROCNO 1 EXPNO 11 NAME 10?smith?0817 Sample Label hexyl_dpd Group sintim User Name smith Heptyl-DPD (1,2-dihydroxyundecane-3,4-dione) and cyclic compounds (113): 180 181 Isopropyl-DPD (1,2-dihydroxy-5-methylhexane-3,4-dione) and cyclic compounds (114): 182 183 Tertbutyl-DPD (1,2-dihydroxy-5,5-dimethylhexane-3,4-dione) and cyclic compounds (115): 184 185 Isobutyl-DPD (1,2-dihydroxy-6-methylheptane-3,4-dione) (116): 186 Neopentyl-DPD (1,2-dihydroxy-6,6-dimethylheptane-3,4-dione) (118): 187 188 189 Cyclopropyl-DPD (1-cyclopropyl-3,4-dihydroxybutane-1,2-dione) and cyclic compounds (119): 190 Cyclobutyl-DPD (1-cyclobutyl-3,4-dihydroxybutane-1,2-dione) 191 192 Cyclopentyl-DPD (1-cyclopentyl-3,4-dihydroxybutane-1,2-dione) 193 Cyclohexyl-DPD (1-cyclohexyl-3,4-dihydroxybutane-1,2-dione) and cyclic compounds (122): 194 195 196 CH2-Cyclohexyl-DPD(1-cyclohexyl-4,5-dihydroxypentane-2,3-dione): 197 Phenyl-DPD (3,4-dihydroxy-1-phenyl-butane-1,2-dione) (125): 198 199 Furanoyl-DPD (1-(furan-2-yl)-3,4-dihydroxybutane-1,2-dione) (126): 200 Methoxyphenyl-DPD (3,4-dihydroxy-1-(4-methoxyphenyl)butane-1,2-dione): 201 Fluorophenyl-DPD (1-(4-fluorophenyl)-3,4-dihydroxybutane-1,2-dione): 202 Deoxy-Isobutyl DPD (134b): 203 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 3.30 9 3.32 6 3.32 8 3.33 7 3.34 5 3.35 3 3.58 8 3.60 2 3.61 8 3.62 3 4.51 6 4.57 4 4.65 4 4.70 4 4.99 3 5.01 1 5.06 2 5.07 9 5.08 9 5.10 7 6.06 9 6.08 7 6.46 5 6.48 2 11.2 4 4.2 6 2.3 5 1.3 0 1.5 1 1.1 8 1.7 6 0.7 6 1.0 0 PC 2.00 GB 0 LB 0.30 Hz SSB 0 WDW EM SF 399.7300000 MHz SI 32768 F2 ? Processing parameters SFO1 399.7324685 MHz PL1 ?4.00 dB P1 10.00 usec NUC1 1H ======== CHANNEL f1 ======== TD0 1 D1 1.50000000 sec TE 295.0 K DE 6.00 usec DW 66.000 usec RG 128 AQ 2.5999219 sec FIDRES 0.192317 Hz SWH 7575.758 Hz DS 2 NS 64 SOLVENT D2O TD 39392 PULPROG zg30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 9.12 Date_ 20091012 F2 ? Acquisition Parameters PROCNO 1 EXPNO 11 NAME 20?smith?1012 Current Data Parameters 204 1-cyclohexyl-3-hydroxybutane-1,2-dione (134c): 205 206 1-(3-methylquinoxalin-2-yl)ethane-1,2-diol (S27): 207 208 1-(3-ethylquinoxalin-2-yl)ethane-1,2-diol (S28): 209 210 1-(3-propylquinoxalin-2-yl)ethane-1,2-diol (S29): 211 212 1-(3-butylquinoxalin-2-yl)ethane-1,2-diol (S30): 213 214 1-(3-pentylquinoxalin-2-yl) ethane-1,2-diol (S31): 215 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 0.92 1 0.93 5 0.94 9 1.41 3 1.42 7 1.45 0 1.46 4 1.86 5 1.88 1 1.89 5 1.89 7 1.91 3 3.00 9 3.02 2 3.02 7 3.04 1 3.04 5 3.05 8 3.80 3 3.81 4 3.82 6 4.02 4 4.03 0 4.04 7 4.05 3 5.16 3 5.17 0 5.17 5 5.18 1 7.27 0 7.72 4 7.72 7 7.73 9 7.74 4 7.75 6 7.75 9 8.02 0 8.02 4 8.03 7 8.03 9 8.06 2 8.06 4 8.07 8 8.08 1 3.3 3 4.6 8 2.3 1 2.0 0 0.9 5 0.9 8 0.9 7 2.1 2 2.0 7 PC 1.00 GB 0 LB 0.30 Hz SSB 0 WDW EM SF 500.1300082 MHz SI 32768 F2 ? Processing parameters SFO1 500.1330008 MHz PL1 0.00 dB P1 11.10 usec NUC1 1H ======== CHANNEL f1 ======== TD0 1 D1 4.00000000 sec TE 296.6 K DE 9.00 usec DW 76.800 usec RG 181 AQ 5.0332146 sec FIDRES 0.099341 Hz SWH 6510.417 Hz DS 2 NS 16 SOLVENT CDCl3 TD 65536 PULPROG zg PROBHD 5 mm BBO2 ne? INSTRUM spect Time 11.45 Date_ 20090810 F2 ? Acquisition Parameters PROCNO 1 EXPNO 1 NAME C5H11_quinoaline Current Data Parameters 216 1-(3-heptylquinoxalin-2-yl) ethane-1,2-diol (S33): 217 218 1-(3-isopropylquinoxalin-2-yl)ethane-1,2-diol (S34): 219 220 1-(3-tert-butylquinoxalin-2-yl)ethane-1,2-diol (S35): 221 222 1-(3-isobutylquinoxalin-2-yl) ethane-1,2-diol (S36): 223 224 1-(3-sec-butylquinoxalin-2-yl) ethane-1,2-diol: 225 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 1.7 5 1.9 7 1.6 5 1.8 2 1.4 3 0.6 6 0.5 8 1.0 3 0.8 1 1.0 2 1.0 0 1.8 8 2.0 0 PC 2.00 GB 0 LB 0.30 Hz SSB 0 WDW EM SF 399.7300000 MHz SI 32768 F2 ? Processing parameters SFO1 399.7324685 MHz PL1 ?4.00 dB P1 10.00 usec NUC1 1H ======== CHANNEL f1 ======== TD0 1 D1 1.50000000 sec TE 295.0 K DE 6.00 usec DW 66.000 usec RG 128 AQ 2.5999219 sec FIDRES 0.192317 Hz SWH 7575.758 Hz DS 2 NS 64 SOLVENT CDCl3 TD 39392 PULPROG zg30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 10.34 Date_ 20091214 F2 ? Acquisition Parameters PROCNO 1 EXPNO 10 NAME 9?smith?1214 Current Data Parameters Sample Label 2methyl_quin Group sintim User Name smith 1-(3-neopentylquinoxalin-2-yl) ethane-1,2-diol: 226 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 10.2 9 1.0 4 1.0 6 1.0 0 1.0 0 1.1 3 2.0 8 1.9 8 PC 2.00 GB 0 LB 0.30 Hz SSB 0 WDW EM SF 399.7300000 MHz SI 32768 F2 ? Processing parameters SFO1 399.7324685 MHz PL1 ?4.00 dB P1 10.00 usec NUC1 1H ======== CHANNEL f1 ======== TD0 1 D1 1.50000000 sec TE 295.2 K DE 6.00 usec DW 66.000 usec RG 456.1 AQ 2.5999219 sec FIDRES 0.192317 Hz SWH 7575.758 Hz DS 2 NS 64 SOLVENT CDCl3 TD 39392 PULPROG zg30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 19.28 Date_ 20091110 F2 ? Acuisition Parameters PROCNO 1 EXPNO 10 NAME 26?smith?1110 Current Data Parameters Sample Label acetyltertbutyl_uinox Group sintim User Name smith 1-(3-cyclopropylquinoxalin-2-yl)ethane-1,2-diol (S39): 227 228 1-(3-cyclobutylquinoxalin-2-yl)ethane-1,2-diol: 229 230 1-(3-cyclopentylquinoxalin-2-yl)ethane-1,2-diol (S41): 231 1-(3-cyclohexylquinoxalin-2-yl)ethane-1,2-diol (S42): 232 233 1-(3-cycloheptylquinoxaline-2-yl)ethane-1,2-diol: 234 235 1-(3-(cyclohexylmethyl)quinoxalin-2-yl)ethane-1,2-diol: 236 237 1-(3-(furan-2-yl)quinoxalin-2-yl)ethane-1,2-diol (S46): 238 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 0.90 8 1.28 0 1.33 9 3.70 3 3.71 6 3.73 2 3.74 5 4.09 8 4.10 6 4.12 7 4.13 5 5.73 6 5.74 4 5.74 9 5.75 8 6.68 9 6.69 4 6.69 8 6.70 3 7.29 0 7.44 1 7.44 3 7.45 0 7.45 2 7.75 1 7.75 3 7.75 5 7.75 7 7.78 2 7.78 6 7.79 6 7.80 0 7.80 2 7.80 6 7.81 6 7.82 1 8.07 6 8.08 1 8.09 7 8.10 0 8.13 6 8.13 9 8.15 5 8.16 0 1.0 4 4.0 8 1.8 9 7.2 5 0.8 7 0.4 8 1.0 0 0.9 9 0.9 4 2.9 4 0.9 3 0.9 9 PC 2.00 GB 0 LB 0.30 Hz SSB 0 WDW EM SF 399.7300000 MHz SI 32768 F2 ? Processing parameters SFO1 399.7324685 MHz PL1 ?4.00 dB P1 10.00 usec NUC1 1H ======== CHANNEL f1 ======== TD0 1 D1 1.50000000 sec TE 294.9 K DE 6.00 usec DW 66.000 usec RG 574.7 AQ 2.5999219 sec FIDRES 0.192317 Hz SWH 7575.758 Hz DS 2 NS 64 SOLVENT CDCl3 TD 39392 PULPROG zg30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 16.41 Date_ 20090916 F2 ? Acquisition Parameters PROCNO 1 EXPNO 10 NAME 16?smith?0916 Current Data Parameters 239 200 180 160 140 120 100 80 60 40 20 ppm 66.94 6 71.65 6 77.11 6 77.43 3 77.63 7 77.75 2 78.15 0 113.00 0 114.65 1 128.68 3 129.46 9 130.60 4 131.00 1 145.42 4 PC 1.40 GB 0 LB 1.00 Hz SSB 0 WDW EM SF 100.5121480 MHz SI 32768 F2 ? Processing parameters SFO2 399.7315989 MHz PL12 17.37 dB PL2 ?4.00 dB PCPD2 89.00 usec NUC2 1H CPDPRG2 waltz16 ======== CHANNEL f2 ======== SFO1 100.5242095 MHz PL1 0.00 dB P1 4.50 usec NUC1 13C ======== CHANNEL f1 ======== TD0 1 d11 0.03000000 sec D1 1.50000000 sec TE 295.7 K DE 6.00 usec DW 18.400 usec RG 287.4 AQ 1.2059124 sec FIDRES 0.414641 Hz SWH 27173.912 Hz DS 16 NS 3800 SOLVENT CDCl3 TD 65536 PULPROG zgdc30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 20.05 Date_ 20090917 F2 ? Acquisition Parameters PROCNO 1 EXPNO 10 NAME 16?smith?0917 Current Data Parameters 1-(3-phenyl-quinoxalin-2-yl)-ethane-1,2-diol (S45): 240 241 242 1-(3-methoxyphenyl-quinoxalin-2-yl)-ethane-1,2-diol (S47): 243 1-(3-fluorophenyl-quinoxalin-2-yl)-ethane-1,2-diol (S48): 244 1-(3-nitrophenyl-quinoxalin-2-yl)-ethane-1,2-diol (S49): 1-(3-isobutylquinoxalin-2-yl) ethanol: 245 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 0.09 8 0.91 8 0.96 0 0.96 2 0.98 0 1.00 0 1.01 6 1.04 1 1.06 0 1.07 7 1.27 5 1.54 9 1.56 5 2.40 9 2.42 6 2.44 3 2.46 0 2.47 7 2.87 4 2.89 2 2.91 8 2.97 5 5.24 3 5.25 9 5.27 5 5.29 1 7.28 9 7.72 0 7.73 2 7.73 7 7.74 6 7.75 6 7.76 1 7.77 3 7.77 8 8.05 7 8.06 3 8.06 6 8.06 9 8.08 1 8.09 1 8.09 5 8.09 8 8.10 5 6.0 9 2.6 5 0.9 4 1.9 0 0.9 1 1.9 9 2.0 0 PC 2.00 GB 0 LB 0.30 Hz SSB 0 WDW EM SF 399.7300000 MHz SI 32768 F2 ? Processing parameters SFO1 399.7324685 MHz PL1 ?4.00 dB P1 10.00 usec NUC1 1H ======== CHANNEL f1 ======== TD0 1 D1 1.50000000 sec TE 295.0 K DE 6.00 usec DW 66.000 usec RG 64 AQ 2.5999219 sec FIDRES 0.192317 Hz SWH 7575.758 Hz DS 2 NS 64 SOLVENT CDCl3 TD 39392 PULPROG zg30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 10.21 Date_ 20091214 F2 ? Acquisition Parameters PROCNO 1 EXPNO 10 NAME 8?smith?1214 Current Data Parameters Sample Label deoisobut_quin Group sintim User Name smith 1-(3-cyclohexylquinoxalin-2-yl) ethanol: 246 10 9 8 7 6 5 4 3 2 1 0 ppm 1.27 9 1.28 8 1.29 8 1.31 4 1.44 2 1.46 2 1.48 5 1.49 2 1.55 5 1.57 1 1.74 8 1.75 4 1.82 9 1.83 7 1.84 8 1.85 5 1.86 3 1.86 8 1.88 0 1.89 6 1.90 8 1.96 0 1.98 8 2.91 2 2.96 5 5.28 8 5.30 4 7.29 0 7.71 2 7.71 7 7.72 7 7.73 7 7.74 2 8.03 4 8.04 0 8.04 1 8.05 3 8.05 8 8.07 1 8.07 5 8.08 1 8.08 7 8.08 9 8.09 6 2.8 1 3.5 8 2.8 3 10.9 6 1.1 4 0.9 4 2.0 0 1.9 7 PC 2.00 GB 0 LB 0.30 Hz SSB 0 WDW EM SF 399.7300000 MHz SI 32768 F2 ? Processing parameters SFO1 399.7324685 MHz PL1 ?4.00 dB P1 10.00 usec NUC1 1H ======== CHANNEL f1 ======== TD0 1 D1 1.50000000 sec TE 295.0 K DE 6.00 usec DW 66.000 usec RG 128 AQ 2.5999219 sec FIDRES 0.192317 Hz SWH 7575.758 Hz DS 2 NS 64 SOLVENT CDCl3 TD 39392 PULPROG zg30 PROBHD 5 mm QNP 1H/1 INSTRUM spect Time 10.02 Date_ 20091214 F2 ? Acquisition Parameters PROCNO 1 EXPNO 10 NAME 7?smith?1214 Current Data Parameters Sample Label deoxycyclohex_quin Group sintim User Name smith 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 References 1. Arias, C. A.; Murray, B. E., Antibiotic-Resistant Bugs in the 21st Century- A Clinical Super-Challenge. New Engl. J. Med. 2009, 360 (5), 439-443. 2. Clatworthy, A. E.; Pierson, E.; Hung, D. T., Targeting virulence: a new paradigm for antimicrobial therapy. Nat. Chem. Biol. 2007, 3 (9), 541-548. 274 3. (a) Sintim, H. O.; Smith, J. A. 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