Tailored Vancomycin Diversification and Synthetic Strategies Brad Cox Michigan State University March 29, 2006
utline Bacterial and antibiotic awareness ecessity for novel antibiotics Antibiotic targets and modes of action Cell-wall biosynthesis Combinatorial natural product diversification Enzymatic diverisification Chemical modifications Synthetic glycopeptide mimics
Bacterial Facts Bacteria are everywhere from volcanoes to acid wells and icebergs inhabit most areas on your body can be pathogens Antibiotics subjection selection resistance Bioteach.ubc.ca
in 2005, 33 million pounds antibiotics consumed Subjection over-prescription Furuya, E.Y.; Lowy, F.D. at. Rev. Microbiol. 2006. 4. 36.
in 2005, 33 million pounds antibiotics consumed Subjection over-prescription short generation times Selection evolution: mutations, gene transfer Furuya, E.Y.; Lowy, F.D. at. Rev. Microbiol. 2006. 4. 36.
in 2005, 33 million pounds antibiotics consumed Subjection over-prescription short generation times Selection evolution: mutations, gene transfer 90% of S. aureus penicillin resistant Resistance call for new antibiotics Furuya, E.Y.; Lowy, F.D. at. Rev. Microbiol. 2006. 4. 36.
Antibiotic Targets DA Replication and Repair fluoroquinones Protein Biosynthesis macrolides, tetracyclins, aminoglycosides, and oxazolidinones RA Transcription rifamycin and actinomycin Cell-wall Biosynthesis Glycopeptides β lactams penicillins, cephalosporins
Glycopeptide Properties eptapeptide scaffold Aromatic residues C 3 2 on-ribosomal amino acid production C 3 ighly oxidized Rigid, domed framework 2 Vancomycin
37% Increase in Prescriptions '04-'05 Isolation from Indian Soil Microbe Amycolatopsis orientalis $160 Million in 2005 Vancomycin "Last Resort" Antibiotic Taken Intraveniously Targets Gram + rganisms Padma, P..; Roa, A.B.; Yadav,J.S.; Reddy, G. Appl. Biochem. Biotech. 2002.102-103. 395. Smith, T.L. ew Eng. J Med. 1999. 340. 493. http://phx.corporate-ir.net http://www.mrw.interscience.wiley.com/kirk/articles/glyccava.a01/pdf_fs.html
37% Increase in Prescriptions '04-'05 Isolation from Indian Soil Microbe Amycolatopsis orientalis $160 Million in 2005 Vancomycin "Last Resort" Antibiotic Taken Intraveniously Targets Gram + rganisms Padma, P..; Roa, A.B.; Yadav,J.S.; Reddy, G. Appl. Biochem. Biotech. 2002.102-103. 395. Smith, T.L. ew Eng. J Med. 1999. 340. 493. http://phx.corporate-ir.net http://www.mrw.interscience.wiley.com/kirk/articles/glyccava.a01/pdf_fs.html
37% Increase in Prescriptions '04-'05 Isolation from Indian Soil Microbe Amycolatopsis orientalis $160 Million in 2005 Vancomycin "Last Resort" Antibiotic Taken Intraveniously Targets Gram + rganisms Padma, P..; Roa, A.B.; Yadav,J.S.; Reddy, G. Appl. Biochem. Biotech. 2002.102-103. 395. Smith, T.L. ew Eng. J Med. 1999. 340. 493. http://phx.corporate-ir.net http://www.mrw.interscience.wiley.com/kirk/articles/glyccava.a01/pdf_fs.html
37% Increase in Prescriptions '04-'05 Isolation from Indian Soil Microbe Amycolatopsis orientalis $160 Million in 2005 Vancomycin "Last Resort" Antibiotic Taken Intraveniously Targets Gram + rganisms Padma, P..; Roa, A.B.; Yadav,J.S.; Reddy, G. Appl. Biochem. Biotech. 2002.102-103. 395. Smith, T.L. ew Eng. J Med. 1999. 340. 493. http://phx.corporate-ir.net http://www.mrw.interscience.wiley.com/kirk/articles/glyccava.a01/pdf_fs.html
37% Increase in Prescriptions '04-'05 Isolation from Indian Soil Microbe Amycolatopsis orientalis $160 Million in 2005 Vancomycin "Last Resort" Antibiotic Taken Intraveniously Targets Gram + rganisms Padma, P..; Roa, A.B.; Yadav,J.S.; Reddy, G. Appl. Biochem. Biotech. 2002.102-103. 395. Smith, T.L. ew Eng. J Med. 1999. 340. 493. http://phx.corporate-ir.net http://www.mrw.interscience.wiley.com/kirk/articles/glyccava.a01/pdf_fs.html
37% Increase in Prescriptions '04-'05 Isolation from Indian Soil Microbe Amycolatopsis orientalis $160 Million in 2005 Vancomycin "Last Resort" Antibiotic Taken Intraveniously Targets Gram + rganisms Padma, P..; Roa, A.B.; Yadav,J.S.; Reddy, G. Appl. Biochem. Biotech. 2002.102-103. 395. Smith, T.L. ew Eng. J Med. 1999. 340. 493. http://phx.corporate-ir.net http://www.mrw.interscience.wiley.com/kirk/articles/glyccava.a01/pdf_fs.html
37% Increase in Prescriptions '04-'05 Isolation from Indian Soil Microbe Amycolatopsis orientalis $160 Million in 2005 Vancomycin "Last Resort" Antibiotic Taken Intraveniously Targets Gram + rganisms Padma, P..; Roa, A.B.; Yadav,J.S.; Reddy, G. Appl. Biochem. Biotech. 2002.102-103. 395 Smith, T.L. ew Eng. J Med. 1999. 340. 493. http://phx.corporate-ir.net http://www.mrw.interscience.wiley.com/kirk/articles/glyccava.a01/pdf_fs.html
Vancomycin Total Syntheses Convergent syntheses Average steps: 95 Average yield: < 1% S Ar Cyclization S Ar Cyclization Macrolactamization 2 Peptide Coupling Suzuki Coupling Boger, D.L. J. Am. Chem. Soc. 1999, 121, 3226. Evans, D.A. Angew. Chem. Int. Ed. 1998, 37, 2700. icolaou, K.C. Chem. Eur. J. 1999, 5, 2548-2667.
Vancomycin Total Synthesis 1 S Ar Cyclization 5 S Ar Cyclization C D E 3 Macrolactamization B 2 A 4 Peptide Coupling 2 Suzuki Coupling 2 Me F F ME M C Me 2 C Boc B() 2 TBS B Me Br D Boc Me 2 C C E 2 Me A Me Me Boger, D.L.; Miyazaki, S.; Kim, S..; Wu, J..; Jin, Q. J. Am. Chem. Soc. 1999, 121, 10004.
Cell-Wall Biosynthesis Ac Ac P _ L-Ala D-Glu P L-Lys Lipid D-Ala D-Ala Lipid II Precursor Glycotransferase _ Ac Ac L-Ala D-Glu L-Lys D-Ala D-Ala Ac Ac L-Ala D-Glu L-Lys 2 D-Ala D-Ala Transpeptidase D-Ala D-Ala L-Lys D-Glu L-Ala Ac Ac Schwarts, B.; Markwalder, J.A.; Wang, Yi. J. Am. Chem. Soc. 2001, 123, 11638.
Vancomycin Primary Mode of Action P _ Lipid II Precursor Lipid P _ Ac D-Ala D-Glu D-Ala L-Lys L-Ala Ac Cell Membrane L-Lys 2 Barna, J.C.J.; Williams, D.. Annu. Rev. Microbiol. 1984, 38, 339.
Glycopeptide Resistance 2 *Loss of 1 bond leads to a 1000-fold affinity decrease L-Lys Cell Membrane Bugg, T.D.; Dutka-Malen, S.; Arthur, M.; Courvalin, P. Biochemistry, 1991, 30, 2017.
eptapeptide Scaffold Engineering 2 Crowley, B.M.; Boger, D.L. J. Am. Chem. Soc. 2006, 128, 2885.
eptapeptide Scaffold Engineering 2 100-fold binding Affinity increase over Vancomycin on VRSA Crowley, B.M.; Boger, D.L. J. Am. Chem. Soc. 2006, 128, 2885.
eptapeptide Scaffold Engineering 2 L-Lys Cell Membrane Crowley, B.M.; Boger, D.L. J. Am. Chem. Soc. 2006, 128, 2885.
Differential Glycopeptide Activities Against S. aureus In vitro activity (MIC 90 ) of glycopeptides Bacterial Species Status MIC 90 (mg/l) Vancomycin ritavancin Telavancin Teicoplanin Dalbavancin S. aureus MSSA 1 1 0.5 1 0.06 MRSA 1 1 0.5 2 0.06 VISA 8 1 2 2 GRSA >128 0.5 2 0.25 MSSA methicillin sensitive S. aureus MRSA methicillin resistant S. aureus VISA vancomycin intermediate S. aureus GRSA glycopeptide resistant S. aureus. Van Bambeke, F. Curr. pin. Pharmacol. 2004, 4, 471.
Glycoside Effects C 3 R 1 Vancomycin R 1 =C 3 R 2 = R 2 Me C 3 R 1 Me R 3 Chlorobiphenyl Vancomycin Chlorobiphenyl Disaccharide R 1 =C 3 R 2 = R 3 = MIC (μg/ml) E. faecium E. faecalis Sensitive Resistant Sensitive Resistant vancomycin 1 2048 4 2048 chlorobiphenyl vancomycin 0.03 16 0.25 16 chlorobiphenyl disaccharide 128 128 128 128 Ge, M.; Chen, Z.; nishi.r.; Kohler, J.; Kahne, D. Science, 1999, 284, 507.
Proposed Inhibition Models of ydrophobically Derivatized Vancomycin Membrane anchoring Dimerization Transglycosylase inhibition
Membrane Anchoring D-Ala D-Ala L-Lys D-Glu L-Ala 2 3 C C 3 Membrane Lipid Layer Lipid layer ydrophobic interactions Sharman, G.J.; Try, A.C.; Dancer, R.J.; Cho, Y.R.; Williams, D.. J. Am. Chem. Soc. 1997, 119, 12041.
Glycopeptide Vessicle Anchoring C 3 2 3 C C 3 R 1 C 3 CE:Chloroeremomycin R 1 = BCE:Biphenyl R 1 = Sharman, G.J.; Try, A.C.; Dancer, R.J.; Cho, Y.R.; Williams, D.. J. Am. Chem. Soc. 1997, 119, 12041.
Glycopeptide Dimerization 2 3 C C 3 3 C C 3 2 Lipid II Lipid II Sharman, G.J.; Try, A.C.; Dancer, R.J.; Cho, Y.R.; Williams, D.. J. Am. Chem. Soc. 1997, 119, 12041.
Glycopeptide Dimerization 3 C 2 C 3 R 1 C 3 Dimerization Constant (M -1 ) Free Antibiotic Tripeptide Complex BCE(D) 1.6±0.2 x 10 5 3.0±1.0 x 10 7 BCE() 2.0±1.0 x 10 5 CE(D) 1.8±0.6 x 10 5 1.1±0.4 x 10 7 CE() 1.6±0.2 x 10 4 C 3 D 2 CE:Chloroeremomycin R 1 = BCE:Biphenyl R 1 = Sharman, G.J.; Try, A.C.; Dancer, R.J.; Cho, Y.R.; Williams, D.. J. Am. Chem. Soc. 1997, 119, 12041.
Transglycosylase Inhibition Chlorobiphenyl- R 2 = C 3 C 3 2 R 2 Vancomycin R 1 = R 1 R 2 = Damaged Vancomycin R 1 = R 2 = C Ac 2 R 3 = Ac C 2 C 2 R 3 P Moenomycin Delipidated Moenomycin R 3 = Chen, L.; Walker, D.; Sun, B.; Kahne, D. Proc. atl. Acad. Sci. 2003, 100, 5658.
Transglycosylase Inhibition C Ac 2 C 2 P Ac Moenomycin C 2 R 3 R 3 = Delipidated Moenomycin R 3 = Compound IC 50 (μg/ml) vancomycin 0.38 damaged vancomycin 144 chlorobiphenyl vancomycin 1.5 damaged chlorobiphenyl vancomycin 3.88 moenomycin 0.0006 damaged moenomycin 2.23 Chen, L.; Walker, D.; Sun, B.; Kahne, D., Proc. atl. Acad. Sci. 2003, 100, 5658.
Transglycosylase Inhibition C 3 C 3 R 2 R 1 2 Vancomycin R 1 = R 2 = Chlorobiphenyl- R 2 = Damaged Vancomycin R 1 = R 2 = Compound IC 50 (μg/ml) vancomycin 0.38 damaged vancomycin 144 chlorobiphenyl vancomycin 1.5 damaged chlorobiphenyl vancomycin 3.88 moenomycin 0.0006 damaged moenomycin 2.23 Chen, L.; Walker, D.; Sun, B.; Kahne, D., Proc. atl. Acad. Sci. 2003, 100, 5658.
Efficacy Comparision of ydrophobic Chain Length MIC (μg/ml) Compound n Methicillin Resistant S. aureus n 3 8 1600 25 12 12.5 4 125 n 8 1 10 14 0.5 5 C 3 C 3 n 10 0.06 12 2.8 vancomycin aglycon Ge, M.; Chen, Z.; nishi.r.; Kohler, J.; Kahne, D. Science, 1999, 284, 507. Mu, Y.; odwell, M.; Pace, J.L.; Shaw, J.P. Bioorg. Med. Chem. Lett. 2004, 14, 735. Kubo, I.; Fujita K.; ihei, K.; Masuoka,. Bioorg. Med. Chem. 2003, 11, 573. Klink, J.W.; Larsen, L.; Perry,.B.; Weavers, R.T.; Cook, G.M.; Bioorg. Med. Chem. 2005, 13, 6651.
Enzymatic Diversification C 3 2 2 3 C C 3 C 3 2
Enzymatic Glycosylation Vancomycin Aglycon P - P - R GtfE TDP 2 Vancomycin Aglycon GtfD TDPsugar TDP-4-epi- Vancosamine UDP Gtf - Glycotransferase Vancomycin Aglycon Losey,.C.; Jiang, J.; Biggins, J.B.; Kahne, D.; Thorson, J.S.; Walsh, C.T. Chem. Biol. 2002, 9, 1305.
Enzymatic Glycosylation GtfA TDP epi-vancosamine 3 C 2 C 3 Chloroorienticin B GtfD TDP vancosamine Gtf - Glycotranferase GtfC TDP epi-vancosamine Vancomycin C 3 2 3 C C 3 2 C 3 Chloroeremomycin berthur, M.; Leimkuhler, C.; Kruger, R.G.; Lu, W.; Kahne, D.; Walsh, C.T. J. Am. Chem. Soc. 2005, 127, 10747.
Chemical Modification 3 C 3 2 C 3 2 2
Glycorandomization 3 R 1 CuI, 70 C, 12 h 35-87% R 1 Fu, X.; Albermann, C.; Jiang, J.; Zhang, C.; Thorson, J. ature Biotech. 2003, 21, 1467. Fu, X.; Albermann, C.; Zhang, C.; Thorson, J. rg. Lett. 2005, 7, 1513.
Derivatized Sugar Effect on Vancomycin Efficacy 1 2 3 4 5 Ph C S MIC (µg/ml) Compound S. aureus E. faecalis E. faecium Van 12 23 23 1 11 10 10 2 7 12 11 3 13 >24 >24 4 >24 16 12 5 10 20 19 Fu, X.; Albermann, C.; Zhang, C.; Thorson, J. rg. Lett. 2005, 7, 1513.
Derivatized Sugar Effect on Vancomycin Efficacy 1 2 3 4 5 Ph C S MIC (µg/ml) Compound S. aureus E. faecalis E. faecium Van 12 23 23 1 11 10 10 2 7 12 11 3 13 >24 >24 4 >24 16 12 5 10 20 19 o increased efficacy Fu, X.; Albermann, C.; Zhang, C.; Thorson, J. rg. Lett. 2005, 7, 1513.
Solid Phase Vancosamine and -terminus Derivitization C 3 R 1 C 3 2 R 2 MIC (μg/ml) Vancosamine R 1 -terminus R 2 E. faecalis E. faecalis Resistant S. aureus F 2.5 >10 2.5 5 10 5 >10 10 5 5 >10 5 Vancomycin C 3 2.5 >10 2.5 Demethylvancomycin 2.5 >10 <0.6 Fmoc C 3 2.5 >10 0.6 Yao,..; Liu, G.; e, W.Y. Bioorg. Med. Chem. Lett. 2005, 15, 2325.
Enzymatic Glucosamine Acylation 2 aatf or tatf (n) CoA n=2-16 (n) Atf - acetyltransferase Kruger, R.G.; Lu, W.; berthur, M.; Kahne, D.; Walsh, C.T. Chem. Biol. 2005, 12, 131.
Lipoglycopeptide Efficacy Comparison C 3 C 3 n X m 2 MIC 50 (μg/ml) Entry X n m MSSA MRSA GRSA 1 S-S 1 6 0.3 0.6 1.2 2 S-S 2 6 0.6 1.2 1.8 3 S-S 1 7 1.2 2.4 0.6 4 1 7 0.4 0.4 1.8 5 C 2 1 7 2.8 1.5 1.6 Vancomycin 0.7 2 >256 Mu, Y.; odwell, M.; Pace, J.L.; Shaw, J.P. Bioorg. Med. Chem. Lett. 2004, 14, 735.
Tissue Distribution C 3 X C 3 2 % dose X in liver in kidney in urine S-S 4.9 0.5 17.3 37 22 16 C 2 70 3.4 0.4 * 50 mg/kg dose Mu, Y.; odwell, M.; Pace, J.L.; Shaw, J.P. Bioorg. Med. Chem. Lett. 2004, 14, 735.
Attempted RCM-Based Vancomycin Mimic Synthesis 2 Me Boc Boc Brink,.T.; Rijkers, D.T.S.; Kemmink, J.; ilbers,.w.; Liskamp, R.M.J. rg. Biomol. Chem. 2004, 2, 2658.
Attempted RCM-Based Vancomycin Mimic Synthesis Mes Mes Ru Ph PCy 3 Boc overall 18.2 % Boc Brink,.T.; Rijkers, D.T.S.; Kemmink, J.; ilbers,.w.; Liskamp, R.M.J. rg. Biomol. Chem. 2004, 2, 2658.
Partial Tripeptide Vancomycin Mimic R 2 R 3 I Sonagashira Cyclization 6-23% R 4 R 1 Boc R 3 R 2 R 5 R 2 Boc R 4 Boc R 1 R 3 R 4 Boc t-bu Macrolactamization 54-61% R 1 Brink,.T.; Rijkers, D.T.S.; Liskamp, M.J. J. rg. Chem. 2006, 71, 1817.
Lactamization of Partial Vancomycin Mimic ATU _ + PF 6 I + Boc Cbz t-bu + _ + 80% 10% Pd(PPh 3 ) 4 10% CuI Et 2 TF Cbz Boc t-bu 1)TFA/C 2 2 1:1 2)ATU/At/DIPEA DMF 0.5mM 54% Cbz Brink,.T.; Rijkers, D.T.S.; Liskamp, M.J. J. rg. Chem. 2006, 71, 1817.
Future Synthetic Strategy I Me Me Me R 1 Boc R 5 Me R 1 R 1 Boc R 5 Brink,.T.; Rijkers, D.T.S.; Liskamp, M.J. J. rg. Chem. 2006, 71, 1817.
Conclusion Abuse of antibiotics ecessity for cycles of new antibiotics Synergistic enzymatic and chemical derivitization of the glycopeptide scaffold Enzymatic diversification Future synthetic efforts for further and more robust diversification 3 2 2 Chemical Modification
Acknowledgements John Frost Karen Frost Group Members Vu Bui Steve Cheley Jiantao Guo Justas Jancauskas Man Kit Lau ingqing Ran eather Stueben Dongming Xie Jinsong Yang Jennifer Froelich Emma s Bakery in kemos