Synthesis

1
Synthesis Modification of the Antimicrobial
Tripeptide Analog Z-Leu-Val-Gly-CHN2

• Breezy J. Heggenstaller
• Juniata College Chemistry Department
• Dr. Richard Hark, Research Advisor

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Z-LVG-CHN2 Mimics Human Cystatin C1

• cystatin C
• cysteine proteinase inhibitor present in
  extracellular fluids

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Cysteine Proteinase Functions

• intracellular catabolism of peptides and proteins
• processing of prohormones and proenzymes
• penetration of normal human tissue by malignant
  cells and possibly microorganisms, including
  viruses

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Z-LVG-CHN2 Antibacterial Activity

• some bacteria produce proteinases
• tested against several bacterial strains
• exhibits activity against all strains of group A
  streptococci

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Z-LVG-CHN2 Antibacterial Activity

• Activity comparable to well-established
  antibiotics
• tetracycline
• bacitracin
• Strains overexpressing cysteine protease show
  less sensitivity to Z-LVG-CHN2

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Contradiction to These Findings

• research by Dr. Michael Boyle, Juniata College
  William J. Von Liebig Chair in Biomedical
  Sciences with Streptococcus pyogenes
• mutants lacking the gene that produce cysteine
  proteinase growth inhibition is still observed
  when treated with Z-LVG-CHN2

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Importance of This Contradiction

• why are these mutants still susceptible?
• an additional site of action must exist

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Approach to Determination of Mechanism of Action

• synthesize Z-LVG-CHN2
• design and synthesize a fluorescent analog
• use this analog to track binding in the cell

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Synthesis of Z-LVG-CHN2

• classical methods for solution peptide synthesis
• standard coupling reagents (ethyl chloroformate,
  DCC)
• characterization NMR, TLC
• purification HPLC, reverse phase column
  chromatography

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General Scheme
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Step 1
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Step 2
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Step 3
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Step 4
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Step 5
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Step 6
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Step 7
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Z-LVG-CHN2
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Progress

• steps 1-3 completed (show data)
• step 4 chemicals ordered
• previous problem

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Recap

• synthesizing Z-LVG-CHN2
• designing and synthesizing a fluorescent analog
• indirectly testing and monitoring to determine
  site of activity

21

• BjÖrck L, Åkesson P, Bohus M, Trojnar J,
  Abrahamson M, Olafsson I, Grubb A. 1989.
  Bacterial growth blocked by a synthetic peptide
  based on the structure of a human proteinase
  inhibitor. Nature 337385-6.
• Hall A, Abrahamson M, Grubb A, Trojnar J, Kania
  P, Kasprzykowska R, Kasprzykowski F. 1992.
  Cystatin C based peptidyl diazomethanes as
  cysteine proteinase inhibitors influence of the
  peptidyl chain length. J Enzyme Inhibition
  6113-123.