Synthesis and Retrosynthesis of Peptidomimetic Inhibitors - Thrombin

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Synthesis and Retrosynthesis of Peptidomimetic
Inhibitors - Thrombin

• Presented by
• Kevin Condel

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Overview / Terminology

• Goal To design and synthesize a peptidomimetic
  that competes to inhibit the enzyme thrombin.
• Thrombin is part of a cascade leading to the
  formation of insoluble fibrin, a material found
  in blood clots. Unregulated clotting may lead to
  cardiac arrest or stroke.
• A Peptidomimetic is any small organic molecule
  that mimics the transition state of a natural
  substrate.
• Peptidomimetics competitively inhibit the enzyme
  process, preventing the natural reaction from
  occurring.
• i.e. - The peptidomimetic binds more readily to
  thrombin than the substrate.
• Hydroxy-aldehydes are important components of
  peptidomimetic inhibitors of the thrombin system.
  This work involves the development of a simple,
  yet effective protocol for the generation of
  hydroxy-aldehydes.

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Thrombin

• Blood clotting must be regulated.
• Errors in blood clotting lead to
• cardiac arrest (in the heart)
• stroke (in the brain)
• Thrombin begins inactive and is shown on the
  bottom-left.
• Inactive thrombin has extra domains, colored
  blue, which are clipped off during activation.
• The purple atoms are Ca2 ions, bound to modified
  glutamate amino acids.
• The strong () charge on these ions tether the
  protein to the surfaces of blood vessels, holding
  thrombin and localizing it to one spot.

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Thrombin

• Since inactive thrombin is held, blood clots will
  generally not spread to other areas.
• Only the thrombin adjacent to the damage will be
  activated.
• Activated thrombin (the upper structure shown
  here) lasts only seconds, serving also to limit
  the clot to the area of damage.
• Thrombin is simply part of a cascade which serves
  to synthesize the cross-linked fibrin polymers
  found in blood clots.

Click to enlarge
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Thrombin as an Enzyme

• Thrombin has an active site consisting of the
  catalytic triad Ser 195, His 57, and Asp 102

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Enzyme Binding Site

• In addition to the active site, thrombin has
  three binding sites, labeled as S1, S2 and S3,
  that determine the strength and specificity of
  binding
• The lipophilicity of S3 has been well determined
• Lipophilicity represents the affinity of a
  molecule or moiety for a lipophilic environment
  (i.e. hydrophobicity)

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Inhibition of the Active Site

• Leeches synthesize proteins that block thrombin
  (and other enzymes), stopping the formation of
  the clot.
• One example, a protein called hirudin, is shown
  here on the left in blue. Notice how it fits the
  active site of thrombin perfectly.

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Peptidomimetics

• Small peptide-like molecules that mimic
  transition state of substrate and work by
  competitively inhibiting the binding of the
  natural substrate (ex. Hirudin from the leech)
• Peptide analog must be stable.
• Drug must be a reversible inhibitor of the enzyme
  but can be irreversible if the enzyme is unique
  to the disease.

Saquinavir
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Project Design

• Goals
• Design a polypeptide isotere based on a natural
  thrombin substrate (natural
  Phe-Pro-Arg tripeptide shown below)
• Optimize a generalized scheme for isotere
  synthesis
• Model the S2 and S3 steric and hydrophobic
  requirements

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Project Setup

• All reactions required an anhydrous environment.
• Nitrogen steadily flushed through the system to
  exclude water vapor.
• Various temperatures were achieved as follows
• -78?C (Dry Ice Acetone)
• 0?C (Ice Bath)

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Overall Reaction
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2-TST
1. 4.22ml n-Butyl-Lithium was injected into a
round bottom flask containing a swirling
solution of 50 ml ether through the septum using
the syringe 2. 2.33ml 2-Bromothiazole in 50 ml
of ether slowly added over 30 min via
a separatory funnel into a controlled -78ºC
nitrogenous environment. 3. Mixture allowed to
stir for 30 min 4. 4.33ml (CH3)SiCl in 50 ml
ether added drop wise for 30 min via the sep
funnel
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2-TST NMR Identification
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Benzaldehyde
1. 7.33g Benzoic Acid was added under inert
nitrogenous conditions to a flask containing 15
mL Tetrahydrofuran (THF). 2. Under 0ºC
conditions, 45 mL of pre-cooled 1.0 M Lithium
Aluminum Hydride (LiAlH4) in THF was added
dropwise with vigorous stirring.
(No isolation)
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Benzaldehyde
3. After the hydrogen had evolved, the solution
was cooled to room temp. and stirred for 30
min. 4. In a separate flask under the same
nitrogenous conditions, 14.3 g Pyridinium
Chlorochromate (PCC) was added to 100mL
Methylene Chloride and stirred into solution.
(No isolation)
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Benzaldehyde
5. The alkoxyaluminum salt in THF created by
mixing LiAlH4 with benzoic acid was next added
dropwise at room temperature via a separatory
funnel 6. The reaction mixture was stirred for
12 hours at room temperature, diluted with
diethyl ether, and filtered and washed to
remove the supernatant liquid. The ether was
then distilled from the filtrate to obtain
benzaldehyde.
(No isolation)
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Benzaldehyde NMR Identification
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Amino Acid Reduction Methodology (Theoretical)

• Use benzaldehyde synthesis schematic to reduce
  the amino acid argenine
• When reduced argenine is combined with 2-TST it
  creates the Arg side chain of the Phe-Pro-Arg
  tripeptide peptidomimetic
• Unmasking protocol removes thiazole ring and
  replaces it with CHO group, creating the active
  site inhibitor.
• Phe-Pro addition will be conducted in future
  experimentation.

Click to enlarge
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Reduction of Argenine (Novel Approach)
1. 12.64g Argenine was added under inert
nitrogenous conditions to a flask containing 20
mL Methyl Sulfoxide as a solvent. 2. Under 0ºC
conditions, 45 mL of pre-cooled 1.0 M Lithium
Aluminum Hydride (LiAlH4) in THF was added
dropwise with vigorous stirring.
(No isolation of alkoxy-salt)
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Reduction of Argenine (Novel Approach)
3. After the hydrogen had evolved (2 h), the
solution was cooled to room temp. and stirred
for 30 min. The intermediate was not
isolated. 4. In a separate flask under the same
nitrogenous conditions, 14.3 g Pyridinium
Chlorochromate (PCC) was added to 100mL
Methylene Chloride and stirred into solution.
(No isolation of alkoxy-salt)
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Reduction of Argenine (Novel Approach)
5. The intermediate in Methyl Sulfoxide created
by mixing LiAlH4 with argenine was next added
dropwise at room temperature via a separatory
funnel. 6. Mixture stirred for 12 hours at room
temperature, diluted with diethyl ether,
filtered and washed. The ether was then
distilled from the filtrate to obtain the
aldehyde of argenine.
(No isolation of alkoxy-salt)
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Methodological Problems with Argenine Reduction

• Need a nonpolar, nonreactive solvent to dissolve
  argenine without interfering with the reaction.
• Methyl Sulfoxide NOT efficient as a solvent for
  this reaction due to its exothermicity.
• i.e (Broken Manifold and intense sulfur scent)
• Length of complete reaction and temperature
  requirements are dependent on the solvent used to
  dissolve argenine.
• With Methyl Sulfoxide, it is proposed that 0ºC
  conditions must exist for at least two hours
  prior to addition of PCC.

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Retrosynthesis and Unmasking Protocol Mechanism
(Theory)
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Retrosynthesis and Unmasking Protocol Mechanism
(Theory)
Previous research has determined that CF3SO3Me4
is added to N-methylate the thiazole ring. NaBH4
is added to reduce the mixture and break the pi
bonds.
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Retrosynthesis and Unmasking Protocol Mechanism
(Theory)
Cu and water are added to hydrolyze the system,
removing the thiazole ring and adding yet
another aldehyde.
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Retrosynthesis and Unmasking Protocol Mechanism
(Theory)
HNR2
HNR2 is added as part of a dehydration reaction
to remove water and add NR2 to the molecule.
This NR2 represents the part of the molecule
that will be interacting with the active site of
thrombin.
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References

• Alessandro Dondoni, et al. Synthesis of TSTs and
  Reactions with Carbonyl Compounds J. Org. Chem.
  1988, 53, 1748-1761
• Benoit Bachand , et al. Synthesis and
  Structure-Reactivity of Potent Bicyclic Lactam
  Thrombin Inhibitors Bioinorg. Med. Chem. 1999,
  9, 913-918
• Jin Soon Cha, et al. Preparation of Aldehydes
  from Carboxylic Acids by Reductive Oxidation with
  Lithium Aluminum Hydride and Pyridinium
  Chlorochromate or Pyridinium Dichromate Bull.
  Korean Chem Soc. 1999, Vol. 20, No. 4

Acknowledgements

• We gratefully acknowledge the support of the
  Welch Foundation in the form of a Departmental
  Research Grant

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Questions?
Thrombin
Theory
Structure
Peptidomimetics
Method
Argenine Reduction
Inhibition
Project Goals
Catalytic Triad
Apparatus
Problems
Binding Sites
Retro- synthesis
Overall Reaction
2-TST Reaction
Benzaldehyde Reaction
NMR Identification
NMR Identification
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Proposed Peptidomimetic
Click to view natural substrate
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Natural Substrate
Click to view proposed peptidomimetic
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PCC Pyridinium Chlorochromate