Transcript No Slide Title

Synthesis and Retrosynthesis of
Peptidomimetic Inhibitors Thrombin
Presented by:
Kevin Condel
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.
• Blood clotting must be regulated.
Thrombin
• 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.
•
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.
Thrombin
Click to enlarge
Thrombin as an
Enzyme
• Thrombin has an active site
consisting of the catalytic triad:
Ser 195, His 57, and Asp 102
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)
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.
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
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
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)
Overall
Reaction
S
S
Br 1. BuLi
2. TMSCl
SiMe 3
N
N
OR
OR
OR
CHO
unmasking
N
2-TST
R
CHO
R
S
OH
CHO
R
OH
S
SiMe3
2-TST
N
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
S
Br
Bu- +Li
S

+
N
N
Bu
Br
Me 3Si
Cl

Li
S
SiMe 3
+
N
Li
Cl
2-TST NMR
Identification
S
SiMe3
N
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.
C
OH
LiAlH4
0ºC
O
RT
PCC
C
H
O
RT, 6-12 h
CH2
OH
(No isolation)
CH2
OH
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.
C
OH
LiAlH4
0ºC
O
RT
PCC
C
H
O
RT, 6-12 h
CH2
OH
(No isolation)
CH2
OH
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.
C
OH
LiAlH4
0ºC
O
RT
PCC
C
H
O
RT, 6-12 h
CH2
OH
(No isolation)
CH2
OH
Benzaldehyde NMR
Identification
C
H
O
Amino Acid Reduction Methodology
(Theoretical)
Click to enlarge
• 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.
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.
+
+
H3N
C
O
C
+
NH
H2N
H3N
OH
LiAlH4
0ºC
RT
+
CH2
NH
H2N
NH2
OH
PCC
C
+
H3N
NH2
(No isolation of
alkoxy-salt)
0ºC 2 h
RT 6-12 h
C
+
C
H
NH
H2N
NH2
O
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.
+
+
H3N
C
O
C
+
NH
H2N
H3N
OH
LiAlH4
0ºC
RT
+
CH2
NH
H2N
NH2
OH
PCC
C
+
H3N
NH2
(No isolation of
alkoxy-salt)
0ºC 2 h
RT 6-12 h
C
+
C
H
NH
H2N
NH2
O
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.
+
+
H3N
C
O
C
+
NH
H2N
H3N
OH
LiAlH4
0ºC
RT
+
CH2
NH
H2N
NH2
OH
PCC
C
+
H3N
NH2
(No isolation of
alkoxy-salt)
0ºC 2 h
RT 6-12 h
C
+
C
H
NH
H2N
NH2
O
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.
Retrosynthesis and Unmasking
Protocol Mechanism (Theory)
+
H3N
C
+
+
C
H3N
H
O
N
RT, 4 h
NH
H2N
Me3Si
+
S
C
OH
S
NH
H2N
NH2
Aldehyde of
Argenine
C
N
NH2
Intermediate
2-TST
RT, 4 h
Retrosynthesis and Unmasking
Protocol Mechanism (Theory)
Me
+
H3N
C
+
+
N
H3N
C
OH
S
NH
H2N
CF3SO3Me4
NaBH4
C
+
N
C
OH
S
NH
H2N
NH2
NH2
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.
Retrosynthesis and Unmasking
Protocol Mechanism (Theory)
Me
+
H3N
C
+
N
H3N
C
OH
NH
O
+
S
Cu++ / Water
C
+
C
C
H
OH
NH
H2N
H2N
NH2
NH2
Cu++ and water are added to hydrolyze the system, removing the
thiazole ring and adding yet another aldehyde.
Retrosynthesis and Unmasking
Protocol Mechanism (Theory)
+
H3N
C
+
C
CH2
O
+
NR2
H3N
C
C
H
OH
NaBH3CN
NH
HNR2
C
+
H2N
NH2
OH
NH
H2N
NH2
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.
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
Questions?
Thrombin
Theory
Structure
Peptidomimetics
Inhibition
Project Goals
Method
Argenine
Reduction
Catalytic
Triad
Apparatus
Problems
Binding
Sites
Overall Reaction
Retrosynthesis
2-TST Reaction
Benzaldehyde Reaction
NMR Identification
NMR Identification
Proposed Peptidomimetic
Click to view natural substrate
Natural Substrate
Click to view proposed peptidomimetic
PCC
Pyridinium Chlorochromate
NH+
CrO3Cl-