Synthesis of Model Compounds that Mimic the Primary and Secondary
Coordination Sphere of Carbonic Anhydrase
1,
Martin Garcia Chavez
Ian
2
Shaw ,
Jeff Barlow
2, Eric
2
Brown*
C.
(1)Manchester University, (2)*Boise State University, Department of Chemistry and Biochemistry, Boise, ID
Abstract TPA as Suppor6ng Ligand for Synthe6c Models Metalloenzymes play a critical role in the daily life of humans, animals, and
plants. An example of this type of enzymes is carbonic anhydrase (CA),
which is present in all living organisms. The main function of CA is the
reversible hydration of CO2 to form bicarbonate, which it performs at a very
high catalytic activity. Additionally, recent studies have determined that CA
is the enzyme in plants that reacts with atmospheric carbonyl disulfide
(CS2) and carbonyl sulfide (COS). The focus of this research is to gain insight
into how carbonyl disulfide activation occurs at the zinc protein site
through a synthetic modeling approach; where low molecular complexes are
designed to mimic the active site and the reactivity of the enzyme. One of
the most intriguing mechanistic questions concerning the catalytic cycle for
COS activation by CA is the desulfurization of the zinc hydrosulfide that is
formed. Our hypothesis is that the secondary amino acid residues (those
that do not directly bind to the zinc center) play a key role by making the
SH a better leaving group. Details of the reactivity and characterization of
model complexes relevant to the catalytic cycle of CA will be presented.
N: Pyridine N atoms that
bind to the zinc atom
NH
(primary coordination
HN
N
N
sphere).
N-H: Hydrogen on amino
N
groups bind to the SH group
of the Zn-SH complex
N
(secondary coordination
sphere).
N-benzyl-6-((((6-(benzylamino)pyridin-2-yl)methyl)(pyridin-2-ylmethyl)amino)methyl)pyridin-2Reasoning
amine
• When ligand is complexed with Zn it has been shown to interact
with heterocumulenes under basic conditions.
• Easy to synthesize and modify to more accurately reflect the
active site of carbonic anhydrase.
• The ligand contains two hydrogen bond donor sites (atoms labeled
in red).
Introduc6on to Carbonic Anhydrase Description
• Metalloenzyme common to animals, plants, and bacteria.
• Active site consists of a Zn(II) ion coordinated
to three histidines and an aqua ligand.
• Both Zn and Cd forms found naturally.
• Active site requires hydrogen bonding residues
for catalytic efficiency.
(1) Graphic of CA active site taken from
Protein Databank: http://
www.rcsb.org /pdb/cgi/explore.cgi?
pdbId=1CA2
Functions
• Has a major role in CO2 transport and regulation
of blood pH levels
• Plays a critical role in COS (carbonyl sulfide) sequestration by
plants
• Has a high catalytic efficiency that approaches the
diffusion-control limit
Effects of Hydrogen Bond Donors Synthesis of the Ligand Synthesis of Compound 1:
O
N
2
Br
H
N
2
+
+
H
O
SH
B- O
Na+ O
NH2
O
O
No Hydrogen Bond Donors
SH Chemical Shift: -1.520 ppm
O
N
CH2Cl2
N
Br
Br
+
N
63.3 %Yield
N
(1)
N
One Hydrogen Bond Donor
SH Chemical Shift: -0.929 ppm
N,N-bis((6-bromopyridin-2-yl)methyl)(pyridin-2-yl)methanamine
Synthesis of Ligand:
E
N
Previous Studies Towards the Isola6on and Characteriza6on of a Zinc Hydrosulfide Complex S
F
C
G
I
B
H
4-5 ppm region
H
D
N
I
K
K
K
K
41.20 % Yield
N
1. Zn(ClO4)2 6H2O, CH3CN
2. (CH3)4NOH
3. S=C=O
N
N
Zn
N
N
J
+
A
G
H
E
S: Residual
Solvent
N
N
N
N
Two Hydrogen Bond Donors
SH Chemical Shift: -0.553 ppm
N
SH
N
N
Hydrosulfide through Use of an Aprotic Solvent
+
ClO4-
H
K
N
H
NH2 NaOH,
toluene/H2O
7 days
Zn
N
F
HN
(1) + 20
δ
HS
N
Research Objec6ves N
N
N
N
NH
• Isolate and characterize Zn intermediates pertinent to the
mechanism proposed.
• Explain the chemistry fundamental to the formation of [ZnII-SH]+.
• Evaluate the effect that hydrogen bond donors have on the
formation of ZnII-SH complexes and their desulfurization.
• Develop a catalytic cycle based on carbonic anhydrase.
Zn
+
N
δ
SH
Zn
N
H
N
N
N
S
Yield = 73%
Research Ques6on Crystal Structure of
[(TPA)Zn-SH]+
Cationic Portion.
Proposed Mechanism
H
H
O
H2O
- H2S
Zn His
His
His
G
+
-H
O
H
O
O
O=C=S
Zn His
His
His
A
O
H2S and CO2
H
Zn His
O
His
His
H
E
S
S
Pathway A
H
- CO2
H2O
O
H
O
Zn His
His
His
F
SH
CO2
S: Residual Solvent
B
A
B, 3H
A, 3H
C, 3H
D, 3H
E, 6H
N
C
E
D
9.00
8.50
*8.00
5.0
N
N
N
N
N
N
1. Zn(ClO4)2 6H2O, Dry MeOH
2. Anhydrous NaHS
N
H
N
H
δ
SH
Zn
N
N
N
N
•  Based on the NMR spectrum, the Zn-SH complex was synthesized
successfully albeit as a mixture of compounds.
•  The SH peak is observed at -0.553 ppm.
•  The complex is labile in the presence of water.
•  Create the zinc hydrosulfide complex in the absence of water.
The zinc hydrosulfide appears to be very sensitive to water.
•  Conduct alkylation reactions with the zinc hydrosulfide complex
to determine the nucleophilicity of the SH group.
•  Determine the effects of hydrogen bond donors on the
desulfurization of the zinc hydrosulfide.
Conclusions • Characterized many compounds relevant to the catalytic cycle of
carbonic anhydrase.
• The secondary coordination sphere of the active site of carbonic
anhydrase plays a key role in the desulfurization step.
• Hydrogen bonding affects the sensitivity of the Zn-SH complex in
the presence of water.
• The sulfonium ion character of the SH bond increases as hydrogen
bonding increases shifting the -SH chemical shift downfield in the
1H NMR.
Acknowledgements 7.50
F, IH
10.0
HN
NH
Zn
N
Pathway B
Research Question:
What effects does hydrogen bonding have on the hydrosulfide
desulfurization in carbonic anhydrase?
www.PosterPresentations.com
+
ClO4
S
Zn His
His
His
D
(2) Anders, E. et al. Chem. Eur. J. 2004, 10, 3091-3105. (3) Anders, E. et al. ChemBioChem 2007, 8, 530-536.
J
Hydrosulfide Complex Synthesis C
S
D
C
H+
S
RESEARCH POSTER PRESENTATION DESIGN © 2011
B I
A
Zn His
His
His
F
H
O
H
Future Studies 6-8.5 ppm region
+1
H
Zn His
His
His
B
S
H
K
0.0
We thank the REU Site: Materials for Energy & Sustainability, Boise
State University, Grant Number DMR 1359344.