Nitro as a novel zinc-binding group in the inhibition of carboxypeptidase A

Article abstract of DOI:10.1016/j.bmc.2008.02.010

2-Substituted 3-nitropropanoic acids were designed and synthesized as inhibitors against carboxypeptidase A (CPA). (R)-2-Benzyl- 3-nitropropanoic acid showed a potent inhibition against CPA (K_i = 0.15 μM). X-ray crystallography discloses that t

Full text of DOI:10.1016/j.bmc.2008.02.010

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3596–3601
Nitro as a novel zinc-binding group in the inhibition
of carboxypeptidase A
Si-Hong Wang,a Shou-Feng Wang,a Wei Xuan,a Zong-Hao Zeng,b
Jing-Yi Jin,c,* Jie Mad and Guan Rong Tiana,*
aDepartment of Chemistry, Yanbian University, Yanji, Jilin 133002, China
bInstitute of Biophysics, Chinese Academy of Science, Beijing 100871, China
cKey Laboratory of Organism Functional Factors of the Changbai Mountains of Ministry of Education, Yanbian University,
Gongyuan Road 977#, Yanji, Jilin 133002, China
dCollege of Chemistry and Chemical Engineer, Qiqihaer University, Qiqihaer, Heilongjiang 161006, China
Received 6 January 2008; revised 1 February 2008; accepted 5 February 2008
Available online 8 February 2008
Abstract—2-Substituted 3-nitropropanoic acids were designed and synthesized as inhibitors against carboxypeptidase A (CPA). (R)-
2-Benzyl- 3-nitropropanoic acid showed a potent inhibition against CPA (Ki = 0.15 lM). X-ray crystallography discloses that the
nitro group well mimics the transition state occurred in the hydrolysis catalyzed by CPA, that is, an O,O0-bidentate coordination
to the zinc ion and the two respective hydrogen bonds with Glu-270 and Arg-127. Because the nitro group is a planar species, we
proposed (R)-2-benzyl-3-nitropropanoic acid as a pseudo-transition-state analog inhibitor against CPA.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
development of enalapril as the ACE inhibitor and
Bay-12-9566 as the MMP inhibitor, respectively.2
Zinc proteases are a family of enzymes having a catalyt-
ically essential zinc ion at their active sites. These en-
zymes play key roles in a wide variety of physiological
and pathological processes, which are thus the most
studied drug design targets, such as angiotensin-convert-
ing enzyme (ACE) and matrix metalloproteases
(MMPs).1,2 The inhibitor design strategy that is widely
applied to these pathologically important zinc proteases
makes use of a zinc-binding group (ZBG) that can form
coordinative bonds to the zinc ion at the active site of
the enzymes.3–8 The carboxylate or sulfhydryl group
are commonly used for such purpose, which are usually
lessons from the inhibition of carboxypeptidase A
(CPA) as the leading representative of zinc proteases.9,10
For example, the carboxylate as the ZBG has been
extensively investigated in the inhibition of CPA by
2-benzylsuccinic acid (BSA),11 and then utilized in the
Being an isostere of the carboxylate,12 the nitro group
also shows a variety of coordinative fashions to the me-
tal ions.13 Compared to the wide applications of the car-
boxylate as the ZBG in the inhibition of zinc proteases,
the utilization of a nitro group as the ZBG has been
rarely explored to the best of our knowledge. We
thought that such investigation using CPA as the proto-
typical enzyme would be of interest as an alternative
inhibitor design rationale that can be applied to other
zinc proteases of medicinal interests. In the presented
paper, we hope to report our studies that involved the
introduction of the nitro group as ZBG in the inhibition
of CPA.
2. Results and discussion
Three racemic forms of 2-substituted 3-nitropropanoic
acids were first synthesized (Scheme 1).14 Their inhibi-
tory activities against CPA were evaluated and their
inhibitory constant (Ki) values are collected in Table 1.
It can be found that (RS)-2-isobutyl-3-nitropropanoic
acid 2 inhibits the CPA-catalyzed hydrolysis more
Keywords: Carboxypeptidase A; Inhibition; Nitro; Zinc-binding group;
X-ray crystallography; Transition-state analog inhibitor.
*
Corresponding authors. Tel.: +86 4332732286; fax: +86 4332732456
(J.-Y.J.); tel.: +86 4332762289; fax: +86 4332732456 (G.R.T.); e-mail
addresses: jyjin-chem@ybu.edu.cn; grtn@ybu.edu.cn
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.010

S.-H. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3596–3601
3597
To explore the binding mode of (R)-3 at the active site of
CPA,20 CPA-(R)-3 crystals for X-ray diffraction were
then obtained by soaking the enzyme crystals into the
solution containing racemic 3 and determined at a reso-
ꢀ
˚
lution of 1:5 A (PDB ID code: 2RFH). The final model
including all residues of CPA and (R)-3 was refined in
ꢀ
Scheme 1. Synthesis of the racemic 2-substituted 3-nitropropanoic
acids as inhibitors against CPA.
˚
the 38:42–1:70 A (Table 2). Figure 1 depicts the stereo-
view of the difference electron density in the region of
the active site of CPA that is occupied by (R)-3. Dis-
tances of important interactions between CPA and
(R)-3 in the complex are listed in Table 3.
Table 1. Inhibitory constants against CPA
Inhibitor
Ki (lM)
(RS)-1
(RS)-2
(RS)-3
(R)-3
62.6
2.08
0.79
0.15
68
The X-ray crystallographic studies disclosed the follow-
ing main structural information. Firstly, it is found that
(R)-3 occupies the S10 subsite of CPA where it makes the
expected interactions: the benzyl moiety resides in the
(S)-3
(R)-BSA
0.4511
Table 2. Data collection and refinement statistics for the CPA-(R)-3
complex
potently than (RS)-2-allyl-3-nitropropanoic acid 1,
which should be ascribed to the preference of CPA to
the substrate having a large hydrophobic side chain.15
Introduction of an aromatic ring further augmented
the binding affinity of the inhibitors to CPA, which
may be resulted from the additional ‘edge-to-face’ inter-
actions of the benzyl side chain of 3 with aromatic en-
zyme residues.15
Space group
P21
Unit cell
ꢀ
˚
a, b, c (A)
42.30, 62.70, 48.97
90.00, 96.99, 90.00
a, b, c (deg)
ꢀ
˚
Resolution range (A)
Number of unique reflections
Overall completeness (%)
Rmerge (%)a
38.42–1.70
27,657
98.7
4.5
22.6
R factorb
Because (RS)-2-benzyl-3-nitropropanoic acid 3 inhibits
CPA most potently, we then synthesized the two opti-
cally active forms (R-, S-) of 3 to explore the stereo-
chemistry associated with the inhibition (Scheme 2).
The optically pure 2-benzyl-3-hydroxypropanoic acid
was obtained according to the literature.16 Introduction
of the nitro group was achieved by the reaction of halo-
gen–NO2 exchange using Amberlite IRA-900 resin in
the nitrite form.17 Finally, it should be noted that hydro-
lysis of 3-nitropropanoic acid methyl ester under the
acidic or basic conditions failed. The needed carboxylic
acids can be obtained through treatment of the ester
with lithium iodide in refluxing anhydrous ethyl acetate,
which has been previously reported to be efficacious for
ester dealkylation of compounds having an X@O group
(X = C or S) at the c-position to the ester moiety.18,19
rms deviationsc
ꢀ
˚
Bonds (A)
0.013
1.3
14.1
Angles (deg)
Dihedrals (deg)
a Rmerge
for
ata
sets
for
replicate
reflections,
P
P
R ¼ kF hi j ꢀhj F h ji j hj F h ji, jFhij = scaled structure factor for
reflection h in data set i, hjFhji = average structure factor for reflec-
tion h calculated from replicate data.
P
P
b R factor, R ¼
j F o j ꢀ j F c j = j F o j; |Fo| and |Fc| are the
observed and calculated structure factors, respectively.
c rms: root mean square.
Consistent to the L-specificity of CPA, (R)-3 is the more
potent inhibitor of CPA than the corresponding S-form.
Especially, the Ki value of (R)-3 against CPA is 0.15 lM,
which is about threefold more potent than (R)-BSA
(Ki = 0.45 lM).11
Scheme 2. Synthesis of both optically pure 2-benzyl-3-nitropropanoic
acids. Reagents: (a) HCL-MeOH; (b) i—MeSO2Cl, DMAP; ii—LiBr;
iii—Amberlite IR-900, NaNO2; (c) LiI, EtOAc.
Figure 1. Difference electron density map for CPA-(R)-3 complex
generated with Fourier coefficient |Fo| ꢀ |Fc| phases calculated from the
final model omitting the bound inhibitor.

3598
S.-H. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3596–3601
oxygen and a glutamate oxygen of Glu-270 (Fig. 2b).22
Table 3. Selected CPA-(R)-3 interactions
ꢀ
In spite of being not a tetrahedron species, the nitrite
in CPA-(R)-3 complex exhibited all above the structur-
ally essential features of the transition state (Fig. 2d).23
Therefore, we suggest (R)-3 as a pseudo-transition-state
analog inhibitor for CPA.
˚
Separation (A)
Atom in (R)-3
Enzyme residue
O1
O2
O1
O3
O4
O3
O4
O3
O4
Arg-145 guanidinium N1
Arg-145 guanidinium N2
Tyr-248 phenolic O
Zn2+
2.99
2.63
3.90
2.10
3.06
4.89
2.87
2.60
4.49
Zn2+
Arg-127 guanidinium N1
Arg-127 guanidinium N2
Glu-270 carboxylate O1
Glu-270 carboxylate O2
3. Conclusion
In conclusion, we successfully introduced the nitro
group as a new ZBG to the inhibition of CPA. X-ray
crystallography of CPA-(R)-3 complex disclosed its
O,O0-bidentate chelation to the zinc ion at the active site
of CPA. Although the nitro group is not a tetrahedron
species, it also showed the similar binding modes as
transition-state analog. We then proposed it as a new
pseudo-transition-state analog inhibitor for CPA.
hydrophobic pocket and the carboxylate makes a salt
link with the guanidinium group of Arg-145. Tyr-248
is found in the ‘so-called’ down position, which is
responsible to the formation of the hydrophobic pocket.
Such binding modes are commonly observed in X-ray
crystal structures of CPA-inhibitor complexes.15 Sec-
ondly, the nitro group of (R)-3 acts as an asymmetric
bidentate ligand of the zinc ion in an O,O0-chelation
ꢀ
4. Experimental
˚
mode (2.10 and 3:06 A). In this sense, the nitro group
should be reasonably regarded as a new ZBG. Finally,
the two oxygen atoms (O3 and O4) of the nitro moiety
in (R)-3 are engaged in hydrogen bonding with one of
the carboxylate oxygen atoms in Glu-270 and one of
the guanidinium nitrogen atoms in Arg-127 with the dis-
ꢀ
BSA complex,21 the similar hydrogen bond between the
inhibitor and Glu-270 could be observed but the hydro-
gen bond with Arg-127 is missing. Therefore, the more
potent inhibition exhibited by (R)-3 than (R)-BSA could
be reasonably ascribed to the additional hydrogen bond
with Arg-127 in the CPA-(R)-3 complex (Fig. 2).
4.1. Materials and methods
All chemicals were purchased from Aldrich and used as
received without further purification. Flash chroma-
tography was performed with 100–200 mesh silica gel
(Qingdao, China) and thin-layer chromatography
(TLC) was carried out on silica coated glass sheets
(Qingdao silica gel 60 F-254). Melting points were taken
on a Thomas–Hoover capillary melting point apparatus
and uncorrected. 1H nuclear magnetic resonance
(NMR) and 13C NMR spectra were recorded with a
Bruker AV 300 (300 MHz) instrument using tetrameth-
ylsilane as the internal standard. IR spectra were re-
corded on a Perkin-Elmer 1300 FT-IR spectrometer.
High-resolution mass spectra were taken on a Shimadzu
GC–MS-QP2010. Elemental analyses were performed at
Changchun Institute of Applied Chemistry, Chinese
Academy of Sciences, Changchun, China.
˚
tances of 2.60 and 2.87 A, respectively. In the CPA-(R)–
It is interesting to discuss the inhibition type shown by
(R)-3 against CPA. It has been generally accepted that
the transition state in the CPA-catalyzed hydrolysis is
structurally featured by the tetrahedral carbon with
the gem-diolate oxygens stabilized by chelation to the
zinc ion and two respective hydrogen bonds with Glu-
270 and Arg-127 (Fig. 2a).15 For the typical transition-
state analog inhibitor against CPA, O-[[(1R)-[[N-phenyl-
methoxycarbonyl]-L-alanyl]amino]ethyl]hydroxyphos-
phinyl]-L-3-phenyllacetate, the salient structural features
are coordination of the phosphinyl moiety to the zinc
and Arg-127 and a short distance between a phosphinyl
CPA was purchased from Sigma Chemical Co. (Allan
form, twice crystallized from bovine pancreas, aqueous
suspension in toluene) and used without further purifi-
cation for kinetic assays. Lithium chloride and Hepes
were obtained from Sigma. O-(trans-p-chlorocinna-
moyl)-L-b-phenyllactate (Cl-CPL) was used as the sub-
strate in the kinetic study.24 All solutions for kinetic
study were prepared by dissolving in doubly distilled
and deionized water. CPA stock solutions were prepared
by dissolving CPA in 0.05 M Tris/0.5 M NaCl, pH 7.5,
buffer solution and their concentrations were estimated
from the absorbance at 278 nm (ꢀ278 = 64,200). The
stock assay solutions were filtered (GHP Acrodic syr-
inge filter, pore size 0.2 lm) before use. Perkin-Elmer
HP 8453 UV–Vis spectrometer was used for UV absor-
bance measurements.
4.2. Synthesis
Figure 2. Schematic representation of the binding mode of (a) the
occurred tetrahedron intermediate in the catalysis of the peptide
substrate; (b) the phosphate-based transition-state analog inhibitor; (c)
2-benzylsuccinic acid; and (d) 2-benzyl-3-nitropropanoic acid to CPA.
4.2.1. General procedure for the synthesis of (RS)-2-
substituted 3-nitropropanoic acid. To a solution of
lithium diisopropylamide (LDA, 2.3 equiv) and hexam-

S.-H. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3596–3601
3599
ethylphosphorous triamide (HMPA, 5.0 equiv) in THF
at ꢀ78 ꢁC was added 3-nitropropanoic methyl ester
(1.0 equiv). After stirring for 1 h, the corresponding
halides (1.25 equiv) were then added. The solution was
kept stirring at the same temperature for 5 h. The reac-
tion was quenched by addition of acetic acid (1.0 equiv)
followed by distilled water. After warmed to room tem-
perature, the solution was then diluted by water and
extracted by diethyl ether for three times. The combined
organic layer was washed with brine and then dried over
anhydrous MgSO4. Evaporation under reduced pressure
provided an oil, which was then purified by column
chromatography (n-hexane/EtOAc = 5:1) to give the
corresponding methyl ester.
aqueous NaHCO3 solution. The organic layer was
washed with brine and then dried over anhydrous
MgSO4. Evaporation under reduced pressure provided
an oil, which was then purified by column chromatogra-
phy (n-hexane/EtOAc = 5:1) to give the corresponding
methyl ester as an oil. Yield: 86%. IR (KBr): 3303,
1735 cmꢀ1 1H NMR (300 MHz, CDCl3) d: 2.18 (t,
.
J = 9.2 Hz, 1H), 2.88 (m, 2H), 3.03 (dd, J = 9.2,
16.9 Hz, 1H), 3.70 (s, 3H), 3.65-3.80 (m, 2H), 7.19–
7.35 (m, 5H).13C NMR (75 MHz, CDCl3) d: 34.41,
49.24, 51.85, 62.24, 126.54, 128.52, 128.91, 138.56,
175.11.
(S)-2-Benzyl-3-hydroxypropanoic acid methyl ester:
23
D
½aꢁ
+39.3ꢁ (c 0.41, CH3OH). HRMS calcd for
C11H14O2: 178.0944. Found: 178.0943.
To an anhydrous solution of ethyl acetate containing the
above methyl ester (1.0 equiv) was added lithium iodide
(7.0–8.0 equiv). The resulting mixture was heated under
reflux for 24 h under N2. After cooling to room temper-
ature, the reaction mixture was treated with water and
then acidified to pH 1.0 with 10% citric acid solution.
After extracted with ethyl acetate, the combined organic
layer was washed with saturated Na2S2O3 solution and
then dried over anhydrous MgSO4. Evaporation under
reduced pressure provided an oil, which was then puri-
fied by column chromatography to give the needed
acids.
(R)-2-Benzyl-3-hydroxypropanoic acid methyl ester:
23
D
C11H14O2: 178.0944. Found: 178.0943.
½aꢁ ¼ ꢀ38:6ꢂ (c 0.47, CH3OH), HRMS calcd for
4.2.3. General procedure for the synthesis of the optically
active 2-benzyl-3-nitropropanoic acid methyl ester. To
5 mL solution of CH2Cl2 containing the optically active
2-benzyl-3-hydroxypropanoic
acid
methyl
ester
(1.0 equiv), dimethylaminopyridine (0.05 equiv) and
pyridine (2.0 equiv) at 0 ꢁC, a solution of methanesulfo-
nyl chloride (2.0 equiv) in CH2Cl2 (1 mL) were added.
After standing at room temperature overnight, the
reaction mixture were then poured into 20 mL 10%
HCl followed by extraction with ethyl acetate (3·
15 mL). The combined organic layer was washed with
aqueous NaHCO3 and brine, and then dried over anhy-
drous MgSO4. Evaporation under reduced pressure gave
an oil, which was then purified by column chromatogra-
phy (silica gel, n-hexane/EtOAc = 5:1) to yield the
needed mesylate.
(RS)-1: IR (KBr): 1364, 1569, 1738, 3460 cmꢀ1 1H
.
NMR (300 MHz, CDCl3) d: 2.28–2.49 (m, 2H), 3.20–
3.39 (m, 1H), 4.57 (dd, J = 5.6, 10.2 Hz, 1H), 4.71 (dd,
J = 5.6, 10.2 Hz, 1H), 5.11–5.18 (m, 2H), 5.76–5.85 (m,
1H). 13C NMR (75 MHz, CDCl3) d: 33.00, 42.30,
74.17, 117.41, 133.54, 173.94. HRMS calcd for
C6H9NO4: 159.0532. Found: 159.0530.
(RS)-2: IR (KBr) 1363, 1571, 1738, 3457 cmꢀ1 1H
.
NMR (300 MHz, CDCl3) d: 0.98 (d, J = 6.1 Hz, 6H),
1.37–1.43 (m, 1H), 1.63-1.73 (m, 2H), 3.26–3.30 (m,
1H), 4.43 (dd, J = 4.8, 9.8 Hz, 1H), 4.71 (dd, J = 4.8,
9.8 Hz, 1H), 8.68 (s, 1H). 13C NMR (75 MHz, CDCl3)
d: 22.06, 22.32, 25.65, 37.99, 40.98, 75.05, 178.19.
HRMS calcd for C7H13NO4: 175.0845. Found:
175.0844.
A solution of tetrahydrofuran containing the above
mesylate and anhydrous lithium bromide (2.0 equiv)
was kept stirring at room temperature. Completion of
the reaction was ensured by the TLC detection. After
addition of ethyl acetate, the organic layer was washed
with water and then dried over anhydrous MgSO4.
Evaporation under reduced pressure gave the crude
product, which was then purified by column chromatog-
raphy (silica gel, n-hexane/EtOAc = 10:1) to yield the
bromide.
(RS)-3: mp 70–71 ꢁC. IR (KBr): 3429, 1718, 1016 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d: 2.88 (dd, J = 5.4,
14.0 Hz, 1H), 3.41 (dd, J = 5.4, 14.0 Hz, 1H), 3.30–
3.70 (m, 1H), 3.90 (dd, J = 5.2, 12.6 Hz, 1H), 4.66 (dd,
J = 5.2, 12.6 Hz, 1H), 7.19–7.39 (m, 5H), 9.22 (br,
1H). 13C NMR (75 MHz, CDCl3) d: 34.78, 44.31,
73.52, 127.57, 128.87, 129.11, 136.08, 178.01. Anal.
C10H11NO4 requires C, 57.41; H, 5.30; N, 6.70. Found:
C, 57.48; H, 5.33; N, 6.65.
Amberlite IRA-900 in the nitrite form (1.5 g) was added
to a solution of benzene containing the above bromide
(112 mg, 0.439 mmol). The resulting slurry was kept stir-
ring at 20 ꢁC for 36 h. After filtration, the filtrate was
evaporated under reduced pressure to give a yellow
oil. Purification by column chromatography (silica gel,
n-hexane/EtOAc = 5:1) to yield the product as an oil.
4.2.2. General procedure for the synthesis of the optically
active 2-benzyl-3-hydroxypropanoic acid methyl ester.
To an ice-chilled stirred solution of 2-benzyl-3-hydroxy-
propanoic acid (1.0 equiv) in MeOH (20 mL) was
added acetyl chloride (2 mL). The mixture was stirred
for 24 h at room temperature, and then evaporated
under reduced pressure. The residue was dissolved in
ethyl acetate (30 mL) followed by neutralization with
Yield: 33 %. IR (KBr): 3304, 1733 cmꢀ1 1H NMR
.
(300 MHz, CDCl3) d: 2.84 (dd, J = 9.0, 14.0 Hz, 1H),
3.16 (dd, J = 8.9, 14.0 Hz, 1H), 3.40–3.60 (m, 1H),
3.75 (s, 3H), 4.35 (dd, J = 4.4, 14.6 Hz, 1H), 4.68 (dd,
J = 4.3, 14.6 Hz, 1H), 7.16–7.38 (m, 5H). 13C NMR
(75 MHz, CDCl3) d: 35.09, 44.49, 52.51, 74.06, 127.38,
128.84, 128.97, 136.40, 172.03.

3600
S.-H. Wang et al. / Bioorg. Med. Chem. 16 (2008) 3596–3601
23
D
(S)-2-Benzyl-3-nitropropanoic acid methyl ester. ½aꢁ
operating at 40 kV · 20 mA and R-Axis IV++ imaging-
plate detector at a wavelength of 1.5418 . A 0.5 mm col-
limator was used to keep the whole crystal bathed in the
X-ray beam. A total of 360 images with 1.0 oscillation
were collected. The collected intensities were indexed,
integrated, corrected for absorption, scaled and merged
using HKL2000. Diffraction images were processed with
the CCP4 program suite. The structure of the native
CPA (PDB code: 5CPA) was used as the starting model.
Model refinement was carried out with the COOT pro-
gram and the REFMAC 5.2. Water molecules were
added to the model with the program CCP-4. The inhib-
itor model was included in the last stage of the
refinement.
ꢀ7.8 (c 0.26, CH3OH). HRMS calcd for C11H13NO3:
207.0895. Found: 207.0894.
23
(R)-2-Benzyl-3-nitropropanoic acid methyl ester. ½aꢁ
D
+8.1 (c 0.24, CH3OH). HRMS calcd for C11H13NO3:
207.0895. Found: 207.0894.
4.2.4. Synthesis of the optically active 2-benzyl-3-nitro-
propanoic acid. The procedures are similar as described
in the general procedures of hydrolysis of the methyl es-
ters containing nitro group.
25
D
(S)-3: mp 70–71 ꢁC. ½aꢁ ꢀ4.3ꢁ (c 0.34, CH3OH).
HRMS calcd for C10H11NO4: 209.0689. Found:
209.0688. Anal. C10H11NO4 requires C, 57.41; H, 5.30;
N, 6.70. Found: C, 57.44; H, 5.31; N, 6.94.
Acknowledgment
25
D
(R)-3: mp 70–71 ꢁC. ½aꢁ +4.5ꢁ (c 0.33, CH3OH).
The work was financially supported by the Science and
Technology Committee of Jilin Province (No.
20060568) and the Open Project Program of Key Labo-
ratory of Organism Functional Factors of the Changbai
Mountains of Ministry of Education, Yanbian Univer-
sity, China.
HRMS calcd for C10H11NO4: 209.0689. Found:
209.0688. Anal. C10H11NO4 requires C, 57.41; H, 5.30;
N, 6.70. Found: C, 57.58; H, 5.33; N, 6.65.
4.3. Determination of Ki value
The enzyme stock solution was added to a solution con-
taining Cl-CPL (final concentrations: 50 and 100 lM)
and inhibitor (five different final concentrations in the
range of 0.5–2.0 Ki) in 0.05 M Tris/0.5 M NaCl, pH
7.5, buffer (1 mL cuvette), and the change in absorbance
at 320 nm was measured immediately. The final concen-
tration of CPA was 20 nM. Initial velocities were then
calculated from the linear initial slopes of the change
in absorbance where the amount of substrate consumed
was less than 10%. The Ki values were then estimated
from the semireciprocal plot of the initial velocity versus
the concentration of the inhibitor according to the meth-
od of Dixon.25
References and notes
1. Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1359.
2. Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem.
2000, 43, 305.
3. Jacobsen, F. E.; Lewis, J. A.; Cohen, S. M. J. Am. Chem.
Soc. 2006, 128, 3156.
4. Rush, T. S.; Powders, R. Curr. Top. Med. Chem. 2004, 4,
1311.
5. Tekeste, T.; Vahrenkamp, H. Inorg. Chem. 2006, 45,
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6. Tekeste, T.; Vahrenkamp, H. Eur. J. Inorg. Chem. 2006,
24, 5158.
7. Jacobsen, F. E.; Cohen, S. M. Inorg. Chem. 2004, 43, 3038.
8. He, H.; Puerta, D. T.; Cohen, S. M.; Rodgers, K. R. Inorg.
Chem. 2005, 44, 7431.
4.4. X-ray crystallography
CPA was washed three times with water to remove tol-
uene used in packaging and then dissolved in 1.2 M
LiCl. After centrifugation, the enzyme solution was then
diluted to 10–15 mg/mL with a buffer solution of 1.2 M
LiCl, 25 mM Tris, pH 7.5. An aliquot of this solution
was then pipetted into a 100 lL glass dialysis button.
A 7 kDa molecular weight cutoff membrane, which
was pre-washed by doubly distilled and deionized water
followed by equilibrated in 0.15 M LiCl, was placed
over the button and secured with a rubber O-ring. The
button was then dialyzed against 0.2 M LiCl, 25 mM
Tris, pH 7.5, and at 4 ꢁC. Crystals appeared after about
4 days on the bottom and edges of the well. The crystals
were then crosslinked using a buffer solution containing
0.15 M LiCl and 0.02% (v/v) glutaraldehyde for 90 min.
The crystals were then transferred to a soaking solution
of the same buffer containing 2 mM racemic 3 and
stored for 17 days in a cold room (4 ꢁC) before data col-
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