2-Aminoimidazole amino acids as inhibitors of the binuclear manganese metalloenzyme human arginase i

Article abstract of DOI:10.1021/jm100306a

Arginase, a key metalloenzyme of the urea cycle that converts l-arginine into l-ornithine and urea, is presently considered a pharmaceutical target for the management of diseases associated with aberrant l-arginine homeostasis, such as asthma, cardiovascular diseases, and erectile dysfunction. We now report the design, synthesis, and evaluation of a series of 2-aminoimidazole amino acid inhibitors in which the 2-aminoimidazole moiety serves as a guanidine mimetic. These compounds represent a new class of arginase inhibitors. The most potent inhibitor identified in this study, 2-(S)-amino-5-(2-aminoimidazol-1-yl) pentanoic acid (A1P, 10), binds to human arginase I with K_d = 2 μM and significantly attenuates airways hyperresponsiveness in a murine model of allergic airways inflammation. These findings suggest that 2-aminoimidazole amino acids represent new leads for the development of arginase inhibitors with promising pharmacological profiles.

Full text of DOI:10.1021/jm100306a

4266 J. Med. Chem. 2010, 53, 4266–4276
DOI: 10.1021/jm100306a
2-Aminoimidazole Amino Acids as Inhibitors of the Binuclear Manganese Metalloenzyme Human
Arginase I
Monica Ilies,† Luigi Di Costanzo,†,§ Michelle L. North,‡ Jeremy A. Scott,‡ and David W. Christianson*,†
†Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, and
‡Department of Medicine, Faculty of Medicine, Divisions of Occupational and Respiratory Medicine, University of Toronto, Toronto, Ontario,
M5S 1A8, Canada. §Present address: RCSB Protein Data Bank, Department of Chemistry and Chemical Biology, Rutgers, The State University
of New Jersey, Piscataway, New Jersey 08854-8087.
Received March 8, 2010
Arginase, a key metalloenzyme of the urea cycle that converts L-arginine into L-ornithine and urea, is
presently considered a pharmaceutical target for the management of diseases associated with aberrant
L-arginine homeostasis, such as asthma, cardiovascular diseases, and erectile dysfunction. We now report
the design, synthesis, and evaluation of a series of 2-aminoimidazole amino acid inhibitors in which the
2-aminoimidazole moiety serves as a guanidine mimetic. These compounds represent a new class of
arginase inhibitors. The most potent inhibitor identified in this study, 2-(S)-amino-5-(2-aminoimidazol-
1-yl)pentanoic acid (A1P, 10), binds to human arginase I with Kd=2 μM and significantly attenuates
airways hyperresponsiveness in a murine model of allergic airways inflammation. These findings suggest
that 2-aminoimidazole amino acids represent new leads for the development of arginase inhibitors with
promising pharmacological profiles.
Introduction
arginase inhibition or posttranscriptional gene silencing of
arginase I significantly decreases airways hyperresponsiveness
induced by IL-13 or aeroallergens in different animal models.11
The role of arginase in asthma has been recently reviewed.12 In
addition to asthma, the biochemical interplay between arginase
and nitric oxide synthase makes arginase an increasingly im-
portant pharmaceutical target for the management of various
diseases associated with aberrant L-arginine homeostasis and/or
NO biosynthesis, such as erectile dysfunction,13,14 pulmonary
diseases such as cystic fibrosis,15 cardiovascular diseases includ-
ing atherosclerosis,16 and cancer tumor growth.17
In the active site of arginase, the binuclear manganese cluster
and residues that interact with the R-amino and R-carboxylate
groups of amino acid inhibitors constitute essential molecular
recognition elements for high affinity binding. X-ray crystallo-
graphic studies of rat arginase I and human arginase I
complexed with inhibitors such as 2-(S)-amino-6-boronohex-
anoic acid (ABH)13a,18,19 and S-(2-boronoethyl)-L-cysteine
(BEC)13b,19 serve to guide continuing studies toward the
structure-based design of new inhibitors that bear alternative
functional groups targeting coordination interactions with the
binuclear manganese cluster. New inhibitors with different
chemical properties may exhibit alternative pharmacokinetic
profiles that enable their study as potential lead compounds
for drug development.
Arginase is a metalloenzyme that catalyzes the hydrolysis of
L-arginine to form L-ornithine and urea. Two distinct isozymes
with different tissue distributions, subcellular localizations,
and metabolic functions have been identified in mammals.1
The cytosolic isozyme, arginase I, is found mainly in the liver
where it catalyzes the final cytosolic step of the urea cycle.
Here, arginase activity allows for the ultimate excretion of
excess nitrogen resulting from protein catabolism in the form
of urea.2 The mitochondrial isozyme, arginase II, is pre-
dominantly expressed in extrahepatic tissues3 where it serves
to regulate L-arginine homeostasis and biosynthetic pathways
dependent on L-arginine and L-ornithine. These pathways
include nitric oxide (NOa) biosynthesis, L-proline biosynthesis
to support collagen production (as an alternative to the
L-glutamate derived biosynthesis of L-proline), and polyamine
biosynthesis to facilitate cellular proliferation (Figure 1).4-6
Arginase I is also expressed in extrahepatic tissues to regulate
L-arginine homeostasis.7 For example, arginase I expression
is significantly increased in the asthmatic lung.8 Arginase I is
more strongly induced by Th2 cytokines such as IL-13 that
have a critical role in the development of asthma in experi-
mental models,9 while arginase I and II single-nucleotide poly-
morphisms may contribute to atopic asthma.10 Additionally,
*To whom correspondence should be addressed. Phone: 215-898-
5714. Fax: 215-573-2201. E-mail: chris@sas.upenn.edu.
We have previously shown that the guanidine side chain of
L-arginine is not a particularly effective metal ligand.20 The
positively charged guanidinium group has a high pKa value of
12.5-13,21 so protonation significantly competes with poten-
tial metal coordination behavior. However, the incorporation
of a guanidinium group within a 2-aminoimidazole hetero-
cycle reduces the pKa by 4-5 units,22 which in turn can
facilitate metal coordination interactions and improve the
aAbbreviations: SAR, structure-activity relationship; NO, nitric oxide;
ABH, 2-(S)-amino-6-boronohexanoic acid; BEC, S-(2-boronoethyl)-
L-cysteine; 2AH, L-2-aminohistidine; AHH, 2-aminohomohistidine; A1P,
2-(S)-amino-5-(2-aminoimidazol-1-yl)pentanoic acid; A4P, 2-amino-5-
(2-aminoimidazol-4-yl)pentanoic acid; APP, (S)-2-amino-5-(imidazol-2-
ylamino)pentanoic acid; DIBAH, diisobutylaluminum hydride; DMAP,
dimethylaminopyridine; DMF, dimethylformamide; TFA, trifluoroacetic
acid.
r
pubs.acs.org/jmc
Published on Web 05/04/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4267
Figure 1. Pathways of L-arginine metabolism.
Table 1. 2-Aminoimidazole and Amino Acid Derivatives Synthesized
as Inhibitors of Human Arginase I
pharmacokinetic profile. Accordingly, 2-aminoimidazole deri-
vatives display a broad range of biological properties and repre-
sent significant precursors for drug design and natural products
synthesis. Interestingly, 2-aminoimidazoles have been isolated
from marine sponges as secondary metabolites known as oroi-
dins, which exhibit a wide range of biological activities.23-25
Although certain 2-aminoimidazole amino acids have been
reported with potential applications in medicinal chemistry,26-28
such compounds have never been investigated as arginase
inhibitors. Here, we report the structure-based design, syn-
thesis, and evaluation of a series of 2-aminoimidazole amino
acid inhibitors of human arginase I. The most active of these
compounds, 2-(S)-amino-5-(2-aminoimidazol-1-yl)pentanoic
acid (A1P, 10), binds to human arginase I with low micro-
molar affinity and significantly reduces airways hyperrespon-
siveness in a murine model of allergic airways inflammation.
On the basis of these findings, we advance that 2-amino-
imidazole amino acids may serve as valuable leads for the
development of a new class of arginase-directed drugs. More-
over, the utilization of a 2-aminoimidazole moiety to target
metal coordination interactions is potentially generalizable
toward the inhibition of other binuclear metallohydrolases.
Results
Chemistry and in Vitro Structure-Activity Relationships
(SARs). 2-Aminoimidazole (Table 1) is a weak noncompeti-
tive inhibitor of human arginase I with Ki =3.6 mM. The
X-ray crystal structure of the enzyme-inhibitor complex
(Figure 2) reveals that 2-aminoimidazole binds in the active
site but does not interact directly with the binuclear manga-
nese cluster. Instead, the inhibitor is aligned parallel to the
imidazole group of H126 with a mean interplane distance
˚
of ∼4 A. Additionally, the 2-amino group donates a hydro-
gen bond to a water molecule that in turn donates a hydrogen
bond to the metal-bridging hydroxide ion. In studying the
structure of this complex, we reasoned that the 2-aminoimi-
dazole moiety could be targeted to interact directly with the
binuclear manganese cluster by incorporating it within the
side chain of an L-amino acid.
The synthesis of the simplest member of the series of
2-aminoimidazole amino acids shown in Table 1, L-2-amino-
histidine (2AH), was achieved using a previously pub-
lished procedure.26 This compound exhibits noncompetitive
a Kinetic assay. Errors are standard deviations of experiments run in
triplicate. b Surface plasmon resonance measurement of the dissociation
constant, Kd.
inhibition against human arginase I with a ∼10-fold inc-
rease in inhibitor binding affinity (Ki=0.3 mM) relative to
2-aminoimidazole.
The X-ray crystal structure of the human arginase I-2AH
complex (Figure 3) reveals that the 2-aminoimidazole group

4268 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Ilies et al.
Figure 2. Stereoview of a simulated annealing gradient map showing 2-aminoimidazole (3.0σ contour, cyan) bound to human arginase I.
Dashed lines indicate manganese coordination (red) and hydrogen bond (green) interactions. Atom color codes are as follows: carbon (yellow),
oxygen (red), nitrogen (blue), manganese (violet).
Figure 3. Stereoview of a simulated annealing omit map showing the inhibitor 2AH and a sulfate ion bound to human arginase I. The inhibitor
(3.0σ contour, cyan) and sulfate ion (3.5σ contour, red) were omitted from the structure factor calculation. The sulfate ion coordinates to the
manganese ions and accepts hydrogen bonds from the side chain of 2AH. Dashed lines indicate manganese coordination (red) and hydrogen
bond (green) interactions. Atom color codes are as follows: carbon (yellow), oxygen (red), nitrogen (blue), manganese (violet), sulfur (green).
is repositioned in the active site, but the side chain is too short
to interact directly with the binuclear manganese cluster.
However, inhibitor binding recruits a tetrahedral anion inter-
preted as sulfate to achieve manganese coordination. As
observed for the binding of other amino acid inhibitors,19,29
the R-amino group donates hydrogen bonds to D183 and two
water molecules, and the R-carboxylate group accepts hydro-
gen bonds from N130, S137, and two water molecules
(Figure 3). Hydrogen bond interactions between the 2-ami-
noimidazole group and the metal-bound sulfate anion stabilize
inhibitor binding and may contribute to the ∼10-fold increase
in affinity relative to 2-aminoimidazole. Although the binding
affinity of 2AH is modest, the intermolecular interactions
observed in the human arginase I-2AH complex illuminate
a novel coordination mode to the binuclear manganese cluster.
Given that the side chain of 2AH is too short to allow the
2-aminoimidazole group to interact directly with the man-
ganese ions, we synthesized the congener 2-aminohomohis-
tidine (AHH, Table 1), which bears a side chain one carbon
longer, using a previously published procedure.27 AHH is a
competitive inhibitor with Ki = 3 mM for the racemic
mixture, indicating no significant improvement in the bind-
ing affinity compared with 2AH.
interactions in the active site as observed for the binding of
other amino acid inhibitors.19,29 Additionally, the 2-aminoi-
midazole group of AHH displaces the metal-bridging hydro-
xide ion of the unliganded enzyme and coordinates to Mn2þ
A
with an average N Mn2þ separation of 2.5 A. The
˚
A
3 3 3
˚
N
Mn2þ separation is 2.8 A, which is too long to be
considered an inner sphere coordination interaction. The
imidazole group of AHH is sandwiched between the imida-
zole groups of H126 and H141 with mean interplane separa-
B
3 3 3
˚
tions of 3.5 and 3.6 A, respectively. The lengthy metal
coordination interactions observed in this enzyme-inhibitor
complex, which should be closer to 2.0 A for optimal
˚
N
Mn2þ interactions, may be responsible for the lower
binding affinity of AHH.
3 3 3
Encouraged by these structural results, we opted to inves-
tigate three different derivatives bearing aminoimidazole side
chains one carbon longer than that of AHH: 2-(S)-amino-
5-(2-aminoimidazol-1-yl)pentanoic acid (A1P), 2-amino-5-
(2-aminoimidazol-4-yl)pentanoic acid (A4P), and (S)-2-ami-
no-5-(imidazol-2-ylamino)pentanoic acid (APP) (Table 1).
We used the protected L-amino acid aldehyde 4 and the
triprotected 2-aminoimidazole 5 as key intermediates in the
syntheses of these derivatives (Scheme 1).
The X-ray crystal structure of the human arginase
I-AHH complex (Figure 4) reveals that the R-amino and
R-carboxylate groups make the expected hydrogen bond
The aldehyde 4 was synthesized from commercially avail-
able (S)-5-tert-butoxy-4-(tert-butoxycarbonylamino)-5-oxo-
pentanoic acid (1) that was first esterified with methyl

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4269
Figure 4. Stereoview of a simulated annealing omit map showing the inhibitor AHH bound to human arginase I. The inhibitor (3.0σ contour,
cyan) was omitted from the structure factor calculation. Dashed lines indicate manganese coordination (red) and hydrogen bond (green)
interactions. Atom color codes are as follows: carbon (yellow), oxygen (red), nitrogen (blue), manganese (violet).
Scheme 1. Synthesis of the Key Intermediates 4 and 6
Scheme 2. Synthesis of A1P
chloroformate, then further N-protected with a second tert-
butoxycarbonyl group (intermediates 2 and 3, respectively).
Selective reduction of the less sterically hindered methyl ester
group of 3 with diisobutylaluminum hydride (DIBAH) at
-78 ꢀC provided the corresponding aldehyde 4 in good over-
all yield.30 The heterocyclic intermediates were generated
by the treatment of 2-aminoimidazole with di-tert-butyl pyro-
carbonate, yielding the derivative 5 that was selectively de-
protected with ammonia in methanol to form 2-[bis(tert-butoxy-
carbonyl)amino]imidazole (6).
(TFA) in methylene chloride generated A1P (10) in good
overall yield. While it is conceivable that racemization
of the amino acid could occur under these conditions,
racemization of a related amino acid was blocked by the
utilization of a bulky tert-butyl protecting group on the
R-carboxylate,33 as we have similarly utilized for the pre-
paration of A1P.
Two different assays indicate that A1P binds to human
arginase I with low micromolar affinity. The fixed-point
kinetic assay using 14C-labeled L-arginine34 demonstrates that
A1P is a competitive inhibitor with Ki=4 μM (Figure 5A).
Surface plasmon resonance analysis35 confirms low micro-
molar affinity with Kd = 2 μM in a 1:1 Langmuir binding
model (Figure 5B). Unfortunately, we were unable to prepare
crystals of human arginase I complexed with A1P by crystal
soaking or cocrystallization approaches.
While the synthesis of A1P has been previously reported,31
we developed an alternative synthetic route with a compar-
able overall yield. We reduced the aldehyde intermediate 4
with DIBAH to form alcohol 7 (Scheme 2). Bromination
of 7 with triphenylphosphine and carbon tetrabromide led
to the corresponding bromo derivative 8 (as previously
reported by our group32), which was then coupled to the
selectively protected 2-aminoimidazole 6 with potassium
carbonate in dimethylformamide (DMF) to yield protected
A1P (9); complete deprotection with trifluoroacetic acid
The N-alkylated derivative (APP, 12) was synthesized by the
reductive coupling of aldehyde intermediate 4 with 2-amino-
imidazole to yield protected APP (11); deprotection with TFA
in methylene chloride generated APP (12) (Scheme 3).

4270 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Ilies et al.
Figure 5. (A) Kinetic replot of the inhibition of human arginase I by A1P yields Ki = 4 μM. (B) Surface plasmon resonance sensorgram
showing the binding of A1P to human arginase I; Kd = 2 μM.
Scheme 3. Synthesis of APP
Scheme 4. Synthesis of A4P
Enzymological measurements indicate that APP binds
to human arginase I with modest affinity (Ki = 500 μM,
Table 1). Alkylation via the 2-amino group increases the
length of the amino acid side chain, which may contribute to
decreased affinity relative to A1P.
The synthesis of A4P (Scheme 4) started from the com-
mercially available 2,6-diaminoheptanedioic acid, which was
diesterified with thionyl chloride in methanol, then N-pro-
tected with di-tert-butyl pyrocarbonate in acetonitrile in the
presence of dimethylaminopyridine (DMAP) to generate the
protected intermediate 13. One ester group of 13 was then
selectively reduced to the corresponding aldehyde 14 by
using 1 equiv of DIBAH. Deprotection with 4 N HCl and
ethyl acetate (EtOAc) generated the diaminoaldehyde 15
that was condensed with cyanamide in water36 at pH 4.5 to
afford A4P (16). Enzymological assay indicates that A4P
lacks inhibitory activity (Ki > 0.8 M).
Arginase Inhibition by A1P in a Murine Model of Allergic
Airways Inflammation. To determine the effectiveness of the
tightest binding 2-aminoimidazole amino acid, A1P, for
reducing airways hyperresponsiveness in vivo, we used an
acute (3-week) ovalbumin (OVA)-sensitization and -chal-
lenge murine model, as previously described.8a In this model,
mice sensitized with OVA but challenged with PBS (OVA/
PBS) serve as controls, while OVA-sensitized and -chal-
lenged mice (OVA/OVA) develop lung-specific airways in-
flammation and airways hyperresponsiveness, as a model of
asthma. We have previously shown that pharmacologic
inhibition of arginase with BEC attenuates maximum central
airways responsiveness (RNmax) to methacholine in this
model.8a Consistent with the effective in vivo inhibition of
arginase, treatment with nebulized A1P (80 μg/g) attenuated
significantly the total resistance of the respiratory system (R)
following methacholine challenge in OVA/OVA mice
(Figure 6A). Similarly, the maximum resistance of the cen-
tral airways (RN) was significantly attenuated by A1P treat-
ment in the same mice (Figure 6B).
Discussion
Imidazole and related azoles are well-known ligands
in metalloenzyme active sites, and ongoing explorations of
azole derivatives as potential pharmacophores have inspired
the current study. For example, spectroscopic studies on the
binding of imidazole and its analogues 1,2,3-triazole, 1,2,4-
triazole, and tetrazole to Co2þ-substituted human carbonic
anhydrase I and bovine carbonic anhydrase II suggest that
1,2,3-triazole forms a pentacoordinated metal complex while
1,2,4-triazole and tetrazole bind in tetrahedral metal com-
plexes.37 The crystal structure of human carbonic anhydrase I
complexed with imidazole reveals the inhibitor bound via one
of its nitrogen atoms asa fifth ligand tothe Zn2þ ion,38 and the
structure of human carbonic anhydrase II complexed with
1,2,4-triazole shows that the heterocycle coordinates to Zn2þ
through the N4 atom.39 In other systems, the X-ray crystal
structure of methionine aminopeptidase-2 in complex with a
1,2,4-triazole derivative reveals a novel binding mode that

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4271
Figure 6. Impact of arginase inhibition by A1P in the acute ovalbumin (OVA)-sensitization and -challenge murine model of allergic airways
inflammation. (A) A1P attenuated significantly the maximum total lung resistance (R) evoked by methacholine challenge in mice sensitized to
OVA and challenged with nebulized vehicle (OVA/PBS) or OVA (OVA/OVA and OVA/OVA A1P). (B) A1P also notably decreased the
maximum methacholine-induced increase in central airways responsiveness (RN) in the same animal model. Values are expressed as the mean (
SE (n = 12/group): (/) P < 0.05; (///) P < 0.0001 to OVA/PBS; (##) P < 0.01 to OVA/OVA.
may account for nanomolar affinity: two of the heterocyclic N
atoms coordinate the Co2þ ion, while the third one makes a
hydrogen bond to a histidine residue in the active site.40
Finally, a common binding mode for imidazole-based heme
oxygenase-1 inhibitors has been recently reported, revealing
that the imidazole group coordinates to the heme iron.41
In contrast with imidazole and the azoles described above,
2-aminoimidazoles have had only limited study in the design
of metalloenzymes inhibitors. To our knowledge, the only
examples reported to date are those studied as inhibitors of
NO synthase.28,42 Optical absorption, magnetic circular di-
chroism, and electronic paramagnetic resonance analyses of
endothelial nitric oxide synthase complexed with imidazole
derivatives suggest the direct binding of 2-aminoimidazole to
the guanidine binding subdomain near the catalytic heme
group.42 Accordingly, such derivatives are considered promis-
ing ligands for exploring the active sites of different forms of
nitric oxide synthase and for the development of isozyme-
selective inhibitors. Some 2-amino-5-azolylpentanoic acids
related to L-ornithine have also been reported as nitric oxide
synthase inhibitors.28 However, we have determined that the
2-aminoimidazole amino acids shown in Table 1 are only
weak or modest inhibitors of recombinant rat neuronal nitric
oxide synthase (nNOS) or recombinant murine inducible
nitric oxide synthase (iNOS), with IC50 values ranging from
65 to 3300 μM (data not shown). Moreover, the new lead
arginase inhibitor identified in the current study, A1P (10), is
only a weak inhibitor of iNOS and nNOS, with IC50 values of
480 and 135 μM, respectively (data not shown). Such selec-
tivity for arginase inhibition may facilitate the use of A1P
and its derivatives for the selective modulation of L-arginine
levels in the management of diseases associated with aberrant
L-arginine homeostasis.
Notably, in vitro SARs based on biological assays and
X-ray crystallographic studies show a clear correlation of
inhibitor potency with the length of the carbon linker separ-
ating the heterocycle and the amino acid moieties; the highest
efficacy observed for A1P is associated with a side chain that is
the same length as that of the natural substrate, L-arginine.
Interestingly, 2-aminoimidazoles smaller thanL-arginine (2AI
and 2AH) are noncompetitive inhibitors, whereas inhibitors
that are more isosteric with L-arginine (AHH and A1P) are
competitive inhibitors. The orientation of the 2-aminoimida-
zole moiety as it is attached to the amino acid side chain
is a critical affinity determinant, as shown by differences in
the inhibitory potencies of A1P, A4P, and APP. Since the
pKa of the 2-aminoimidazole moiety is relatively invariant to
alkylation on imidazole N or C atoms,22 differences in pKa
cannot contribute to differences in inhibitor affinity. Accord-
ingly, the optimal affinity of A1P in the series of compounds
shown in Table 1 correlates with a 2-aminoimidazole orienta-
tion that is most closely isosteric with the guanidinium moiety
of the arginase substrate L-arginine. We speculate that this
optimizes enzyme-inhibitor metal coordination interactions.
Human asthma patients have increased levels of expression
and activity of arginase I in the airways,8 similar to the mouse
model of OVA-induced airways inflammation43 used in this
study. Recently, we have demonstrated that arginase inhibi-
tion by the boronic acid derivative BEC reduces airways
hyperresponsiveness in murine models of allergic airways
inflammation.8a The current study indicates that A1P is
similarly effective in vivo as an arginase inhibitor in this acute
asthma model.
Summary and Conclusions
This is the first investigation of arginase inhibition using
the 2-aminoimidazole moiety as a “warhead” to target the
binuclear manganese cluster. Importantly, A1P is a mimetic
of the substrate L-arginine in which the side chain Nε and Nη
atoms are covalently linked via the imidazole heterocycle:
Incorporation of the guanidinium nitrogen atoms within the
imidazole moiety serves to depress the pKa of the side chain
to ∼7-8, which can also lead to improved bioavailability.
Our SAR analysis demonstrates that the 2-aminoimidazole
moiety can target metal coordination and hydrogen bond
interactions in the enzyme active site. The most potent
inhibitor studied, A1P, exhibits low micromolar binding
affinity. Moreover, A1P inhibits arginase in vivo and sig-
nificantly reduces airways hyperresponsiveness to metha-
choline in OVA-sensitized and -challenged Balb/C mice.
Only three other types of amino acid inhibitors are known
to bind to arginase with low micromolar affinity or better:
those bearing boronic acid side chains,13b,18 one bearing an
N-hydroxyguanidinium side chain,44 and one bearing an N-
hydroxyamino side chain (N-hydroxy-L-lysine).45 Therefore,
we now add the 2-aminoimidazole amino acid A1P to this
important group of lead compounds for the development of

4272 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Ilies et al.
new arginase inhibitors that may exhibit alternative and
improved pharmacokinetic profiles.
HC-5/C-4 of imidazole), 1.38 (s, 18H, 2Boc). 13C NMR (DMSO-
d6): δ 156.2 (2CO, 2Boc), 140.7 (C-2 of imidazole), 126.5 (C-4/C-5
of imidazole), 116.3 (C-5/C-4 of imidazole), 82.6 (2CMe3, 2Boc),
27.4 (6CH3, 2Boc). MS ESþ m/z 284.2 (M þ H)þ.
Experimental Section
2-[Bis(tert-butyloxycarbonyl)amino]-5-[2-bis(tert-butyloxycar-
bonyl)aminoimidazol-1-yl]pentanoic Acid tert-Butyl Ester (9).
Anhydrous K2CO3 (0.77 g, 5.6 mmol) was suspended in DMF
(7.5 mL) and purged with N2 for 5 min. 6 (0.80 g, 2.8 mmol) was
added in one portion under N2 purging, followed by the bromide
8 (1.3 g, 2.9 mmol) in DMF (3 mL). The mixture was warmed to
60 ꢀC while stirring vigorously and purging with N2. Then the N2
line was removed and stirring was continued overnight at 60 ꢀC
under inert atmosphere. The reaction mixture was allowed to
cool at room temperature, poured into cold water (50 mL), and
extracted with CH2Cl2 (5 ꢀ 40 mL). The combined organic
extracts were washed with water (50 mL), brine (50 mL), dried
(Na2SO4), filtered, and concentrated. Flash chromatography on
silica gel (hexane/EtOAc gradients) afforded 9 (1.1 g, 60%) as a
light-brown oil. 1H NMR (CDCl3): δ 7.00 (d, J=1.2, 1H, HC-4/
C-5 of imidazole), 6.89 (d, J=1.2, 1H, HC-5/C-4 of imidazole),
4.69 (dd, J=8.4, 5.6, 1H, CHCOO), 3.73 (dd, J=7.1, 5.8, 2H,
CH2N), 2.1-1.81 (m, 4H, CHCH2 þ CHCH2CH2), 1.48 (s,
18H, 2Boc), 1.42 (s, 18H, 2Boc), 1.41 (s, 9H, 3CH3 of COOtBu).
13C NMR (CDCl3): δ 169.5 (CO, COOtBu), 152.9 (3CO, 3Boc),
150.1 (CO, Boc), 138.2 (C-2 of imidazole), 127.2 (C-4/C-5 of
imidazole), 118.7 (C-5/C-4 of imidazole), 83.4 (2CMe3, 2Boc),
81.9 (2CMe3, 2Boc), 77.6 (CMe3, COOtBu), 58.5 (CHCOO),
45.3 (CH2N), 28.4 (6CH3, 2Boc), 28.2 (6CH3, 2Boc), 28.1 (3CH3
of COOtBu), 27.2 (CHCH2), 26.9 (CHCH2CH2). MS ESþ m/z
655.4 (M þ H)þ.
Synthesis of 2-Aminoimidazole Amino Acids. General Proce-
dures. All reagents were of at least 95% purity, purchased from
Sigma Aldrich Co., Fisher Scientific, or Alfa Aesar and used as
received. All solvents were of HPLC grade and purchased from
Fisher Scientific or Sigma Aldrich Co. For anhydrous condi-
tions, solvents were freshly distilled under N2 (Et2O from Na,
CH2Cl2 from P2O5, and THF from Na/benzophenone). Reac-
tions were monitored by TLC with Sigma-Aldrich aluminum
˚
plates (silica gel F254, 60 A, 0.25 mm), visualized by quenching
under UV light, equilibrating in a glass chamber containing
iodine, and/or staining with ninhydrin solution. Flash column
chromatography was performed using Fisher Scientific silica gel
60 (230-400 mesh). HPLC was performed on a Waters system
with a 1525 binary pump and a 2487 dual λ absorbance UV
detector (detection at 254 nm), using a Symmetry (C18) 5 μM
column (4.6 mm ꢀ 150 mm) (MeCN/H2O/TFA, 10:90:0.1;
isocratic elution). Mass spectrometry was carried out on Waters
Acquity SQD UPLC-MS (low resolution) and on LCT Premier
XE Micromass/Waters MS Technologies (high resolution)
spectrometers. Purities of all synthesized and tested compounds
were greater than 95%.
1H and 13C NMR spectra were recorded on Bruker DMX 360
and DRX 500 spectrometers at 360 and 500 MHz, respectively,
for 1H and at 90.6 and 125.6 MHz, respectively, for 13C NMR.
Assignments were made on the basis of chemical shifts, signal
intensity, COSY, and HMQC sequences. 1H and 13C NMR
chemical shifts (δ) are reported in ppm relative to the residual
solvent peaks. 1H NMR coupling constants (J) are reported in
Hz, and multiplicities are denoted as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; bs, broad singlet.
2-(S)-Amino-5-(2-aminoimidazol-1-yl)pentanoic Acid Ammo-
nium Salt (A1P, 10). The Boc-protected derivative 9 (0.65 g,
1 mmol) was dissolved in CH2Cl2 (4 mL), cooled at 0 ꢀC (water/
ice bath), and treated dropwise with TFA (4 mL) with constant
stirring. The flask was allowed to reach room temperature and
the mixture stirred for another 4 h until TLC monitoring
(MeOH/CH2Cl2, 5:95) revealed complete deprotection. Eva-
poration, washing with CHCl3, and drying under vacuum
yielded 10 (0.39 g, 92%) as a bis-trifluoroacetate salt. Further
purification by flash column chromatography (CHCl3/MeOH/
NH3 gradients) afforded 10 (0.31 g) as a colorless oil. 1H NMR
(D2O): δ 6.80 (s, 1H, HC-4 of imidazole), 6.70 (s, 1H, HC-5 of
imidazole), 3.86 (m, 2H, CH2N), 3.66 (m, 1H, CHCOO), 1.83
(m, 4H, 2CH2). 13C NMR (D2O): d 176.2 (CO), 147.5 (C-2 of
imidazole), 119.1 (C-4 of imidazole), 116.4 (C-5 of imidazole),
54.5 (CHCOO), 44.1 (CH2N), 28.2 (CHCH2CH2CH2N), 24.6
(CHCH2CH2CH2N). A1P-Naþ (pH 14): 1H NMR (D2O þ
NaOD): δ 6.61 (s, 1H, HC-4 of imidazole), 6.46 (s, 1H, HC-5 of
imidazole), 3.63 (m, 2H, CH2N), 3.11 (m, 1H, CHCOO), 1.60
(m, 2H, CHCH2CH2CH2N), 1.44 (m, 2H, CHCH2CH2CH2N).
2-Bis(tert-butyloxycarbonyl)amino-1-tert-butyloxycarbonyli-
midazole (5). 2-Aminoimidazolium sulfate (5.5 g, 41.5 mmol)
was dissolved in water (25 mL) and treated with a solution of
NaOH (1.7 g, 42.5 mmol) in water (3 mL). The water was
removed under reduced pressure. The residue was extracted
with MeOH (30 mL), and the methanolic extracts were rotoe-
vaporated to dryness. The resulting light-brown oil was sus-
pended in MeCN (30 mL). Then NEt3 (20 mL) and DMAP (1 g)
were added. The suspension was cooled to 0 ꢀC. Then di-tert-
butyl pyrocarbonate (27.6 g, 166 mmol) in MeCN (30 mL) was
added dropwise. The mixture was stirred at 20 ꢀC for 16 h, after
which di-tert-butyl pyrocarbonate (6.9 g, 41.5 mmol) in MeCN
(15 mL) was added. The resulting suspension was refluxed for
8 h. The solvent was rotoevaporated and the residue partitioned
between CH2Cl2 (200 mL) and water (100 mL). The aqueous
layer was reextracted with CH2Cl2 (4 ꢀ 50 mL), and the
combined organic extracts were washed with water (100 mL),
saturated aqueous NaHCO3 (100 mL), brine (100 mL), dried
(Na2SO4), filtered, and concentrated under reduced pressure.
Purification by flash column chromatography on SiO2 (hexane/
EtOAc gradients) afforded pure product 5 (10.1 g, 63%) as a
colorless oil. 1H NMR (CDCl3): δ 7.39 (d, J=1.9, 1H, HC-4 of
imidazole), 6.93 (d, J=1.9, 1H, HC-5 of imidazole), 1.58 (s, 9H,
N-1Boc), 1.43 (s, 18H, 2-NBoc2). 13C NMR (CDCl3): δ 149.4
(2CO, 2-NBoc2), 146.4 (CO, N1-Boc), 138.3 (C-2 of imidazole),
126.7 (C-4/C-5 of imidazole), 118.5 (C-5/C-4 of imidazole), 86.1
(CMe3, N-1Boc), 83.6 (2CMe3, 2-NBoc2), 27.9 (9CH3, 3Boc).
MS ESþ m/z 384.2 (M þ H)þ
A1P 2HCl (pH 1): 1H NMR (D2O): δ 6.75 (bs, 2H, HC-4, HC-5
3
of imidazole), 3.96 (t, J=5.8, 1H, CHCOO), 3.84 (t, J=6.4, 2H,
CH2N), 1.84 (m, 4H). HRMS m/z 199.1188 (calcd for M þ H,
199.1195).
2-[Bis(tert-butyloxycarbonyl)amino]-5-(imidazol-2-ylamino)-
pentanoic Acid tert-Butyl Ester (11). 2-Aminoimidazolium sul-
fate (2 g, 15 mmol) was dissolved in water (10 mL) and treated
with NaOH (0.6 g, 15 mmol) in water (3 mL). The solvent was
removed under vacuum and the evaporation residue washed
with EtOH (50 mL), then sonicated in EtOH (50 mL) for 10 min
to ensure the complete extraction of 11 from the inorganic salt
matrix. The resulting light-brown solution was filtered and the
inorganic precipitate washed with EtOH (3 ꢀ 10 mL). Rotoe-
vaporation to dryness of the combined EtOH extracts yielded
the 2-aminoimidazole free base (1.25 g, 99%) as a brown oil. The
crude product was retaken in MeOH (10 mL), aldehyde 4 (3 g,
7.75 mmol) was added, and the resulting mixture was treated
with NaBH3CN (300 mg, 4.7 mmol) and activated molecular
2-Bis(tert-butyloxycarbonyl)aminoimidazole (6). To a solu-
tion of 5 (1.48 g, 3.9 mmol) in MeOH (20 mL) at room
temperature, NH3 in MeOH (3 mL, 7N) was added. The mixture
was stirred at room temperature for 48 h. Then the solvent was
removed in vacuo. Purification by flash column chromatogra-
phy on SiO2 (hexane/EtOAc gradient) yielded 6 (0.83 g, 56%) as
a white solid. 1H NMR (DMSO-d6): δ 12.13 (s, 1H, NH of
imidazole), 7.07 (s, 1H, HC-4/C-5 of imidazole), 6.80 (s, 1H,
˚
sieves (4 A, 2 g). The suspension was stirred at room temperature
until TLC indicated no further advancement of the reaction

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4273
(48 h). Column chromatography of the residue (silica gel,
hexane/EtOAc gradient) yielded 11 (1.05 g, 30%) as a purple
oil (the alcohol 7 was the major byproduct and could be
recovered: 1.8 g, 60% conversion). 1H NMR (CDCl3): δ 10.03
(s, 1H, NH of imidazole), 6.55 (s, 1H, HC-4/C-5 of imidazole),
6.49 (s, 1H, HC-5/C-4 of imidazole), 5.22 (t, J=6.1, 1H, NH-2 of
imidazole), 4.64 (dd, J=9.8, 4.8, 1H, CHCOO), 3.30 (dd, J=7.0,
6.5, 2H, CH2N), 2.10-1.67 (m, 4H, CHCH2 þ CHCH2CH2),
1.50 (s, 18H, 2Boc), 1.43 (s, 9H, 3CH3 of COOtBu). 13C NMR
(CDCl3): δ 169.8 (CO, COOtBu), 153.4 (2CO, 2Boc), 148.1 (C-2
of imidazole), 121.8 (C-4 of imidazole), 111.0 (C-5 of imidazole),
84.0 (2CMe3, 2Boc), 82.1 (CMe3, COOtBu), 58.7 (CHCOO),
43.3 (CH2N), 28.3 (6CH3, 2Boc), 28.2 (3CH3 of COOtBu), 26.6
(CHCH2), 25.8 (CHCH2CH2). HRMS m/z 455.2856 (calcd for
M þ H, 455.2870).
and the resulting light-brown oil was dried overnight under
vacuum, then resolubilized in water (18 mL). The pH was
adjusted to 4.5 with 2 N NaOH, and cyanamide (1.04 g) was
added in one portion. The reaction mixture was refluxed at 95 ꢀC
for 3 h, then rotoevaporated to dryness. Purification by flash
column chromatography (CHCl3/MeOH/NH4OH gradients)
generated 16 (0.23 g, 30%) as a light-yellow oil. 1H NMR
(D2O): δ 6.77 (s, 1H, HC-4 of imidazole), 3.75 (m, 1H, CHCOO),
2.55 (m, 2H, CHCH2CH2CH2-imidazole), 1.88 (m, 2H, CHCH2-
CH2CH2-imidazole), 1.68 (m, 2H, CHCH2CH2CH2-imidazole).
13C NMR (D2O): δ 175.1 (CO), 147.1 (C-2 of imidazole), 128.4
(C-4 of imidazole), 109.4 (C-5 of imidazole), 54.6 (CHCOO),
29.9 (CHCH2CH2CH2-imidazole), 23.9 (CHCH2CH2CH2-imi-
dazole), 23.3 (CHCH2CH2CH2-imidazole). HRMS m/z 199.1189
(calcd for M þ H, 199.1195).
Kinetic Assays. 2-Aminoimidazole amino acids affinities for
human arginase I were determined using the fixed point assay
with [14C-guanidino]-L-arginine34 as previously reported.46 Re-
combinant human arginase I was prepared as previously de-
scribed.19 The final working concentrations in the reaction tubes
were 1 mM for the unlabeled L-arginine and 0.07 μg/μL for
human arginase I. Data analysis was based on the kinetic replot
v0/vi as a function of inhibitor concentration, where v0 and vi were
the observed velocities in the absence and presence of inhibitor,
respectively. The mode of inhibition (i.e., noncompetive or
competitive) was determined from Lineweaver-Burk plots of
kinetic data at several different inhibitor concentrations.
Surface Plasmon Resonance. The binding affinity of A1P was
determined on a Biacore 3000 instrument, following a pre-
viously reported procedure,32 except that all measurements were
made at pH 8.5 and the analyte concentrations ranged from 0 to
800 μM. Binding data were analyzed using a 1:1 Langmuir
interaction model.47
Crystallography. Crystals of human arginase I complexed
with 2AH were prepared by soaking 2AH into preformed
crystals of the native enzyme, which were prepared by the
hanging drop vapor diffusion method at 21 ꢀC. Drops contain-
ing 3 μL of protein solution [3.5 mg/mL protein, 50.0 mM bicine
(pH 8.5), 2 mM thymine, 100 μM MnCl2] and 3 μL of precipitant
solution [0.1 M HEPES (pH 7.0), 28% Jeffamine] were equili-
brated against a 1 mL reservoir of precipitant buffer. Crystals
generally formed within 3-4 days. Crystals were harvested and
soaked in a precipitant solution augmented with 20 mM 2AH
for 1 week and then cryoprotected in a precipitant solution
containing 32% Jeffamine prior to flash cooling in liquid
nitrogen.
(S)-2-Amino-5-(imidazol-2-ylamino)pentanoic Acid(APP) (12).
The same general procedure as for the synthesis of 10 afforded 12
as a bis-trifluoroacetate salt (90%). 1H NMR (D2O): δ 6.87 (bs,
2H, HC-4, HC-5 of imidazole), 4.01 (m, 1H, CHCOO), 3.32 (m,
2H, CH2N), 2.05 (m, 2H, CHCH2), 1.80 (m, 2H, CHCH2CH2).
13C NMR (D2O): δ 165.5 (CO), 146.7 (C-2 of imidazole), 113.4
(C-4 of imidazole), 112.9 (C-5 of imidazole), 60.6 (CHCOO), 42.5
(CH2N), 29.5 (CHCH2CH2CH2N), 26.5 (CHCH2CH2CH2N)
(signals from TFA were omitted).
Dimethyl 2,6-Bis(tert-butoxycarbonylamino)heptanedioate (13).
2,6-Diaminopimelic acid (6 g, 31.5 mmol) was dissolved in MeOH
(50 mL) under N2 purging in an oven-dried flask. The suspen-
sion was cooled to -5 to -10 ꢀC (ice/acetone bath) and treated
dropwise with thionyl chloride (5.1 mL, 69.4 mmol). The mixture
was stirred at -5 to -10 ꢀC for 1 h, then overnight at room
temperature, when 1H NMR and TLC monitoring (CHCl3/
MeOH/NH4OH, 2:8:1) indicated completion of the reaction.
The solvent was rotoevaporated and the crude product washed
with Et2O, then dried overnight under vacuum to yield the
corresponding amino ester (8.5 g) as a dihydrochloride salt.
NEt3 (12.3 mL) and DMAP (2.9 g) were added to a suspension
of the amino ester in MeCN (80 mL). The suspension was cooled
to 0 ꢀC. Then di-tert-butyl pyrocarbonate (25.8 g, 118.1 mmol) in
MeCN (30 mL) was added dropwise. The mixture was stirred at
room temperature for 48 h. Solvent rotoevaporation and purifica-
tion by flash column chromatography (silica gel, hexane/EtOAc
gradients) afforded 13 as a light-yellow oil (88%). 1H NMR
(CDCl3): δ 5.09 (m, 2H, 2CHCOO), 4.26 (m, 2H, 2NH), 3.72
(s, 6H, 2COOCH3), 1.82 (m, 2H, CAH2CH), 1.63 (m, 2H,
CBH2CH), 1.43 (s, 18H, 2Boc), 1.39 (m, 2H, CH2CH2CH). 13C
NMR (CDCl3): δ 173.5 (2CO, 2COOMe), 155.8 (2CO, 2Boc),
80.34 (2CMe3, 2Boc), 53.5 (CAH), 52.7 (CBH), 32.7 (CAH2CH),
32.6 (CBH2CH), 28.6 (6CH3, 2Boc), 21.8 (CAH2CH2CH), 21.6
(CBH2CH2CH). MS ESþ m/z 419.2 (M þ H)þ.
Crystals of the human arginase I-AHH and human arginase
I-2 aminoimidazole complexes were prepared by cocrystalliza-
tion in hanging drops at 21 ꢀC. Drops containing 3 μL of protein
solution [3.5 mg/mL protein, 50 mM bicine (pH 8.5), 2 mM
AHH or 2 mM 2-aminoimidazole, 100 μM MnCl2] and 3 μL of
precipitant solution [0.1 M HEPES (pH 7.0), 28% Jeffamine]
were equilibrated against a 1 mL reservoir of precipitant solu-
tion. Crystals appeared overnight and grew with typical dimen-
sions of 0.5 mm ꢀ 0.2 mm ꢀ 0.2 mm within 1 week. Crystals were
soaked for 24 h in a precipitant solution augmented with 5 mM
AHH or 2-aminoimidazole and cryoprotected as described
above.
Methyl 2,6-Bis(tert-butoxycarbonylamino)-7-oxoheptanoate
(14). 13 (1.2 g, 2.86 mmol) was dissolved in Et2O (30 mL) with
stirring at room temperature and nitrogen purging, then cooled
to -78 ꢀC (acetone/dry ice bath). DIBAH (1 M) in hexane (1.86
mL, 1.86 mmol) was added dropwise. 1H NMR monitoring
indicated completion of the reaction after 1 h. The reaction
mixture was quenched with water (5 mL) and filtered. The
filtrate was rotoevaporated to dryness, washed with CHCl3,
and dried overnight under vacuum to afford 14 (0.99 g, 89%) as
a colorless oil. 1H NMR (CDCl3): δ 9.57 (s, 1H, CHO), 5.09 (m,
2H, CHCHO þ CHCOOMe), 4.27 (m, 2H, 2NH), 3.73 (s, 3H,
COOCH3), 1.80 (m, 2H, CAH2CH), 1.64 (m, 2H, CBH2CH),
1.44 (s, 18H, 2Boc), 1.40 (m, 2H, CH2CH2CH). 13C NMR
(CDCl3): δ 200.0 (CHO), 173.4 (CO, COOMe), 155.9 (2CO,
2Boc), 80.3 (2CMe3, 2Boc), 59.8 (CHCHO), 53.4 (CAH), 52.6
(CBH), 32.8 (CAH2CH), 32.6 (CBH2CH), 28.6 (6CH3, 2Boc),
21.7 (CAH2CH2CH), 21.5 (CBH2CH2CH), 21.3 (CCH2CH2-
CH). MS ESþ m/z 389.2 (M þ H)þ.
X-ray diffraction data from all crystals were collected at the
National Synchrotron Light Source beamline X6A. Diffraction
intensities measured from human arginase I-inhibitor com-
plexes exhibited symmetry consistent with apparent space group
˚
˚
P6 (unit cell parameters a=b=90.1 A, c=69.7 A). Intensity data
integration and reduction were performed using the HKL2000
suite of programs.48 Data reduction statistics are recorded in
Table 2. As with crystals of other human arginase I complexes,49
deviations from ideal Wilson statistics were observed with ÆI2æ/
ÆIæ2=1.5, indicating perfect hemihedral twinning.50 The struc-
ture of each enzyme-inhibitor complex was solved by molecular
replacement using the program Phaser51 with chain A of the
2-Amino-5-(2-aminoimidazol-4-yl)pentanoic Acid (A4P, 16). 14
(1.6 g, 4.12 mmol) was stirred in EtOAc (32 mL) and 4 N HCl (30
mL) at room temperature for 2 h. The solvent was rotoevaporated,

4274 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Table 2. Data Collection and Refinement Statistics
Ilies et al.
guidelines of the Canadian Council on Animal Care. The acute
model utilized a 3-week OVA-sensitization and -challenge pro-
tocol, as described previously.8a Briefly, female Balb/C mice (6-
8 weeks old, Charles River Laboratories, Saint-Constant, PQ)
were sensitized to chicken OVA on days 0 and 7 (ip, 25 μg of
chicken OVA; grade V, >98% pure; Sigma Chemical Company,
Mississauga, Ontario, Canada), with 1 mg of aluminum hydro-
xide gel (Invitrogen, Grand Island, NY). Beginning on day 14,
animals were randomized to repeated exposure to nebulized 6%
OVA in PBS or PBS alone for 25 min/day on days 14-20, using
human
arginase
I-2AI
human
arginase
I- 2AH
human
arginase
I-AHH
Data Collection
˚
resolution, A
50.0-1.90
91110/50007
50.0-1.47
196796/107754
50.0-1.90
95366/50344
total/unique reflections
measured
a,b
Rmerge
0.061 (0.265)
13.9 (4.0)
99.1 (98.7)
0.059 (0.616)
21.1 (2.3)
99.4 (99.6)
0.086 (0.310)
15.4 (3.8)
99.8 (100.0)
I/σ(I) a
ꢀ
an AeroNebLab nebulizer (SciReq Inc., Montreal PQ).
completeness, % a
In Vivo Arginase Inhibition with A1P and Methacholine
Challenge. On day 21, 24 h after the final OVA or PBS challenge,
mice were anesthetized with 50 mg/kg ketamine (Bioniche,
Belleville, Ontario, Canada) and 10 mg/kg xylazine (Bayer Inc.,
Toronto, Ontario, Canada) prior to intubation with an 18G
stainless steel cannula (BD Biosciences Canada, Mississauga,
Ontario, Canada) for in vivo, ventilator-based assessment of
methacholine-responsiveness using the FlexiVent system Scireq.
After determination of baseline pulmonary resistance para-
meters, A1P (80 μg/g body weight) was delivered via nebulization
directly into the ventilatory circuit of a randomly selected sub-
group of OVA/OVA mice. In a previous study we showed that
arginase inhibition did not exhibit any effect on OVA/PBS mice.
Fifteen minutes after delivery of the drug, baseline pulmonary
resistance was reassessed and the methacholine dose-response
curve was initiated, as previously described.8a In brief, mice
were challenged with methacholine (0-100 mg/mL in sterile
PBS: Sigma) nebulized directly into the ventilatory circuit
(AeroNebLab nebulizer). All data points were collected using
the FlexiVent software (Scireq Inc.) and analyzed off-line using
Excel (Microsoft Corp., Redmond, WA). We have previously
shown8a that this treatment-based protocol completely abrogates
the allergic airways-induced hyperresponsiveness to methacho-
line in several murine models of allergic airways inflammation.
Refinement
reflections used in
refinement/test set
47507/2500
102365/5389
49288/2184
c
Rtwin
0.168
0.176
4772
246
12
0.149
0.162
4772
460
24
0.141
0.190
4762
338
26
c
Rtwin/free
Protein atomsd
water moleculesd
inhibitor atomsd
manganese ionsd
sulfate ionsd
4
4
4
2
Root-Mean-Square Deviation
˚
bond length, A
0.007
2.1
3MJL
0.011
2.2
3MFW
0.008
2.1
3MFV
bond angle, deg
PDB accession code
a
˚
Number in parentheses refer to the outer 0.1 A shell of data.
P
P
b Rmerge
=
|I - ÆIæ|/ I, where I is the observed intensity and ÆIæ is
P
the average intensity calculated for replicate data. c Rtwin
=
|[|Fcalc/A|2 þ
P
|Fcalc/B|2]1/2 - |Fobs||/ |Fobs| for reflections contained in the working set.
|Fcalc/A| and |Fcalc/B| are the structure factor amplitudes calculated for the
separate twin domains A and B, respectively. Rtwin underestimates the
residual error in the model over the two twin-related reflections by a factor
of approximately 0.7. The same expression describes Rtwin/free, which was
calculated for test set reflections excluded from refinement. d Per asym-
metric unit.
Acknowledgment. This work was supported by the America
Asthma Foundation and NIH Grant GM49758. We thank
Dr. Steven Seeholzer, Dr. Hua Ding, and the Protein Core
Facility at Children’s Hospital of Philadelphia, PA, for assis-
tance with surface plasmon resonance measurements, and
Dr. Rakesh Kohli for recording the HRMS spectra of the
compounds. We also thank the National Synchrotron Light
Source, beam time X6A, for access to X-ray crystallographic
data collection facilities. D.W.C. is a Senior Fellow of the
American Asthma Foundation. M.L.N. is a recipient of CIHR
and Ontario Thoracic Society Doctoral Awards. J.A.S. is a
Scientist in the Keenan Research Centre in the Li Ka Shing
Knowledge Institute of St. Michael’s Hospital.
unliganded human arginase I (PDB accession code 2ZAV, less
water molecules)49 used as a search probe against twinned data.
In order to calculate electron density maps, structure factor
amplitudes (|Fobs|) derived from twinned intensity data (|Iobs|)
were deconvoluted into structure factor amplitudes correspond-
ing to individual twin domains A and B (|Fobs/A| and |Fobs/B|,
respectively) using the structure-based algorithm of Redinbo
and Yeates50 implemented in CNS.52
Crystallographic refinement of each enzyme-inhibitor com-
plex against twinned data was performed as previously de-
scribed using CNS.49 In the later stages of each refinement,
after the majority of water molecules were located, gradient omit
maps revealed the inhibitor bound in the active site of each
monomer in the asymmetric unit.
Inhibitors were refined with full occupancy and atomic B
factors consistent with the average B-factor calculated for the
entire protein. For the refinement of the human arginase I-
2-aminoimidazole and human arginase I-2AH complexes, full-
matrix least-squares refinement was performed against twinned
data using the program Shelx53 in the final stages of refinement.
Isotropic B factors were used in refinement with the exception of
the manganese ions and sulfur atoms in the human arginase
I-2AH complex, which were refined anisotropically (Table 2).
Disordered segments were excluded from the final models of
the human arginase I-AHH complex (M1-S5 and N319-K322),
the human arginase I-2-aminoimidazole complex (M1-S5 and
N319-K322), and the human arginase I-2AH complex (M1-K4
and N319-K322). The quality of each refined model was
assessed using PROCHECK.54
References
(1) (a) Borcic, O.; Straus, B. Separation of arginase isoenzymes from
human tissues by agar gel electrophoresis. J. Clin. Chem. Clin.
Biochem. 1976, 14, 533–535. (b) Grody, W. W.; Dizikes, G. J.;
Cederbaum, S. D. Human arginase isozymes. Isozymes: Curr. Top.
Biol. Med. Res. 1987, 13, 181–214. (c) Jenkinson, C. P.; Grody, W. W.;
Cederbaum, S. D. Comparative properties of arginases. Comp. Bio-
chem. Physiol. 1996, 114B, 107–132. (d) Ash, D. E.; Cox, J. D.;
Christianson, D. W. Manganese and Its Role in Biological Processes. In
Metal Ions in Biological Systems; Sigel, A., Sigel, H., Eds.; M.
Dekker: New York, 2000; Vol. 37, pp 407-428.
(2) Morris, S. M., Jr. Regulation of enzymes of the urea cycle and
arginine metabolism. Annu. Rev. Nutr. 2002, 22, 87–105.
(3) (a) Morris, S. M., Jr.; Bhamidipati, D.; Kepka-Lenhart, D. Human
type II arginase: sequence analysis and tissue-specific expression.
Gene 1997, 193, 157–161. (b) Gotoh, T.; Araki, M.; Mori, M.
Chromosomal localization of the human arginase II gene and tissue
distribution of its mRNA. Biochem. Biophys. Res. Commun. 1997,
233, 487–491.
Acute Murine OVA-Sensitization and Challenge Model of
Allergic Airways Inflammation. These protocols were approved
by the University of Toronto Faculty Advisory Committee on
Animal Services and were conducted in accordance with the
(4) (a) Christianson, D. W. Arginase: structure, mechanism, and
physiological role in male and female sexual arousal. Acc. Chem.

Article
Res. 2005, 38, 191–201. (b) Dowling, D. P.; Di Costanzo, L.;
Gennadios, H. A.; Christianson, D. W. Evolution of the arginase fold
and functional diversity. Cell. Mol. Life Sci. 2008, 65, 2039–2055.
(5) (a) Wu, G.; Morris, S. M., Jr. Arginine metabolism: nitric oxide
and beyond. Biochem. J. 1998, 336, 1–17. (b) Morris, S. M., Jr.
Arginine metabolism: boundaries of our knowledge. J. Nutr. 2007,
137, 1602S–1609S. (c) Morris, S. M., Jr. Recent advances in arginine
metabolism: roles and regulation of the arginases. Br. J. Pharmacol.
2009, 157, 922–930.
(6) (a) Smith, R. J.; Phang, J. M. The importance of ornithine as a
precursor for proline in mammalian cells. J. Cell. Physiol. 1979, 98,
475–482. (b) Albina, J. E.; Abate, J. A.; Mastrofrancesco, B. Role of
ornithine as a proline precursor in healing wounds. J. Surg. Res. 1993,
55, 97–102. (c) Nieves, C., Jr.; Langkamp-Henken, B. Arginine and
immunity: a unique perspective. Biomed. Pharmacother. 2002, 56,
471–482.
(7) (a) Kim, P. S.; Iyer, R. K.; Lu, K. V.; Yu, H.; Karimi, A.; Kern, R.
M.; Tai, D. K.; Cederbaum, S. D.; Grody, W. W. Expression of the
liver form of arginase in erythrocytes. Mol. Gen. Metab. 2002, 76,
100–110. (b) Wei, L. H.; Wu, G; Morris, S. M., Jr.; Ignarro, L. J.
Elevated arginase I expression in rat aortic smooth muscle cells
increases cell proliferation. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
9260–9264.
(8) (a) North, M. L.; Khanna, N.; Marsden, P. A.; Grasemann, H.;
Scott, J. A. Functionally important role for arginase 1 in the airway
hyperresponsiveness of asthma. Am. J. Physiol.: Lung Cell. Mol.
Physiol. 2009, 296, L911–L920. (b) Bergeron, C.; Boulet, L.-P.; Page,
N.; Laviolette, M.; Zimmermann, N.; Rothenberg, M. E.; Hamid, Q.
Influence of cigarette smoke on the arginine pathway in asthmatic
airways: increased expression of arginase I. J. Allergy Clin. Immunol.
2007, 119, 391–397. (c) Zimmermann, N.; King, N. E.; Laporte, J.;
Yang, M.; Mishra, A.; Pope, S. M.; Muntel, E. E.; Witte, D. P.; Pegg,
A. A.; Foster, P. S.; Hamid, Q.; Rothenberg, M. E. Dissection of
experimental asthma with DNA microarray analysis identifies arginase
in asthma pathogenesis. J. Clin. Invest. 2003, 111, 1863–1874.
(9) (a) Pauleau, A.-L.; Rutschman, R.; Lang, R.; Pernis, A.;
Watowich, S. S.; Murray, P. J. Enhancer-mediated control of
macrophage-specific arginase I expression. J. Immunol. 2004, 172,
7565–7573. (b) Zimmermann, N.; Mishra, A.; King, N. E.; Fulkerson,
P. C.; Doepker, M. P.; Nikolaidis, N. M.; Kindinger, L. E.; Moulton,
E. A.; Aronow, B. J.; Rothenberg, M. E. Transcript signatures in experi-
mental asthma: identification of STAT6-dependent and -independent
pathways. J. Immunol. 2004, 172, 1815–1824.
(10) (a) Li, H.; Romieu, I.; Sienra-Monge, J.-J.; Ramirez-Aguilar, M.;
Estela del Rio-Navarro, B.; Kistner, E. O.; Gjessing, H. K.; Lara-
Sanchez, I. C; Chiu, G. Y.; London, S. J. Genetic polymorphisms in
arginase I and II and childhood asthma and atopy. J. Allergy Clin.
Immunol. 2006, 117, 119–126. (b) Litonjua, A. A.; Lasky-Su1, J.;
Schneiter, K.; Tantisira, K. G.; Lazarus, R.; Klanderman, B.; Lima, J. J.;
Irvin, C. G; Peters, S. P.; Hanrahan, J. P.; Liggett, S. B.; Hawkins, G. A.;
Meyers, D. A.; Bleecker, E. R.; Lange, C.; Weiss, S. T. ARG1 is a novel
bronchodilator response gene. Screening and replication in four asthma
cohorts. Am. J. Respir. Crit. Care Med. 2008, 178, 688–694.
(11) (a) Yang, M.; Rangasamy, D.; Matthaei, K. I.; Frew, A. J.;
Zimmermann, N.; Mahalingam, S.; Webb, D. C.; Tremethick,
D. J.; Thompson, P. J.; Hogan, S. P.; Rothenberg, M. E.; Cowden,
W. B.; Foster, P. S. Inhibition of arginase I activity by RNA
interference attenuates IL-13-induced airways hyperresponsive-
ness. J. Immunol. 2006, 177, 5595–5603. (b) Maarsingh, H.; Zuidhof,
A. B.; Bos, I. S. T.; van Duin, M.; Boucher, J.-L.; Zaagsma, J.; Meurs, H.
Arginase inhibition protects against allergen-induced airway obstruc-
tion, hyperresponsiveness, and inflammation. Am. J. Respir. Crit.
Care Med. 2008, 178, 565–573.
(12) (a) Meurs, H.; Maarsingh, H.; Zaagsma, J. Arginase and asthma:
novel insights into nitric oxide homeostasis and airway hy-
perresponsiveness. Trends Pharmacol. Sci. 2003, 24, 450–455. (b)
Zimmermann, N.; Rothenberg, M. E. The arginine-arginase balance in
asthma and lung inflammation. Eur. J. Pharmacol. 2006, 533, 253–
262. (c) Munder, M. Arginase: an emerging key player in the mamma-
lian immune system. Br. J. Pharmacol. 2009, 158, 638–651.
(d) Munder, M. Role of arginase in asthma: potential clinical applica-
tions. Expert Rev. Clin. Pharmacol. 2010, 3, 17–23.
(13) (a) Cox, J. D.; Kim, N. N.; Traish, A. M.; Christianson, D. W.
Arginase-boronic acid complex highlights a physiological role in
erectile function. Nat. Struct. Biol. 1999, 6, 1043–1047. (b) Kim, N.
N.; Cox, J. D.; Baggio, R. F.; Emig, F. A.; Mistry, S. K.; Harper, S. L.;
Speicher, D. W.; Morris, S. M.; Ash, D. E.; Traish, A.; Christianson,
D. W. Probing erectile function: S-(2-boronoethyl)-L-cysteine binds to
arginase as a transition state analogue and enhances smooth muscle
relaxation in human penile corpus cavernosum. Biochemistry 2001, 40,
2678–2688. (c) Cama, E.; Colleluori, D. M.; Emig, F. A.; Shin, H.; Kim,
S. W.; Kim, N. N.; Traish, A.; Ash, D. E.; Christianson, D. W. Human
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4275
arginase II: crystal structure and physiological role in male and female
sexual arousal. Biochemistry 2003, 42, 8445–8451.
(14) (a) Bivalacqua, T. J.; Hellstrom, W. J. G.; Kadowitz, P. J.;
Champion, H. C. Increased expression of arginase II in human
diabetic corpus cavernosum: in diabetic-associated erectile dys-
function. Biochem. Biophys. Res. Commun. 2001, 283, 923–927.
(b) Bivalacqua, T. J.; Burnett, A. L.; Hellstrom, W. J. G.; Champion,
H. C. Overexpression of arginase in the aged mouse penis impairs erectile
function and decreases eNOS activity: influence of in vivo gene therapy of
anti-arginase. Am. J. Physiol.: Heart Circ. Physiol. 2007, 292, H1340–
H1351.
ꢀ
(15) Grasemann, H.; Schwiertz, R.; Matthiesen, S.; Racke, K.; Ratjen,
F. Increased arginase activity in cystic fibrosis airways. Am. J.
Respir. Crit. Care Med. 2005, 172, 1523–1528.
(16) (a) Yang, Z.; Ming, X.-F. Endothelial arginase: a new target in
atherosclerosis. Curr. Hypertens. Rep. 2006, 8, 54–59. (b) Nelin, L.
D.; Stenger, M. R.; Malleske, D. T.; Chicoine, L. G. Vascular arginase
and hypertension. Curr. Hypertens. Rev. 2007, 3, 242–249. (c)
Santhanam, L.; Lim, H. K.; Lim, H. K.; Miriel, V.; Brown, T.; Patel,
M.; Balanson, S.; Ryoo, S.; Anderson, M.; Irani, K.; Khanday, F.;
Di Costanzo, L.; Nyhan, D.; Hare, J. M.; Christianson, D. W.; Rivers,
R.; Shoukas, A.; Berkowitz, D. E. Inducible NO synthase-dependent
S-nitrosylation and activation of arginase 1 contribute to age-related
endothelial dysfunction. Circ. Res. 2007, 101, 692–702. (d) Santhanam,
L.; Christianson, D. W.; Nyhan, D.; Berkowitz, D. E. Arginase and
vascular aging. J. Appl. Physiol. 2008, 105, 1632–1642. (e) Ryoo, S.;
Gupta, G.; Benjo, A.; Lim, H. K.; Camara, A.; Sikka, G.; Lim, H. K.;
Sohi, J.; Santhanam, L.; Soucy, K.; Tuday, E.; Baraban, E.; Ilies, M.;
Gerstenblith, G.; Nyhan, D.; Shoukas, A.; Christianson, D. W.; Alp, N. J.;
Champion, H. C.; Huso, D.; Berkowitz, D. E. Endothelial arginase II.
A novel target for the treatment of atherosclerosis. Circ. Res. 2008, 102,
923–932.
(17) (a) Singh, R.; Pervin, S.; Karimi, A.; Cederbaum, S.; Chaudhuri, G.
Arginase activity in human breast cancer cell lines: Nω-hydroxy-L-
arginine selectively inhibits cell proliferation and induces apoptosis
in MDA-MB-468 cells. Cancer Res. 2000, 60, 3305–3312. (b) Chang,
C.-I.; Liao, J. C.; Kuo, L. Macrophage arginase promotes tumor cell
growth and suppresses nitric oxide-mediated tumor cytotoxicity. Can-
cer Res. 2001, 61, 1100–1106. (c) Lind, S. D. Arginine and cancer.
J. Nutr. 2004, 134, 2837S–2841S. (d) Bronte, V.; Zanovello, P.
Regulation of immune responses by L-arginine metabolism. Nat. Rev.
Immunol. 2005, 5, 641–654. (e) Zea, A. H.; Rodriguez, P. C.; Atkins, M.
B.; Hernandez, C.; Signoretti, S.; Zabaleta, J.; McDermott, D.; Quiceno,
D.; Youmans, A.; O'Neill, A.; Mier, J.; Ochoa, A. C. Arginase-producing
myeloid suppressor cells in renal cell carcinoma patients: a mechanism
of tumor evasion. Cancer Res. 2005, 65, 3044–3048. (f) Ochoa, A. C.;
Zea, A. H.; Hernandez, C.; Rodriguez, P. C. Arginase, prostaglandins,
and myeloid-derived suppressor cells in renal cell carcinoma. Clin.
Cancer Res. 2007, 13, 721s–726s. (g) Marx, J. Cancer's bulwark against
immune attack: MDS cells. Science 2008, 319, 154–156.
(18) Baggio, R.; Elbam, D.; Kanyo, Z. F.; Carroll, P. J.; Cavalli, R. C.;
Ash, D. E.; Christianson, D. W. Inhibition of Mn2þ2-arginase by
borate leads to the design of a transition state analog inhibitor,
2(S)-amino-6-boronohexanoic acid. J. Am. Chem. Soc. 1997, 119,
8107–8108.
(19) Di Costanzo, L.; Sabio, G.; Mora, A.; Rodriguez, P. C.; Ochoa,
A. C.; Centeno, F.; Christianson, D. W. Crystal structure of human
˚
arginase I at 1.29-A resolution and exploration of inhibition in the
immune response. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13058–
13063.
(20) Di Costanzo, L.; Flores, L. V., Jr.; Christianson, D. W. Stereo-
chemistry of guanidine-metal interactions: implications for L-argi-
nine-metal interactions in protein structure and function. Proteins
2006, 65, 637–642.
(21) Creighton, T. E. Proteins: Structures and Molecular Properties;
W. H. Freeman: New York, 1992.
(22) Storey, B. T.; Sullivan, W. W.; Moyer, C. L. The pKa values of some
2-amino imidazolium ions. J. Org. Chem. 1964, 29, 3118–3120.
(23) Sullivan, J. D.; Giles, R. L.; Looper, R. E. 2-Aminoimidazoles from
Leucetta sponges: synthesis and biology of an important pharma-
cophore. Curr. Bioact. Compd. 2009, 5, 39–78.
(24) (a) Mourabit, A. A.; Potier, P. Sponge’s molecular diversity
through the ambivalent reactivity of 2-aminoimidazole: a universal
chemical pathway to the oroidin-based pyrrole-imidazole alkaloids
and their palau’amine congeners. Eur. J. Org. Chem. 2001, 2, 237–
243. (b) Hoffmann, H.; Lindel, T. Synthesis of the pyrrole-imidazole
alkaloids. Synthesis 2003, 12, 1753–1783. (c) Rogers, S. A.; Bero,
J. D.; Melander, C. Chemical synthesis and biological screening of 2-
aminoimidazole-based bacterial and fungal antibiofilm agents. Chem-
BioChem 2010, 11, 396–410.
(25) (a) Benoit-Vical, F.; Salery, M.; Soh, P. N.; Ahond, A.; Poupat, C.
Girolline: a potential lead structure for antiplasmodial drug

4276 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Ilies et al.
research. Planta Med. 2008, 74, 438–444. (b) Mancini, I.; Guella, G.;
Debitus, C.; Waikedre, J.; Pietra, F. From inactive nortopsentin D,
a novel bis(indole) alkaloid isolated from the axinellid sponge Drag-
macidon sp. from deep waters south of New Caledonia, to a strongly
cytotoxic derivative. Helv. Chim. Acta 1996, 79, 2075–2082.
(26) Nagai, W.; Kirk, K. L.; Cohen, L. A. Synthesis of 2-amino-L-
histidine and 2-aminohistamine. J. Org. Chem. 1973, 38, 1971–1974.
(27) Friedel, M.; Lindel, T. Synthesis of L-aminohomohistidine (L-Ahh).
Tetrahedron Lett. 2004, 45, 2779–2781.
(28) Ulhaq, S.; Chinje, E. C.; Naylor, M. A.; Jaffar, M.; Stratford, I. J.;
Threadgill, M. D. S-2-Amino-5-azolylpentanoic acids related to
L-ornithine as inhibitors of the isoforms of nitric oxide synthase
(NOS). Bioorg. Med. Chem. 1998, 6, 2139–2149.
(29) (a) Zakharian, T. Y.; Di Costanzo, L.; Christianson, D. W.
Synthesis of (2S)-2-amino-7,8-epoxyoctanoic acid and structure
of its metal-bridging complex with human arginase I. Org. Biomol.
Chem. 2008, 6, 3240–3243. (b) Zakharian, T. Y.; Di Costanzo, L.;
Christianson, D. W. (S)-2-Amino-6-nitrohexanoic acid binds to human
arginase I through multiple nitro-metal coordination interactions in the
binuclear manganese cluster. J. Am. Chem. Soc. 2008, 130, 17254–
17255.
(30) Adamczyk, M.; Johnson, D. D.; Reddy, R. E. Collagen crosslinks:
a convenient synthesis of tert-butyl-(2S)-2-[(tert-butoxycarbonyl)-
amino]-4-(2-oxiranyl)butanoate. Tetrahedron: Asymmetry 1999,
10, 775–781.
T. A., Jr.; Veber, D. F.; Xiong, W. C.; Yamamoto, Y.; Yamashita,
K.; Yang, G.; Thompson, S. K. Highly potent inhibitors of
methionine aminopeptidase-2 based on a 1,2,4-triazole pharmaco-
phore. J. Med. Chem. 2007, 50, 3777–3785.
(41) Rahman, M. N.; Vlahakis, J. Z.; Szarek, W. A.; Nakatsu, K.; Jia, Z.
X-ray crystal structure of human heme oxygenase-1 in complex
with 1-(adamantan-1-yl)-2-(1H-imidazol-1-yl)ethanone: a com-
mon binding mode for imidazole-based heme oxygenase-1 inhibi-
tors. J. Med. Chem. 2008, 51, 5943–5952.
(42) Berka, V.; Palmer, G.; Chen, P.-F.; Tsai, A.-L. Effects of various
imidazole ligands on heme conformation in endothelial nitric oxide
synthase. Biochemistry 1998, 37, 6136–6144.
(43) (a) Kenyon, N. J.; Bratt, J. M.; Linderholm, A. L.; Last, M. S.;
Last, J. A. Arginases I and II in lungs of ovalbumin-sensitized mice
exposed to ovalbumin: sources and consequences. Toxicol. Appl.
Pharmacol. 2008, 230, 269–275. (b) Bratt, J. M.; Franzi, L. M.;
Linderholm, A. L.; Last, M. S.; Kenyon, N. J.; Last, J. A. Arginase
enzymes in isolated airways from normal and nitric oxide synthase
2-knockout mice exposed to ovalbumin. Toxicol. Appl. Pharmacol.
2009, 234, 273–280.
(44) Custot, J.; Moali, C.; Brollo, M.; Boucher, J. L.; Delaforge, M.;
Mansuy, D.; Tenu, J. P.; Zimmermann, J. L. The new R-amino acid
Nω-hydroxy-nor-L-arginine: a high-affinity inhibitor of arginase
well adapted to bind to its manganese cluster. J. Am. Chem. Soc.
1997, 119, 4086–4087.
(31) Ulhaq, S.; Naylor, M. A.; Chinje, E. C.; Threadgill, M. D.;
Stratford, I. J. S-2-Amino-5-(2-nitroimidazol-1-yl)pentanoic acid:
a model for potential bioreductively activated prodrugs for inhibi-
tors of nitric oxide synthase (NOS) activity. Anti-Cancer Drug Des.
1997, 12, 61–65.
(32) Cama, E.; Shin, H.; Christianson, D. W. Design of amino acid
sulfonamides as transition-state analogue inhibitors of arginase.
J. Am. Chem. Soc. 2003, 125, 13052–13057.
(45) Custot, J.; Boucher, J.-L.; Vadon, S.; Guedes, C.; Dijols, S.;
Delaforge, M.; Mansuy, D. Nω-Hydroxyamino-R-amino acids as
a new class of very strong inhibitors of arginases. J. Biol. Inorg.
Chem. 1996, 1, 73–82.
(46) Di Costanzo, L.; Moulin, M.; Haertlein, M.; Meilleur, F.; Chris-
tianson, D. W. Expression, purification, assay, and crystal struc-
ture of perdeuterated human arginase I. Arch. Biochem. Biophys.
2007, 465, 82–89.
(33) Ciapetti, P.; Falorni, M.; Mann, A.; Taddei, M. 2-Amino-4-
bromobutanoic acid. A versatile reagent for the synthesis of
nonnatural amino acids. Molecules Online 1998, 2, 86–93.
(47) Schuck, P. Use of surface plasmon resonance to probe the equili-
brium and dynamic aspects of interactions between biological
macromolecules. Annu. Rev. Biophys. Biomol. Struct. 1997, 26,
541–566.
€
(34) Ruegg, U. T.; Russell, A. S. A rapid and sensitive assay for
arginase. Anal. Biochem. 1980, 102, 206–212.
(48) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. Methods Enzymol. 1997, 276, 307–
326.
(49) Di Costanzo, L.; Pique, M. E.; Christianson, D. W. Crystal
structure of human arginase I complexed with thiosemicarbazide
reveals an unusual thiocarbonyl μ-sulfide ligand in the binuclear
manganese cluster. J. Am. Chem. Soc. 2007, 129, 6388–6389.
(50) Yeates, T. O. Detecting and overcoming crystal twinning. Methods
Enzymol. 1997, 276, 344–358.
(35) Pearson, J. T.; Hill, J. J.; Swank, J.; Isoherranen, N.; Kunze, K. L.;
Atkins, W. M. Surface plasmon resonance analysis of antifungal
azoles binding to CYP3A4 with kinetic resolution of multiple
binding orientations. Biochemistry 2006, 45, 6341–6353.
(36) Lancini, G. C.; Lazzari, E. A new synthesis of alkyl and aryl
2-aminoimidazoles. J. Heterocycl. Chem. 1966, 3, 152–154.
(37) (a) Alberti, G.; Bertini, I.; Luchinat, C.; Scozzafava, A. A new class
of inhibitors capable of binding both the acidic and alkaline forms
of carbonic anhydrase. Biochim. Biophys. Acta 1981, 668, 16–26.
(b) Bertini, I.; Luchinat, C. Cobalt(II) as a probe of the structure and
function of carbonic anhydrase. Acc. Chem. Res. 1983, 16, 272–279.
(38) Kannan, K. K.; Chakravarty, S.; Satyamurthy, P.; Ramanadham,
M.; Kumar, V.; Yadav, V. S. Structure and function of carbonic
anhydrase: Synchrotron X-ray diffraction studies of human car-
bonic anhydrase I and inhibitor complexes. Synchrotron Radiat.
Biosci. 1994, 86–95.
(51) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn,
M. D.; Storoni, L. C.; Read, R. Phaser crystallographic software.
J. Appl. Crystallogr. 2007, 40, 658–674.
€
(52) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.;
Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Crystallography & NMR system: a new software suite for macro-
molecular structure determination. Acta Crystallogr. D 1998, 54,
905–921.
(39) Mangani, S.; Liljas, A. Crystal structure of the complex between
human carbonic anhydrase II and the aromatic inhibitor 1,2,4-
triazole. J. Mol. Biol. 1993, 232, 9–14.
(53) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A
2008, 64, 112–122.
(40) Marino, J. P., Jr.; Fisher, P. W.; Hofmann, G. A.; Kirkpatrick, R.
B.; Janson, C. A.; Johnson, R. K.; Ma, C.; Mattern, M.; Meek, T.
D.; Ryan, M. D.; Schulz, C.; Smith, W. W.; Tew, D. G.; Tomazek,
(54) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton,
J. M. PROCHECK: a program to check the stereochemical quality
of protein structures. J. Appl. Crystallogr. 1993, 26, 283–291.