Synthesis and evaluation of trans 3,4-cyclopropyl L-arginine analogues as isoform selective inhibitors of nitric oxide synthase

Article abstract of DOI:10.1016/S0968-0896(02)00554-0

Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized. These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity.

Full text of DOI:10.1016/S0968-0896(02)00554-0

Bioorganic & Medicinal Chemistry 11 (2003) 869–873
Synthesis and Evaluation of trans 3,4-Cyclopropyl
L-Arginine Analogues as Isoform Selective Inhibitors
of Nitric Oxide Synthase
Dan Fishlock,a Basil Perdicakis,b Heather J. Montgomery,a J. Guy Guillemette,a
Eric Jervisb and Gilles A. Lajoiea,*
aDepartment of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
bDepartment of Chemical Engineering, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
Received 29 August 2002; revised 15 October 2002; accepted 29 October 2002
Abstract—Four optically pure conformationally restricted l-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl l-arginine,
and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) l-ornithine 4 were synthesized. These compounds were tested as poten-
tial inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of
NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and
anti-trans-3,4-cyclopropyl N-(1-iminoethyl) l-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were
time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3
was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4,
and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl
ring in the l-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
cible NOS (iNOS) is transcriptionally regulated. Once
expressed, iNOS is active, essentially unregulated, and
produces NO at potentially cytotoxic levels, resulting in
local tissue damage. The selective inhibition of iNOS
has thus been an area of active research.
Nitric oxide (NO) is involved in the regulation of
diverse physiological processes such as smooth muscle
contractility, platelet reactivity, central and peripheral
neurotransmission.1,2 At high concentrations NO func-
tions as a defensive cytotoxin against tumor cells and
pathogens.3 While crucial for many physiological func-
tions, inappropriate excess of this mediator has been
associated with a number of pathologies including dia-
betes, rheumatoid arthritis, carcinogenesis, septic shock,
multiple sclerosis, transplant rejection and stroke.4
A number of conformationally restricted a-amino acid
NOS inhibitors have been reported in the literature,5ꢀ8
though none has shown dramatically improved potency
or selectivity. These efforts have involved the introduc-
tion of unsaturation or aromaticity into the amino acid
backbone, or a saturated ring within the amino acid or
guanidinium functionality. The introduction of a cyclo-
propyl ring into the amino acid backbone has not been
examined.
NO is produced in different mammalian tissues by three
distinct nitric oxide synthase isoforms (NOS; EC
1.14.13.39). These enzymes are homodimeric proteins
that catalyze a five-electron oxidation of l-arginine to
yield NO and l-citrulline. Two of the three NOS iso-
forms, endothelial (eNOS) and neuronal (nNOS), are
constitutively expressed enzymes, while the third, indu-
Some cyclopropyl amino acids have been synthesized or
isolated from natural sources, but not 3,4-cyclopropyl
arginine derivatives.9ꢀ11 A series of 3,4-cyclopropyl
analogues of arginine were designed in order to evaluate
what effect the subtle difference in cyclopropane con-
formation might have on inhibitory activity and selec-
tivity. The cyclopropyl methylene may introduce some
steric bulk that could interact favorably and differentially
*Corresponding author. Tel.: +519-661-3054x5860; fax: +519-661-
3175; e-mail: glajoie@uwo.ca
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00554-0

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D. Fishlock et al. / Bioorg. Med. Chem. 11 (2003) 869–873
with the binding pockets of the NOS isoforms. Such
selectivity has been observed with cyclopropyl gluta-
mate isomers with NMDA and neuronal glutamate
receptors.12,13
zoic acid (Scheme 2). Isolated yields following flash
chromatography were excellent (85–93%). Staudinger
reduction of each azide to the corresponding amine 12/
13 also proceeded in excellent yields, providing the pro-
tected diamino acid substrates.
In order to evaluate their potency and selectivity for
human NOS isoform inhibition, the optically pure syn 1
and anti 2 isomers of trans-3,4-cyclopropyl l-arginine
and syn 3 and anti-trans-3,4-cyclopropyl N-(1-imino-
ethyl) l-ornithine 4 were synthesized.
Shearer et al.22 has reported the highly reactive S-2-
naphthylmethyl thioacetamidate hydrobromide 14 as an
acetamidation reagent. Simply stirring each of the pro-
tected cyclopropyl amines 12/13 with 14 produces the
corresponding acetamidine HBr salt that was isolated
by extraction. The aqueous phase was lyophilized and
the product immediately deprotected under standard
conditions. Acid hydrolysis of the OBO ester was per-
formed using very mild and brief conditions to minimize
decomposition to an acetamide by-product. Cation
exchange chromatography provided the free syn 3 and
anti 3,4-cyclopropyl-N-iminoethyl-l-ornithine 4 that
were assayed for activity against the NOS isoforms,
along with the arginine analogues 1 and 2.
Results and Discussion
Both guanidine and acetamidine side chains were
desired for direct comparison, in order assess the effect
on potency and selectivity of the amino acid backbone
structure versus the side-chain reactive functionality.
Our group has recently reported the stereoselective
synthesis of l-cyclopropyl glutamates 6/714 as well as
the syn and anti isomers of trans-cyclopropyl arginine 1/
2.15 These were accessed via the 1,3-dipolar addition of
diazomethane to a chiral dehydroglutamate derivative
5, followed by photolysis of the resultant pyrazoline
(Scheme 1). This approach exploits the 4-methyl-2,6,7-
trioxabicyclo-[2.2.2] ortho (OBO) ester function, used to
protect the carboxylic acid of amino acids. The OBO
protection scheme prevents epimerization at the a-car-
bon of serine allowing the direct oxidation of the
hydroxyl side chain to the corresponding aldehyde
without loss of chirality. This serine aldehyde equivalent
has been used to obtain a variety of non-natural
a-amino acids.16ꢀ21
Arginine analogue 1 did not inhibit any of the NOS
isozymes, but was found to be a substrate, with Km
values greater than those found for l-arginine (8.5ꢁ1
mM vs 2.8ꢁ1.6 mM for nNOS, respectively). Compound
2 was found to be a mixed inhibitor of the NOS iso-
forms and not a substrate. It was not isoform selective,
nor did it exhibit any time dependent inhibition char-
acteristics. Further investigations were not performed
on compound 2 due to its poor inhibitory properties
and its lack of isozyme selectivity.
Compound 3 was found to be a competitive inhibitor of
all NOS isozymes. It strongly inhibited iNOS in a time
dependent manner, but its pre-incubation with nNOS
and eNOS did not result in increase inhibition as evi-
denced by the IC50-PI/[E] (pre-incubated IC50) values
given in Table 1. Compound 3 exhibited nNOS/iNOS,
eNOS/iNOS, and eNOS/nNOS selectivities of 109, 59,
and 1, respectively (Table 1).
This methodology was applied to the synthesis of syn-
and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) l-orni-
thine 3 and 4, via the Cbz protected E-3,4-l-di-dehy-
drogluatamate OBO derivative 5. Cyclopropanation
and purification of each diastereomer 6 and 7 by crys-
tallization, followed by DIBALreduction of each to 8
and 9 was performed as previously described.14,15
Compound 4 was found to be a weak competitive inhi-
bitor of the cNOS isoforms and a slow binding inhibitor
of iNOS with an IC50-PI/[E] value of 383ꢁ35 compared
to over 4500 for the cNOS isoforms (Table 1). Com-
Conversion to the azides 10, 11 was achieved by Mitsu-
nobu reaction of the corresponding alcohol with hydra-
Scheme 1. Access to syn- and anti-trans-(3,4)-cyclopropyl l-arginine.

D. Fishlock et al. / Bioorg. Med. Chem. 11 (2003) 869–873
871
Scheme 2. a) DIBAL-H, CH2Cl2, ꢀ78ꢂC; b) DEAD, Ph3P, HN3, CH2Cl2; c) Ph3P, THF, then H2O, reflux; d) S-2-naphthylmethyl thioacetamidate
hydrobromide, EtOH; e) i) H2, Pd/C; ii) 0.1% TFA/H2O; iii) Cs2CO3/H2O; iv) Dowex 50W X4-100.
Table 1. Inhibition kinetics of iNOS, nNOS and eNOS by compounds 3, 4 and 1400 W
Inhibitors
iNOS
nNOS
eNOS
IC50-PIb/[E]
Selectivitya
IC50/[E]
IC50-PIb/[E]
IC50/[E]
IC50-PIb/[E]
IC50/[E]
nNOS/iNOS
eNOS/iNOS
3
4
825ꢁ115
22405ꢁ609
N.D.
3.0ꢁ0.3
383ꢁ26
2.4ꢁ0.4
584ꢁ261
331ꢁ13
7259ꢁ1368
53ꢁ9
305ꢁ57
6071ꢁ834
N.D.
178ꢁ75
4905ꢁ620
916ꢁ170
109
19
22
59
13
382
15065ꢁ3396
1400 Wc
N.D.
N.D., not determined. 95% Confidence intervals are shown.
aSelectivity is defined as the ratio of the IC50-PI/[E] (eNOS) or IC50-PI/[E] (eNOS) to IC50-PI/[E] (iNOS).
bAssayed after 15 min of preincubation (PI) with enzyme, as described in the Experimental. Enzyme concentrations were 28.7 nM for iNOS and
nNOS, and 100 nM for eNOS.
cData obtained from Montgomery et al.23
pound 4 exhibited nNOS/iNOS, eNOS/iNOS, and
eNOS/nNOS selectivities of 19, 13, and 1, respectively
(Table 1).
The incorporation of constraining elements into argi-
nine analogues,5,7,26 l-N-iminoethylornithine analo-
gues6 or homocysteines8 have presumably prevented
these inhibitors from adopting the correct binding
orientation to fit within the enzyme active site. It is
noteworthy that these studies were performed on race-
mic mixtures of enantiomers. In contrast, syn-trans-3,4-
cyclopropyl l-arginine 3, was found to be a classical
competitive inhibitor of cNOS enzymes and a competi-
tive slow tight binding inhibitor of iNOS. The presence
of a cyclopropyl group places the binding groups in
different orientations and these geometries provide
some selectivity between the isoforms. Whether the
cyclopropyl methylene group interacts with hydro-
phobic residues in the active site of each or any of the
isoforms awaits structural studies of the inhibitor bound
enzymes. Our results provide the basis for a novel class
of NOS inhibitors composed of conformationally
restricted compounds.
The alteration of inhibitory potency and isoform selec-
tivity of the diastereomers 3 and 4 is of particular inter-
est. Compound 3 was found to be a 10- to 100-fold
more potent inhibitor of the NOS isozymes than com-
pound 4 (Table 1). The iNOS isoform selectivities of
compound 3 improved by a factor of 5 over the corre-
sponding selectivities for compound 4. No significant
change in eNOS/nNOS selectivity was observed
between compounds 3 and 4. Compound 3 exhibited a
tight binding nature with iNOS as evidenced by the
IC50-PI/[E] ratio that is less than 10, whereas compound
4 did not.24
A comparison of the inhibitory effects of compound 3
and the potent iNOS selective slow tight binding inhi-
bitor N-(3-(aminomethyl)benzyl)acetamidine (1400 W)25
reveals several interesting similarities.23,25 1400 W and
compound 3 exhibited IC50-PI values of similar order of
magnitude for all NOS isoforms. Both inhibitors are
slow binding inhibitors of iNOS, but not of the cNOS
enzymes.25 The nNOS/iNOS selectivity of compound 3
was 5-fold better than that for 1400 W, but the eNOS/
iNOS and eNOS/nNOS selectivities of 1400W are 6 and
32 times greater than that for compound 3. In fact 1400
W selectively inhibits nNOS over eNOS whereas com-
pound 3 inhibits eNOS and nNOS to the same extent.
Experimental
Materials
All reagents were purchased from Aldrich Canada, Ltd.
and used directly unless otherwise stated. CH2Cl2 was
distilled from CaH2 and THF from Na/benzophenone.
H2O was deionized and distilled. All reactions were
performed under argon atmosphere in oven-dried glass-

872
D. Fishlock et al. / Bioorg. Med. Chem. 11 (2003) 869–873
ware. TLC was performed using Merk aluminum
backed silica gel 60 254, and visualized using 5%
(NH4)6MoO24/0.2% Ce(SO4)2/5% H2SO4. Chromato-
graphy was performed using silica gel (230–400 mesh).
NMR spectra were recorded in D2O or CDCl3 pre-fil-
tered through basic alumina to remove traces of acid.
Optical rotations were measured on a Perkin–Elmer 241
digital polarimeter. Elemental analyses were performed
by MHW Laboratories, Phoenix, AZ. HR-FAB was
performed by Tim Jones at Brock University, Ontario.
HR-QTOF was performed by Amanda Doherty-Kirby
at University of Western Ontario.
(silica, 3:1 CHCl3/MeOH with 0.2% Et3N) provided the
amine as a clear oil in yields ranging 85–93%.
Cbz-L-ꢀ,ꢁ-(syn)-cyclopropyl-ornithine-OBO ester, 12.
25
D
½aŠ
ꢀ10.3 (c=1.0, EtOAc); 1H NMR (CDCl3,
300 MHz) d 7.32 (m, 5H), 5.11 (d, J=12.0 Hz, 1H), 5.04
(d, J=12.0 Hz, 1H), 5.0 (d, J=9.1 Hz, 1H), 3.88 (s, 6H),
3.54 (m, 1H), 2.65 (dd, J=6.1, 12.2 Hz, 1H), 2.28 (dd,
J=8.0, 12.7 Hz, 1H), 0.89 (m, 1H), 0.83 (m, 1H), 0.78 (s,
3H), 0.52 (m, 1H), 0.38 (m, 1H); 13C NMR (CDCl3,
75 MHz) d 156.5, 136.7, 128.5, 128.1, 108.8, 72.7, 66.7,
56.9, 46.1, 30.7, 18.1, 17.9, 14.4, 9.3. HR-MS (QTOF)
expected for C19H27N2O5: 363.1920. Found: 363.1922.
Procedure A: conversion of alcohols to N3: Cbz-L-trans
ꢀ,ꢁ-(anti)-cyclopropyl-pentahomoserine(N3)-OBO ester,
10. Cbz-l-b,g-(syn)-cyclopropyl-pentahomoserine-OBO
ester 8 (400 mg, 1.1 mmol) was dissolved in dry CH2Cl2
(15 mL) under argon and Ph3P (511 mg, 1.94 mmol) was
added. This solution was cooled to 0 ꢂC, and DEAD
(0.31 mL, 1.94 mmol) was added dropwise over 20 min
and stirred an additional 5 min. Fresh HN327,28 (0.77 M
solution in benzene, 5.9 mL, 4.52 mmol) was then added
dropwise over 1 h while maintaining the reaction mixture
at 0 ꢂC. The reaction mixture was allowed to warm to
room temperature and stirred for 16 h. The solvent was
removed in vacuo, trapping the excess HN3 with a
NaOH trap. Purification by column chromatography
(silica, 1:1 EtOAc:Hex with 0.2% triethylamine) pro-
vided 363 mg of azide 10 (85% yield) as a clear oil.
Cbz-L-ꢀ,ꢁ-(anti)-cyclopropyl-ornithine-OBO ester, 13.
25
D
½aŠ
ꢀ18.3 (c=1.03, EtOAc); 1H NMR (CDCl3,
300 MHz) d 7.32 (m, 5H), 5.05 (m, 2H+1H), 3.86 (s,
6H), 3.35 (app t, J=9.3 Hz, 1H), 2.69 (dd, J=5.7, 13.0,
1H), 2.27 (dd, J=8.1, 13.0, 1H), 1.71 (br s), 0.94 (m,
1H), 0.77 (m, 1H), 0.77 (s, 3H), 0.54 (m, 1H), 0.23 (m,
1H); 13C NMR (CDCl3, 75 MHz) d 136.6, 128.5, 128.1,
108.8, 72.8, 66.9, 58.1, 46.3, 30.7, 21.9, 18.5, 14.4, 7.5.
Anal. calcd for C19H26O5N2: C, 62.97; H, 7.26; N, 7.73.
Found: C, 62.74; H, 7.22; N, 7.61.
Procedure C: trans ꢀ,ꢁ-(syn)-cyclopropyl-iminoethyl L-
ornithine, 3. Cbz-l-b,g-(syn)-cyclopropyl-ornithine-
OBO ester 12 (635 mg, 0.17 mmol) was dissolved in
EtOH (2 mL) at 0 ꢂC, to which S-2-naphthylmethyl
thioacetamidate hydrobromide 14 (51 mg, 0.17 mmol)
was added portionwise with stirring. This mixture was
stirred a total of 6 h, and then the solvent evaporated to
provide a pale yellow oil. The residue was suspended in
distilled H2O (5 mL ) and E2tO (5 mL), and extracted
with additional H2O (2Â5 mL). The aqueous fractions
were combined and lyophilized to provide 79 mg of white
powder. This fully protected amino acid was dissolved in
1:1 EtOH/EtOAc with an equal mass of 10% (w/w) Pd/
C. The suspension was stirred vigorously under a pure
H2 atmosphere for 16 h. The mixture was filtered and the
solvent evaporated. The clear oil was dissolved in 0.1%
TFA in CH2Cl2 and the solvent immediately evaporated
in vacuo. The clear oil was suspended in 10% Cs2CO3
and stirred for 16 h, then lyophilized. The residue was
dissolved in minimal purified H2O, acidified with double
distilled 6 N HCl to pH 4 then loaded onto Dowex 50W
X-4-100 cation exchange resin. After washing with pure
H2O, the product was eluted with 0.5 N NH4OH. Frac-
tions were combined and lyophilized to provide 26 mg of
the desired amino acid (82%).
25
½aŠ ꢀ7.8 (c=0.9, EtOAc); TLC (1:1 EtOAc/Hex)
D
1
Rf=0.59; H NMR (CDCl3, 300 MHz) d 7.27 (m, 5H),
5.06 (m, 1H+2H), 3.88 (s, 6H), 3.51 (app t, J=9.0 Hz,
1H), 3.10 (dd, J=6.4, 12.8 Hz, 1H), 2.87 (dd, J=7.7, 12.8
Hz, 1H), 1.13 (m, 1H, 0.99 (m, 1H)), 0.78 (s, 3H), 0.64 (m,
1H), 0.47 (m, 1H); 13C NMR (CDCl3, 75 MHz) d 156.5,
136.7, 128.5, 128.1, 108.5, 72.8, 66.9, 57.0, 54.9, 30.7, 18.1,
14.4, 13.9, 9.4. Anal. calcd for C19H24O5N4: C, 58.75; H,
6.23; N, 14.42. Found: C, 58.65; H, 6.22; N, 14.46.
Cbz - L - trans ꢀ,ꢁ - (anti) - cyclopropyl - pentahomoseri-
ne(N3)-OBO ester, 11. As described in procedure A,
Cbz-l -b,g-(anti)-cyclopropyl-pentahomoserine-OBO
ester 9 (146 mg, 0.402 mmol) provided 145 mg of azide
product 11 (93% yield) as a clear oil.
25
½aŠ ꢀ22.1 (c=0.95, EtOAc); TLC (1:1 EtOAc/Hex)
D
1
Rf=0.59); H NMR (CDCl3, 300 MHz) d 7.33 (m, 5H),
5.08 (m, 1H), 4.89 (m, 1H), 3.89 (s, 6H), 3.40 (app br t,
J=9.3 Hz, 1H), 3.13–3.02 (m, 2H), 1.19 (m, 1H), 0.97
(m, 1H), 0.78 (s, 3H), 0.70 (m, 1H), 0.37 (m, 1H); 13C
NMR (CDCl3, 75 MHz) d 136.6, 128.6, 128.1, 108.8,
72.8, 67.0, 57.7, 55.0, 30.7, 18.2, 17.0, 14.5, 7.5. Anal.
calcd for C19H24O5N4: C, 58.75; H, 6.23; N, 14.42.
Found: C, 58.68; H, 6.23; N, 14.21.
25
Mp 187–194 ꢂC dec.; ½aŠ +48.9 (c=1.15, H2O); 1H
NMR (D2O, 300 MHz) d D3.01 (dd, J=7.2, 13.9 Hz, 1H),
2.95 (dd, J=7.4, 14.1 Hz, 1H), 2.66 (d, J=8.6 Hz, 1H),
2.03 (s, 3H), 0.93 (m, 1H), 0.81 (m, 1H), 0.58 (m, 1H),
0.42 (m, 1H); 13C NMR (D2O, 75 MHz) d 180.4, 164.4,
58.6, 45.7, 18.3, 21.4, 14.7, 8.4. HR-MS (Q-TOF)
expected for C8H16N3O2: 186.1243. Found: 186.1241.
Procedure B: Staudinger reduction of N3 to NH2. In a
typical procedure, azide was dissolved in dry CH2Cl2
and Ph3P (4 molar equiv) was added. The solution was
stirred at room temperature for 24 h under argon, and
then H2O (ꢃ15 molar equiv) was added. This mixture
was heated at reflux for 5 h, allowed to cool and the
solvent evaporated in vacuo. Column chromatography
trans ꢀ,ꢁ-(Anti)-cyclopropyl-iminoethyl L-ornithine, 4.
Procedure C was applied to Cbz-l-b,g-(anti)-cyclopro-
pyl-ornithine-OBO ester 13 (80 mg, 0.22 mmol) and
provided 28 mg of the desired amino acid (70%).

D. Fishlock et al. / Bioorg. Med. Chem. 11 (2003) 869–873
873
25
D
Mp 192–197 ꢂC dec.; ½aŠ +19.0 (c=0.5, H2O); 1H
References and Notes
NMR (D2O, 300 MHz) d 3.09 (dd, J=7.0, 13.9 Hz, 1H),
2.98 (dd, J=7.6, 14.0 Hz, 1H), 2.90 (d, J=9.8 Hz, 1H),
2.05 (s, 3H), 1.20 (m, 1H), 0.93 (m, 1H), 0.64 (m, 2H);
13C NMR (D2O, 75 MHz) d 175.8, 164.5, 58.5, 45.3,
19.3, 18.4, 15.6, 9.0; HR-MS (Q-TOF) expected for
C8H16N3O2: 186.1243. Found: 186.1249.
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24. Copeland, R. Enzymes: A Practical Introduction to Struc-
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New York, 2000.
25. Garvey, E. P.; Oplinger, J. A.; Furfine, E. S.; Kiff, R. J.;
Laszlo, F.; Whittle, B. J.; Knowles, R. G. J. Biol. Chem. 1997,
272, 4959.
Enzyme kinetics
The activity of NOS was determined using the hemo-
globin capture assay30,31 that yield results consistent
with the direct radioactive assay that monitors the for-
mation of l-[3H]-citrulline from l-[3H]-arginine.29
Quadruplicate reactions were initiated by the addition
of l-arginine and monitored on a 96-well plate reader
(Spectramax 190, Molecular Devices, Sunnyvale, CA) at
26 ꢂC. Nitric oxide-mediated oxidation of oxyhemoglo-
bin was monitored at 401 nm (e=0.1034 OD/nano-
mole).30 Experiments were conducted in 100 mL
volumes that contained 50 mM Tris–HCl, pH 7.5, 10–
100 nM of NOS isozyme, 2.5–50 uM l-arginine, 500
mM NADPH, 5.0 mM H4B, 1.0 mM CaM, 1.0 mM
CaCl2, 16 mM DTT, 1.0 mM FAD, 1.0 mM FMN, 100
units/mLsuperoxide dismutase (SOD), 50 units/mL
catalase, 10.0 mM bovine oxyhemoglobin, and 0.2 mg/
mLbovine serum albumin. Various concentrations of
the optically pure conformationally restricted synthe-
sized cyclopropane based l-arginine analogues were
added to the reaction mixtures to test for NOS inhibi-
tion. To determine if inhibitors displayed time depen-
dent inhibition IC50 values were calculated by pre-
incubating enzyme in the presence of varying amounts
of cyclopropane based l-arginine analogues, 500 mM
NADPH, 10.0 mM FAD, 10.0 mM FMN, 50.0 mM H4B,
157.0 mM DTT, 100 units/mLSOD and 50 units/mL
catalase for 15 min at 26 ꢂC.25 Blank wells that did not
contain enzyme demonstrated similar behaviour to wells
with a sufficiently high concentration of inhibitor to
fully inhibit NOS activity. Blank wells were run in par-
allel with reaction mixtures and kinetic data was cor-
rected for the loss of signal in the blank.
26. Shearer, B. G.; Lee, S.; Oplinger, J. A.; Frick, L. W.;
Garvey, E. P.; Furfine, E. S. J. Med. Chem. 1997, 40, 1901.
27. Adams, R. Organic Reactions, Vol. III; John Wiley &
Sons: London, 1946; 327.
28. Brauer, G. Handbook of Preparative Inorganic Chemistry,
Vol. I; Academic Press: New York, 1963; 472.
29. Montgomery, H. J.; Romanov, V.; Guillemette, J. G. J.
Biol. Chem. 2000, 275, 5052.
30. Gross, S. S. Methods Enzymol. 1996, 268, 159.
31. Hevel, J. M.; Marletta, M. A. Methods Enzymol. 1994,
233, 250.
Acknowledgements
This research was supported by a grant from the Nat-
ural Sciences and Engineering Research Council of
Canada and the Heart and Stroke Foundation.