Synthesis and evaluation of peptidomimetics as selective inhibitors and active site probes of nitric oxide synthases

Article abstract of DOI:10.1021/jm000127z

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L- citrilline and nitric oxide (NO). Selective inhibition of the isoforms of NOS could have great therapeutic potential in the treatment of certain disease states arising from pathologically elevated synthesis of NO. Recently, we reported dipeptide amides containing a basic amine side chain as potent and selective inhibitors of neuronal NOS (Huang, H.; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147). The most potent nNOS inhibitor among these compounds is L-Arg(NO₂)-L-Dbu-NH₂ (1) (K(i) = 130 nM), which also exhibits the highest selectivity over eNOS (>1500-fold) with excellent selectivity over iNOS (190-fold). Here we describe the design and synthesis of a series of peptidomimetic analogues of this dipeptide as potential selective inhibitors of nNOS. The biochemical evaluation of these compounds also revealed the binding requirements of the dipeptide inhibitors with NOS. Incorporation of protecting groups at the N- terminus of the dipeptide amide 1 (compounds 4 and 5) resulted in dramatic decreases in the inhibitory potency of nNOS. Masking the NH group of the peptide bond (peptoids 6-8 and N-methylated compounds 9-11) also gave much poorer nNOS inhibitors than 1. Both of the results demonstrate the importance of the α-amine of the dipeptide and the NH moiety of the peptide bond for binding at the active site. Modifications at the C-terminus of the peptide included converting the amide to the methyl ester (12), tert-butyl ester (13), and carboxylic acid (14) and also descarboxamide analogues (15-17), which revealed less restricted binding requirements for the C-terminus of the dipeptide. Further optimization should be possible when we learn more about the binding requirements at the active sites of NOSs.

Full text of DOI:10.1021/jm000127z

2938
J . Med. Chem. 2000, 43, 2938-2945
Syn th esis a n d Eva lu a tion of P ep tid om im etics a s Selective In h ibitor s a n d Active
Site P r obes of Nitr ic Oxid e Syn th a ses
Hui Huang,‡,§ Pavel Marta´sek,†,# Linda J . Roman,†,| and Richard B. Silverman*,‡
Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University,
Evanston, Illinois 60208-3113, and Department of Biochemistry, The University of Texas Health Science Center,
San Antonio, Texas 78284-7760
Received March 17, 2000
Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrilline and nitric
oxide (NO). Selective inhibition of the isoforms of NOS could have great therapeutic potential
in the treatment of certain disease states arising from pathologically elevated synthesis of
NO. Recently, we reported dipeptide amides containing a basic amine side chain as potent
and selective inhibitors of neuronal NOS (Huang, H.; Martasek, P.; Roman, L. J .; Masters, B.
S. S.; Silverman, R. B. J . Med. Chem. 1999, 42, 3147). The most potent nNOS inhibitor among
these compounds is L-ArgNO -L-Dbu-NH2 (1) (Ki ) 130 nM), which also exhibits the highest
2
selectivity over eNOS (>1500-fold) with excellent selectivity over iNOS (190-fold). Here we
describe the design and synthesis of a series of peptidomimetic analogues of this dipeptide as
potential selective inhibitors of nNOS. The biochemical evaluation of these compounds also
revealed the binding requirements of the dipeptide inhibitors with NOS. Incorporation of
protecting groups at the N-terminus of the dipeptide amide 1 (compounds 4 and 5) resulted in
dramatic decreases in the inhibitory potency of nNOS. Masking the NH group of the peptide
bond (peptoids 6-8 and N-methylated compounds 9-11) also gave much poorer nNOS inhibitors
than 1. Both of the results demonstrate the importance of the R-amine of the dipeptide and
the NH moiety of the peptide bond for binding at the active site. Modifications at the C-terminus
of the peptide included converting the amide to the methyl ester (12), tert-butyl ester (13), and
carboxylic acid (14) and also descarboxamide analogues (15-17), which revealed less restricted
binding requirements for the C-terminus of the dipeptide. Further optimization should be
possible when we learn more about the binding requirements at the active sites of NOSs.
In tr od u ction
NOS requires 2 equiv of molecular oxygen and 1.5 equiv
of reduced nicotinamide adenine dinucleotide phosphate
(NADPH) as cosubstrates.9 All isoforms of NOS contain
an N-terminal oxygenase domain with binding sites for
L-arginine, cofactors (6R)-5,6,7,8-tetrahydrobiopterin
(H4B) and heme, and a linked C-terminal reductase
domain with NADPH, FAD, FMN, and calmodulin
binding sites.9
Nitric oxide (NO) has been of great interest to
medicinal chemists since it was discovered as an im-
portant biological second messenger over a decade ago.1
A family of enzymes, the nitric oxide synthases (NOS,
E.C. 1.14.13.39), catalyzes the stepwise oxidation of
L-arginine to L-citrulline and nitric oxide.2 To date, three
structurally distinct NOS isoforms have been identified.3
One constitutively expressed isoform is located in neu-
ronal tissue4 (nNOS) and is involved in neurotransmis-
sion and long-term potentiation.5 The other constitutive
isoform is endothelial NOS (eNOS), which is involved
in the regulation of smooth muscle relaxation and
vascular tone.6 NO produced by the inducible isoform
(iNOS) in activated macrophage cells plays a key role
in normal immune responses by functioning as a cyto-
toxic agent.7 The isoforms of NOS share only ap-
proximately 50% primary sequence homology, which
suggests that they may differ from each other in
regulatory aspects;8 however, there is very high se-
quence identity across species. The catalytic reaction of
Overproduction of NO has been implicated in a wide
variety of disease states including septic shock, inflam-
matory, and neurodegenerative diseases; selective in-
hibitors of the isoforms of NOS could have great
therapeutic potential in the treatment of these dis-
eases.10,11 Prototypical NOS inhibitors were mostly
analogues of the natural substrate, L-arginine, including
Nω-methyl-L-arginine (L-NMA),12 Nω-nitro-L-arginine (L-
NNA),13 and Nδ-(iminoethyl)-L-ornithine (L-NIO).14 Most
of these amino acid-based inhibitors are irreversible
inactivators of NOS with minimal selectivity among the
isoforms. Modifications of the guanidine moiety of
L-arginine led to some potent NOS inhibitors, such as
S-alkyl-L-thiocitrulline,15 amidines,16 guanidines,17 and
isothioureas.18 Apart from the L-arginine binding site
of NOS, other binding sites have been targeted when
designing the inhibitors of NOS: various indazoles or
imidazoles inhibit NOS acting as ligands to the heme
prosthetic group,19 and 4-aminopteridine derivatives
bind specifically at the H4B binding site of NOS.20
* Address correspondence to Prof. Richard B. Silverman at the
Department of Chemistry. Phone: (847) 491-5653. Fax: (847) 491-
7713. E-mail: Agman@chem.nwu.edu.
‡ Northwestern University.
† The University of Texas Health Science Center.
§ Carried out all of the experiments in this study.
#
Developed the eNOS overexpression system in E. coli, purified the
eNOS, and isolated the eNOS.
We have previously reported a library of dipeptide
amides containing nitroarginine as inhibitors of nNOS.21
| Developed the overexpression system for nNOS in E. coli and
purified the enzyme.
10.1021/jm000127z CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/30/2000

Active Site Probes of NOS
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2939
Sch em e 1
Ch a r t 1
Excellent inhibitory potency and selectivity for nNOS
over eNOS and iNOS are achieved with the dipeptide
amides that have an amine-containing side chain. The
most potent nNOS inhibitor among these compounds
is the dipeptide amide containing L-2,4-diaminobutyric
acid (Dbu), L-ArgNO -L-Dbu-NH2 (1) (Ki ) 130 nM),
2
which also exhibits the highest selectivity over eNOS
(>1500-fold) with a 192-fold selectivity over iNOS. The
length of the amine side chain seems to have only a
minor effect on the potency for all isoforms of NOS:
L-ArgNO -L-Orn-NH2 (2) and L-ArgNO -L-Lys-NH2 (3)
inhibit nNOS with Ki values of 330 and 450 nM,
respectively. Unlike nitroarginine, none of these dipep-
tides exhibited time-dependent inhibition of any of the
isoforms. To investigate the SAR studies and improve
the potential bioavailability of the dipeptide inhibitors,
a series of peptidomimetic analogues were synthesized
and evaluated in the present study. Protecting the
R-amino group of the dipeptide amide 1 with an acetyl
and Cbz (benzyloxycarbonyl) group gives compounds 4
and 5. Peptoid analogues (6-8) and N-methylated
compounds (9-11) mask the NH group of the peptide
bond. Compounds 12-14 are the methyl ester, tert-butyl
ester, and carboxylic acid of the dipeptide 1, respec-
tively. Removal of the C-terminal carboxamide moiety
results in descarboxamide analogues 15-17. (See Chart
1 for structures of compounds 1-17.) These modifica-
tions of the original potent dipeptide inhibitors also
reveal the binding requirements of the dipeptide with
NOS.
2
2
N-Methylated diepeptides (9-11) were synthesized on
a solid support as shown in Scheme 3. The R-amino
group of the amino acids (Dbu, Orn, and Lys) was
protected and activated by 2-nitrophenylsulfonyl chlo-
ride, followed by methylation using the Mitsunobu
reaction.23 The second amino acid (L-ArgNO ) was coupled
2
to the resin after removal of the 2-nitrophenylsulfonyl
group.
The dipeptide methyl ester, L-ArgNO -L-Dbu-OCH3
2
(12), was obtained by the coupling of Boc-L-ArgNO and
Ch em istr y
2
L-Dbu(Boc)-OCH3, which was prepared by the meth-
ylation of commercially available Fmoc-L-Dbu(Boc)-OH
followed by removal of the Fmoc group. The synthetic
The two R-amino-protected dipeptide amides (4, 5)
were synthesized on solid phase as shown in Scheme 1.
Initially, acetylation of L-ArgNO was carried out prior
2
route of L-ArgNO -L-Dbu tert-butyl ester (13) is shown
2
to the peptide coupling. However, it was realized later
that the acetyl-protected amino acid epimerized easily
during the peptide coupling reaction. Acetylation of the
dipeptide on the Rink resin worked well.
in Scheme 4; the key step is the amide degradation of
NR-Cbz-L-Gln tert-butyl ester to NR-Cbz-L-Dbu tert-butyl
ester. The NR-Cbz group was removed by carefully
monitoring the reaction, because the Nδ-Fmoc group also
can be cleaved during catalytic hydrogenation, although
in a slower reaction. The dipeptide carboxylic acid 14
was obtained from the dipeptide tert-butyl ester 13 by
acid deprotection (Scheme 4).
A peptoid differs from a peptide as a result of the
transfer of the side chain of the amino acid from the
R-carbon to the nitrogen of the peptide bond. Peptoids
represent a new class of imino acid residues that are
not found in nature and have shown good proteolytic
stability.22 The syntheses of the peptoid analogues 6-8
were carried out on solid phase, as shown in Scheme 2.
The only difficulty with this synthesis was the formation
of the monoprotected diamines, which were prepared
by carefully adjusting the pH of the reaction mixture.
The synthesis of compounds 15-17 was carried out
on Wang resin (Scheme 5). The resin was activated by
p-nitrophenyl chloroformate followed by coupling with
L-ArgNO methyl ester. Monoprotected diamines were
2
coupled to the resin after the hydrolysis of the methyl

2940 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Huang et al.
Sch em e 2
Sch em e 3
Sch em e 4
Sch em e 5
ester. Compounds 15-17 were also synthesized in a
solution-phase reaction, which gave pure compounds
after chromatography. All of the compounds synthesized
on solid phase were >80% pure by HPLC. They were
purified using prep-HPLC prior to the enzyme assay.
Resu lts a n d Discu ssion
The Ki data for these peptidomimetic compounds are
given in Table 1 along with the data for the three potent
and selective dipeptide amides inhibitors (1-3). None

Active Site Probes of NOS
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2941
Ta ble 1. NOS Inhibition by the Nω-Nitroarginine-Containing
nitrogen of the peptide bond. This makes the peptide
bond less susceptible to esterases, but it totally changes
the spatial geometry of the peptide. The peptoid ana-
logues of the dipeptide inhibitors (6-8) were found to
be poorer inhibitors of nNOS and iNOS than were the
corresponding dipeptides (1-3), while having little
difference toward eNOS inhibition. This results in the
dramatic decreases of isoform selectivity of nNOS over
eNOS (from 1538-fold to 3-4-fold). The length of the
amine side chain has only a small effect on the inhibi-
tory potency of the isoforms of NOS: shorter is slightly
better. The spatial change of the peptoids moves the
amino group of the side chain away from the possible
hydrogen bond or ionic donor of the enzyme (see Figure
1), which may explain the decreases in inhibitory
potency.
Dipeptides, Dipeptide Esters, and Peptidomimeticsa
selectivityc
Ki (µM)b
eNOS/ iNOS/
nNOS iNOS eNOS nNOS nNOS
compound
1, L-ArgNO -L-Dbu-NH2
0.13
0.33
0.45
30.4
52
19
30.6
47
25 200
97 245
1538
742
313
9
16
3
3.5
4
40
135
86
798
28
192
294
231
86
31
40
33
23
142
283
260
46
40
43
185
256
308
d
2
2, L-ArgNO -L-Orn-NH2
d
2
3, L-ArgNO -L-Lys-NH2
104 141
2600 263
1600 827
760 55.5
1000 108
1100 197
340 95
1300 623
1900 631
27 463
413 288
1210 98
100 199
118 213
108 70
d
2
4, Ac-L-ArgNO -L-Dbu-NH2
2
5, Cbz-L-ArgNO -L-Dbu-NH2
2
6, peptoid-Dbu
7, peptoid-Orn
8, peptoid-Lys
9, L-ArgNO -(N-CH3)-L-Dbu-NH2
2.4
2
10, L-ArgNO -(N-CH3)-L-Orn-NH2 4.6
2
11, L-ArgNO -(N-CH3)-L-Lys-NH2
7.3
0.58
2
12, L-ArgNO -L-Dbu-OCH3
2
13, L-ArgNO -L-Dbu-OC(CH3)3
10.2
28.4
0.54
0.46
0.35
2
14, L-ArgNO -L-Dbu-OH
3.5
2
15, L-ArgNO -NH-(CH2)2-NH2
368
463
200
2
It is also possible that the potency decreases of the
peptoid analogues result from the masking of the NH
group of the peptide bond, which may be a crucial
hydrogen bond donor. Simple N-methylation of the
peptide bond of the dipeptide inhibitors supports this
hypothesis. The inhibitory potency of the N-methylated
dipeptide amides (9-11) drops about 20-fold for both
nNOS and iNOS, compared with the corresponding
dipeptide amides (1-3), but the potency changes much
less toward eNOS. This suggests that the NH group of
the peptide bond may be involved in hydrogen bonding
to nNOS and iNOS, but not so much to eNOS, and that
this interaction is not as strong as the ionic interactions
of the two amino groups with the active site. A com-
parison between 9-11 and the peptoid analogues 6-8
shows an increase in potency of 9-11 toward nNOS,
comparable inhibitory activities toward iNOS, but de-
creased potency of 9-11 against eNOS. Again, there is
an increase in potency toward eNOS by peptide bond
N-alkylation. However, a geometric change caused by
N-alkylation cannot be ruled out as the reason for the
potency decreases with nNOS and iNOS. Although there
is not much difference in the inhibition of the NOS
isozymes by these N-alkylated dipeptide amides, the one
with the shortest amine side chain is a little more potent
than the other two.
16, L-ArgNO -NH-(CH2)3-NH2
2
17, L-ArgNO -NH-(CH2)4-NH2
2
a
The enzymes used for the Ki measurements are bovine brain
nNOS, recombinant murine iNOS, and recombinant bovine eNOS.
b
The Ki values represent duplicate measurements; standard
deviations of (8-12% were observed. c The ratio of Ki(eNOS or
iNOS) to Ki(nNOS); all are nNOS-selective. Data taken from
d
ref 21.
F igu r e 1. Hypothetical model of the dipeptide amide inhibitor
binding at the active site of nitric oxide synthase.
of these dipeptide analogues shows slow, tight binding
inhibitory pattern like L-nitroarginine. Incorporation of
the acetyl and Cbz protecting groups into the N-
terminus of the dipeptide amide L-ArgNO -L-Dbu-NH2 (1)
2
results in dramatic decreases in potency of nNOS and
iNOS inhibition while having minimal influence on
eNOS inhibition. Even though these two protecting
groups differ greatly in size, there is little difference in
the inhibition of the isoforms of NOS by compounds 4
and 5. This result suggests that a charged N-terminus
may be significant for the interaction of the dipeptide
inhibitors with nNOS and iNOS, but not with eNOS,
and that there is not much steric crowding at the
N-terminus. Our study on the D-forms of the dipeptide
amide inhibitors21,24 indicates that there are two im-
portant ionic interactions between the two amino resi-
dues of the dipeptide (the R-amino group of the N-ter-
minal amino acid and the ω-amino group of Dbu) and
the NOS active site (Figure 1), which is consistent with
the findings here. The requirement for this charged
N-terminus for binding at the active sites of nNOS and
iNOS appears to be more demanding than that of eNOS,
which is beneficial for the future design of isoform
selective NOS inhibitors.
To investigate the importance of the C-terminal
carboxamide moiety of the dipeptide inhibitors for
binding, several modifications were made. A methyl
ester has a size and charge similar to those of the
carboxamide, and the dipeptide methyl ester 12 is also
a potent inhibitor of nNOS with only a 4-fold decrease
in potency as compared to the corresponding dipeptide
amide 1. The inhibitory potency remains the same for
iNOS and decreases 2-fold for eNOS, which results in
an nNOS/eNOS selectivity (800-fold) only one-half of
that with the carboxamide. To increase the stability of
the ester toward possible esterase hydrolysis, the tert-
butyl ester 13 was synthesized. This compound is 78
times less potent an inhibitor of nNOS and 17-fold less
potent toward iNOS but comparable to the correspond-
ing carboxamide in inhibitory properties toward eNOS.
Steric hindrance of the tert-butyl group is most likely
the reason for such dramatic potency decreases. To
investigate the importance of an electrostatic effect at
the C-terminus, the corresponding dipeptide (carboxylic
acid, 14) was synthesized. This compound is a much
poorer inhibitor of both nNOS (218-fold) and iNOS (48-
A peptide is changed to a peptoid by moving the side
chain of the amino acid from the R-carbon to the

2942 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Huang et al.
Gen er a l P r oced u r e for Solid -P h a se P ep tid e a n d P ep -
tid om im etic Syn th esis. Rink resin (300 mg, 0.8 mmol/g) was
swelled in 2 mL of DMF. The Fmoc group was removed by
treatment of the resin with 20% piperidine in DMF (4 mL) for
30 min, followed by successive washings with DMF (3 times),
methanol (2 times), and vacuum-drying. The Fmoc protected
C-terminal amino acid (3 equiv) was coupled to the resin using
diisopropylcarbodiimide (DIC; 3 equiv) and HOBt (3 equiv) as
the coupling reagents. The mixture was agitated for 5 h at
room temperature. The resin was washed successively with
DMF, methanol, and DMF, followed by Fmoc deprotection and
washing. The second amino acid was coupled to the resin in
the same way. After Fmoc deprotection, washing, and drying,
the dipeptide was cleaved from the resin using TFA/CH2Cl2
(1:1 v/v) for 1 h. The resin was removed by filtration, and the
filtrate was concentrated to dryness. The oily residue was
dissolved in a small amount of water, which was washed with
ether, and lyophilized.
fold) but is a slightly better inhibitor of eNOS than 1.
One explanation could be a repulsive ionic interaction
between the carboxylic acid moiety of the dipeptide and
the active site. The cause is probably not an intramo-
lecular hydrogen bond between the carboxylic acid and
the amino side chain of the dipeptide, because that very
different geometry should have had an effect on eNOS
as well.
Finally, we wondered if the carboxamide group was
necessary at all. The descarboxamide analogues, L-
ArgNO -NH(CH2)nNH2 (n ) 2-4, 15-17), were designed
2
and synthesized. These compounds retain the amino
side chain of the potent dipeptides but have more
flexibility. The Ki data of these compounds in Table 1
show that they also are potent and selective inhibitors
of nNOS with potencies and selectivities similar to those
of dipeptide inhibitors 1-3. Interestingly, nNOS potency
increases slightly with the length of the amine chain,
which is opposite that observed in 1-3. It is apparent
that the C-terminal carboxamide moiety of the dipeptide
inhibitors is not essential for the active site binding.
N-(O-Acetyl)h yd r oxysu ccin im id e (AcOSu ). To a stirred
CH2Cl2 solution containing acetic acid (2 g, 33.3 mmol) were
added N-hydroxysuccinimide (3.8 g, 33.3 mmol) and DCC (7.5
g, 36.6 mmol). The milky solution was stirred overnight. The
urea was filtered off and washed with CH2Cl2. The combined
filterates were concentrated under vacuum. The product was
crystallized from ethanol as a white powder (4 g, 76% yield):
1H NMR (CDCl3) δ 2.86 (brs, 4H), 2.37 (s, 3H).
In conclusion, peptidomimetic approaches have been
applied to the potent and selective dipeptide amide
inhibitors of nNOS 1-3. Incorporation of protecting
groups at the N-terminus of the dipeptide amide 1
(compounds 4 and 5) and masking of the NH group of
the peptide bond (peptoids 6-8 and N-methylated
compounds 9-11) result in dramatic decreases in the
inhibitory potency of nNOS as compared to 1, which
demonstrates the importance of the R-amino group of
the dipeptide and the NH moiety of the peptide bond
for binding at the active site. Conversion of the car-
boxamide to the methyl ester (12) resulted in a small
decrease in potency and selectivity, but conversion to
either the tert-butyl ester (13) or carboxylic acid (14)
resulted in a dramatic loss of both potency and selectiv-
ity. The descarboxamide analogues (15-17) had only a
small effect on both potency and selectivity. The ability
to delete the carboxamide group opens up a variety of
important peptidomimetic approaches that can be taken.
Nr-Acetyl-L-Ar gNO -L-Dbu -NH2 (4). This dipeptide amide
2
was synthesized as described in the general procedure. Fol-
lowing the peptide coupling N-acetylation was conducted for
2 days according to the procedure of Pacofsky et al:25 1H NMR
(D2O) δ 4.48 (dd, 1H), 4.29 (dd, 1H), 3.32 (t, 2H), 3.09 (m, 2H),
2.17-2.28 (m, 1H), 2.03-2.17 (m, 1H), 2.05 (s, 3H), 1.65-1.91
(m, 4H); HRMS (M + 1) calcd for C12H24N8O5 361.1943, found
316.1926. Anal. (C12H24N8O5‚1.5TFA) C, H, N.
Nr-Cbz-L-Ar gNO -L-Dbu -NH2 (5). This dipeptide amide was
2
synthesized as described in the general procedure using
commercially available NR-Cbz-L-ArgNO
:
1H NMR (D2O) δ 7.35
2
(m, 5H), 5.16 (s, 2H), 4.50 (dd, 1H), 4.35 (dd, 1H), 3.35 (t, 2H),
3.19 (m, 2H), 2.20-2.31 (m, 1H), 2.07-2.20 (m, 1H), 1.55-
1.71 (m, 2H), 1.71-1.91 (m, 2H); HRMS (M + 1) calcd for
C
18H28N8O6 453.2205, found 453.2212. Anal. (C18H28N8O6‚
1TFA‚0.5H2O) C, H, N.
Nr-(ter t-Bu toxyca r bon yl)a lk a n ed ia m in es. The mono-
protected alkanediamines were prepared according to the
procedure of Videnov et al.26 with some modifications. The
diamines (80 mmol) were dissolved in water (100 mL) in the
presence of phenolphthalein. Concentrated HCl was added
until the red color disappeared. The colorless solution was
titrated with 1 N NaOH until the red color stayed. In the case
of 1,2-ethanediamine, the color of the solution was pale pink.
To each mixture was added a solution of (Boc)2O (4.4 g, 20
mmol) in 2-propanol (100 mL). The reactions with 1,2-
ethanediamine and 1,3-propanediamine were stirred at room
temperature for 2 days, while the reaction with 1,4-butane-
diamine was stirred for 5 days. The reactions were monitored
by TLC (n-butanol:acetic acid:water 4:1:1). Water and 2-pro-
panol were removed by evaporation. The NaCl was precipi-
tated by ethanol, filtered, and washed. The filtrate was
concentrated in a vacuum, and the residue was dissolved in 1
N NaOH solution. The aqueous solution was extracted with
ethyl acetate (EtOAc) twice. The organic layer was extracted
twice with 10% citric acid solution. The combined aqueous
solution was basified with 1 N NaOH again and extracted with
EtOAc several times. Yellowish oily products were obtained
after evaporation of the solvent and used in solid-phase
synthesis without further purification. NR-Boc-1,2-ethanedi-
amine (1.8 g, 56% yield): 1H NMR (CDCl3) δ 4.95 (brs, 1H),
3.19 (dt, 2H), 2.81 (t, 2H), 1.48 (s, 9H), 1.34 (s, 2H). NR-Boc-
1,3-propanediamine (1.5 g, 43% yield): 1H NMR (CDCl3) δ 4.94
(brs, 1H), 3.22 (dt, 2H), 2.79 (t, 2H), 1.63 (tt, 2H), 1.56 (brs,
2H), 1.48 (s, 9H). NR-Boc-1,4-butanediamine (0.3 g, 8% yield):
1H NMR (CDCl3) δ 5.12 (brs, 1H), 2.95 (dt, 2H), 2.54 (t, 2H),
1.17-1.45 (m, 13H).
Exp er im en ta l Section
Ma ter ia ls. All amino acids and coupling reagents were
purchased from Advanced ChemTech, Inc. NADPH, calmodu-
lin, and human ferrous hemoglobin were obtained from Sigma
Chemical Co. Tetrahydrobiopterin (H4B) was purchased from
Alexis Biochemicals. HEPES, DTT, and conventional organic
solvents were purchased from Fisher Scientific. All other
chemicals were purchased from Aldrich, unless otherwise
stated.
An a lytica l Meth od s. The dipeptides and peptidomimetics
were purified on an Alltech Hyperprep PEP HPLC column
(250 × 22 mm) using a gradient of 100% solvent A (0.1% TFA
in H2O) to 60% of solvent B (0.08% TFA in CH3CN) over 30
min at a flow rate of 7.5 mL/min. Optical spectra and enzyme
assays were performed on a Perkin-Elmer Lambda 10 UV/vis
spectrophotometer. 1H NMR spectra were recorded on a Varian
VXR-300 spectrometer in the solvent indicated. Chemical
shifts are reported as δ values in parts per million relative to
TMS in CDCl3 or to DSS in D2O. Electrospray mass spectra
were performed on a Micromass Quattro II spectrometer.
Elemental analyses were obtained from Oneida Research
Services, Inc., Whiteboro, NY. Thin-layer chromatography was
carried out on E. Merck precoated silica gel 60 F254 plates.
Amino acids were visualized with a ninhydrin spray reagent
or a UV/vis lamp. E. Merck silica gel 60 (230-400 mesh) was
used for flash chromatography.

Active Site Probes of NOS
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2943
P ep t oid An a logu es 6-8. The procedure for solid-phase
synthesis of the peptoid analogues was similar to the general
procedure mentioned above with two exceptions: each coupling
step was conducted overnight; 2-bromoacetic acid, NR-Boc-
was warmed to room temperature, a solution of 1 N NaCO3
(100 mL) was added carefully. The organic layer was sepa-
rated, and washed with water, then with 1 N NaCO3 solution.
The solvent was evaporated, resulting in a transparent oily
product (1.5 g, 25% yield): 1H NMR (CDCl3) δ 7.30 (m, 5H),
6.21 (brs, 1H), 5.97 (brs, 1H), 5.83 (brs, 1H), 5.08 (s, 2H), 4.20
(m. 1H), 2.25 (m, 2H), 2.05-2.20 (m, 1H), 1.82-1.97 (m, 1H),
1.43 (s, 9H).
2-N-(Ben zoxyca r bon yl)-4-N-(9-flu or en ylm eth yloxyca r -
bon yl)-L-2,4-d ia m in obu tyr ic Acid ter t-Bu tyl Ester (19).
The amide moiety of 18 (1.5 g 4.5 mmol) was converted into
an amine and was protected with an Fmoc group in a one-pot
reaction according to the procedure of Kazmierski.28 The
product was purified by flash chromatography (hexanes:EtOAc
3:1) and was obtained as a white solid in a 46% yield (1.31 g):
1H NMR (CDCl3) δ 7.77 (d, 2H), 7.61 (d, 2H), 7.35 (m, 9H),
5.56 (brs, 1H), 5.14 (s, 2H), 4.37 (m, 2H), 4.24 (t, 1H), 3.52 (m,
1H), 3.10 (m, 1H), 2.12 (m, 1H), 1.70 (m, 1H), 1.45 (s, 9H).
alkanediamines, and Boc-L-ArgNO were in 5 equiv excess.
2
P ep toid -Dbu (6): 1H NMR (D2O) δ 4.60 (t, 1H), 4.00-4.30
(m, 2H), 3.90 (m, 1H), 3.45 (m, 1H), 3.22 (m, 4H), 1.75-1.98
(m, 2H), 1.50-1.75 (m, 2H); HRMS (M + 1) calcd for C10H22N8O4
319.1837, found 319.1823. Anal. (C10H22N8O4‚2TFA‚0.5H2O)
C, H, N.
P ep toid -Or n (7): 1H NMR (D2O) δ 4.57 (t, 1H), 3.95-4.23
(m, 2H), 3.45 (m, 2H), 3.23 (m, 2H), 2.92 (m, 2H), 1.75-2.00
(m, 4H), 1.45-1.75 (m, 2H); HRMS (M + 1) calcd for C11H24N8O4
333.1993, found 333.1975. Anal. (C11H24N8O4‚2TFA‚0.5H2O)
C, H, N.
1
P ep toid -Lys (8): H NMR (D2O) δ 4.55 (t, 1H), 3.91-4.20
(m, 2H), 3.44 (m, 1H), 3.30 (m, 3H), 2.98 (m, 2H), 1.83-2.03
(m, 2H), 1.54-1.80 (m, 6H); HRMS (M + 1) calcd for C12H26N8O4
347.2150, found 347.2183. Anal. (C12H26N8O4‚2TFA‚0.5H2O)
C, H, N.
4-N-(9-F lu or en ylm eth yloxyca r bon yl)-L-2,4-d ia m in obu -
tyr ic Acid ter t-Bu tyl Ester (20). Compound 19 (1.31 g, 2
mmol) was dissolved in methanol (20 mL) and was hydroge-
nated with a 5% Pd/C catalyst until the Cbz group was
removed. The catalyst was filtered through a Celite pellet, and
the methanol was evaporated in a vacuum. To avoid cleavage
of the Fmoc group, which usually reacts much slower under
the same conditions, TLC (hexanes:EtOAc:NH4OH 1:1:0.1) was
taken every 0.5 h to monitor the progress of the reaction until
compound 19 was gone and most of the product was compound
20: 1H NMR (CDCl3) δ 7.78 (d, 2H), 7.59 (d, 2H), 7.39 (t, 2H),
7.30 (t, 2H), 5.62 (brs, 1H), 4.40 (d, 2H), 4.37 (m, 2H), 4.24 (t,
1H), 3.52 (m, 1H), 3.10 (m, 1H), 2.12 (m, 1H), 1.70 (m, 1H),
1.45 (s, 9H). This yellow oily compound (0.77 g, 95% yield) was
used in the next step without further purification.
N-Meth yla ted Dip ep tid e Am id es 9-11. Fmoc-L-Dbu-
(Boc)-OH, Fmoc-L-Orn(Boc)-OH, and Fmoc-L-Lys(Boc)-OH were
coupled to the Rink resin as described in the general procedure.
N-Methylation was carried out according to the procedure of
Yang et al.23
L-Ar gNO -(N-CH3)-L-Dbu -NH2 (9): 1H NMR (D2O) δ 4.95
2
(dd, 1H), 4.54 (t, 1H), 3.75 (t, 2H), 2.99 (s, 3H), 2.94 (m, 2H),
2.20 (m, 1H), 2.08 (m, 1H), 1.89 (m, 2H), 1.67 (m, 2H); HRMS
(M + 1) calcd for C11H24N8O4 333.1993, found 333.1977. Anal.
(C11H24N8O4‚2TFA‚H2O) C, H, N.
L-Ar gNO -(N-CH3)-L-Or n -NH2 (10): 1H NMR (D2O) δ 4.89
2
(dd, 1H), 4.54 (t, 1H), 3.29 (t, 2H), 3.02 (s, 3H), 2.97 (m, 2H),
1.91 (m, 4H), 1.64 (m, 4H); HRMS (M + 1) calcd for C12H26N8O4
347.2150, found 347.2183. Anal. (C12H26N8O4‚2TFA‚0.5H2O)
C, H, N.
NO
L-Ar gNO -L-Dbu ter t-Bu tyl Ester (13). Fmoc-L-Arg -OH
2
2
(864 mg, 1.96 mmol) and L-Dbu(Fmoc) tert-butyl ester (20; 770
mg, 1.78 mmol) were coupled to form Fmoc-L-ArgNO -L-Dbu-
L-Ar gNO -(N-CH3)-L-Lys-NH2 (11): 1H NMR (D2O) δ 4.87
2
2
(Fmoc) tert-butyl ester as described.29 The Fmoc-L-ArgNO -L-
2
(dd, 1H), 4.53 (t, 1H), 3.31 (t, 2H), 3.04 (s, 3H), 2.96 (m, 2H),
1.91 (m, 4H), 1.69 (m, 4H), 1.35 (m, 2H); HRMS (M + 1) calcd
for C13H28N8O4 361.2307, found 361.2305. Anal. (C13H28N8O4‚
2TFA‚H2O) C, H, N.
Dbu(Fmoc) tert-butyl ester (600 mg, 40% yield) was purified
by flash chromatography (1% methanol in EtOAc): 1H NMR
(CDCl3) δ 7.74 (m, 4H), 7.55 (m, 4H), 7.39 (m, 4H), 7.28 (m,
4H), 6.02 (brs, 1H), 5.44 (brs, 1H), 4.49 (brs, 1H), 4.30 (m, 4H),
4.12 (brs, 1H), 3.05-3.40 (m, 4H), 2.01 (m, 1H), 1.83 (m, 1H),
1.65 (m, 4H), 1.40 (s, 9H). The Fmoc-protected dipeptide (600
mg, 0.78 mmol)) was treated with 20% piperidine in DMF (10
mL). After being stirred at room temperature for a 0.5 h, the
solution was dried under high vacuum (at least 8 h, to make
sure all of the piperidine and DMF were evaporated). The
residue was dissolved in water and washed with ether twice.
The aqueous layer was acidified to pH 4 using 10% HCl and
lyophilized to dryness (170 mg, 50% yield): 1H NMR (D2O) δ
4.51 (m, 1H), 4.09 (t, 1H), 3.34 (m, 2H), 3.10 (t, 2H), 2.27 (m,
1H), 2.07 (m, 1H), 1.91 (m, 2H), 1.72 (m, 2H), 1.44 (s, 9H);
HRMS (M + 1) calcd for C14H29N7O5 376.2303, found 376.2325.
Anal. (C14H29N7O5‚2TFA‚H2O) C, H, N.
L-Ar gNO -L-Dbu Meth yl Ester (12). An ether solution of
2
CH2N2 was dropped slowly into a stirred solution of Fmoc-L-
Dbu(Boc)-OH (380 mg, 0.86 mmol) in ether. The reaction was
stirred for 10 min more and stopped by evaporation of the
ether. The oily product, Fmoc-L-Dbu(Boc)-OCH3 (390 mg,
quantitative yield), was used in the next step without purifica-
tion. After removal of the Fmoc group by catalytic hydrogena-
tion, L-Dbu(Boc)-OCH3 was purified chromatographically
(EtOAc:CH3OH:NH4OH 20:1:0.1), giving L-Dbu(Boc)-OCH3
(190 mg, 97% yield): 1H NMR (CDCl3) δ 5.24 (brs, 1H), 3.67
(s, 3H), 3.45 (dd, 1H), 3.29 (m, 1H), 3.19 (m, 1H), 1.90 (m, 1H),
1.63 (m, 1H), 1.38 (s, 9H). Boc-L-ArgNO -OH (295 mg, 0.92
2
mmol) and L-Dbu(Boc)-OCH3 (190 mg, 0.84 mmol) were
coupled to form the dipeptide, Boc-L-ArgNO -L-Dbu(Boc)-OCH3
2
L-Ar gNO -L-Dbu -OH (14). Compound 13 (100 mg, 0.23
2
(350 mg, 80% yield), according to the procedure of Silverman
et al.:27 1H NMR (CDCl3) δ 7.55 (brs, 1H), 5.48 (brs, 1H), 4.87
(brs, 1H), 4.64 (m. 1H), 4.32 (brs, 1H), 3.73 (s, 3H), 3.22-3.55
(m, 3H), 3.19 (m, 1H), 1.50-2.19 (m, 6H), 1.41 (s, 18H).
mmol) was treated with TFA (4 mL) for 1 h. The solution was
concentrated to dryness. The oily residue was dissolved in a
small amount of water, which was washed with ether and
lyophilized to a white foam (80 mg, 92% yield): 1H NMR (D2O)
δ 4.53 (dd, 1H), 4.06 (t, 1H), 3.27 (m, 2H), 3.07 (t, 2H), 2.26
(m, 1H), 2.07 (m, 1H), 1.92 (m, 2H), 1.70 (m, 2H); HRMS
(M + 1) calcd for C10H21N7O5 320.1677, found 320.1684. Anal.
(C10H21N7O5‚2TFA‚H2O) C, H, N.
l-ArgNO -L-Dbu methyl ester (12) was obtained after deprotec-
2
tion of the Boc group using TFA/CH2Cl2 (1:1 v/v) (104 mg, 96%
yield): 1H NMR (D2O) δ 4.38 (dd, 1H), 3.98 (t, 1H), 3.68 (s,
3H), 3.20 (t, 2H), 2.98 (t, 2H), 2.20 (m, 1H), 1.99 (m, 1H), 1.80
(m, 2H), 1.62 (m, 2H); HRMS (M + 1) calcd for C11H23N7O5
334.1833, found 334.1834. Anal. (C11H23N7O5‚2TFA‚H2O) C,
H, N.
Nr-(Ben zoxyca r b on yl)-L-glu t a m in e ter t-Bu t yl E st er
(18). This compound was synthesized according to the proce-
dure of Anderson et al.27 To a suspension of Cbz-L-Gln (5 g,
17.8 mmol) in CH2Cl2 (10 mL) in a pressure bottle was added
concentrated sulfuric acid (100 µL). While the bottle was cooled
in liquid nitrogen, precooled isobutylene liquid (5.51 g, 98
mmol) was added to the mixture. The pressure bottle was
sealed and kept at room temperature for 6 days. The bottle
was cooled again before the lid was opened. After the mixture
L-Ar gNO -NH(CH2)n NH2 (n ) 2-4, 15-17). To an ice-cooled
2
mixture of Wang resin (2.4 g, 0.76 mmol/g, 100-200 mesh)
and 4-nitrophenyl chloroformate (1.10 g, 3 equiv) in CH2Cl2
(15 mL) was added 4-methylmorpholine (602 µL, 3 equiv). The
mixture was stirred overnight from 0 °C to room temperature.
The resin was drained, washed successively with CH2Cl2,
methanol, water, and methanol and dried in vacuum. The resin
was treated with L-ArgNO methyl ester (3 equiv), HOBt (3
2
equiv), and diisopropylethylamine (DIEA; 5 equiv) in DMF/
CH2Cl2 (1:1, 15 mL) overnight at room temperature. After
removal of the excess reagents, the resin was washed thor-

2944 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Huang et al.
(8) Knowles, R. G.; Moncada, S. Nitric Oxide Synthases in Mam-
mals. Biochem. J . 1994, 198, 249-258.
oughly with DMF, CH2Cl2, and methanol and dried. The resin
was treated with lithium hydroxide (5 equiv) in THF/H2O (5:1
v/v, 15 mL) for 3 h at room temperature followed by washing
with THF, H2O, and methanol and then dried in vacuum. The
resin was divided into three portions, each of which was
coupled with a different NR-Boc-alkanediamine using a 4 equiv
excess of coupling reagents. After washing and drying, the
resin was treated with TFA/CH2Cl2 (1:1 v/v) for 30 min. The
products were purified by HPLC. These compounds were also
synthesized by solution phase according to the peptide syn-
thesis procedure as described.29
(9) Griffith, O. W.; Stuehr, D. J . Nitric Oxide Synthases: Properties
and Catalytic Mechanism. Annu. Rev. Physiol. 1995, 57, 707-
736.
(10) Marletta, M. A. Approaches Toward Selective Inhibition of Nitric
Oxide Synthase. J . Med. Chem. 1994, 37, 1899-1907.
(11) Moncada, S.; Higgs, E. A. Molecular Mechanism and Therapeutic
Strategies related to Nitric Oxide. FASEB J . 1995, 9, 1319-
1330.
(12) Olken, N. M.; Marletta, M. A. NG-Methyl-L-Arginine Functions
as an Alternative Substrate and Mechanism-based Inhibitor
of Nitric Oxide Synthase. Biochemistry 1993, 32, 9677-9685.
(13) Furfine, E. S.; Harmon, M. F.; Paith, J . E.; Garvey, E. P.
Selective Inhibitor of Constitutive Nitric Oxide Synthase by NG-
Nitroarginine. Biochemistry 1993, 32, 8512-8517.
L-Ar gNO -NH(CH2)2NH2 (15): 1H NMR (D2O) δ 4.08 (t, 1H),
2
3.32 (m, 4H), 3.15 (m, 2H), 1.98 (m, 2H), 1.64-1.88 (m, 2H);
HRMS (M + 1) calcd for C8H19N7O3 262.1622, found 262.1639.
Anal. (C8H19N7O3‚2.5TFA) C, H, N.
(14) McCall, T. B.; Feelisch, M.; Palmer, R. M. J .; Moncada, S.
Identification of Nδ-(Iminoethyl)-L-Ornithine as an Irreversible
Inhibitor of Nitric Oxide Synthase in Phagocytic Cells. Br. J .
Pharmacol. 1991, 102, 234-238.
(15) Narayanan, K.; Spack, L.; McMillan, K.; Kilbourn, R. G.;
Hayward, M. A.; Masters, B. S. S.; Griffith, O. W. S-Alkyl-L-
thiocitrullines. J . Biol. Chem. 1995, 270, 11103-11110.
(16) Collins, J . L.; Shearer, B. G.; Oplinger, J . A.; Lee, S.; Garvey, E.
P.; Salter, M.; Duffy, C.; Burnette, T. C.; Furfine, E. S. N-
Phenylamidines as Selective Inhibitors of Human Neuronal
Nitric Oxide Synthase: Structure-activity Studies and Dem-
onstration of in vivo Activity. J . Med. Chem. 1998, 41, 2858-
2871.
(17) Wolff, D. J .; Lubeskie, A. Aminoguanidine is an Isoform-selective,
Mechanism-based Inactivator of Nitric Oxide Synthase. Arch.
Biochem. Biophys. 1995, 316, 290-301.
(18) J ang, D.; Szabo, C.; Murell, G. A. C. S-Substituted Isothioureas
are Potent Inhibitors of Nitric Oxide Biosynthesis in Cartilage.
Eur. J . Pharmacol. 1996, 312, 341-347.
(19) Griffith, O. W.; Gross, S. S. Inhibitors of Nitric Oxide Synthases.
In Methods in Nitric Oxide Research; Feelisch, M., Stamler, J .
S., Eds.; J ohn Wiley and Sons: New York, 1996; pp 187-208.
(20) Fro¨hlich, L. G.; Kotsonis, P.; Traub, H.; Taghavi-Moghadam, S.;
Al-Masoudi, N.; Hofmann, H.; Strobel, H.; Matter, H.; Pfleiderer,
W.; Schmidt, H. H. H. W. Inhibition of Neuronal Nitric Oxide
Synthase by 4-Amino Pteridine Derivatives: Structure-Activity
Relationship of Antagonists of (6R)-5,6,7,8-Tetrahydrobiopterin
Cofactor. J . Med. Chem. 1999, 42, 4108-4121.
(21) Huang, H.; Martasek, P.; Roman, L. J .; Masters, B. S. S.;
Silverman, R. B. Nω-Nitroarginine-Containing Dipeptide Amides.
Potent and Highly Selective Inhibitors of Neuronal Nitric Oxide
Synthase. J . Med. Chem. 1999, 42, 3147.
L-Ar gNO -NH(CH2)3NH2 (16): 1H NMR (D2O) δ 3.98 (t, 1H),
2
3.30 (m, 4H), 2.97 (t, 2H), 1.32-1.96 (m, 4H), 1.60-1.72 (m,
2H); HRMS (M + 1) calcd for C9H21N7O3 276.1779, found
276.1786. Anal. (C9H21N7O3‚2TFA‚0.8H2O) C, H, N.
L-Ar gNO -NH(CH2)4NH2 (17): 1H NMR (D2O) δ 3.95 (t, 1H),
2
3.28 (m, 4H), 2.98 (m, 2H), 1.90 (m, 2H), 1.50-1.73 (m, 6H);
HRMS (M + 1) calcd for C10H23N7O3 290.1935, found 290.1926.
Anal. (C10H23N7O3‚2TFA‚0.5H2O) C, H, N.
En zym e a n d Assa y. All of the NOS isoforms used are
recombinant enzymes overexpressed in E. coli from difference
sources; there is very high sequence identity for the isoforms
from different sources. The murine macrophage iNOS was
expressed and isolated according to the procedure of Hevel et
al.30 The rat nNOS was expressed31 and purified as described.32
The bovine eNOS was isolated as reported.33 Nitric oxide
formation from NOS was monitored by the hemoglobin capture
assay as described previously.34
Deter m in a tion of Ki Va lu es. The apparent Ki values were
obtained by measuring percent inhibition in the presence of
10 µM L-arginine with at least three concentrations of inhibi-
tor. The parameters of the following inhibition equation35 were
fitted to the initial velocity data: % inhibition ) 100[I]/[[I] +
Ki(1 + [S]/Km)]. Km values for L-arginine were 1.3 µM (nNOS),
8.3 µM (iNOS), and 1.7 µM (eNOS). The selectivity of an
inhibitor was defined as the ratio of the respective Ki values.
(22) Miller, S. M.; Simon, R. J .; Ng, S.; Zuckerman, R. N.; Kerr, J .
M.; Moos, W. H. Comparison of the proteolytic susceptibilities
of homologous L-amino acid, D-amino acid, and N-substituted
glycine peptide and peptoid oligomers. Drug Dev. Res. 1995, 35,
20-32.
(23) Yang, L.; Chiu, K. Solid-Phase Synthesis of Fmoc N-Methyl
Amino Acids: Application of the Fukuyama Amine Synthesis.
Tetrahedron Lett. 1997, 38, 7307-7310.
(24) Huang, H.; Martasek, P.; Roman, L. J .; Silverman, R. B.
Syntheses and Evaluation of Dipeptide Amides Containing Nω-
Nitroarginine and D-2,4-Diaminobutyric Acid as Inhibitors of
Neuronal Nitric Oxide Synthase. Bioorg. Med. Chem., submitted.
(25) Pacofsky, G. J .; Lackey, K.; Alligood, K. J .; Berman, J .; Charifson,
P. S.; Crosby, R. M.; Dorsey, G. F.; Feldman, P. L.; Gilmer, T.
M.; Hummel, C. W.; J ordan, S. R.; Mohr, C.; Shewchuk, L. M.;
Sternbach, D. D.; Rodriguez, M. Potent Dipeptide Inhibitors of
the pp6060c-src SH2 Domain. J . Med. Chem. 1998, 41, 1894-
1908.
Ack n ow led gm en t. We thank Professor Michael A.
Marletta (University of Michigan) for the recombinant
E. coli cells which express murine macrophage iNOS.
We also thank Dr. J ose´ Antonio Go´mez-Vidal for helpful
discussions. We are grateful to the National Institutes
of Health for financial support of this work to R.B.S.
(GM 49725), M.A.M. (CA 50414), and B.S.S.M. (GM
52419) and to the Robert A. Welch Foundation to
B.S.S.M. (AQ 1192) in whose lab P.M. and L.J .R. work.
Su p p or tin g In for m a tion Ava ila ble: Analytical data for
compounds 4-17. This material is available free of charge via
the Internet at http://pubs.acs.org.
(26) Videnov, G.; Aleksiev, B.; Stoev, M.; Paipanova, T.; J ung, G.
Tmob Side Chain-Protected S-Cysteine and Homo-S-Cysteine-
sulfonamides, Their NR-Protected and Nω-Aminoethylated De-
rivatives. Liebigs Ann. Chem. 1993, 941-945.
Refer en ces
(1) Kerwin, J . F., J r.; Lancaster, J . R., J r.; Feldman, P. L. Nitric
Oxide: A New Paradigm for Second Messengers. J . Med. Chem.
1995, 38, 4342-4362.
(2) Kerwin, J . F., J r.; Heller, M. The Arginine-Nitric Oxide Path-
way: A target for New Drugs. Med. Res. Rev. 1994, 14, 23-74.
(3) Stuehr, D. J .; Griffith, O. W.; Mammalian Nitric Oxide Syn-
thases. Adv. Enzymol. Relat. Areas Mol. Biol. 1992, 65, 287-
346.
(4) Schmidt, H. H. H. W.; Murad, F. Purification and Characteriza-
tion of Human NO Synthase. Biochem. Biophys. Res. Commun.
1991, 181, 1372-1377.
(5) Schmidt, H. H. H. W.; Walter, U. NO at Work. Cell 1994, 78,
919-925.
(6) Fo¨rstermann, U.; Pollock, J . S.; Schmidt, H. H. H. W.; Heller,
M.; Murad, F. Calmodulin-Dependent Endothelium-Derived
Relaxing Factor/Nitric Oxide Synthase Activity Is Present in the
Particulate and Cytosolic Fractions of Bovine Aortic Endothelial
Cells. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1788-1792.
(7) MacMicking, J .; Xie, Q. W.; Nathan, C. Nitric Oxide and
Macrophage Function. Annu. Rev. Immunol. 1997, 15, 323-350.
(27) Anderson, G. W.; Callahan, F. M. tert-Butyl Esters of Amino
Acids and Peptides and Their Use in Peptide Synthesis. J . Am.
Chem. Soc. 1960, 82, 3359.
(28) Kazmierski, W. M. Metal Chelating Amino Acids in the Design
of Peptides and Proteins. Synthesis of NR-Fmoc/But Protected
Amino Acids Incorporating Aminodiacettic Acid Moiety. Tetra-
hedron letters 1993, 34, 4493-4496.
(29) Silverman, R. B.; Huang, H.; Marletta, M. A.; Martasek, P.
Selective Inhibition of Neuronal Nitric Oxide Synthase by Nω-
Nitroarginine- and Phenylalanine-Containing Dipeptides and
Dipeptide Esters. J . Med. Chem. 1997, 40, 2813-2817.
(30) Hevel, J . M.; White, K. A.; Marletta, M. A. Purification of the
Inducible Murine Macrophage Nitric Oxide Synthase. J . Biol.
Chem. 1991, 266, 22789-22791.
(31) Roman, L. J .; Sheta, E. A.; Martasek, P.; Gross, S. S.; Liu, Q.;
Masters, B. S. S. High-Level Expression of Functional Rat
Neuronal Nitric Oxide Synthase in Escherichia coli. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 8428-8432.

Active Site Probes of NOS
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2945
(32) Gerber, N. C.; Montellano, P. R. Neuronal Nitric Oxide Syn-
thase: Expression in Escherichia coli, Irreversible Inhibition by
Phenyldiazene, and Active Site Topology. J . Biol. Chem. 1995,
270, 17791-17796.
(34) Hevel, J . M.; Marletta, M. A. Nitric Oxide Synthase Assays.
Methods Enzymol. 1994, 133, 250-258.
(35) Segel, I. H. Enzyme Kinetics; J ohn Wiley and Sons: New York,
1975; p 105.
(33) Martasek, P.; Liu, Q.; Roman, L. J .; Gross, S. S.; Sessa, W. C.;
Masters, B. S. S. Characterization of Bovine Endothelial Nitric
Oxide Synthase Expressed in Escherichia coli. Biochem. Biophys.
Res. Commun. 1996, 219, 359-365.
J M000127Z