Selective inhibition of neuronal nitric oxide synthase by N(ω)- nitroarginine- and phenylalanine-containing dipeptides and dipeptide esters

Article abstract of DOI:10.1021/jm970200u

A series of N(ω)-nitroarginine (Arg(NO₂))- and phenylalanine- containing dipeptides and dipeptide esters were synthesized as potential selective inhibitors of neuronal nitric oxide synthase (nNOS). All of the dipeptides and dipeptide esters are

Full text of DOI:10.1021/jm970200u

J . Med. Chem. 1997, 40, 2813-2817
2813
Articles
Selective In h ibition of Neu r on a l Nitr ic Oxid e Syn th a se by Nω-Nitr oa r gin in e-
a n d P h en yla la n in e-Con ta in in g Dip ep tid es a n d Dip ep tid e Ester s
Richard B. Silverman,*,‡ Hui Huang,‡,| Michael A. Marletta,†, and Pavel Martasek§,∇
Departments of Chemistry and of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University,
Evanston, Illinois 60208-3113, Interdepartmental Program in Medicinal Chemistry and Department of Biological Chemistry,
University of Michigan, Ann Arbor, Michigan 48109, and Department of Biochemistry, The University of Texas Health Science
Center, San Antonio, Texas 78284-7760
Received March 26, 1997X
A series of Nω-nitroarginine (ArgNO )- and phenylalanine-containing dipeptides and dipeptide
2
esters were synthesized as potential selective inhibitors of neuronal nitric oxide synthase
(nNOS). All of the dipeptides and dipeptide esters are competitive inhibitors of nNOS,
macrophage nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS), except
for the ones that contain D-ArgNO (8-10, 12, 13), which are uncompetitive inhibitors of iNOS
2
but competitive inhibitors of nNOS and eNOS. None of the dipeptides or dipeptide esters tested
(1, 2, 12, 13) exhibited time-dependent inhibition of any of the NOS isoforms, unlike Nω-nitro-
L-arginine itself, which does, although it is reversible. The order of the amino acids in the
dipeptide or dipeptide ester is important to selectivity, and the selectivity depends on the
chirality of the amino acids. In the case of the corresponding benzyl esters (5 vs 6), both
dipeptides favor iNOS over nNOS and eNOS inhibition. All of the dipeptide methyl esters
containing a D-amino acid, however, exhibit an inhibitory preference for nNOS over iNOS and
eNOS. The most impressive selectivities observed are 1800- and 800-fold for 12 and 13,
respectively, in favor of nNOS over iNOS; unfortunately, the selectivities of these compounds
for nNOS over eNOS are only 2.5 and 5.3, respectively.
In tr od u ction
Overproduction of NO, consequently, has been impli-
cated in a wide variety of diseases.3 NO overproduction
by nNOS has been implicated in strokes,4 septic shock,5
seizures,6 schizophrenia,7 migraine headaches,8 and
Alzheimer’s disease.9 iNOS overproduction of NO has
been associated with tolerance to and dependence on
morphine,10 development of colitis,11 tissue damage and
inflammation,12 overproduction of osteoclasts, leading
to osteoporosis, Paget’s disease, and rheumatoid arthri-
tis,13 and destruction of photoreceptors in the retina.14
This suggests that inhibition of NOS would have a
significant beneficial effect on disease states arising
from the overproduction of NO. A wide variety of
compounds have been shown to inhibit NOS.15,18 How-
ever, because of the general importance of NO to human
health, selective inhibition of the isoforms of NOS is
essential. Selectivity for the three isoforms already has
been reported to some degree. The early inhibitors were
analogues of L-arginine. Nω-Methyl-L-arginine and Nω-
ethyl-L-arginine, however, show only about a factor of
2 selectivity; Nω-methyl-L-arginine is selective for eNOS
and nNOS over iNOS and Nω-ethyl-L-arginine is selec-
tive for iNOS over nNOS and eNOS.16 Nω-Nitro-L-
arginine is about 300-fold selective for nNOS over
iNOS.17 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine
and S-ethylisothiourea had 10-40-fold selectivity in
favor of iNOS,18 and 2-iminoazaheterocycles showed
selectivities of 1.1-9 in favor of iNOS over eNOS and
nNOS.19 Various indazole analogues had selectivities
of 5-10 for either nNOS or iNOS.20 Imidazole ana-
logues exhibited selectivities in the range of 3-6-fold
in favor of iNOS.21 Aminoguanidine shows a 50-fold
selectivity for iNOS over nNOS and 500-fold over
Nitric oxide (NO) has been shown to be an important
biological second messenger which is biosynthesized by
a family of enzymes called nitric oxide synthases (EC
1.14.13.39). Three principal isoforms of this enzyme
have been isolated and characterized, each associated
with different physiological functions.1 The isoform
from endothelial cells (eNOS) is involved in the regula-
tion of smooth muscle relaxation, blood pressure lower-
ing, and inhibition of platelet aggregation. Neuronal
nitric oxide synthase (nNOS) is important for long-term
potentiation, and an inducible form (iNOS), which acts
in host defense, is generated by activated macrophage
cells during an immune response. The endothelial and
neuronal isoforms are expressed constitutively and
require Ca2+ and calmodulin for activity; the macroph-
age form is induced by cytokines or bacterial lipopolysac-
charides and is Ca2+ and calmodulin independent. All
forms of the enzyme require NADPH, tetrahydrobiop-
terin, heme, and FAD and FMN. Despite the extraor-
dinary importance of NO to the apparent health of
organisms, it also has been associated with a large
number of harmful effects as a result of its reactive free
radical properties; in fact, NO has a lifetime of only a
few seconds at physiological pH and temperature.2
† University of Michigan.
‡ Northwestern University.
§ The University of Texas Health Science Center.
| Carried out all of the chemical and enzymological studies described
here.
Supplied the recombinant E. coli cells expressed with murine
macrophages iNOS.
∇ Supplied the recombinant eNOS used in these studies.
X Abstract published in Advance ACS Abstracts, August 1, 1997.
S0022-2623(97)00200-8 CCC: $14.00 © 1997 American Chemical Society

2814 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Silverman et al.
Sch em e 1
found in the Supporting Information. None of the
dipeptides or dipeptide esters tested (1, 2, 12, 13)
exhibited time-dependent inhibition of any of the iso-
forms, unlike Nω-nitro-L-arginine itself, which we found
had Ki and kinact values of 9.3 µM and 0.132 min-1
,
respectively, with nNOS. None of the kinetic plots
showed curvature, suggesting that there was no time
dependence to the inhibition.
The order of the amino acids in the dipeptide is
important to selectivity, but it depends on the chirality
of the amino acids. Thus, for the dipeptide and dipep-
tide methyl ester pairs 1 vs 2 and 3 vs 4, having
L-ArgNO at the N-terminus favors nNOS over iNOS and
2
eNOS inhibition, but when the L-Phe is at the N-
terminus, it favors iNOS over nNOS and eNOS inhibi-
tion. In the case of the corresponding benzyl ester (5
vs 6), both dipeptides favor iNOS over nNOS and eNOS
inhibition. All of the dipeptide methyl esters containing
a D-amino acid, however, exhibit an inhibitory prefer-
ence for nNOS over iNOS and eNOS. The most impres-
sive selectivities observed are 1800- and 800-fold for 12
and 13, respectively, in favor of nNOS over iNOS.
These results indicate that there is a difference in the
binding sites of nNOS vs iNOS that can be taken
advantage of by appropriate design of D-amino acid-
containing peptide esters. Unfortunately, the selectivity
of these analogues for nNOS over eNOS is only 2.5 and
5.3, respectively. The selectivity of eNOS over iNOS for
12 and 13, however, is 714- and 152-fold, respectively.
Because of the unnatural chirality of the amino acids
and the incorporation of an ester functionality, the most
nNOS- vs iNOS-selective compounds are peptidomi-
metic analogues, which may make them orally bioavail-
able. The enormous selectivity of 12 and 13 suggests
that other dipeptides and dipeptide esters of unnatural
amino acids should be tested.
eNOS.22 Several series of isothiourea analogues favored
inhibition of iNOS over eNOS by factors of between 2-
and 6-fold with one analogue being 19-fold selective;
bisisothioureas were more selective with one analogue
showing a selectivity of 190-fold in preference for
iNOS.23 S-Methyl- and S-ethyl-L-thiocitrulline were 10-
and 50-fold, respectively, more selective for nNOS than
eNOS.24 L-N6-(1-Iminoethyl)lysine, another inhibitor of
NOS, favors the inhibition of iNOS by a factor of 30 over
eNOS and by 13 over nNOS.25
Because of the relatively high selectivity of Nω-nitro-
L-arginine for nNOS over iNOS,19 and the observation
that it is a time-dependent inhibitor of nNOS but a
reversible inhibitor of iNOS,19,26 we decided to determine
if Nω-nitroarginine-containing dipeptides and dipeptide
esters would have increased selectivity for nNOS. Since
L-arginine methyl ester, L-argininamide, and L-arginine-
containing dipeptides are substrates for NOS,27,28 it was
thought that the dipeptide esters may be functional as
well. Furthermore, the fact that Nω-nitro-L-arginine
inhibits nNOS in vivo following intraperitoneal injec-
tion30 suggests that it crosses the blood-brain barrier.
Here we describe results of our studies of selective
inhibition of all three isoforms by Nω-nitroarginine- and
phenylalanine-containing dipeptides and dipeptide es-
ters.
Exp er im en ta l Section
Gen er a l Ch em ica l Meth od s. Rea gen ts. NR-Boc-ω-nitro-
D-arginine and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylu-
ronium hexafluorophosphate (HBTU) were purchased from
NOVA Biochemicals (San Diego, CA). L-ArgNO -L-Phe-OMe (3)
2
was purchased from BACHEM Feinchemikalien AG (Buben-
dorf, Switzerland) and was used without further purification.
All other N-Boc-L- or D-amino acids and L- or D-amino acid
esters were purchased from Sigma (St. Louis, MO). 1-[3-
(Dimethylamino)propyl]-3-ethylcarbodiimide (EDC), 1-hydrox-
ybenzotriazole hydrate (HOBT), N,N-diisopropylethylamine
(DIEA), and trifluoroacetic acid (TFA) were obtained from
Aldrich Chemical Co. (Milwaukee, WI). Acids, bases, and
conventional organic solvents were purchased from Fischer
Scientific Co.
An a lytica l Ch em ica l Meth od s. Optical rotations were
determined with an AA-100 polarimeter (Optical Activity Ltd.,
England). 1H-NMR spectra were recorded on a Varian Gemini-
300 spectrometer in the solvent indicated. 1H-NMR chemical
shifts in D2O are reported relative to internal sodium 3-(tri-
methylsilyl)-1-propanesulfonate (DSS). Fast atom bombard-
ment (FAB) mass spectra were performed on a VG-Instru-
ments 70-250-SE high-resolution mass spectrometer. Elemental
analyses were obtained from Oneida Research Services, Inc.,
Whitesboro, NY. Thin-layer chromatography was carried out
on Merck silica gel 60-F254, 0.25 mm thickness. Amino acids
were visualized with a ninhydrin/pyridine spray reagent or a
UV/vis lamp. High-performance liquid chromatography was
performed on a Beckman System Gold instrument (Model 125P
solvent module and Model 166 detector). Samples were
analyzed by elution from a Vydac RP C18 90A (4 × 250 mm)
pharmaceutical column with a flow rate of 0.75 mL/min.
Samples (10 µL) were injected and eluted using the following
Resu lts a n d Discu ssion
Ch em istr y. All of the dipeptides were synthesized
manually by the route shown in Scheme 1. Chiral
HPLC, which can separate all four epimers of each
dipeptide, indicated the presence of only one epimer for
each compound, and all dipeptides were pure by NMR
and elemental analysis and were optically active (Table
1). A variety of other conditions led to epimerization
in the product, which resulted in two product peaks
(diastereomers) in the chiral HPLC system used.
En zym ology. All of the dipeptides and dipeptide
esters are competitive inhibitors of the three isoforms
of NOS, except for the ones that contain D-ArgNO (8-
2
10, 12, 13), which are uncompetitive inhibitors of iNOS
but competitive inhibitors of nNOS and eNOS (Table
2). The kinetic data were obtained by Dixon analysis,29
and a replot of these data was obtained by the method
of Cornish and Bowden30 to determine the type of
inhibition. Plots for the competitive inhibition of nNOS
by D-phenylalanyl-D-nitroarginine methyl ester (12) and
for the uncompetitive inhibition of iNOS by D-ni-
troargininyl-D-phenylalanine methyl ester (9) can be

Selective Inhibition of NO Synthases
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2815
Ta ble 1. Analytical Data for the Nitroarginine-Containing Dipeptides and Dipeptide Esters
FAB
HRMS
1H-NMR chemical shift
inhibitor
(δ, D2O)
(M + 1)
anal. (C, H, N)
[R]23.5
D
1, L-ArgNO -L-Phe
7.25 (m, 5H), 4.62 (t, 1H),
3.94 (t, 1H), 3.18 (m, 2H),
3.05 (m, 2H), 1.79 (m, 2H),
1.53 (m, 2H)
7.30 (m, 5H), 4.31 (t, 1H),
4.25 (t, 1H), 3.21 (m, 4H),
1.85 (m, 1H), 1.74 (m, 1H),
1.56 (m, 2H)
7.30 (m, 5H), 4.36 (m, 1H),
4.26 (t, 1H), 3.70 (s, 3H),
3.19 (m, 4H), 1.82 (m, 1H),
1.71 (m, 1H), 1.54 (m, 2H)
7.21 (m, 5H), 7.10 (m, 5H),
4.96 (d, 2H), 4.68 (t, 1H),
3.95 (t, 1H), 3.04 (m, 4H),
1.78 (m, 2H), 1.51 (m, 2H)
7.28 (m, 10H), 5.15 (d, 2H),
4.40 (t, 1H), 4.19 (t, 1H),
3.10 (m, 4H), 1.78 (m, 2H),
1.42 (m, 2H)
7.25 (m, 5H), 4.88 (dd, 1H),
3.87 (t, 1H), 3.75 (s, 3H),
3.38 (dd, 1H), 2.96 (t, 2H),
2.85 (dd, 1H), 1.52 (m, 2H),
0.99 (m, 2H)
7.25 (m, 5H), 4.88 (dd, 1H),
3.87 (t, 1H), 3.75 (s, 3H),
3.36 (dd, 1H), 2.97 (t, 2H),
2.85 (dd, 1H), 1.52 (m, 2H),
0.99 (m, 2H)
7.25 (m, 5H), 4.68 (m, 1H),
3.93 (t, 1H), 3.28 (s, 3H),
3.19 (m, 2H), 3.03 (m, 2H),
1.80 (m, 2H), 1.54 (m, 2H)
7.25 (m, 5H), 4.21 (m, 2H),
3.69 (s, 3H), 3.20 (dd, 1H),
3.01 (m, 3H), 1.63 (m, 1H),
1.42 (m, 1H), 1.08 (m, 1H),
0.94 (m, 1H)
7.25 (m, 5H), 4.20 (m, 2H),
3.68 (s, 3H), 3.20 (dd, 1H),
3.01 (m, 3H), 1.64 (m, 1H),
1.43 (m, 1H), 1.09 (m, 1H),
0.95 (m, 1H)
7.30 (m, 5H), 4.36 (m, 1H),
4.26 (t, 1H), 3.70 (s, 3H),
3.19 (m, 4H), 1.82 (m, 1H),
1.71 (m, 1H), 1.54 (m, 2H)
7.30 (m, 5H), 4.31 (t, 1H),
4.25 (t, 1H), 3.21 (m, 4H),
1.85 (m, 1H), 1.74 (m, 1H),
1.56 (m, 2H)
367
367
381
457
457
381
C
C
C
C
C
C
15H22N6O5‚1.5CF3COOH‚0.5H2O
+17.9 (c 1.70, water)
+15.7 (c 1.5, water)
+6.4 (c 1, water)
2
2, L-Phe-L-ArgNO
15H22N6O5‚2HCl
2
4, L-Phe-L-ArgNO -OMe
16H24N6O5‚2 CF3COOH
22H28N6O5‚2 CF3COOH
22H28N6O5‚2 CF3COOH
16H24N6O5‚2 CF3COOH
2
5, L-ArgNO -L-Phe-OBn
+12.5 (c 1.2, MeOH)
-5.5 (c 1.6, MeOH)
+27.0 (c 1, water)
2
6, L-Phe-L-ArgNO -OBn
2
7, L-ArgNO -D-Phe-OMe
2
8, D-ArgNO -L-Phe-OMe
381
C
16H24N6O5‚2 CF3COOH
-27.2 (c 1, water)
2
9, D-ArgNO -D-Phe-OMe
381
381
C
C
16H24N6O5‚ 2HCl H2O
16H24N6O5‚2 CF3COOH
-12.9 (c 1.40, MeOH)
+56.8 (c 1, water)
2
10, L-Phe-D-ArgNO -OMe
2
11, D-Phe-D-ArgNO -OMe
381
C
C
16H24N6O5‚1.5 CF3COOH
16H24N6O5‚2CF3COOH
-56.6 (c 1, water)
2
12, D-Phe-D-ArgNO -OMe
381
367
-6.5 (c 1, water)
2
13, D-Phe-D-ArgNO
C15H22N6O5‚2HCl
-15.6 (c 1.5, water)
2
Ta ble 2. Ki Values for Inhibition of NOS Isoforms by Nitroarginine-Containing Dipeptides and Dipeptide Esters
selectivity
inhibitor
nNOS (µM)
iNOS (µM)
eNOS (µM)
nNOS/iNOS
nNOS/eNOS
iNOS/eNOS
1, L-ArgNO -L-Phe
18
140
14
370
55
110
92
290
150
90
160
93
45
204
18
395
490
400
1350
125
250
525
1400
375
150
8500
5
8.9
0.66
3.2
0.55
0.33
0.41
1.1
21.9
3.5
28.6
3.6
2.3
2.3
5.7
4.8
2.5
1.7
21.3
2.5
5.3
2.5
5.3
8.9
6.6
6.9
5.6
5.3
0.22
0.058
0.02
7.1
0.0014
0.0066
2
2, L-Phe-L-ArgNO
2
3, L-ArgNO -L-Phe-OMe
2
4, L-Phe-L-ArgNO -OMe
2
5, l-ArgNO -L-Phe-OBn
2
6, L-Phe-L-ArgNO -OBn
45
100
2
7, L-ArgNO -D-Phe-OMe
2
8, D-ArgNO -L-Phe-OMe
6400a
6500a
7500a
1200
3600a
13600a
22.1
43.3
83.3
3
1800
800
2
9, D-ArgNO -D-Phe-OMe
2
10, L-Phe-D-ArgNO -OMe
2
11, D-Phe-L-ArgNO -OMe
400
2
17
2
12, D-Phe-D-ArgNO -OMe
2
13, D-Phe-D-ArgNO
90
2
a
Uncompetitive inhibition.

2816 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Silverman et al.
gradient protocol: 0-5 min, solvent A (0.06% TFA in water);
5-60 min, linear gradient from solvent A to solvent B (0.05%
TFA in acetonitrile).
Chiral HPLC was carried out on a Beckman System Gold
HPLC with a 150 × 4.0 mm Crownpak CR (+) column (Chiral
Technologies, Inc., Exton, PA) using a gradient of 100% solvent
A to 90% solvent A/10% solvent B over 30 min at a flow rate
of 0.5 mL/min. Solvent A is water taken to pH 2.0 with
perchloric acid, and solvent B is methanol.
En zym e P r ep a r a tion . nNOS was obtained from bovine
brain as described.19 A typical preparation had a specific
activity of 250 nmol of nitric oxide (mg of protein)-1 min-1 at
30 °C. iNOS was purified according to the procedures of Hevel
et al.31 with a specific activity of 500 nmol of nitric oxide (mg
of protein)-1 min-1 at 30 °C. eNOS, expressed in Escherichia
coli, was purified as described and supplemented with L-
arginine and BH4;32 the specific activity was 235 nmol of NO
mg-1 min-1 at 25 °C.
Ch em ica l Syn th esis. N-Boc-d ip ep tid e Meth yl Ester .
N-Boc-protected amino acid (1.5 mmol), amino acid methyl
ester (1.5 mmol), HOBT (1.5 mmol), and EDC (1.65 mmol) were
mixed at 0 °C (ice-water bath) in freshly distilled methylene
chloride (4 mL). The suspension was stirred for 10 min to
which was added dropwise DIEA (3.0 mmol) under N2. The
mixture was stirred for 2 h from 0 °C to room temperature.
Shorter reaction time and concentrated reaction solution
helped to minimize the chance of racemization. A large
amount of water (30 mL) was added to quench the reaction;
then the crude product was extracted with ethyl acetate (30
mL). After being washed sequentially with water (30 mL),
5% (v/v) HCl solution (2 × 20 mL), water (20 mL), 5% (w/v)
NaHCO3 solution (2 × 20 mL), water (20 mL), and saturated
NaCl solution (20 mL), the organic layer was dried over
MgSO4. Crude dipeptide was obtained as a white powder by
rotary evaporation of the ethyl acetate solution. The powder
was dissolved in CHCl3 and loaded onto a silica gel column (3
× 50 cm). Pure N-Boc-dipeptide methyl ester was eluted with
20:1 CHCl3/CH3OH, monitoring by TLC. The yields were
about 50%.
N-Boc-d ip ep tid e Ben zyl Ester . To a stirred suspension
of amino acid benzyl ester (1.5 mmol) and N-Boc-amino acid
(1.3 equiv) in 2 mL of CH2Cl2 at 0 °C was added a methylene
chloride solution (2 mL) of HBTU/HOBT (1.3 equiv each). After
the mixture stirred for a few more minutes, 6 equiv of DIEA
was added dropwise under N2. The reaction mixture became
homogenous after 2 h. Ethyl acetate was added to dilute the
reaction solution, and the same workup procedure was per-
formed as described above.
An a lytica l Bioch em ica l Meth od s. Nitric oxide formation
was measured using the hemoglobin capture assay.33 A typical
assay mixture for nNOS contained 3-15 µM L-arginine, 1.6
mM CaCl2, 11.6 µg/mL calmodulin, 100 µM DTT, 100 µM
NADPH, 6.5 µM BH4, and 3 mM oxyhemoglobin in 100 mM
Hepes (pH 7.5). The reaction mixture for iNOS assay included
10-60 µM L-arginine, 100 µM DTT, 100 µM NADPH, 6.5 µM
BH4, and 3 µM oxyhemoglobin in 100 mM Hepes (pH 7.5). The
production of nitric oxide by eNOS was measured as de-
scribed.34 Briefly, the assay mixture contained 80 µM oxyhe-
moglobin, 3-25 µM L-arginine, 100 µM DTT, 10 µM CaCl2, 1
µg/mL calmodulin, 5 µM BH4, 100 µM NADPH, and 50 mM
Hepes (pH 7.5). All assays were in a final volume of 600 µL
and were initiated with enzyme. Nitric oxide reacts with
oxyHb to yield methemoglobin which is detected at 401 nm (ꢀ
35
) 19 700 M-1 cm-1
)
on a Perkin-Elmer Lambda 1 UV/vis
spectrophotometer. The concentration of arginine is higher
for iNOS than for nNOS or eNOS because there is substrate
inhibition with nNOS and eNOS but not iNOS. Protein
concentration of enzyme was determined with the Bradford
Assay (Bio-Rad) using bovine serum albumin as the standard.
In h ibition Meth od s. The reversible inhibition of NOS by
the dipeptide analogues was studied under initial rate condi-
tions with the hemoglobin assay as described above. The Ki
values were determined from Dixon plots29 with various
L-arginine and inhibitor concentrations. The type of reversible
inhibition was determined from Cornish-Bowden replots30 of
the data in the Dixon plots.
Ackn owledgm en t. We thank Francisco Javier Blan-
co for his preliminary work on this project. We are
grateful to the National Institutes of Health for financial
support of this work to R.B.S. (GM49725) and M.A.M.
(CA50414) and to the National Institutes of Health
(GM52419) and the Robert A. Welch Foundation (AQ-
1192) for financial support to Bettie Sue Siler Masters
(University of Texas Health Science Center) in whose
lab P.M. expressed and purified the E. coli eNOS used
in these studies.
Dip ep t id e E st er s 4-12. The Boc groups of the methyl
ester- and benzyl ester-protected dipeptides were removed by
TFA or HCl (3 N in ethyl acetate). The Boc-protected amino
acid ester was treated with acid (3 mL). The solution was
stirred for 1 h (shorter time for HCl deprotection) under N2.
The acid was evaporated at room temperature, and the residue
was dissolved in water (10 mL). The aqueous solution was
washed with ethyl ether (3 × 10 mL) and vacuum evaporated.
When it was difficult to remove the water, the oily residue
was dissolved in methanol and evaporated. The yield from
the deprotection step was about 80%. The TFA or HCl salts
of the dipeptide esters were used for all of the elemental
analyses. The analytical data for the compounds are listed in
Table 1.
Su ppor tin g In for m ation Available: Dixon and Cornish-
Bowden plots for the competitive inhibition of nNOS by
D-phenylalanyl-D-nitroarginine methyl ester (12) and for the
uncompetitive inhibition of iNOS by D-nitroargininyl-D-phe-
nylalanine methyl ester (9) (4 pages). Ordering information
is given on any current masthead page.
Dip ep tid es 1, 2, a n d 13. To a solution of N-Boc-dipeptide
methyl ester (1 mmol) in methanol (5 mL) was added 1 N
NaOH solution (10 mL). The mixture was stirred for 2 h at
room temperature, and then the methanol was evaporated.
The aqueous solution was washed with ethyl acetate (2 × 10
mL) and acidified with 1 N citric acid. The precipitate that
formed was extracted with ethyl acetate (2 × 20 mL). The
extract was washed with water (2 × 20 mL) and dried over
MgSO4. The N-Boc-dipeptide acid was obtained in a yield of
70% after evaporation of the solvent. A further deprotection
of the Boc group by TFA or HCl was carried out as described
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