S. D. Luzzi, M. A. Marletta / Bioorg. Med. Chem. Lett. 15 (2005) 3934–3941
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(%) for C20H24N2O5S: C 59.39, H 5.98, N 6.93. Found:
C 59.39, H 6.08, N 6.84.
Anal. Calcd (%) for C9H21N4O4.5: C 42.01, H 8.23, N
21.78; Found: C 42.36, H 8.21, N 21.74.
4.1.4. Nd-Methyl-L-ornithine (5). A solution of 4 (2.66 g,
0.007 mol) in 48% HBr (21 mL) was heated to reflux for
2 h. The solution was cooled to room temperature and
filtered. The filtrate was evaporated to give a viscous
red liquid, which was dissolved in water (25 mL) and ap-
plied to a 26 mL of AG50W-X8 (H+) cation exchange
resin. The column was washed with water until the pH
of the eluent was neutral and then eluted with 2 N
NH4OH. Tubes containing (5) were combined and evap-
orated to give a brown oil, which was dissolved in water
(15 mL). The pH was adjusted to 5.8 using HCl and the
solution was boiled for 30 s with activated charcoal. The
charcoal was filtered off and the filtrate was evaporated
to dryness giving 5 (804 mg, 83%) as a solid; mp 217 ꢁC;
1H NMR (400 MHz, D2O/TPS) d (ppm): 3.32 (t, 1H),
2.96 (t, 2H), 2.62 (s, 3H), 1.67 (4H, m); 13C NMR
(400 MHz, D2O/TPS) d 183.9, 58.0, 51.7, 35.5, 33.7,
25.1; FABMS calcd for C6H15N2O2 ([M+H]+), found
m/z 147.
4.1.6. Nd-Methyl-N-cyano-L-ornithine B,B-boroxazoli-
done (8). To a solution of 7 (419 mg, 2.85 mol) in dry
DME (17 mL) was added dry triethylamine (412 lL,
2.98 mmol). A solution of CNBr (622 mg, 5.87 mmol)
in dry DME (10 mL) was added and the solution was
stirred under argon for 3 h. The reaction mixture was
concentrated, applied to a silica gel column, and eluted
with EtOAc to give 7 (89 mg, 25%) as a white solid;
mp 143–145 ꢁC; 1H NMR (400 MHz, DMSO) d
(ppm): 6.52 (t, J = 8.0 Hz, 1H), 5.60 (t, J = 8.0 Hz),
3.51 (m, 1H), 2.98 (t, J = 6.8 Hz, 2H), 2.81 (s, 3H),
1.81–1.88 (m, 1H), 1.65–1.72 (m, 2H), 1.14–1.57 (m,
1H), 0.68–0.73 (m, 6H), 0.15–0.28 (m, 4H); 13C NMR
(400 MHz, DMSO) d 173.83, 118.56, 54.05, 51.66,
38.20, 27.49, 23.73, 12.79, 12.06, 8.93; FABMS calcd
for C11H22BN3O2 (
18+) found m/z 240; Anal. Calcd
(%) for C11H22BN3O2: C 55.25, H 9.27, N 17.57. Found:
C 54.91, H 9.54, N 17.86.
4.1.7. NG-Hydroxy-Nd-methyl-L-arginine (dMHA) (9).
To a round-bottomed flask containing 8 (25 mg,
0.104 mmol) was added triethylamine (73 lL,
0.523 mmol). To this flask was added MeOH (750 lL),
followed immediately by NH2OH Æ HCl (33 mg,
0.475 mmol). The reaction was stirred for 2 h and then
concentrated on a rotary evaporator, loaded onto a
4 · 17 cm microgranular cellulose column, and eluted
with 5:3 MeCN: 0.1% TFA(aq) to give 9 (13 mg, 86%)
4.1.5. Nd-Methyl-L-arginine (dMA) (6). Compound 5
was dissolved in water and the pH was adjusted to 3
using concentrated HCl. The solution was evaporated
to dryness to yield 995 mg of 5 as the HCl salt. The solid
was dried overnight on a vacuum line and ground with
glass stirring rod until a dry, finely divided powder
was obtained. To a suspension of the HCl salt of 5
(650 mg, 0.004 mol) in dry DME (5.6 mL) was added
1 M triethylborane in THF (4.5 mL, 0.0045 mol). The
mixture was heated to reflux under argon for 48 h.
The resulting suspension was filtered, rinsed twice with
petroleum ether, and dried. The white compound
(381 mg, 0.002 mol) was dissolved in anhydrous DMF
(3.15 mL) to which N,N0-bis-tert-butoxycarbonylthiou-
rea (492 mg, 1.78 mmol) and triethylamine (819 lL,
5.9 mmol) had been added. The reaction was cooled to
0 ꢁC with an ice bath and HgCl2 (532 mg, 0.002 mol)
was added. After 1.5 h of reaction, EtOAc (25 mL)
was added and the mixture was filtered through Celite
to remove mercury salts. The EtOAc layer was washed
twice with water and once with brine and then dried
(MgSO4). Evaporation of the EtOAc gave a yellow oil,
which was applied to a silica gel column, and eluted with
EtOAc. Ninhydrin positive fractions were combined and
evaporated. Hydrolysis of the boroxazolidone was
effected by the addition of 1.5 M HCl (10 mL) and heat-
ing the acidic solution at 100 ꢁC for 1 h. Evaporation of
the solvent resulted in a clear oil to which was added
TFA (10 mL). This solution was stirred for 2 h before
being loaded onto AG50WX-8 (H+), washed with 5 col-
umn volumes of water, and eluted with 1 M NH4OH.
Ninhydrin positive tubes were combined and evaporated
to give a clear oil. The oil was dissolved in water and the
pH was adjusted to 2.4 with acetic acid and evaporated
to give 6 (218 mg, 48%) as a white crystalline solid; mp
195 ꢁC; 1H NMR (400 MHz, D2O/TPS) d (ppm): 4.12 (t,
J = 6.0 Hz, 1H), 3.43 (t, 2H, J = 8.4 Hz), 3.05 (s, 3H),
1.91 (4H, m); 13C NMR (400 MHz, D2O/TPS) d
172.20, 156.76, 52.97, 49.71, 36.12, 26.95, 22.48; HRMS
calcd for C7H17N4O2+: 189.1352. Found: 189.1352.
1
as a clear oil: H NMR (400 MHz, D2O/TPS) d (ppm):
4.12 (t, J = 8.0 Hz, 1H), 3.41 (t, J = 8.0 Hz, 2H), 3.02
(s, 3H), 1.85 (m, 4H); 13C NMR (400 MHz, D2O/TPS)
d 171.8, 158.7, 52.7, 49.6, 35.7, 26.7, 22.1; HRMS calcd
for C7H17N4O3+: 205.1295. Found: 205.1301.
4.1.8. Expression and purification of inducible nitric oxide
synthase. Expression and purification of iNOS were car-
ried out, as described previously,6,21 with minor modifi-
cations that follow. All buffers used in the lysis,
purification, and concentration steps contained 10 lM
H4B. Cell pellets from 3 L culture were resuspended in
lysis buffer containing 50 mM Hepes (pH 7.4), 10% gly-
cerol, 1 mM Pefabloc SC, 10 lg/mL benzamidine,
5 lg/mL leupeptin, and 1 lg/mL each of pepstatin,
chymostatin, and antipain. The resuspended cells were
lysed with a high-pressure homogenizer. Centrifugation
for 1 h at 42,000 rpm yielded a supernatant that was
immediately purified using 20,50-ADP-Sepharose affinity
chromatography and DEAE Bio-Gel A anion exchange
chromatography, as previously described.6 The eluate
was concentrated using a centrifugal filtration device
and loaded onto a gel filtration column (S200 16/60),
which was equilibrated with 100 mM Hepes (pH 7.4),
100 mM NaCl, 10 lM H4B, and 10% glycerol. Fractions
containing iNOS were pooled, concentrated, and frozen
in aliquots at ꢀ80 ꢁC. iNOS purified in this manner is
>95% pure by SDS–PAGE with Coomassie staining
Å
and had a specific activity of ꢁ122 lmol NO/h/mg
measured by the oxyhemoglobin assay (see below).42 Pro-
tein concentration was determined using the Bradford
protein assay using bovine serum albumin as a standard.