Asymmetric synthesis of conformationally restricted L-arginine analogues as active site probes of nitric oxide synthase

Article abstract of DOI:10.1021/jo982161f

Using the catalytic asymmetric sharpless carbamate aminohydroxylation, conformationally restricted L-arginine and L-homoarginine derivatives (5-8) were prepared in good enantiomeric excess to investigate the binding requirements of L-arginine-based compounds with nitric oxide synthase. The L- arginine derivatives (5 and 6) inhibited both the inducible and neuronal isoforms of nitric oxide synthase with little isoform selectivity (5, IC₅₀ = 42 and 144 μM, 6, 8 and 12 μM, respectively). The guanidine-containing compound (5) did not act as a nitric oxide producing substrate for nitric oxide synthase. The ability of these compounds to interact with the enzyme supports the idea that L-arginine-based inhibitors bind to the enzyme in a folded conformation. The L-homoarginine derivatives (7 and 8) did not interact with the enzyme as either substrates or inhibitors. The two-carbon L-arginine homologue (9), prepared from L-phenylalanine, demonstrated the greatest isoform selective inhibition of the compounds examined (IC₅₀(iNOS) = 19 and IC₅₀(nNOS) = 147 μM, IC₅₀(nNOS)/IC₅₀i(NOS) = 7.7). These results suggest isoform selective inhibition may be related to the folded conformations required for binding of these higher L-arginine homologues.

Full text of DOI:10.1021/jo982161f

J . Org. Chem. 1999, 64, 3467-3475
3467
Asym m etr ic Syn th esis of Con for m a tion a lly Restr icted L-Ar gin in e
An a logu es a s Active Site P r obes of Nitr ic Oxid e Syn th a se
Robert N. Atkinson,† Lisa Moore,‡ J oseph Tobin,‡ and S. Bruce King*,†
Department of Chemistry, Wake Forest University, and Department of Anesthesiology, Wake Forest
University School of Medicine, Winston-Salem, North Carolina 27106
Received October 27, 1998
Using the catalytic asymmetric Sharpless carbamate aminohydroxylation, conformationally
restricted L-arginine and L-homoarginine derivatives (5-8) were prepared in good enantiomeric
excess to investigate the binding requirements of L-arginine-based compounds with nitric oxide
synthase. The L-arginine derivatives (5 and 6) inhibited both the inducible and neuronal isoforms
of nitric oxide synthase with little isoform selectivity (5, IC50 ) 42 and 144 µM, 6, 8 and 12 µM,
respectively). The guanidine-containing compound (5) did not act as a nitric oxide producing
substrate for nitric oxide synthase. The ability of these compounds to interact with the enzyme
supports the idea that L-arginine-based inhibitors bind to the enzyme in a folded conformation.
The L-homoarginine derivatives (7 and 8) did not interact with the enzyme as either substrates or
inhibitors. The two-carbon L-arginine homologue (9), prepared from L-phenylalanine, demonstrated
the greatest isoform selective inhibition of the compounds examined (IC50(iNOS) ) 19 and IC50-
(nNOS) ) 147 µM, IC50(nNOS)/IC50i(NOS) ) 7.7). These results suggest isoform selective inhibition
may be related to the folded conformations required for binding of these higher L-arginine
homologues.
Sch em e 1
In tr od u ction
Nitric oxide (NO) plays various roles in a number of
physiological processes.1 The biochemical production of
nitric oxide results from the stepwise oxidation of the
terminal guanidino group of L-arginine to initially pro-
duce L-NG-hydroxyarginine followed by a second oxidation
to form nitric oxide and L-citrulline (Scheme 1).2 The
nitric oxide synthases (NOS) catalyze this conversion that
requires 2 equiv of molecular oxygen and 1.5 equiv of
reduced nicotine adenine dinucleotide phosphate (NAD-
PH) as cosubstrates (Scheme 1).3 Three distinct mam-
malian NOS isoforms exist: endothelial NOS (eNOS) and
neuronal NOS (nNOS), which are constitutively ex-
pressed, and inducible NOS (iNOS).3 The selective inhi-
bition of the various NOS isoforms represents a potential
therapeutic approach for disease states where an over-
production of nitric oxide from a particular isoform is
implied.4 A pictorial model of the NOS active site based
upon experiments with various inhibitors and substrates
includes an amino acid, guanidinium, and heme binding
sites.5,6,7 Recent X-ray crystallographic structures of the
inducible NOS oxygenase domain with both inhibitors
and substrate provide more detailed active site structural
information regarding this isoform including the identi-
fication of a glutamic acid residue (Glu371) as a critical
binding point for both substrates and inhibitors.8,9 De-
spite the discovery of a number of isoform specific
inhibitors and the recent X-ray crystallographic studies,
the structural basis of isoform selective NOS inhibition
remains poorly understood.
The X-ray crystallographic structure of the inducible
NOS oxygenase domain with L-arginine indicates that
L-arginine binds to this truncated isoform in a somewhat
folded (not fully extended) conformation.9 Results from
a recent study with a group of conformationally restricted
L-phenylalanine-derived inhibitors (including 1 and 2),
which display modest selective inhibition of the constitu-
tive NOS isoforms, also suggest that L-arginine-derived
inhibitors preferentially bind to the enzyme in a folded
conformation.10 One-carbon homologues of L-arginine also
interact with nitric oxide synthase as L-homoarginine
* Phone: 336-758-5774. Fax: 336-758-4656. e-mail: kingsb@wfu.edu.
† Department of Chemistry.
‡ Department of Anesthesiology.
(1) Kerwin, J . F., J r.; Lancaster, J . R., J r.; Feldman, P. F. J . Med.
Chem. 1995, 38, 4343-4362.
(2) Stuehr, D. J .; Kwon, N. S.; Nathan, C. F.; Griffith, O. W.;
Feldman, P. L.; Wiseman, J . J . Biol. Chem. 1991, 266, 6259-6263.
(3) Griffith, O. W.; Stuehr, D. J . Annu. Rev. Physiol. 1995, 57, 707-
736.
(4) Fukuto, J . M.; Chaudhuri, G. Annu. Rev. Pharmacol. Toxicol.
1995, 35, 165-194.
(8) Crane, B. R.; Arvai, A. S.; Gachhui, R.; Wu, Chaoqun; Ghosh,
D. K.; Getzoff, E. D.; Stuehr, D. J .; Tainer, J . A. Science 1997, 278,
425-431.
(9) Crane, B. R.; Arvai, A. S.; Ghosh, D. K.; Wu, C.; Getzoff, E. D.;
Stuehr, D. J .; Tainer, J . A. Science 1998, 279, 2121-2126.
(10) Shearer, B. G.; Lee, S.; Franzmann, K. W.; White, H. A. R.;
Sanders, D. C. J .; Kiff, R. J .; Garvey, E. P.; Furfine, E. S. Biorg. Med.
Chem. Lett. 1997, 7, 1763-1768.
(5) Sennequier, N.; Stuehr, D. J . Biochemistry 1996, 35, 5883-5892.
(6) Feldman, P. L.; Chi, S.; Sennequier, N.; Stuehr, D. J . Biorg. Med.
Chem. Lett. 1996, 6, 111-114.
(7) Garvey, E. P.; Oplinger, J . A.; Tanoury, G. J .; Sherman, P. A.;
Fowler, M.; Marshall, S.; Harmon, M. F.; Paith, J . E.; Furfine, E. S. J .
Biol. Chem. 1994, 269, 26669-26676.
10.1021/jo982161f CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/29/1999

3468 J . Org. Chem., Vol. 64, No. 10, 1999
Atkinson et al.
acts as an NO-producing substrate and NG-L-(1-imino-
ethyl)lysine (3) preferentially inhibits the inducible iso-
form of NOS as compared to NG-L-(1-iminoethyl)ornithine
(4), which demonstrates little isoform selectivity.11,12
L-Homoarginine and 3 must adopt a less than fully
extended conformation to interact with the enzyme in a
fashion similar to L-arginine or 4, and these results
suggest that differences between the folded conformations
of homologous inhibitors may partially explain the struc-
tural basis of NOS isoform selective inhibition.
Sch em e 2
To further examine the effects of conformational
restriction on the interaction of homologous substrates
and inhibitors with NOS, we have prepared and bio-
chemically evaluated derivatives 5-10 as probes of the
NOS active site. The guanidine-containing derivatives (5,
7, and 9) represent constrained analogues of L-arginine,
L-homoarginine, and a two-carbon homologue of L-argi-
nine. The thiourea-containing derivatives (6, 8, and 10)
represent conformationally restricted homologues of the
known NOS inhibitor L-thiocitrulline.13
acids. The Sharpless reaction introduces the nitrogen
atom protected as either a BOC or Cbz carbamate, an
attractive feature for further synthetic manipulations.
The asymmetric preparation of the L-phenylglycines (5-
8) using the Sharpless asymmetric aminohydroxylation
demonstrates one of the first applications of this newly
described methodology to the synthesis of biologically
important compounds.14
The Sharpless carbamate aminohydroxylation com-
bined with an alcohol-carboxylic acid oxidation forms a
newly described synthetic strategy for the preparation
of enantiopure R-arylglycines from styrenes.14 Use of the
bis(dihydroquinyl)phthalazine ((DHQ)2-PHAL) ligand es-
tablishes the S-configuration of the amino alcohol ste-
reocenter and leads to the formation of natural L-amino
Resu lts a n d Discu ssion
Compounds 5 and 6 were prepared from commercially
available 3-nitrostyrene (Aldrich, Scheme 2). Selective
nitro group reduction using sodium hydrosulfite and a
phase- and electron-transfer catalyst to form amine (11)
followed by Cbz protection produced 12 in good yield.15
Gram-scale Sharpless asymmetric aminohydroxylation of
12 using the bis(dihydroquinyl)phthalazine ((DHQ)2-
PHAL) ligand yielded the BOC-protected amino alcohol
(13) in 66% yield and 96% ee after removal of small
(11) Yokoi, I. Kabuto, H.; Habu, H.; Inada, K.; Toma, J .; Mori, A.
Neuropharmacology 1994, 33, 1261-1265.
(12) Moore, W. M.; Webber, R. K.; J erome, G. M.; Tjoeng, F. S.;
Misko, T. P. Currie, M. G. J . Med. Chem. 1994, 37, 3886-3888.
(13) (a) Narayanan, K.: Griffith, O. W. J . Med. Chem. 1994, 37,
885-887. (b) Abu-Soud, H. M.; Feldman, P. L.; Clark, P.; Stuehr, D.
J . J . Biol. Chem. 1994, 269, 32318-32326.
(14) Reddy, K. L.; Sharpless, K. B. J . Am. Chem. Soc. 1998, 120,
1207-1217.
(15) Park, K. K.; Oh, C. H.; J oung, W. K. Tetrahedron Lett. 1993,
34, 7445-7446.

Asymmetric Synthesis of Restricted L-Arginine Analogues
J . Org. Chem., Vol. 64, No. 10, 1999 3469
Sch em e 3
amounts of the unwanted regioisomer and diol by flash
chromatography.14 Oxidation of 13 with sodium hy-
pochlorite, 2,2,6,6,-tetramethyl-1-piperidinyloxy free radi-
cal (TEMPO), and potassium bromide gave the carboxylic
acid (14) in 74% yield (Scheme 2).16 Hydrogenolysis of
the Cbz group of 14 followed by condensation with N,N′-
bis(BOC-1-guanyl)pyrazole in THF produced the pro-
tected guanidine (15) in 82% yield (Scheme 2).17 Acidic
deprotection of 15 formed the desired L-arginine deriva-
tive (5) in 85% yield and 96% ee (Scheme 2). Treatment
of 14 with diazomethane in ether yielded the correspond-
ing methyl ester (16, Scheme 2) in 97% yield. Hydro-
genolysis of the Cbz group of 16 followed by condensation
with thiophosgene and ammonia gave the thiourea (17)
in 81% yield. Deprotection of 17 using boron tribromide
(BBr3) afforded the L-thiocitrulline derivative (6) in 82%
yield and 75% ee (Scheme 2).18
Scheme 3 depicts the similar preparation of the L-
homoarginine derivatives (7 and 8) from commercially
available 4-aminostyrene (Aldrich). Cbz protection of the
amine to give 18 followed by gram-scale Sharpless
asymmetric aminohydroxylation using the ((DHQ)2-
PHAL) ligand formed the BOC-protected amino alcohol
(19) in 59% yield and 75% ee (Scheme 3).14 The TEMPO-
catalyzed oxidation of 19 proved unsuccessful, and 19 was
converted to the aldehyde (20) using the Dess-Martin
periodinane. Difficulties in the oxidation of alcohols
containing electron rich aromatic rings using this method
have been noted, and the poor solubility of 19 in acetone,
the reaction solvent, further complicated this oxidation.16
Oxidation of 20 with sodium chlorite, sodium dihydrogen
phosphate, and 2-methyl-2-butene formed the carboxylic
acid (21) in 78% yield (Scheme 3).19 Hydrogenolysis of
the Cbz group of 21 followed by condensation with N,N′-
bis(BOC-1-guanyl)pyrazole in THF produced the pro-
tected guanidine (22) in 81% yield (Scheme 3).17 Acidic
deprotection of 22 formed the desired L-homoarginine
derivative (7) in 68% yield and 75% ee (Scheme 3).
Treatment of 21 with diazomethane in ether yielded the
corresponding methyl ester (23, Scheme 3) in 84% yield.
Hydrogenolysis of the Cbz group of 23 followed by
condensation with thiophosgene and ammonia gave the
thiourea (24) in 69% yield and 60% ee. Deprotection of
24 using BBr3 afforded the L-homothiocitrulline deriva-
tive (8) in 52% yield and 60% ee (Scheme 3).18
4). Treatment of 28 with thiophosgene and ammonia gave
the thiourea (30) in 89% yield. Acidic deprotection of 30
afforded the derivative (10) in 98% yield and 99% ee
(Scheme 4).
Compounds 9 and 10 were prepared from L-phenyla-
lanine as outlined in Scheme 4. Nitration of L-phenyla-
lanine yielded 4-nitro-L-phenylalanine monohydrate (25)
in 53% yield (Scheme 4).20 Sequential protection of the
amino and carboxylic acid groups of 25 with BOC
anhydride and N,N-DMF-di-t-Bu acetal produced 26 and
27 in 92 and 74% yields, respectively (Scheme 4).
Catalytic hydrogenation of 27 formed amine (28) in 94%
yield. Nitro group reduction of 26 followed by condensa-
tion with N,N′-bis(BOC-1-guanyl)pyrazole in THF pro-
duced the protected guanidine (29) in 93% yield (Scheme
4).17 Acidic deprotection of 29 formed the desired L-
arginine derivative (9) in 98% yield and 99% ee (Scheme
These results indicate that carbamate-containing ole-
fins act as suitable substrates for the Sharpless carbam-
ate aminohydroxylation for the preparation of amino-
substituted R-arylglycines and do not interfere with the
catalytic cycle. While the regioselectivity of the amino-
hydroxylation of the meta- and para-substituted N-Cbz
styrenes (12 and 18) compares well with the results of
other reported styrenes,14 the enantioselectivity of ami-
nohydroxylation differed markedly (96% vs 75% ee) with
the enantiomeric excess of 19 being improved to 81% by
a single recrystallization. Both the regioselectivity and
enantioselectivity of the Sharpless aminohydroxylation
depend on the substrate structure, the asymmetric
ligand, and the reaction solvent.14 The influence of
substituent position (ortho vs meta vs para) on the
enantioselectivity of aminohydroxylation of substituted
styrenes has not been reported, and at this time the
(16) Anelli, P. L.; Biffi, C.; Montanari, F.; Quici, S. J . Org. Chem.
1987, 52, 2559-2562.
(17) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Tetrahedron Lett.
1993, 34, 3389-3392.
(18) Felix, A. M. J . Org. Chem. 1974, 39, 1427-1429.
(19) Boger, D. L.; Borzilleri, R. M.; Nukui, S. J . Org. Chem. 1996,
61, 3561-3565.
(20) Houghten, R. A.; Rapoport, H. J . Med. Chem. 1974, 17, 556-
558.

3470 J . Org. Chem., Vol. 64, No. 10, 1999
Atkinson et al.
Ta ble 1. In d u cible a n d Neu r on a l NOS In h ibition a n d
Sch em e 4
Selectivity by Com p ou n d s 5-10
IC50 (µM)
selectivity
compd
iNOS
nNOS
IC50(nNOS)/IC50(iNOS)
5
6
42
8
144
12
3.4
1.5
7
8
9
>1000
>1000
19
>1000
600
147
7.7
10
>1000
>1000
respectively). Chiral HPLC determination of the enan-
tiomeric excess of the individual synthetic intermediates
in these routes indicated that the loss of stereochemical
integrity occurred during the thiourea-forming step.
Treatment of the intermediate isothiocyanates formed by
catalytic hydrogenation and thiophosgene condensation
of 16 and 23 with excess ammonia resulted in complete
racemization. Despite careful monitoring of this conden-
sation by thin-layer chromatography to judge the reaction
progress and the use of 1 equiv of ammonia, partial
racemization occurred, suggesting that ammonia-medi-
ated acid-base reactions contributed to the loss of
stereochemical integrity of these compounds.
Preliminary biochemical evaluation in the presence of
both inducible nitric oxide synthase (iNOS) and neuronal
nitric oxide synthase (nNOS, a constitutive isoform)
indicated that none of the synthetic guanidine-containing
L-arginine derivatives (5, 7, or 9) supported nitrite
synthesis. The identification of nitrite, the stable oxida-
tive decomposition product of nitric oxide,22 provides
evidence for nitric oxide formation, and these results
demonstrate the inability of these compounds to act as
nitric oxide producing substrates. These results support
the reported strict structural requirements for nitric
oxide producing substrates of nitric oxide synthase.5 A
recently proposed mechanism suggests that proton dona-
tion from the guanidinium group of L-arginine plays an
important role in nitric oxide formation from the nitric
oxide synthase catalyzed oxidation of L-arginine and the
decreased basicity of these aryl guanidines (pKa ) 10.7
for phenylguanidine vs pKa ) 12.5 for L-arginine)23 may
partially explain their inability to act as substrates.9
Alternatively, the failure of these compounds to act as
NO-producing substrates may be due to their decrease
of the NOS heme midpoint reduction potential as dem-
onstrated for a number of other NOS inhibitors.24
Using the radioactive L-citrulline assay to assay NOS
activity,25 the L-thiocitrulline derivative (6) demonstrated
the greatest amount of inhibition of both the inducible
and neuronal isoforms of NOS (IC50 ) 8 and 12 µM,
respectively, Table 1). In comparison, the Ki values for
iNOS and nNOS inhibition by 1 have been determined
as 2.60 and 0.37 µM, respectively, and the Ki value for
iNOS inhibition by thiocitrulline has been determined
as 9 µM.10,13 The L-arginine derivative (5) also inhibited
both isoforms of the enzyme (IC50 ) 42 and 144 µM,
respectively, Table 1). Both 5 and 6 demonstrated a slight
observed enantioselectivity difference in the reaction of
12 and 18 appears best explained by a structural differ-
ence between the substrates.
The conversion of carboxylic acids 14 and 21 to the
guanidines 5 and 7 proceeded without the loss of enan-
tiomeric excess, highlighting the utility of this methodol-
ogy for the asymmetric preparation of enantiopure
L-phenylglycines. Retention time analysis following chiral
HPLC separation using a CROWNPAK CR-(+) column
(Chiral Technologies, Inc., Exton, PA) indicated the major
formation of the L-amino acid (S-configuration) for each
of the final amino acid products (5-10). Using this
column, natural and unnatural L-amino acids (S-config-
uration) consistently elute slower than D-amino acids (R-
configuration), allowing for the assignment of absolute
configuration.21 These results support the predicted
formation of the amino alcohols (13 and 19) with the
S-configuration from the aminohydroxylation of 12 and
18 using the bis(dihydroquinyl)phthalazine ((DHQ)2-
PHAL) ligand.14
(22) Ignarro, L. J .; Fukuto, J . M.; Griscavage, J . M.; Rogers, N. E.;
Byrns, R. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8103-8107.
(23) J encks, W. P.; Regenstein, J . Handbook of Biochemistry and
Molecular Biology; Fasman, G. D., Ed.; CRC Press: Cleveland, 1976;
pp 305-351.
(24) Presta, A.; Weber-Main, A. M.; Stankovich, M. T.; Stuehr, D.
J . J . Am. Chem. Soc. 1998, 120, 9460-9465.
(25) Moali, C.; Boucher, J . L.; Sari, M. A.; Stuehr, D. J .; Mansuy,
D. Biochemistry 1998, 37, 10453-10460.
A loss of enantiomeric excess occurred during the
preparation of the thioureas 6 and 8 (75 and 60% ee’s,
(21) Application Guide for Chiral Column Selection, 2nd ed.; Chiral
Technologies Inc.: Exton, PA.

Asymmetric Synthesis of Restricted L-Arginine Analogues
J . Org. Chem., Vol. 64, No. 10, 1999 3471
selectivity for the inducible isoform of the enzyme (IC50-
(nNOS)/IC50(iNOS) ) 3.4 and 1.5, respectively, Table 1).
These results support previous evidence that L-arginine
derivatives bind to the nitric oxide synthase active site
in a folded conformation as 5 and 6 represent the only
compounds in this study capable of adopting a folded
conformation similar to that found in 1. The ability of
these compounds to assume a fully extended conforma-
tion by rotation about the bond between the R carbon and
the aromatic ring may partially explain the decrease in
activity compared to 1.
Conformational restriction of L-homoarginine (com-
pounds 7 and 8) produced deleterious effects with regard
to the inhibition of either isoform of the enzyme (Table
1). These results contrast the ability of 2, a L-homoargi-
nine analogue with more conformational flexibility be-
tween the guanidinium and amino acid groups, to mod-
estly inhibit both inducible and neuronal NOS (Ki ) 100
and 44 µM, respectively).10 Other nonconformationally
restricted L-homoarginine derivatives, such as 3 and
L-homothiocitrulline, also demonstrate significant inhibi-
tion of NOS.12,13a While these other L-homoarginine
analogues interact with the enzyme, these results clearly
indicate that the orientation of the functional groups in
7 and 8 does not permit binding of these compounds to
NOS, and this lack of activity prevents the evaluation of
isoform selectivity.
The conformationally restricted two-carbon L-arginine
homologue (9) inhibited both the inducible and neuronal
isoforms of NOS (IC50 ) 19 and 147 µM, Table 1) and
displayed the greatest isoform selectivity of any of the
compounds examined (IC50(nNOS)/IC50(iNOS) ) 7.7,
Table 1). These results indicate that longer L-arginine
homologues can selectively interact with NOS isoforms
by adopting a folded conformation. D,L-Homo-NG-(1-
iminoethyl)lysine, the one-carbon homologue of 3, also
preferentially inhibits inducible NOS compared to con-
stitutive NOS (IC50 ) 73 and 808 µM, respectively, IC50-
(nNOS)/IC50(iNOS) ) 11).12 These results suggest that
differences in the folded conformations of L-arginine
derivatives and L-arginine homologues partially explain
the isoform selectivity observed by these longer homo-
logues. The thiourea (10) did not demonstrate any
interaction with the enzyme, possibly due to the loss of
hydrogen-bonding interactions from the replacement of
the guanidine group of 9 with the thiourea group of 10.
To conclude, the catalytic asymmetric Sharpless ami-
nohydroxylation provided a convenient route for the
preparation of the R-arylglycines (5-8). The ability of the
L-arginine derivatives 5 and 6 to inhibit both the induc-
ible and neuronal isoforms of NOS provides further
evidence that L-arginine-derived compounds bind to the
active site in a folded conformation. The ability of the
conformationally restricted two-carbon L-arginine homo-
logue (9) to preferentially inhibit the inducible isoform
suggests that the folded conformations adopted by ho-
mologous inhibitors partially controls isoform selective
inhibition of NOS. However, the recent report of the Km
values of L-homoarginine with iNOS and nNOS (33 and
23 µM, respectively) weakens the proposal that chain
length confers specificity.25 The inability of 7 and 8 to
interact with NOS also limits the confidence of any
conclusions regarding the relationship of chain length
and conformation on isoform selectivity. Modified sub-
strates, such as 2, 5, 7, and 9, may bind to the NOS
isoforms differently, and the active site deformability of
each isoform should be considered in the design of future
inhibitors. Further studies with other compounds com-
bined with X-ray crystallographic data from the other
NOS isoforms will provide useful information toward
understanding the structural basis of NOS isoform
selective inhibition.
Exp er im en ta l Section
Ch em istr y: Gen er a l. Melting points were determined on
a Mel-Temp apparatus and are uncorrected. Analytical thin-
layer chromatography (TLC) was performed on silica gel 60
F-254 precoated plates. Flash chromatography was performed
on silica gel 60 (230-400 mesh). Proton and carbon NMR
spectra were taken in commercial deuterated solvents on a
Varian VXR 200 multinuclear spectrometer. Infrared spectra
were obtained on a Perkin-Elmer 1600 Series FTIR spectrom-
eter. Organic solvents were distilled from a drying agent prior
to use. Commercially available reagents were used without
further purification.
3-Am in ostyr en e (11). A solution of K2CO3 (14.86 g, 107.5
mmol) and Na2S2O4 (16.85 g, 96.8 mmol) in water (75 mL) was
added dropwise to a mixture of 3-nitrostyrene (3.00 mL, 21.5
mmol) and 1,1′-dioctyl-4,4′-bipyridinium bromide (0.58 g, 1.0
mmol) in CH2Cl2/H2O (150 mL/20 mL) under an argon
atmosphere. The reaction mixture was heated at 35 °C for 8 h
with constant stirring. The organic layer was collected, and
the aqueous layer was extracted with CH2Cl2 (3 × 50 mL).
The combined organic layers were dried (MgSO4), filtered, and
concentrated in vacuo. The crude product was purified by flash
chromatography (1:4 EtOAc:hexanes) to afford 11 (2.31 g, 90%)
as a yellow oil: Rf 0.35 (1:4 EtOAc:hexanes); 1H NMR (CDCl3,
200 MHz) δ 7.13 (t, J ) 8 Hz, 1H), 6.83 (d, J ) 8 Hz, 1H), 6.73
(s, 1H), 6.62 (m, 2H), 5.70 (d, J ) 18 Hz, 1H), 5.20 (d, J ) 11
Hz, 1H), 3.85 (br. s, 2H); 13C NMR (CDCl3, 50 MHz) δ 146.4,
138.1, 136.7, 128.9, 116.2, 114.3, 113.1, 112.2; IR (CDCl3) 3462
cm-1; LRMS (EI) m/z 120 (M + H)+.
N-Ben zyloxyca r bon yl-3-a m in ostyr en e (12). To a solu-
tion of 11 (1.00 g, 8.4 mmol) and 2,6-lutidine (1.95 mL, 16.8
mmol) in CH2Cl2 (30 mL) at 0 °C was added benzyl chlorofor-
mate (1.80 mL, 12.6 mmol) over 10 min. The reaction was
allowed to warm to room temperature and after completion
as judged by thin-layer chromatography was washed with H2O
(20 mL). The organic layer was collected, dried (MgSO4),
filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography (1:7 EtOAc:hexanes) to
afford 12 (2.04 g, 96%) as an amber oil: Rf ) 0.34 (1:7 EtOAc:
1
hexanes); H NMR (CDCl3, 200 MHz) δ 7.50 (s, 1H), 7.41 (m,
5H), 7.29 (d, J ) 6 Hz, 2H), 7.17 (s, 1H), 6.90 (br. s, 1H), 6.71
(dd, J ) 11, 18 Hz, 1H), 5.79 (d, J ) 18 Hz, 1H), 5.29 (d, J )
11 Hz, 1H), 5.23 (s, 2H); 13C NMR (CDCl3, 50 MHz) δ 153.4,
138.0, 137.9, 136.2, 135.7, 128.7, 128.2, 127.9, 127.8, 121.0,
118.0, 116.3, 114.0, 66.6; IR (CDCl3) 3434, 1735, cm-1; LRMS
(FAB) m/z 253 (M+), 276 (M + Na)+. Anal. Calcd for C16H15
-
NO2: C, 75.86; H, 5.96; N, 5.52. Found: C, 75.87; H, 5.99; N,
5.46.
1(S)-N-[(ter t-Bu t yloxy)ca r b on yl]-1-[3-N-(Ben zyloxy)-
ca r bon yla m in op h en yl]-2-h yd r oxyeth yla m in e (13). A so-
lution of NaOH (0.482 g, 12.1 mmol) in H2O (30 mL) followed
by tert-butyl hypochlorite (1.437 mL, 12.1 mmol) was added
to a solution of tert-butyl carbamate (1.435 g, 12.3 mmol) in
n-propyl alcohol (15 mL) and cooled to -5 °C. (DHQ)2-PHAL
(0.1846 g, 0.237 mmol) in n-propyl alcohol (15 mL) followed
by 12 (1.00 g, 3.95 mmol) in n-propyl alcohol (30 mL) and OsO4
(0.040 g, 0.158 mmol) in H2O (1 mL) was added to this cooled
mixture. The reaction mixture was stirred at -5 °C overnight
and quenched with Na2SO3. The reaction mixture was ex-
tracted with EtOAc (3 × 50 mL), and the combined organic
layers were dried (MgSO4), filtered, and concentrated in vacuo.
The crude product was purified by flash chromatography (2:3
EtOAc:hexanes) to afford 13 (1.00 g, 66%) as a white solid:
mp ) 113.0-113.5 °C. The enantiomeric excess (ee) of 13 was
determined to be 96% by HPLC (CHIRALCEL OD-H, 0.4 cm
φ × 25 cm, hexane:isopropyl alcohol, 65:35, 1.0 mL/min, UV

3472 J . Org. Chem., Vol. 64, No. 10, 1999
Atkinson et al.
254 nm, tR (R-enantiomer) 5.6 min, (S-enantiomer) 10.3 min);
1.39 (s, 9H); 13C NMR (CDCl3, 50 MHz) δ 171.3, 163.3, 154.7,
153.4, 153.1, 137.4, 137.1, 129.4, 123.5, 122.6, 120.7, 83.7, 80.0,
79.5, 57.3, 52.6, 28.1, 28.0, 27.9; IR (CDCl3) 3440, 1718, 1704
cm-1; LRMS (FAB) m/z 523.3 (M + H)+. Anal. Calcd for
C25H38N4O8: C, 57.46; H, 7.32; N, 10.72. Found: C, 57.41; H,
7.40; N, 10.61.
Rf ) 0.25 (2:3 EtOAc:hexanes); [R]20 +31.7 (c ) 1.26, EtOH);
D
1H NMR (CDCl3, 200 MHz) δ 7.33 (m, 5H), 7.25 (m, 3H), 6.95
(m, 2H) 5.34 (d, J ) 6 Hz, 1H), 5.14 (s, 2H), 4.65 (br. m, 1H),
3.73 (br. m, 2H), 2.63 (br. m, 1H), 1.38 (s, 9H); 13C NMR
(CDCl3, 50 MHz) δ 156.0, 153.5, 140.6, 138.1, 135.9, 128.9,
128.3, 128.0, 121.3, 117.8, 116.9, 79.7, 66.6, 65.7, 56.4, 28.1;
IR (CDCl3) 3600, 3435, 1730, 1712 cm-1; LRMS (FAB) m/z 387
(M + H)+. Anal. Calcd for C21H26N2O5: C, 65.27; H, 6.78; N,
7.25. Found: C, 65.23; H, 6.81; N, 7.23.
3-Gu a n yl-L-p h en ylglycin e Hyd r och lor id e (5). A solution
of 15 (0.400 g, 0.787 mmol) in HCl/dioxane (4 M, 10 mL,
Aldrich) was stirred at 23 °C for 12 h under an argon
atmosphere. The mixture was concentrated in vacuo, dissolved
in H2O (10 mL), passed through a 6 mL Supelco LC-18 solid-
phase extractor, and lyopholyzed to yield 5 (0.188 g, 85%) as
a white solid. The enantiomeric excess of 5 was determined to
be 96% by HPLC (CROWNPAK CR(+), 0.4 cm φ × 15 cm,
aqueous HClO4, pH ) 1.5, 0.8 mL/min, UV 254 nm, tR
(R-enantiomer) 2.9 min, (S-enantiomer) 6.8 min); [R]20D +65.4
(c ) 0.43, H2O); 1H NMR (D2O, 200 MHz) δ 7.31 (m, 4H), 5.03
(s, 1H); 13C NMR (D2O, 50 MHz) δ 170.8, 156.4, 135.5, 133.8,
131.4, 127.5, 127.4, 125.6, 56.5; IR (film, 3M IR-card, polyeth-
ylene mesh) broad 3614-2350, 1732 cm-1; LRMS (FAB) m/z
209.1 (M + H)+; HRMS (FAB) calcd for C9H13N4O2 209.1038,
found 209.1307.
(S)-N-[(ter t-Bu t yloxy)ca r b on yl]-[3-N-(Ben zyloxy)ca r -
bon yla m in op h en yl]glycin e (14) a n d Meth yl (S)-N-[(ter t-
Bu t yloxy)ca r b on yl]-[3-N-(Ben zyloxy)ca r b on yl a m in o-
p h en yl]glycin e (16). A 5% aqueous solution of NaHCO3 (5
mL), KBr (0.012 g, 0.103 mmol) and TEMPO (0.182 g, 1.17
mmol) was added to a solution of 13 (0.400 g, 1.03 mmol) in
acetone (5 mL) cooled to 0 °C. To this mixture was added an
aqueous NaOCl solution (1.59 mL, 1.36 mmol, 5.25% Clorox
Bleach) dropwise over 10 min. After 1 h, additional aqueous
NaOCl solution (0.76 mL, 0.647 mmol) was added dropwise
over 5 min. The reaction mixture was stirred for an additional
hour, 20 mL of H2O was added, and the aqueous mixture was
extracted with EtOAc (3 × 50 mL). The combined organic
layers were collected, dried (MgSO4), filtered, and concentrated
in vacuo. The crude product was purified by flash chromatog-
raphy (EtOAc) to afford 14 (0.3093 g, 74%) as a yellowish
Met h yl (S)-N-[(ter t-Bu t yloxy)ca r b on yl]-[3-t h iou r ea -
p h en yl]glycin e (17). A mixture of 16 (1.00 g, 2.41 mmol) and
10% Pd-C (50 mg) in MeOH (20 mL) was shaken in a Parr
hydrogenator under 60 psi of H2 for 12 h. The mixture was
filtered through Celite to remove the catalyst, and the filtrate
was concentrated under vacuum to give an oil. This oil was
dissolved in CH2Cl2 (20 mL) and added to a mixture of CaCO3
(0.361 g, 3.62 mmol), H2O (20 mL), CH2Cl2 (20 mL), and
thiophosgene (0.220 mL, 2.89 mmol). This mixture was vigor-
ously stirred at 23 °C, and after 1 h the organic phase was
separated and the aqueous phase was extracted with CH2Cl2
(3 × 10 mL). The combined organic layers were dried (MgSO4),
filtered, and concentrated, yielding a yellow oil. A solution of
NH3 (2.0 M, 3.62 mmol) in EtOH (11.81 mL) was added to the
crude isothiocyanate with vigorous stirring. The reaction was
monitored closely by thin-layer chromatography and concen-
trated in vacuo, and the crude product was purified by flash
chromatography (1:1 EtOAc:pentane) to afford 17 (0.661 g,
81%) as a white foam. The enantiomeric excess of 17 was
determined to be 88% by HPLC (CHIRALPAK AD, 0.4 cm φ
× 25 cm, hexane:EtOH:DEA, 85:15:0.1, 1.5 mL/min, UV 254
nm, tR (S-enantiomer) 14.9 min, (R-enantiomer) 28.2 min); Rf
foam: Rf ) 0.22 (EtOAc); [R]20 +68.3 (c ) 0.75, EtOH).
D
A solution of CH2N2 (0.5 M, 12.5 mmol) in Et2O (25 mL)
was added dropwise to a cooled solution (0 °C) of 14 (1.00 g,
2.50 mmol) in Et2O (10 mL). The reaction mixture was
concentrated in vacuo, and the crude product was purified by
flash chromatography (1:4 EtOAc:pentane) to afford 16 (1.001
g, 97%) as a white foam. The enantiomeric excess of 16 was
determined to be 96% by HPLC (CHIRALPAK AD, 0.4 cm φ
× 25 cm, hexane:isopropyl alcohol, 85:15, 1.25 mL/min, UV
254 nm, tR (R-enantiomer) 11.5 min, (S-enantiomer) 14.5 min);
Rf ) 0.22 (1:4 EtOAc:pentane); [R]20 +90.4 (c ) 1.50, CH2-
D
1
Cl2); H NMR (CDCl3, 200 MHz) δ 7.48-7.27 (m, 10H), 7.00
(d, J ) 8 Hz, 1H), 5.67 (d, J ) 7 Hz, 1H), 5.22 (d, J ) 7 Hz,
1H), 5.15 (s, 2H), 3.62 (s, 3H), 1.35 (s, 9H); 13C NMR (CDCl3,
50 MHz) δ 171.6, 154.7, 153.2, 138.7, 137.1, 135.9, 129.4, 128.4,
128.1, 121.9, 118.6, 117.1, 80.1, 66.7, 57.5, 52.6, 28.0; IR
(CDCl3) 3433, 1740, 1704 cm-1; LRMS (FAB) m/z 421 (M +
Li)+. Anal. Calcd for C22H26N2O6: C, 63.75; H, 6.32; N, 6.75.
Found: C, 63.70; H, 6.33; N, 6.84.
) 0.26 (1:1 EtOAc:pentane); [R]20 +93.6 (c ) 0.30, CH2Cl2);
D
(S)-N-[(ter t-Bu tyloxy)ca r bon yl]-[3-N, N′-bis(ter t-bu tyl-
oxy)ca r bon yl(gu a n yl)a m in op h en yl]glycin e (15). A mix-
ture of 14 (0.700 g, 1.75 mmol) and 10% Pd-C (50 mg) in
MeOH (20 mL) was shaken in a Parr hydrogenator under 60
psi of H2 for 12 h. The mixture was filtered through Celite to
remove the catalyst, and the filtrate was concentrated under
vacuum to give an oil. N,N′-Bis(Boc-1-guanyl)pyrazole (0.813
g, 2.63 mmol) in dry THF (10 mL) was added under argon and
the mixture reacted for 3 days and concentrated in vacuo. The
crude product was purified by flash chromatography (2:3:
0.0.025 EtOAc:pentane:AcOH) to afford 15 (0.732 g, 82%) as
a white foam: Rf 0.28 (2:3:0.0.025 EtOAc:pentane:AcOH);1H
NMR (CDCl3, 200 MHz) δ 10.32 (br s, 1H), 7.77 (d, J ) 8 Hz,
1H), 7.30 (m, 2H), 7.07 (d, J ) 8 Hz, 1H), 5.53 (d, J ) 7 Hz,
1H), 5.13 (d, J ) 7 Hz, 1H), 1.48 (s, 9H), 1.46 (s, 9H), 1.40 (s,
9H); 13C NMR (CDCl3, 50 MHz) δ 172.6, 162.6, 154.8, 153.2,
153.0, 138.4, 137.5, 129.4, 123.5, 122.6, 120.7, 83.7, 80.2, 79.6,
56.5, 28.1, 27.9, 27.6; IR (CDCl3) 3435, 3260, 1712 cm-1; LRMS
(FAB) m/z 523.3 (M + H)+. Carboxylic acid (15) was further
characterized by conversion to the methyl ester. A solution of
CH2N2 (0.5 M, 5.00 mmol) in Et2O (10 mL) was added dropwise
to a cooled solution (0 °C) of 15 (0.100 g, 0.197 mmol) in Et2O
(10 mL). After addition, the reaction mixture was concentrated
in vacuo and crude product was purified by flash chromatog-
raphy (1:7 EtOAc:pentane) to afford the methyl ester of 15
1H NMR (CDCl3, 200 MHz) δ 8.47 (br s, 1H) 7.43-7.16 (m,
4H), 6.33 (br s, 2H), 5.82 (d, J ) 6 Hz, 1H), 5.31 (d, J ) 6 Hz,
1H), 3.71 (s, 3H), 1.38 (s, 9H); 13C NMR (CDCl3, 50 MHz) δ
180.3, 171.0, 154.7, 138.3, 137.1, 130.0, 125.1, 124.1, 122.9,
80.1, 56.9, 52.7, 27.9; IR (CDCl3) 3506, 3433, 1740, 1704 cm-1
;
LRMS (FAB) m/z 340.1 (M
15H21N3O4S: C, 53.08; H, 6.23; N, 12.38; S, 9.44. Found: C,
+
H)+. Anal. Cacld for
C
53.34; H, 6.41; N, 11.75; S, 9.01.
3-Th iou r ea -L-p h en ylglycin e Tr iflu or oa ceta te (6). A
solution of BBr3 (1.0 M, 2.65 mmol) in CH2Cl2 (2.65 mL) was
added to a cooled (-78 °C, acetone dry ice solution) solution
of 17 (0.180 g, 0.531 mmol) in CH2Cl2 (20 mL) over 5 min with
stirring. The reaction mixture was allowed to warm to 25 °C,
stirred for 1 h, and quenched by the careful dropwise addition
of water (30 mL). The aqueous phase was separated and the
organic phase extracted with H2O (3 × 20 mL). The combined
aqueous layers were lyopholized to dryness, and the crude
product (82% yield) was purified by preparative HPLC (Econo-
sil-C-18 10 µ, 280 × 10 mm, aqueous 0.1% TFA, 4.0 mL/min,
UV 254 nm, tR 12.2 min.) to afford 6 (0.071 g, 29%) as a white
solid. The enantiomeric excess of 6 was determined to be 75%
by HPLC (CROWNPAK CR(+), 0.4 cm φ × 15 cm, aqueous
HClO4, pH ) 1.5, 1.3 mL/min, UV 254 nm, tR (R-enantiomer)
2.6 min, (S-enantiomer) 10.5 min); [R]20 +64.2 (c ) 0.35,
D
1
MeOH); H NMR (CD3OD, 200 MHz) δ 7.28 (m, 4H), 4.46 (s,
(0.0988 g, 96%) as a white foam: Rf ) 0.23 (1:7 EtOAc:
1H); 13C NMR (D2O, 50 MHz) δ 179.6, 172.9, 137.7, 135.7,
130.9, 127.0, 126.8, 125.4, 58.1; IR (film, 3M IR-card, polyeth-
ylene mesh) broad 3462-2583, 1733 cm-1; LRMS (FAB) m/z
226.1 (M + H)+; HRMS (FAB) calcd for C9H12N3O2S 226.0650,
found 226.0639.
1
pentane); [R]20 +83.5 (c ) 0.700, CH2Cl2); H NMR (CDCl3,
D
200 MHz) δ 10.30 (br. s, 1H), 7.71 (d, J ) 8 Hz, 1H), 7.35-
7.24 (m, 2H), 7.08 (d, J ) 8 Hz, 1H), 5.54 (d, J ) 7 Hz, 1H),
5.24 (d, J ) 7 Hz, 1H), 3.68 (s, 3H), 1.50 (s, 9H), 1.47 (s, 9H),

Asymmetric Synthesis of Restricted L-Arginine Analogues
J . Org. Chem., Vol. 64, No. 10, 1999 3473
N-Ben zyloxyca r bon yl-4-a m in ostyr en e (18). Benzyl chlo-
roformate (5.46 mL, 38.3 mmol) was added to a solution of
4-aminostyrene (3.00 mL, 25.5 mmol) and 2,6-lutidine (5.91
mL, 51.1 mmol) at 0 °C over 10 min. The reaction mixture
was allowed to warm to room temperature and after comple-
tion (judged by thin layered chromatography) was washed with
H2O (50 mL). The organic layer was collected, dried (MgSO4),
filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography (1:10 EtOAc:pentane) to
afford 18 (6.13 g, 95%) as a white solid: mp ) 86.0-88.5 °C:
flash chromatography (1:3 EtOAc:pentane) to afford 23 (0.1735
g, 84%) as a white foam. The enantiomeric excess of 23 was
determined to be 75% by HPLC (CHIRALPAK AD, 0.4 cm φ
× 25 cm, hexane:isopropyl alcohol, 85:15, 1.5 mL/min, UV 254
nm, tR (R-enantiomer) 20.5 min, (S-enantiomer) 35.8 min); Rf
) 0.30 (1:3 EtOAc:pentane); [R]20 +72.0 (c ) 1.70, CH2Cl2);
D
1H NMR (CDCl3, 200 MHz) δ 7.32-7.20 (m, 10H), 6.87 (s, 1H),
5.52 (d, J ) 7 Hz, 1H), 5.21 (d, J ) 7 Hz, 1H), 5.15 (s, 2H),
3.65 (s, 3H), 1.38 (s, 9H); 13C NMR (CDCl3, 50 MHz) δ 171.6,
154.7, 153.3, 138.1, 135.8, 131.4, 128.4, 128.1, 126.1, 127.7,
118.8, 80.0, 66.8, 56.9, 52.5, 28.1; IR (CDCl3) 3418, 1740, 1711
cm-1; LRMS (FAB) m/z 415 (M + H)+. Anal. Cacld for
1
Rf 0.42 (1:10 EtOAc:pentane); H NMR (CDCl3, 200 MHz) δ
7.33 (m, 9H) 6.66 (m, 3H), 5.65 (d, J ) 17 Hz, 1H), 5.18 (m,
3H); 13C NMR (CDCl3, 50 MHz) δ 153.3, 137.2, 135.9, 135.8,
132.6, 128.3, 128.1, 128.0, 126.6, 118.6, 112.4, 66.7; IR (CDCl3)
3690, 1732 cm-1; LRMS (FAB) m/z 253 (M+). Anal. Calcd for
C
22H26N2O6: C, 63.75; H, 6.32; N, 6.75. Found: C, 63.62; H,
6.35; N, 6.68.
(S)-N-[(ter t-Bu tyloxy)ca r bon yl]-[4-N, N′-bis(ter t-bu tyl-
oxy)ca r bon yl(gu a n yl)a m in op h en yl]glycin e (22). A mix-
ture of 21 (0.714 g, 1.78 mmol) and 10% Pd-C (50 mg) in
MeOH (20 mL) was shaken in a Parr hydrogenator under 60
psi of H2 for 12 h. The mixture was filtered through Celite to
remove the catalyst and the filtrate concentrated under
vacuum to give an oil. N,N′-Bis(BOC-1-guanyl)pyrazole (1.103
g, 3.56 mmol) in dry THF (10 mL) was added to this oil under
argon. After 4 days the reaction mixture was concentrated in
vacuo and the crude product was purified by flash chroma-
tography (1:1:0.5% EtOAc:pentane:AcOH) to afford 22 (0.738
C
16H15NO2: C, 75.87; H, 5.97; N, 5.53. Found: C, 75.77; H,
5.97; N, 5.51.
1(S)-N-[(ter t-Bu t yloxy)ca r b on yl]-1-[4-N-(Ben zyloxy)-
ca r bon yla m in op h en yl]-2-h yd r oxyeth yla m in e (19). A so-
lution of NaOH (1.446 g, 36.2 mmol) in H2O (60 mL) followed
by tert-butyl hypochlorite (4.3131 mL, 36.2 mmol) was added
to a solution of tert-butyl carbamate (4.307 g, 37.3 mmol) in
n-propyl alcohol (30 mL) with stirring and was cooled to -5
°C. (DHQ)2-PHAL (0.553 g, 0.710 mmol) in n-propyl alcohol
(30 mL) followed by 18 (3.00 g, 11.8 mmol) in n-propyl alcohol
(60 mL) and OsO4 (0.120 g, 0.472 mmol) in H2O (6 mL) was
added to this solution. The reaction mixture was stirred at
-5 °C overnight and quenched with Na2SO3. The reaction
solution was extracted with EtOAc (3 × 50 mL), and the
combined organic layers were dried (MgSO4), filtered, and
concentrated in vacuo. The crude product was purified by flash
chromatography (2:3 EtOAc:pentane) to afford 19 (2.71 g, 59%)
as a white solid: mp ) 170.0-172.0 °C. The enantiomeric
excess of 19 was determined to be 75% by HPLC (CHIRALCEL
OD-H, 0.4 cm φ × 25 cm, hexane:isopropyl alcohol, 65:35, 1.5
mL/min, UV 254 nm, tR (S-enantiomer) 3.6 min, (R-enanti-
g, 81%) as a white foam: Rf 0.33 (1:1:0.5% EtOAc:pentane:
1
AcOH); [R]20 +69.3 (c ) 1.50, CH2Cl2); H NMR (CDCl3, 200
D
MHz) δ 10.30 (br s, 1H), 7.47 (d, J ) 8 Hz, 2H), 7.30 (d, J )
8 Hz, 2H), 5.68 (d, J ) 6 Hz, 1H), 5.13 (d, J ) 6 Hz, 1H), 1.48
(s, 18H), 1.39 (s, 9H); 13C NMR (CDCl3, 50 MHz) δ 172.7, 162.7,
154.9, 153.3, 153.0, 136.2, 134.4, 127.5, 122.1, 83.6, 81.1, 79.6,
56.1, 28.1, 27.8, 27.7; IR (CDCl3) 3434, 1713 cm-1; LRMS (FAB)
m/z 509.3 (M + H)+. Carboxylic acid 22 was further character-
ized by conversion to the methyl ester as follows. A solution
of CH2N2 (0.5 M, 5.00 mmol) in Et2O (10 mL) was added
dropwise to a cooled solution (0 °C) of 22 (0.150 g, 0.302 mmol)
in Et2O (10 mL). The reaction mixture was concentrated in
vacuo, and the crude product was purified by flash chroma-
tography (1:5 EtOAc:pentane) to afford the methyl ester of 22
(0.148 g, 94%) as a white foam: Rf ) 0.33 (1:5 EtOAc:pentane);
omer) 9.1 min); Rf ) 0.19 (2:3 EtOAc:pentane); [R]20 +46.19
D
1
(c ) 1.05, acetone); H NMR (acetone-d6, 200 MHz) δ 8.73 (s,
1H), 7.40 (m, 9H), 6.25 (d, J ) 6 Hz, 1H), 5.15 (s, 2H), 4.64
(br. m, 1H), 3.70 (d, J ) 6 Hz, 2H), 2.90 (br. s, 1H), 1.36 (s,
9H); 13C NMR (acetone-d6, 50 MHz) δ 156.2, 154.3, 138.9,
137.8, 136.8, 129.2, 128.8, 128.7, 128.1, 118.9, 78.7, 66.7, 66.3,
57.3, 7.5; IR (CDCl3) 3689-3600, 1700 cm-1; LRMS (FAB) m/z
387 (M + H)+. Anal. Calcd for C21H26N2O5: C, 65.27; H, 6.78;
N, 7.25. Found: C, 65.26; H, 6.71; N, 7.24.
[R]20 +68.3 (c ) 1.30, CH2Cl2); 1H NMR (CDCl3, 200 MHz) δ
D
10.31 (br s, 1H), 7.55 (d, J ) 8 Hz, 2H), 7.28 (d, J ) 8 Hz, 2H),
5.52 (d, J ) 7 Hz, 1H), 5.23 (d, J ) 7 Hz, 1H), 3.68 (s, 3H),
1.50 (s, 9H), 1.47 (s, 9H), 1.40 (s, 9H); 13C NMR (CDCl3, 50
MHz) δ 171.3, 163.3, 154.5, 153.3, 153.1, 136.7, 133.0, 127.5,
122.4, 83.6, 79.9, 79.5, 56.9, 52.5, 28.1, 27.9 (2C); IR (CDCl3)
3440, 1711 cm-1; LRMS (FAB) m/z 523.3 (M + H)+. Anal. Calcd
for C25H38N4O8: C, 57.46; H, 7.32; N, 10.72. Found: C, 58.12;
H, 7.47; N, 10.30.
Ald eh yd e 20, (S)-N-[(ter t-Bu t yloxy)ca r b on yl]-[3-N-
(Ben zyloxy)car bon ylam in oph en yl]glycin e (21) an d Meth -
yl (S)-N-[(ter t-Bu tyloxy)ca r bon yl]-[4-N-(Ben zyloxy)ca r -
bon yla m in op h en yl]glycin e (23). Dess-Martin periodinane
reagent (0.330 g, 0.778 mmol) was added to a solution of 19
(0.150 g, 0.389 mmol) in CH2Cl2 (50 mL) at 0 °C with stirring.
The reaction mixture was gradually warmed to room temper-
ature, stirred for an additional 30 min, and quenched with a
saturated aqueous solution of NaHCO3 containing 1.5 g of
Na2S2O3 (25 mL). The organic layer was collected, and the
aqueous layer was washed with CH2Cl2 (3 × 25 mL). The
combined organic layers were dried (MgSO4), filtered, and
concentrated in vacuo. The crude product was purified by flash
chromatography (1:3 EtOAc:pentane) to afford 20 (0.1344 g,
90%) as a yellow oil: Rf ) 0.21 (1:3 EtOAc:pentane).
4-Gu a n yl-L-p h en ylglycin e Hyd r och lor id e (7). A solution
of 22 (0.276 g, 0.544 mmol) in HCl/dioxane (4 M, 10 mL,
Aldrich) was stirred at 23 °C for 12 h under an argon
atmosphere. The mixture was concentrated in vacuo, dissolved
in H2O (10 mL), passed through a 6 mL Supelco LC-18 solid-
phase extractor, and lyopholyzed to yield 7 (0.162 g, 68%) as
a white solid. The enantiomeric excess of 7 was determined to
be 75% by HPLC (CROWNPAK CR(+), 0.4 cm φ × 15 cm,
aqueous HClO4, pH ) 1.5, 0.8 mL/min, UV 254 nm, tR
(R-enantiomer) 2.7 min, (S-enantiomer) 9.3 min); [R]20D +71.2
1
(c ) 1.62, MeOH); H NMR (D2O, 200 MHz) δ 7.38 (d, J ) 6
A solution of NaClO2 (Aldrich, 80%, 0.659 g, 5.83 mmol) and
NaH2PO4 (0.626 g, 4.54 mmol) in H2O (8 mL) was added
dropwise to a solution of 20 (0.250 g, 0.648 mmol) and
2-methyl-2-butene (11.4 mL, 108 mmol) in t-BuOH (20 mL)
at 25 °C. After stirring for 30 min, the reaction mixture was
concentrated in vacuo, yielding a crude residue. The residue
was diluted with H2O (20 mL) and extracted with EtOAC (3
× 50 mL). The combined organic layers were dried (MgSO4),
filtered, and concentrated in vacuo, and the crude product was
purified by flash chromatography (EtOAc) to afford 21 (0.201
g, 78%) as a white foam: Rf ) 0.33 (0.1% AcOH in EtOAc).
A solution of CH2N2 (0.5 M, 0.75 mmol) in Et2O (1.5 mL)
was added dropwise to a cooled solution (0 °C) of 21 (0.200 g,
0.500 mmol) in Et2O (10 mL). The reaction mixture was
concentrated in vacuo, and the crude product was purified by
Hz, 2H), 7.21 (d, J ) 6 Hz, 2H), 5.06 (s, 1H); 13C NMR (D2O,
50 MHz) δ 170.5, 156.1, 136.2, 130.7, 129.9, 126.4, 56.0; IR
(film, 3M IR-card, polyethylene mesh) broad 3614-2328, 3410,
1732 cm-1; LRMS (FAB) m/z 209.1 (M + H)+, 231.1 (M + Na)+;
HRMS (FAB) calcd for C9H12N4O2Na 231.0857, found 231.0854.
Met h yl (S)-N-[(ter t-Bu t yloxy)ca r b on yl]-[4-t h iou r ea -
p h en yl]glycin e (24). A mixture of 23 (0.133 g, 0.321 mmol)
and 10% Pd-C (10 mg) in MeOH (20 mL) was shaken in a
Parr hydrogenator under 60 psi of H2 for 12 h. The mixture
was filtered through Celite to remove the catalyst and the
filtrate concentrated under vacuum to give an oil. This oil was
dissolved in CH2Cl2 (20 mL) and added to a mixture of CaCO3
(0.0482 g, 0.482 mmol), H2O (20 mL), CH2Cl2 (20 mL), and
thiophosgene (0.0306 mL, 0.401 mmol). This mixture was
vigorously stirred at 23 °C, and after 1 h the organic phase

3474 J . Org. Chem., Vol. 64, No. 10, 1999
Atkinson et al.
was separated and the aqueous phase extracted with CH2Cl2
(3 × 10 mL). The combined organic layers were dried (MgSO4),
filtered, and concentrated, yielding a yellow oil. A solution of
NH3 (2.0 M, 0.241 mL) in EtOH (10.241 mL) was added to the
crude isothiocyanate and stirred vigorously. Upon completion
of reaction (determined by thin-layer chromatography), the
solution was concentrated in vacuo and the crude product was
purified by flash chromatography (2:1 EtOAc:pentane) to afford
24 (0.0751 g, 69%) as a white foam. The enantiomeric excess
was determined to be 60% by HPLC (CHIRALPAK AD, 0.4
cm φ × 25 cm, hexane:EtOH:DEA, 85:15:0.1, 1.5 mL/min, UV
254 nm, tR (R-enantiomer) 22.1 min, (S-enantiomer) 35.7 min);
(S )-N -[(t er t -B u t y lo x y )c a r b o n y l]-[4-a m in o p h e n y l]-
a la n in e ter t-Bu tyl Ester (28). A mixture of 27 (1.25 g, 3.41
mmol) and 10% Pd-C (0.100 g) in MeOH (20 mL) was shaken
in a Parr hydrogenator under 60 psi of H2
for 1 h. The mixture
was filtered through Celite to remove the catalyst and the
filtrate concentrated under vacuum to give an oil. The crude
product was purified by flash chromatography (1:2 EtOAc:
pentane) to afford 28 (1.07 g, 94%) as an amber syrup: Rf )
1
0.31 (1:2 EtOAc:pentane); [R]20 +33.1 (c ) 2.80, CH2Cl2); H
D
NMR (CDCl3, 200 MHz) δ 6.91 (d, J ) 8 Hz, 2H), 6.57 (d, J )
8 Hz, 2H), 4.93 (d, J ) 8 Hz, 1H), 4.34 (dt, J ) 6, 8 Hz, 1H),
3.54 (broad, 2H), 2.90 (d, J ) 6, 2H), 1.39 (s, 9H), 1.38 (s,
9H);13C NMR (CDCl3, 50 MHz) δ 170.8, 154.8, 145.1, 123.8,
125.2, 114.6, 81.2, 79.0, 54.7, 37.0, 27.9, 27.5; IR (CDCl3) 3484,
1726 cm-1; LRMS (FAB) m/z 337.2 (M + H)+. Anal. Calcd for
Rf 0.37 (2:1 EtOAc:pentane); [R]20 +67.7 (c ) 0.75, CH2Cl2);
D
1H NMR (CDCl3, 200 MHz) δ 8.49 (br. s, 1H) 7.41 (d, J ) 8
Hz. 2H), 7.23 (d, J ) 8 Hz, 2H), 6.24 (br s, 2H), 5.73 (d, J ) 7
Hz, 1H), 5.30 (d, J ) 7 Hz, 1H), 3.70 (s, 3H), 1.39 (s, 9H); 13
C
C
18H28N2O4: C, 64.26; H, 8.38; N, 8.32. Found: C, 64.36; H,
8.45; N, 8.34.
NMR (CDCl3, 50 MHz) δ 180.9, 171.0, 154.8, 136.7, 136.0,
128.7, 125.0, 80.5, 56.9, 52.9, 28.2; IR (CDCl3) 3687, 1740, 1704
cm-1; LRMS (FAB) m/z 340.1 (M + H)+. Anal. Calcd for
C15H21N3O4S: C, 53.08; H, 6.24; N, 12.38; S, 9.44. Found: C,
53.46; H, 6.52; N, 11.63; S, 9.20.
(S)-N-[(ter t-Bu tyloxy)ca r bon yl]-[4-N, N′-bis(ter t-bu tyl-
oxy)ca r bon yl(gu a n yl)a m in op h en yl]a la n in e (29). A mix-
ture of 26 (2.997 g, 9.67 mmol) and 10% Pd-C (0.100 g) in
MeOH (20 mL) was shaken in a Parr hydrogenator under 60
psi of H2 for 1 h. The mixture was filtered through Celite to
remove the catalyst and the filtrate concentrated under
vacuum to give an oil. N,N′-Bis(Boc-1-guanyl)pyrazole (5.98
g, 19.3 mmol) in dry THF (50 mL) was added to the oil under
argon. After 2 days the solution was concentrated in vacuo
and purified by flash chromatography (1:1:0.1 EtOAc:pentane:
AcOH) to afford 29 (4.743 g, 93%) as a white foam: Rf 0.28
4-Th iou r ea -L-p h en ylglycin e Tr iflu or oa ceta te (8). A
solution of BBr3 (1.0 M, 3.83 mmol) in CH2Cl2 (3.83 mL) was
added to a cooled (-78 °C, acetone dry ice solution) solution
of 24 (0.260 g, 0.766 mmol) in CH2Cl2 (20 mL) over 5 min with
stirring. The reaction mixture was allowed to warm to 25 °C,
stirred for 1 h, and quenched by the careful dropwise addition
of water (30 mL). The aqueous phase was separated and the
organic phase extracted with H2O (3 × 20 mL). The combined
aqueous layers were lyopholized to dryness, and the crude
product was purified by preparative HPLC (Econosil-C-18 10
µ, 280 × 10 mm, aqueous 0.1% TFA, 4.0 mL/min, UV 254 nm,
tR 8.5 min) to afford 8 (0.183 g, 52%) as a white solid. The
enantiomeric excess of 8 was determined to be 60% by HPLC
(CROWNPAK CR(+), 0.4 cm φ × 15 cm, aqueous HClO4, pH
) 1.5, 1.5 mL/min, UV 254 nm, tR (R-enantiomer) 2.1 min,
(1:1:0.1 EtOAc:pentane:AcOH); [R]20 +27.8 (c ) 2.50, CH2-
D
Cl2); 1H NMR (CDCl3, 200 MHz) δ 10.26 (broad, 1H), 8.94 (br
s, 1H), 7.58 (s, 1H), 7.47 (d, J ) 8 Hz, 2H), 7.11 (d, J ) 8 Hz,
2H), 6.31 (br s, 2H), 5.00 (d, J ) 7 Hz, 1H), 4.52 (dt, J ) 5, 7
Hz, 1H), 3.08 (t, J ) 5 Hz, 2H), 1.48 (s, 9H), 1.47 (s, 9H), 1.41
(s, 9H); 13C NMR (CDCl3, 50 MHz) δ 174.9, 163.1, 155.3, 153.5,
153.1, 135.2, 132.8, 129.8, 122.4, 83.6, 79.8, 79.6, 54.0, 37.0,
28.2, 27.9, 27.7; LRMS (FAB) m/z 523.3 (M + H)+. Carboxylic
acid 29 was further characterized by conversion to the methyl
ester as follows. A solution of CH2N2 (0.5 M, 5.00 mmol) in
Et2O (10 mL) was added dropwise to a cooled solution (0 °C)
of 29 (0.500 g, 0.958 mmol) in Et2O (10 mL). The reaction
mixture was concentrated in vacuo and the crude product
purified by flash chromatography (1:4 EtOAc:pentane) to afford
(S-enantiomer) 12.1 min); [R]20 +40.0 (c ) 0.200, 0.1% TFA);
D
1H NMR (D2O, 200 MHz) δ 7.38 (d, J ) 9 Hz, 2H), 7.25 (d, J
) 9 Hz, 2H), 4.99 (s, 1H); 13C NMR (D2O, 50 MHz) δ 180.0,
171.1, 138.6, 130.7, 129.5, 126.4, 56.4; IR (film, 3M IR-card,
polyethylene mesh) broad 3644-2409, 1726 cm-1; LRMS (FAB)
m/z 248.1 (M + Na)+, 232.1 (M + Li)+; HRMS (FAB) calcd for
C9H11N3O2SNa 248.0469, found 248.0462.
the methyl ester of 29 (0.5029 g, 98%) as a white foam: Rf )
1
0.24 (1:4 EtOAc:pentane); [R]20 +33.5 (c ) 1.00, CH2Cl2); H
D
(S )-N -[(t er t -B u t y lo x y )c a r b o n y l]-[4-n it r o p h e n y l]-
a la n in e ter t-Bu tyl Ester (27). A saturated aqueous solution
of NaHCO3 (10 mL) was added to a solution of 4-nitropheny-
lalanine monohydrate (25, 2.00 g, 8.77 mmol) in 1,4-dioxane/
H2O (30 mL/20 mL) at 0 °C. Di-tert-butyl dicarbonate (3.83 g,
17.5 mmol) was added to this mixture, and the mixture was
allowed to warm to room temperature with stirring. After 24
h, the volume of the clear solution was reduced by 1/2 in vacuo
and acidified with 2 M KHSO4 to pH 2-3 (using pH paper as
an indicator). This solution was extracted with EtOAc (3 ×
50 mL), and the combined organic layers were dried (MgSO4),
filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography (1:4 EtOAc:hexanes to
EtOAC gradient) to afford 26 (2.50 g, 92%) as a white foam:
NMR (CDCl3, 200 MHz) δ 10.30 (s, 1H), 7.53 (d, J ) 8 Hz,
2H), 7.05 (d, J ) 8 Hz, 2H), 4.92 (d, J ) 8 Hz, 1H), 4.52 (dt, J
) 8, 6 Hz, 1H), 3.68 (s, 3H), 3.02 (d, J ) 6 Hz, 2H) 1.50 (s,
9H), 1.48 (s, 9H), 1.40 (s, 9H); 13C NMR (CDCl3, 50 MHz) δ
172.1, 163.4, 155.0, 153.3, 153.1, 135.7, 132.1, 129.6, 122.0,
83.6, 79.8, 79.5, 54.2, 52.1, 37.4, 28.2, 28.0, 27.9; IR (CDCl3)
3433, 1711 cm-1; LRMS (FAB) m/z 537.3 (M + H)+. Anal. Calcd
for C26H40N4O8: C, 58.19; H, 7.51; N, 10.44. Found: C, 58.25;
H, 7.50; N, 10.40.
4-Gu a n yl-L-p h en yla la n in e Hyd r och lor id e (9). A solu-
tion of 29 (2.00 g, 3.83 mmol) in HCl/dioxane (4 M, 20 mL,
Aldrich) was stirred at 23 °C for 24 h under an argon
atmosphere. The mixture was concentrated in vacuo, dissolved
in H2O (20 mL), passed through a 6 mL Supelco LC-18 solid-
phase extractor, and lyopholyzed to yield 9 (1.102 g, 98%) as
an off-white solid. The enantiomeric excess of 9 was deter-
mined to be >99% by HPLC (CROWNPAK CR(+), 0.4 cm φ ×
15 cm, aqueous HClO4, pH ) 1.5, 0.8 mL/min, UV 254 nm, tR
(S-enantiomer) 6.9 min); [R]20D +2.05 (c ) 2.00, H2O); 1H NMR
(D2O, 200 MHz) δ 8.00 (s, 1H), 7.23 (d, J ) 8 Hz, 2H), 7.13 (d,
J ) 8 Hz, 2H), 4.19 (t, J ) 6, 1H), 3.14 (m, 2H); 13C NMR
(D2O, 50 MHz) δ 171.1, 156.0, 133.7, 133.6, 131.0, 126.2, 53.9,
35.0; IR (film, 3M IR-card, polyethylene mesh) broad 3614-
2335, 1732 cm-1; LRMS (FAB) m/z 223.1 (M + H)+; HRMS
(FAB) calcd for C10H15N4O2 223.1195, found 223.1199.
(S)-N-[(ter t-Bu t yloxy)ca r b on yl]-[4-t h iou r ea p h en yl]-
a la n in e ter t-Bu tyl Ester (30). A solution of 28 (1.00 g, 2.97
mmol) in CH2Cl2 (20 mL) was added to a mixture of CaCO3
(0.594 g, 5.94 mmol), H2O (20 mL), CH2Cl2 (20 mL), and
thiophosgene (0.339 mL, 4.45 mmol). This mixture was vigor-
ously stirred at 23 °C, and after 1 h the organic phase was
Rf 0.15 (EtOAc); [R]20 +36.5 (c ) 1.00, CH2Cl2).
D
N,N-Dimethylformamide di-tert-butyl acetal (4.63 mL, 19.4
mmol) was added dropwise over 20 min to a solution of 26
(1.50 g, 4.83 mmol) in dry toluene (20 mL) at 80 °C. The
reaction mixture was stirred for 40 min at 80 °C and then
cooled to room temperature. The mixture was concentrated
in vacuo and the crude product purified by flash chromatog-
raphy (1:7 EtOAc:pentane) to give 27 (1.30 g, 74%) as a white
foam: Rf 0.35 (1:7 EtOAc:pentane); [R]20D +40.0 (c ) 1.09, CH2-
Cl2); 1H NMR (CDCl3, 200 MHz) δ 8.12 (d, J ) 9 Hz, 2H), 7.32
(d, J ) 9 Hz, 2H), 5.06 (d, J ) 7 Hz, 1H), 4.45 (dt, J ) 6, 7 Hz,
1H), 3.13 (ddd, J ) 6, 4, 10 Hz, 2H), 1.38 (s, 18H); 13C NMR
(CDCl3, 50 MHz) δ 169.9, 154.6, 146.4, 144.4, 130.0, 122.9,
82.0, 79.2, 54.2, 37.8, 27.8, 27.4; IR (CDCl3) 3433, 1726 cm-1
LRMS (FAB) m/z 367.2 (M
H)+. Anal. Calcd for
18H26N2O6: C, 59.00; H, 7.15; N, 7.64. Found: C, 58.76; H,
7.07; N, 7.56.
;
+
C

Asymmetric Synthesis of Restricted L-Arginine Analogues
J . Org. Chem., Vol. 64, No. 10, 1999 3475
separated and the aqueous phase was extracted with CH2Cl2
(3 × 25 mL). The combined organic layers were dried (MgSO4),
filtered, and concentrated in vacuo, yielding a yellow oil. A
solution of NH3 (7.0 M, 29.7 mmol) in EtOH (14.24 mL) was
added to the crude isothiocyanate and stirred vigorously. When
the reaction was complete (judged by thin-layer chromatog-
raphy), it was concentrated in vacuo and purified by flash
chromatography (1:1 EtOAc:pentane) to afford 30 (1.04 g, 89%)
Mea su r em en t of Nitr ite P r od u ction by iNOS. Experi-
ments to determine whether any of the synthetic analogues
could be utilized by iNOS as substrates were performed using
the Griess assay. Inducible NOS (25 nM) was incubated at 37
°C for 30 min with 1 mM dithiothreitol; 4 µM each of FAD,
FMN, and H4B; 1 mM each of NADPH and test compound in
a 96-well plate with a final volume of 100 µL. Reactions were
initiated with the addition of NADPH and were performed in
triplicate. After depleting any remaining NADPH by incuba-
tion with lactate dehydrogenase (1000 unit/mL) and sodium
pyruvate (50 mM) for 15 min at 37 °C, Griess reagent was
added and the absorbance at 550 nm was measured with a
microplate reader (Spectramax, Molecular Devices). Nitrite
production was compared to controls containing either no
substrate analogues or L-arginine (1 mM).
Ra d ioa ctive L-Citr u llin e Assa y for NOS Activity. A
diluted solution of commercial iNOS or the cytosolic fraction
of rat cerebellum containing nNOS (25 µL) was added to serial
tubes containing 1 µM radiolabeled 14C-arginine and CaC12
(1 mM) and NADPH (1 mM) and varying concentrations (1 ×
10-6 to 1 × 10-3 M) of the compounds to be tested in a final
volume of 200 µL. The reactions proceeded at 22 °C for 30 min
and were terminated by addition of 2 mL of buffer containing
30 mM hydroxyethyl piperazine diethanesulfonic acid (HEPES)
and 3 mM ethylenediamine tetraacetic acid (EDTA), pH 5.5.
The reaction mixtures were applied to chromatography col-
umns preapplied with 0.5 mL DOWEX AG50WX-8 ion-
exchange resin (Na+ form, pH 7.0). The columns were rinsed
with water (2 × 1 mL) to ensure complete elution of citrulline.
The eluant containing 14C-citrulline was quantified by liquid
scintillation spectroscopy with an efficiency of 90-94%. Reac-
tion velocities of arginine conversion to an equimolar ratio of
NO and 14C-citrulline were calculated and data expressed as
enzyme activity per milligram of protein (picomoles citrulline
per milligram protein per minute). Inhibition of enzyme
activity is expressed as percent inhibition of control (100%).
All reactions were performed in duplicate or triplicate.
as a white foam: Rf ) 0.38 (1:1 EtOAc:pentane); [R]20 +40.4
D
1
(c ) 1.00, CH2Cl2); H NMR (CDCl3, 200 MHz) δ 8.57 (br s,
1H), 7.20 (d, 2H, J ) 8 Hz), 7.12 (d, 2H, J ) 8 Hz), 6.31 (br s,
2H), 5.24 (d, 1H, J ) 8 Hz), 4.40 (dt, 1H, J ) 6, J ) 8), 2.99
(m, 2H), 1.38 (s, 9H), 1.36 (s, 9H); 13C NMR (CDCl3, 50 MHz)
δ 179.9, 170.6, 154.7, 135.2, 134.8, 130.2, 123.9, 81.5, 79.0, 54.4,
37.1, 27.7, 27.3; IR (CDCl3) 3506, 1711 cm-1; LRMS (FAB) m/z
396.2 (M + H)+. Anal. Calcd for C19H29N3O4S: C, 57.69; H,
7.39; N, 10.62; S, 8.10. Found: C, 57.93; H, 7.44; N, 10.44; S,
7.90.
4-Th iou r ea -L-p h en yla la n in e Hyd r och lor id e (10). A so-
lution of 30 (1.00 g, 2.53 mmol) in HCl/dioxane (4 M, 15 mL,
Aldrich) was stirred at 23 °C for 8 h under an argon
atmosphere. The mixture was concentrated in vacuo, dissolved
in H2O (10 mL), passed through a 6 mL Supelco LC-18 solid-
phase extractor, and lyopholyzed to yield 10 (0.777 g, 98%) as
an off-white solid. The enantiomeric excess of 10 was deter-
mined to be >99% by HPLC (CROWNPAK CR(+), 0.4 cm φ ×
15 cm, aqueous HClO4, pH ) 1.5, 0.8 mL/min, UV 254 nm, tR
(S-enantiomer) 9.6 min); [R]20D -5.68 (c ) 1.09, H2O); 1H NMR
(D2O, 200 MHz) δ 7.10 (d, J ) 7 Hz, 2H), 6.99 (d, J ) 7 Hz,
2H), 4.11 (t, J ) 5 Hz, 1H), 3.06 (m, 2H);13C NMR (D2O, 50
MHz) δ 178.9, 170.6, 136.2, 132.7, 130.6, 125.7, 53.8, 34.9; IR
(film, 3M IR-card, polyethylene mesh) broad 3636-2401, 1733
cm-1; LRMS (FAB) m/z 240.1 (M + H)+; HRMS calcd for
C
10H14N3O2S 240.0806, found 240.0797.
Bioch em istr y: Gen er a l. Dithiothreitol, nicotine adenine
dinucleotide phosphate (NADPH), flavin adenine dinucleotide
(FAD), flavin mononucleotide (FMN), (6R)-5,6,7,8-tetrahydro-
biopterin (H4B), L-arginine, lactate dehydrogenase, sodium
pyruvate, and N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfon-
ic acid (HEPES) were purchased from Sigma. Inducible nitric
oxide synthase (mouse, 100 000 × g supernatant) was pur-
chased from Cayman Chemical, Ann Arbor, MI. Neuronal NOS
was isolated from rat cerebellum as previously described.26
Ack n ow led gm en t. This work was supported by a
grant (9630310N, S.B.K.) from the American Heart
Association. The donors of the Petroleum Research Fund
(PRF 32927-G1) and Wake Forest University (Cross
Campus Collaborative Research Fund) are gratefully
acknowledged for financial support. Mass spectrometry
was performed by the Nebraska Center for Mass
Spectrometry.
(26) Bredt, D. S.; Schmidt, H. H. H. W. In Methods in Nitric Oxide
Research; Feelisch, M., Stamler, J . S., Eds.; Wiley: Chichester, 1996;
Chapter 17.
(27) Tobin, J . R.; Martin, L. D.; Breslow, M. J .; Traystman, R. J .
Anesthesiology 1994, 81, 1264-1267.
J O982161F