Efficient synthesis of optically pure Nω-alkylated L-arginines

Article abstract of DOI:10.1055/s-2008-1067165

A number of N^ω-substituted L-arginines have been described to date, particularly with regard to nitric oxide synthase (NOS) modulators. Elaborate multistep syntheses and low yields limit the scope of preparing these modified L-arginines. We describe a synthetic methodology that delivers N ^ω-alkylated L-arginine derivatives from protected L-ornithine in a three-step sequence with excellent overall yields (81-90%) and in high purity. Analysis of the synthesized amino acids on a Crownpak Cr(+) column revealed no significant racemization, that is, >99.9% ee for all final compounds. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2008-1067165

PAPER
2391
Efficient Synthesis of Optically Pure Nw-Alkylated L-Arginines
Synthesisof
O
pticeallyPure w
N
n
-Alkylated
L-Arginines is Schade, Jürke Kotthaus, Bernd Clement*
Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel,
Gutenbergstraße 76, 24118 Kiel, Germany
Fax +49(431)8801352; E-mail: bclement@pharmazie.uni-kiel.de
Received 26 February 2008; revised 15 April 2008
methylarginine dimethylaminohydrolase (DDAH) activi-
ty, for example, inhibit DDAH and thereby attenuate NO
production instead of completely inhibit NOSs.5 Howev-
er, the development of modulators of endogenous NO lev-
Abstract: A number of Nw-substituted L-arginines have been de-
scribed to date, particularly with regard to nitric oxide synthase
(NOS) modulators. Elaborate multistep syntheses and low yields
limit the scope of preparing these modified L-arginines. We de-
scribe a synthetic methodology that delivers Nw-alkylated L-argin- els implies to derivatize L-arginine, the pivotal amino acid
ine derivatives from protected L-ornithine in a three-step sequence
in NO-related metabolism. In particular, the investigation
with excellent overall yields (81–90%) and in high purity. Analysis
of novel inhibitors of NOSs has led to several L-arginine
of the synthesized amino acids on a Crownpak Cr(+) column re-
analogues, such as Nw-allyl-L-arginine or Nw-propargyl-L-
vealed no significant racemization, that is, >99.9% ee for all final
arginine.6,7 In contrast, not many potent inhibitors of the
compounds.
attractive molecular target DDAH, besides Nw-methoxy-
Key words: nitric oxide, Nw-alkyl, L-arginine, NOS, DDAH
ethyl-L-arginine, are known8,9 (Table 1).
Table 1 Selected Nw-Substituted L-Arginines as Known NO Modu-
lators
Nitric oxide (NO) is involved in various physiological
R1
R2
N
processes, such as maintenance of vascular tone, neuronal
signaling, and host response to infection.1 Over- or under-
production of NO contributes to numerous diseases.2
Therefore, it is vital that endogenous NO levels are strictly
regulated. Figure 1 depicts the predominant enzymes in
this regulation process, which consequently present to be
the major targets for potential NO modulators.
N
O
H2N
OH
NH2
Entry
R1
R2
Compound
NOS inhibitors
Due to its ubiquitous occurrence and versatile physiolog-
ical roles, simple elevation or reduction of NO is likely to
cause side-effects. Thus, it may be beneficial to rather
smoothly affect NO levels by indirect modulation of di-
1
Me
Me
H
NMMA10
ADMA11
6c6
2
Me
H
3
H2C=CHCH2
4
HC≡CCH2
H
6e7
endogenous methyl-L-arginines
Nω,ω-dimethyl-L-Arg (ADMA)
Nω-monomethyl-L-Arg (NMMA)
NOS substrate
5
MeO
H
H
6a12
6b8
DDAH
DDAH inhibitor
6
MeOCH2CH2
NOS
NOS
Nω-OH-L-Arg
NO
+
L-Citr
L-Arg
In our studies, we aim to investigate L-arginine based
DDAH-inhibitors and also test selectivity of known NOS
modulators for NOS over DDAH. A modification at the
Nd-position of L-arginine on the NO-generating system
has already been extensively investigated in our laborato-
ry.13 However, an efficient synthetic scheme is required
that would deliver a set of differentially substituted
L-arginines at the Nw-position.
reduction
CYP450
arginase
L-Orn
+
urea
Figure 1 Physiological regulation of endogenous NO-levels.
L-Citr = L-citrulline, L-Orn = L-ornithine, NOS = nitric oxide syn-
thase (EC 1.14.13.39), DDAH = dimethylarginine dimethylaminohy-
drolase (EC 3.5.3.18), arginase (EC 3.5.3.1), microsomal/mitochondrial
reduction: see ref. 3, CYP450-mediated oxidation: see ref. 4.
There are several guanylating agents which deliver more
or less substituted guanidines. Frequently used agents are
pyrazole-1-carboxamidines, thioureas, and isothioureas.14
The cyanamide-guanidine strategy is limited by relatively
low reactivity of the cyanamide towards amines. For ex-
ample, protected Nw-propargyl-L-arginine was prepared
SYNTHESIS 2008, No. 15, pp 2391–2397
Advanced online publication: 08.07.2008
DOI: 10.1055/s-2008-1067165; Art ID: T03108SS
© Georg Thieme Verlag Stuttgart · New York
x
x.
x
x
.
2
0
0
8

2392
D. Schade et al.
PAPER
from its cyanamide in 32% yield.7 We also attempted to required preparation of the guanylating reagent in two
prepare Nw-methoxy-L-arginine from its cyanamide by re- steps.
action with methoxylamine hydrochloride according to
Another general procedure for the synthesis of Nw-alkyl
Wagenaar et al., which was not reproducible with respect
and Nw-aryl-L-arginines was described by Chen et al. who
to the stated yield of 97%.15 By using K2CO3 as the base
employed S-methylisothioureas as the guanylating
and a different solvent system for column chromatogra-
phy we were able to build up the N-methoxyguanidine
moiety at most with an 80% yield (Scheme 1).
agent.19 Although good overall yields were stated, the pre-
requisite to prepare the isothioureas in a two-step se-
quence from alkyl- or arylisothiocyanates seems
disadvantageous. Additionally, ready commercial avail-
ability of the required isothiocyanates is limited and/or
can be partially expensive.
*HCl
OMe
N
OMe
O
H2N
N
N
OtBu
R
An attractive approach to guanidines was described by
Linton et al. who reacted carbamoylthioureas with alkyl-
and arylamines in the presence of N-ethyl-N¢-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDCI) as the
desulfurizing agent.20
H
a: Et3N in EtOH13
b: K2CO3 in EtOH
H2N
N
H
NHBoc
1
2
reflux, 5 h
= R
b: 80% (a: 40%)
Scheme 1 ‘Cyanamide-guanidine’ strategy: Preparation of meth-
oxyguanidine moiety in protected Nw-methoxy-L-arginine
Martin et al. already successfully applied this strategy to
the preparation of Nw-hydroxy-L-arginines and very re-
cently to Nw-amino-L-arginines.21,22 This prompted us to
employ the concept for the preparation of several Nw-
alkyl-L-arginines. Initial isolation of Na-Boc-Nw-Cbz-L-
thiocitrulline tert-butyl ester 4 provided a suitable build-
ing block for all guanidines (Scheme 2). The convenient
reaction of thiourea 4 with 1.5 equivalents of amine and
1.5 equivalents of EDCI in anhydrous CH2Cl2 overnight
allowed complete consumption of 4 as indicated by TLC.
Other groups reported yields for similar guanidines in the
range of 60%.16 Furthermore, within the syntheses of Nd-
substituted L-arginine analogues, we prepared hydroxy-
guanidines in 35–80% yields by treating cyanamides with
hydroxylamine.17 Consequently, this approach appears
more or less limited to few N-nucleophiles.
Rossiter et al. described the preparation of chain-short-
ened L-arginines by means of using N-alkyl-substituted
N,N¢-bis-Boc-1-carboxamidines.8 The need to initially
prepare different types of these reagents under Mitsunobu
conditions and low overall yields (20–50%) may limit the
scope of this approach. The most potent arginine-based
DDAH-inhibitor Nw-methoxyethyl-L-arginine was pre-
pared this way in 41% overall yield. An optimized synthe-
sis of Nw-allyl-L-arginine was described by Bernatowicz
et al. who also utilized a substituted pyrazole-1-carbox-
amidine.18 Although overall yields were good (70%), dis-
advantages are the long reaction times (5 d) and again the
Only the reaction of 4 with b-alanine amide to 5f did not
reach completion overnight – probably due to inferior sol-
ubility compared to the other amines employed – but an
additional half-equivalent of the amine, EDCI and DIPEA
resulted in quantitative conversion into the guanidine
moiety. Protected L-arginines 5 could be subsequently
deprotected under mild conditions with TFA/thioanisole
within 30 minutes.23 After removal of TFA in vacuo and
separation of the amino acids between water and Et2O to
remove unpolar thioanisole and side-products, flash chro-
R1
R2
DIPEA
EDCI
R1
R2
N
N
O
S
O
H
N
O
CbzNCS
H2N
OtBu
NHBoc
OtBu
CbzHN
N
OtBu
CbzHN
CH2Cl2
H
CH2Cl2
overnight
NHBoc
NHBoc
0 °C to r.t., 2 h
3
4, 93%
5a–i, 90–99%
30 min
TFA, thioanisole
R1
R2
R1
R2
H
a
b
c
d
e
H
H
H
H
H
OMe
f
O
R1
R2
N+
CF3COO–
O
NH2
OMe
g
h
i
H2N
N
H
OH
Nω
O
+
NH3
CF3COO–
Nω
6a–i, 96–99%
CF3
H
Scheme 2 Three-step synthesis of Nw-substituted L-arginines
Synthesis 2008, No. 15, 2391–2397 © Thieme Stuttgart · New York

PAPER
Synthesis of Optically Pure Nw-Alkylated L-Arginines
2393
substituents at the Nw-position would dramatically impair
the separation of the D/L-enantiomers. Essentially, based
on the determined detection limit, we concluded optical
purities of 99.9% ee for all the synthesized L-arginines
6.The problematic nature of obtaining correct elemental
analyses of highly hygroscopic amino acids also
prompted us to carefully examine the general purity of
the synthesized final compounds by HPLC. Therefore,
L-arginines 6 were analyzed on a RP-18 column after pre-
column-derivatization with o-phthaldialdehyde (o-PA).
Only Nw-(2-carboxamidoethyl)-L-arginine (6f) turned out
to be less than 99% pure. This may be optimized by iso-
lating its protected precursor 5f in higher purity, but with
concomitant losses in yields.
Table 2 Overall Yields for Nw-Substituted L-Arginines
Entry
Product 6
Overall yield (%)a
Purity (%)b
99.2
1
2
3
4
5
6
7
8
9
a
b
c
d
e
f
87 (2913)c
89 (446)
87 (7016)
89
99.6
99.4
99.5
85 (325)
81
99.5
96.0
g
h
i
88
99.4
82
99.6
In summary, we have demonstrated the applicability of
the ‘carbamoylthiourea-guanidine’ strategy to the prepa-
ration of various Nw-substituted L-arginines in excellent
overall yields and high purity. Chromatography on a
chiral stationary phase demonstrated no risk for racemiza-
tion within this synthetic sequence and thereby underlines
its utility for the preparation of various L-arginine ana-
logues as probes for enzymes of the NO-generating sys-
tem. It is further noteworthy that the described
methodology proved suitable for the preparation of a se-
ries of unsymmetrically substituted, nonamino acid
guanidines (data not shown).
90
99.6
a Literature overall yield is given in parentheses.
b Determined by HPLC, using o-PA (o-phthaldialdehyde) precolumn
derivatization ( 0.1%).
c Not reproducible in own attempts (see also Scheme 1).
matography on a RP-18 column provided Nw-substituted
L-arginines 6 (Table 2).
For the accurate evaluation of enzymatic studies, such as
the determination of Ki or Km values, it is of pivotal impor-
tance that the tested compounds are enantiopure. There-
fore, we tested all synthesized amino acids for
enantiomeric purity on a Crownpak Cr(+) column. Fur-
thermore, this should provide valuable information about
the employed chemistry or, respectively, its risk for race-
mization (Figure 2). We did not synthesize D-enantiomers
for this purpose but varied several chromatographic pa-
rameters that are known to affect the resolution of two
enantiomers, that is, column temperature, pH of eluent,
and flow-rate, and always observed highly symmetric
peaks. In general, the separation factor of basic amino
acids on this chiral stationary phase is very high (we de-
termined a = 2.83 for D/L-arginine, a = 2.59 for Nd-meth-
yl-D/L-arginine17). Thus, it appears very unlikely that
Melting points are uncorrected. 1H and 13C NMR spectra were ob-
tained on a Bruker ARX 300 spectrometer at 300 K. Chemical shifts
(d values) are reported in ppm relative to TMS or 3-(trimethylsilyl)-
1-propanesulfonic acid-d6 sodium salt (TPS) as an internal standard,
or in 1H spectra of compounds 6 relative to the residual solvent sig-
nal. All coupling constants (J values) were obtained by first order
analysis of the multiplets and are quoted in Hz. Low-resolution
mass spectra were recorded on a Bruker-Esquire-LC with an elec-
trospray ionization (ESI). Recordings of high-resolution mass spec-
tra were conducted at the Institute of Analytical Chemistry,
University of Leipzig, Germany on a Bruker 7.4 Tesla FTICR mass
spectrometer BioApex II equipped with an ESI-ion source (Agi-
lent). Chromatography on chiral stationary phase: Waters BreezeTM
HPLC system: Waters 1525 binary pump, Waters 2487 dual wave-
Figure 2 Chromatogram of optically pure Nw-substituted L-arginines 6 on a Crownpak Cr(+) column: retention times: 6a, 6.6 min; 6f, 6.8
min; 6g, 7.5 min; 6e, 9.8 min; 6b, 11.2 min; 6c, 12.5 min; 6h, 12.7 min; 6i, 15.1 min; 6d, 16.0 min (for details see the experimental section).
Synthesis 2008, No. 15, 2391–2397 © Thieme Stuttgart · New York

2394
D. Schade et al.
PAPER
length absorbance detector, Waters 717 Plus autosampler; station-
ary phase: Crownpak Cr(+) column (250 × 4.6 mm) thermostated at
15 °C; the eluent consisted of aqua bidest. and HClO4, pH 1.5; flow
rate was kept at 0.6 mL/min, except for 6d (1 mL/min); detection at
200 nm. For determination of % ee, L-arginines 6 were dissolved in
aqua bidest. (1 mM) and 10 mL was injected. Purity assessment of
L-arginines 6 by RP-HPLC: autosampler Waters 717 plus, Waters
600 Controller, Waters 470 scanning fluorescence detector, set at
lex: 338 nm, lem: 425 nm; stationary phase: NovaPak RP18 (4 × 150
mm, VDS Optilab, 4 mm) with a Phenomenex C18 (4 × 3.0 mm)
guard column, thermostated at 30 °C; eluent A consisted of 86% 10
mM K3PO4 buffer (pH 4.65), 8% MeCN, 6% MeOH; eluent B con-
sisted of 40% MeCN, 30% MeOH, 30% aqua bidest.; elution con-
ditions: flow rate was kept at 1 mL/min; 0–2 min, isocratic with
100% eluent A; 2–3.5 min, linear gradient to 90% eluent A and 10%
eluent B; 3.5–12 min, isocratic with 90% eluent A and 10% eluent
B; 12–16 min, linear gradient to 25% eluent A and 75% eluent B;
16–25 min, isocratic with 25% eluent A and 75% eluent B; 25–28
min, linear gradient to 100% eluent A; 28–35 min, reequilibration
with 100% eluent A; precolumn derivatization: the autosampler was
set to mix 7 mL of o-PA reagent with 8 mL of sample (0.1 mM) and
allowed to react for 4 min at r.t. before injection. o-PA reagent was
prepared by dissolving o-PA (50 mg) in MeOH (1 mL), followed by
adding 0.2 M of potassium borate buffer (pH 9.4, 9 mL) and 2-mer-
captoethanol (53 mL). Elemental analyses were performed on a
CHNS analyzer (HEKAtech GmbH) and were within 0.4% for all
synthesized compounds, except for 5f and the highly hygroscopic
L-arginines 6.
13C NMR (75 MHz, CDCl3): d = 24.8 (g-CH2), 28.7 [C(CH3)3], 29.0
[C(CH3)3], 30.9 (b-CH2), 45.8 (NCH2), 54.2 (a-CH), 68.8 (CH2Ph),
80.4 [C(CH3)3], 82.8 [C(CH3)3], 129.0, 129.4, 129.5 (ArCH), 135.2
(ArC), 153.2 (thiourea-C), 156.0 (CO-Boc), 172.2 (CO2-t-Bu),
179.8 (CO-Cbz).
MS (ESI): m/z = 482 [M + H]+, 426 [M – C4H8 + H]+, 370 [M –
2 × C4H8 + H]+, 326 [M – 2 × C4H8, – CO2 + H]+.
Protected L-Arginines 5; General Procedure
A literature protocol for the preparation of carbamoylguanidines
was used for the herein presented protected L-arginines.20 DIPEA
(1.5 equiv), amine (1.5 equiv), and EDCI (1.5 equiv) were reacted
with thiourea 5 (0.5 mmol) in anhyd CH2Cl2 (5 mL). For amines that
were applied as hydrochlorides (i.e., methoxylamine, but-3-enyl-
amine, and b-alanine amide) 3 equiv of DIPEA were used. Unless
otherwise noted, reactions were complete after stirring overnight.
The organic phase was diluted with CH2Cl2 (10 mL) and washed
with 1% aq HCl (5 mL), H2O (5 mL), and brine (5 mL). The result-
ing oils were purified by column chromatography using CH2Cl2–
MeOH as the eluent.
Nw-Benzyloxycarbonyl-Na-tert-butyloxycarbonyl-Nw¢-methoxy-
L-arginine tert-Butyl Ester (5a)
Eluent: CH2Cl2–MeOH (99:1); Rf = 0.3; yield: 235 mg (95%); col-
orless oil.
1H NMR (300 MHz, CDCl3): d = 1.44 [s, 9 H, C(CH3)3], 1.46 [s, 9
H, C(CH3)3], 1.58–2.02 (m, 4 H, b,g-CH2), 3.11 (m, 2 H, NCH2),
3.66 (s, 3 H, OCH3), 4.18 (m, 1 H, a-CH), 5.11 (m, 1 H, NH), 5.13
(s, 2 H, CH2Ph), 6.25 (m, 1 H, NH), 7.36 (m, 5 H, ArH), 7.91 (br s,
1 H, NHBoc).
13C NMR (75 MHz, CDCl3): d = 25.6 (g-CH2), 28.7 [C(CH3)3], 29.0
[C(CH3)3], 30.9 (b-CH2), 41.2 (NCH2), 54.5 (a-CH), 62.0 (OCH3),
68.3 (OCH2), 80.3 [C(CH3)3], 82.5 [C(CH3)3], 129.0, 129.3, 129.4
(ArCH), 135.8 (ArC), 148.8, 153.6, 156.0, 172.5 (CO2-t-Bu).
All starting materials were commercially available and used with-
out further purification. Na-Boc-L-ornithine tert-butyl ester hydro-
chloride 3 and b-alanine amide hydrochloride were purchased from
Bachem. All solvents were distilled and dried according to standard
procedures. Aqua bidest. refers to doubly distilled water from a
Finnaqua 75-E-4 system. CbzNCS19 was prepared according to a
procedure of Martin et al. but optimized using freshly powdered
KSCN,18 which resulted in higher yields and shorter reaction times.
Reactions were monitored by TLC on precoated silica gel plates
(SiO2 60, F254). All compounds could be visualized by ninhydrin
spray and heating at 120 °C. Purification of synthesized compounds
was performed by column chromatography using silica gel (particle
size 40–63 mm). L-Arginines 6 were purified by flash chromatogra-
phy (CombiFlash®RETRIEVE) on a RP-18 RediSep® column (43
g) using aqua bidest. (containing 0.1% TFA) as the eluent (30 mL/
min).
MS (ESI): m/z = 495 [M + H]+.
Nw-Benzyloxycarbonyl-Na-tert-butyloxycarbonyl-Nw¢-(2-meth-
oxyethyl)-L-arginine tert-Butyl Ester (5b)
Eluent: CH2Cl2–MeOH (95:5); Rf = 0.3; yield: 259 mg (99%); col-
orless oil.
1H NMR (300 MHz, CDCl3): d = 1.44 [s, 9 H, C(CH3)3], 1.45 [s, 9
H, C(CH3)3], 1.51–1.93 (m, 4 H, b,g-CH2), 3.29 (m, 2 H, NCH2),
3.37 (s, 3 H, OCH3), 3.42, 3.50 (2 m, 4 H, NCH2CH2O), 4.16 (m, 1
H, a-CH), 5.10 (br s, 3 H, CH2Ph, NH), 6.21 (br s, 1 H, NH), 7.28
(m, 3 H, ArH), 7.39 (m, 2 H, ArH), 9.11 (br s, 1 H, NHBoc).
13C NMR (75 MHz, CDCl3): d = 25.8 (g-CH2), 28.7 [C(CH3)3], 29.0
[C(CH3)3], 31.2 (b-CH2), 41.3 (NCH2), 54.2 (a-CH), 59.6 (OCH3),
67.1 (CH2Ph), 80.5 [C(CH3)3], 82.8 [C(CH3)3], 128.2, 128.6, 128.9
(ArCH), 138.5 (ArC), 156.2 (guanidine-C), 162.0 (CO-Boc), 164.8
(CO-Cbz), 172.2 (CO2-t-Bu).
Nw-Benzyloxycarbonyl-Na-tert-butyloxycarbonyl-L-thiocitrul-
line tert-Butyl Ester (4)21
Na-tert-Butyloxycarbonyl-L-ornithine tert-butyl ester hydrochlo-
ride (3; 2.28 g, 7 mmol) was dissolved in anhyd CH2Cl2 (20 mL) and
the corresponding base was set free by treatment with gaseous NH3.
The suspension was filtered and the organic solvent removed in
vacuo. The resulting oil was dissolved in anhyd CH2Cl2 (250 mL)
and cooled to 0 °C. A 0.5 M CH2Cl2 solution of CbzNCS (14 mL, 7
mmol) were added dropwise over 30 min and the mixture was
stirred for 2 h while warming to r.t. until TLC indicated complete
consumption of starting material. CH2Cl2 was evaporated to one-
third of the volume and was washed with 1% aq HCl (25 mL), H2O
(25 mL), and brine (25 mL). The organic phase was dried (Na2SO4)
and concentrated to a yellow oil. The crude product (>96% pure by
TLC) was purified by column chromatography (cyclohexane–
EtOAc, 4:1) to obtain 3.13 g (93%) of a light-yellow oil that solidi-
fied upon standing in the refrigerator.
MS (ESI): m/z = 523 [M + H]+.
Nw-Allyl-Nw¢-benzyloxycarbonyl-Na-tert-butyloxycarbonyl-L-
arginine tert-Butyl Ester (5c)
Eluent: CH2Cl2–MeOH (95:5); Rf = 0.3; yield: 240 mg (95%); col-
orless oil.
1H NMR (300 MHz, CDCl3): d = 1.45 [s, 9 H, C(CH3)3], 1.47 [s, 9
H, C(CH3)3], 1.59–1.92 (m, 4 H, b,g-CH2), 3.37 (m, 2 H, NCH2),
3.92 (m, 2 H, NCH2CH=CH2), 4.18 (m, 1 H, a-CH), 5.13 (s, 2 H,
CH2Ph), 5.16–5.38 (m, 3 H, CH=CH2, NH), 5.89 (ddt, J = 17.2,
10.3, 5.2 Hz, 1 H, CH=CH2), 7.32 (m, 3 H, ArH), 7.42 (m, 2 H,
ArH), 8.79 (br s, 1 H, NHBoc).
1H NMR (300 MHz, CDCl3): d = 1.44 [s, 9 H, C(CH3)3], 1.46 [s, 9
H, C(CH3)3], 1.64–1.75 (m, 4 H, b,g-CH2), 3.66 (m, 2 H, NCH2),
4.20 (m, 1 H, a-CH), 5.08 (m, 1 H, NH), 5.10 (s, 2 H, CH2Ph), 7.36
(m, 5 H, ArH), 8.15 (br s, 1 H, NHCbz), 9.65 (br s, 1 H, NHBoc).
13C NMR (75 MHz, CDCl3): d = 25.8 (g-CH2), 28.7 [C(CH3)3], 29.0
[C(CH3)3], 31.5 (b-CH2), 41.6 (NCH2), 44.5 (NCH2), 53.8 (a-CH),
Synthesis 2008, No. 15, 2391–2397 © Thieme Stuttgart · New York

PAPER
Synthesis of Optically Pure Nw-Alkylated L-Arginines
2395
67.3 (CH2Ph), 80.6 [C(CH3)3], 83.0 [C(CH3)3], 117.7 (CH=CH2),
128.3, 128.7, 129.0 (ArCH), 134.5 (CH=CH2), 138.3 (ArC), 156.2
(guanidine-C), 160.8 (CO-Boc), 164.8 (CO-Cbz), 172.2 (CO2-
t-Bu).
1H NMR (300 MHz, CDCl3): d = 1.46 [s, 9 H, C(CH3)3], 1.48 [s, 9
H, C(CH3)3], 1.56–1.92 (m, 4 H, b,g-CH2), 3.23 (m, 2 H, NCH2),
3.42 (pseudo t, 4 H, 2 × NCH2), 3.71 (pseudo t, 4 H, 2 × OCH2),
4.16 (m, 1 H, a-CH), 5.09 (br s, 1 H, NH), 5.13 (s, 2 H, CH2Ph), 7.34
(m, 3 H, ArH), 7.42 (m, 2 H, ArH), 8.27 (br s, 1 H, NHBoc).
13C NMR (75 MHz, CDCl3): d = 26.8 (g-CH2), 28.7 [C(CH3)3], 29.0
[C(CH3)3], 30.9 (b-CH2), 45.8 (NCH2), 48.4 (NCH2), 53.9 (a-CH),
67.2 (OCH2), 67.5 (OCH2), 80.6 [C(CH3)3], 82.9 [C(CH3)3], 128.4,
128.9, 129.0 (ArCH), 138.0 (ArC), 155.4 (guanidine-C), 163.2
(CO-Boc), 165.0 (CO-Cbz), 171.4 (CO2-t-Bu).
MS (ESI): m/z = 505 [M + H]+.
Nw-Benzyloxycarbonyl-Nw¢-(but-3-enyl)-Na-tert-butyloxycarbo-
nyl-L-arginine tert-Butyl Ester (5d)
Eluent: CH2Cl2–MeOH (93:7); Rf = 0.17; yield: 251 mg (97%); col-
orless oil.
1H NMR (300 MHz, CDCl3): d = 1.45 [s, 9 H, C(CH3)3], 1.47 [s, 9
H, C(CH3)3], 1.57–1.92 (m, 4 H, b,g-CH2), 2.35 (pseudo q, J = 6.8
Hz, 2 H, CH2CH=CH2), 3.31 (m, 4 H, 2 × NCH2), 4.17 (m, 1 H, a-
CH), 5.10–5.19 (m, 3 H, CH=CH2, NH), 5.12 (s, 2 H, CH2Ph), 5.80
(ddt, J = 17.1, 10.2, 6.8 Hz, 1 H, CH=CH2), 7.29 (m, 3 H, ArH),
7.39 (m, 2 H, ArH), 8.91 (br s, 1 H, NHBoc).
13C NMR (75 MHz, CDCl3): d = 25.7 (g-CH2), 28.7 [C(CH3)3], 29.0
[C(CH3)3], 31.5 (b-CH2), 34.2 (CH2CH=CH2), 41.0 (NCH2), 41.2
(NCH2), 53.8 (a-CH), 67.1 (CH2Ph), 80.6 [C(CH3)3], 83.0
[C(CH3)3], 118.3 (CH=CH2), 128.2, 128.6, 128.9 (ArCH), 135.5
(CH=CH2), 138.4 (ArC), 156.3 (guanidine-C), 160.8 (CO-Boc),
164.8 (CO-Cbz), 172.1 (CO2-t-Bu).
MS (ESI): m/z = 535 [M + H]+.
Nd-{[(N-Benzyloxycarbonylamino)-2,5-dihydro-1H-pyrrol-1-
yl]methylidene}-Na-tert-butyloxycarbonyl-L-ornithine tert-Bu-
tyl Ester (5h)
Eluent: CH2Cl2–MeOH (95:5); Rf = 0.31; yield: 240 mg (92%);
light yellow oil.
1H NMR (300 MHz, CDCl3): d = 1.45 [s, 9 H, C(CH3)3], 1.47 [s, 9
H, C(CH3)3], 1.55–1.90 (m, 4 H, b,g-CH2), 3.23 (m, 2 H, NCH2),
4.15 (m, 1 H, a-CH), 4.26 (s, 4 H, 2 × NCH2CH=), 5.13 (br s, 3 H,
CH2Ph, NH), 5.80 (s, 2 H, 2 × CH2CH=), 7.30 (m, 3 H, ArH), 7.42
(m, 2 H, ArH).
MS (ESI): m/z = 519 [M + H]+.
13C NMR (75 MHz, CDCl3): d = 26.4 (g-CH2), 28.7 [C(CH3)3], 29.0
[C(CH3)3], 31.0 (b-CH2), 44.1 (NCH2), 54.0 (a-CH), 55.6
(NCH2CH=), 67.3 (CH2Ph), 80.5 [C(CH3)3], 82.9 [C(CH3)3], 125.8
(CH2CH=), 128.1, 128.6, 128.9 (ArCH), 138.7 (ArC), 156.1 (guani-
dine-C), 161.1 (CO-Boc), 161.7 (CO-Cbz), 172.2 (CO2-t-Bu).
Nw-Benzyloxycarbonyl-Na-tert-butyloxycarbonyl-Nw¢-propar-
gyl-L-arginine tert-Butyl Ester (5e)
Eluent: CH2Cl2–MeOH (97:3); Rf = 0.21; yield: 233 mg (93%);
white foamy wax.
MS (ESI): m/z = 517 [M + H]+.
1H NMR (300 MHz, CDCl3): d = 1.46 [s, 9 H, C(CH3)3], 1.48 [s, 9
H, C(CH3)3], 1.54–1.90 (m, 4 H, b,g-CH2), 2.30 (br s, 1 H, C≡CH),
3.37 (m, 2 H, NCH2), 4.12 (m, 3 H, NCH2C≡CH, a-CH), 5.13 (s, 2
H, CH2Ph), 5.18 (m, 1 H, NH), 7.31 (m, 3 H, ArH), 7.41 (m, 2 H,
ArH), 9.03 (br s, 1 H, NHBoc).
Nw-Benzyloxycarbonyl-Na-tert-butyloxycarbonyl-Nw¢,w¢-(2,2,2-
trifluoroethyl)-L-arginine tert-Butyl Ester (5i)
Eluent: CH2Cl2–MeOH (98:2); Rf = 0.43; yield: 271 mg (98%); col-
orless wax.
13C NMR (75 MHz, CDCl3): d = 25.8 (g-CH2), 28.7 [C(CH3)3], 29.0
[C(CH3)3], 31.6, 41.3 (NCH2), 53.7 (a-CH), 67.3, 73.0 (C≡CH),
80.7 [C(CH3)3], 83.1 [C(CH3)3], 128.2, 128.7, 128.9 (ArCH), 138.3
(ArC), 156.4 (guanidine-C), 160.5 (CO-Boc), 164.7 (CO-Cbz),
172.1 (CO2-t-Bu).
1H NMR (300 MHz, CDCl3): d = 1.45 [s, 9 H, C(CH3)3], 1.48 [s, 9
H, C(CH3)3], 1.64–1.94 (m, 4 H, b,g-CH2), 3.27 (m, 1 H), 3.43 (m,
1 H), 3.94 (m, 1 H), 4.18 (m, 1 H, a-CH), 4.36 (m, 1 H), 5.14 (s, 2
H, CH2Ph), 5.26 (m, 1 H), 5.99 (br s, 1 H, NH), 7.31 (m, 3 H, ArH),
7.41 (m, 2 H, ArH), 9.19 (br s, 1 H, NHBoc).
MS (ESI): m/z = 503 [M + H]+.
13C NMR (75 MHz, CDCl3): d = 25.1 (g-CH2), 27.9 [C(CH3)3], 28.2
[C(CH3)3], 31.2 (b-CH2), 40.8 (NCH2), 42.4 (q, JC,F = 34.5 Hz,
CH2CF3), 52.7 (a-CH), 67.0 (CH2Ph), 80.3 [C(CH3)3], 82.6
[C(CH3)3], 124.1 (q, JC,F = 278.9 Hz, CF3), 127.8, 128.0, 128.3
(ArCH), 137.1 (ArC), 156.0 (guanidine-C), 159.4 (CO-Boc), 163.0
(CO-Cbz), 171.3 (CO2-t-Bu).
Nw-Benzyloxycarbonyl-Na-tert-butyloxycarbonyl-Nw¢-(2-carba-
moylethyl)-L-arginine tert-Butyl Ester (5f)
After stirring overnight, additional 0.5 equiv of b-alanine amide hy-
drochloride, EDCI, and 1 equiv DIPEA were added and stirred for
4 h. Eluent: CH2Cl2–MeOH (92:8); Rf = 0.34; yield: 241 mg (90%);
white foamy wax.
MS (ESI): m/z = 547 [M + H]+.
1H NMR (300 MHz, CDCl3): d = 1.45 [s, 9 H, C(CH3)3], 1.48 [s, 9
H, C(CH3)3], 1.56–1.92 (m, 4 H, b,g-CH2), 2.50 (t, 3J = 5.6 Hz, 2 H,
CH2CONH2), 3.24 (m, 2 H, NCH2), 3.28 (m, 2 H, NCH2), 4.15 (m,
1 H, a-CH), 5.12 (s, 2 H, CH2Ph), 5.36 (br s, 1 H, NH), 6.11 (br s,
2 H, CONH2), 7.32 (m, 3 H, ArH), 7.45 (m, 2 H, ArH), 9.05 (br s, 1
H, NHBoc).
13C NMR (75 MHz, CDCl3): d = 25.7 (g-CH2), 28.6 [C(CH3)3], 28.9
[C(CH3)3], 30.7 (b-CH2), 36.2 (CH2), 37.6 (CH2), 41.3 (NCH2),
54.3 (a-CH), 67.1 (CH2Ph), 80.5 [C(CH3)3], 82.7 [C(CH3)3], 128.2,
128.5, 128.9 (ArCH), 138.3 (ArC), 156.3 (guanidine-C), 160.9
(CO-Boc), 164.5 (CO-Cbz), 172.4 (CO2-t-Bu), 175.5 (CONH2).
Deprotected L-Arginines 6; General Procedure21
Protected L-arginines 5 (0.4 mmol) were stirred in a mixture of TFA
(8 mL) and thioanisole (2.4 mL, 50 equiv) for 30 min. TFA was dis-
tilled out under reduced pressure and H2O (5 mL) and Et2O (15 mL)
were added. The organic phase was extracted with H2O (2 × 5 mL).
The aqueous phase was concentrated on a rotary evaporator and the
resulting oil was dissolved in aqua bidest. containing 0.1% of TFA
(1 mL) and purified by flash chromatography on a RP-18 column.
Ninhydrin-positive fractions were combined, concentrated in vacuo
to ca. 10 mL and lyophilized.
Nw-Methoxy-L-arginine Bis(trifluoroacetate) (6a)15
Yield: 171 mg (99%); colorless oil; Rf = 0.56 (i-PrOH–H2O–AcOH,
6:3:1). The spectroscopic data were in good agreement with those
reported.
1H NMR (300 MHz, D2O): d = 1.78 (m, 2 H, g-CH2), 1.99 (m, 2 H,
b-CH2), 3.31 (t, 2 H, 3J = 6.8 Hz, NCH2), 3.75 (s, 3 H, OCH3), 4.06
(t, 3J = 6.2 Hz, 1 H, a-CH).
MS (ESI): m/z = 536 [M + H]+.
Nd-{[(N-Benzyloxycarbonylamino)morpholino]methylidene}-
Na-tert-butyloxycarbonyl-L-ornithine tert-Butyl Ester (5g)
Eluent: CH2Cl2–MeOH (95:5); Rf = 0.25; yield: 265 mg (98%); yel-
low wax.
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2396
D. Schade et al.
PAPER
Nw-(2-Carbamoylethyl)-L-arginine Bis(trifluoroacetate) (6f)
Yield: 185 mg (97%); colorless oil; Rf = 0.43 (i-PrOH–H2O–AcOH,
6:3:1).
13C NMR (75 MHz, D2O/TPS): d = 26.4 (g-CH2), 29.7 (b-CH2),
43.0 (NCH2), 55.5 (a-CH), 67.3 (OCH3), 160.1 (guanidine-C),
174.8 (CO2H).
HRMS (ESI): m/z calcd for C7H17N4O3 [M + H]+: 205.12952;
found: 205.12951.
1H NMR (300 MHz, D2O): d = 1.73 (m, 2 H, g-CH2), 1.98 (m, 2 H,
b-CH2), 2.57 (t, 3J = 6.4 Hz, 2 H, CH2CONH2), 3.26 (t, 3J = 6.5 Hz,
2 H, NCH2), 3.48 (t, 3J = 6.3 Hz, 2 H, NwCH2), 4.07 (t, 3J = 6.1 Hz,
1 H, a-CH).
13C NMR (75 MHz, D2O/TPS): d = 26.5 (g-CH2), 29.6 (b-CH2),
36.8 (CH2CONH2), 40.1 (NwCH2), 43.1 (NCH2), 55.4 (a-CH),
158.6 (guanidine-C), 174.6 (CONH2), 179.0 (CO2H).
Nw-(2-Methoxyethyl)-L-arginine Bis(trifluoroacetate) (6b)8
Yield: 179 mg (97%); colorless oil; Rf = 0.5 (i-PrOH–H2O–AcOH,
6:3:1). The spectroscopic data were in good agreement with those
reported.
1H NMR (300 MHz, D2O): d = 1.76 (m, 2 H, g-CH2), 2.00 (m, 2 H,
b-CH2), 3.29 (m, 2 H, NCH2), 3.39 (s, 3 H, OCH3), 3.40 (m, 2 H,
NwCH2), 3.62 (m, 2 H, OCH2), 4.09 (m, 1 H, a-CH).
HRMS (ESI): m/z calcd for C9H20N5O3 [M + H]+: 246.15607;
found: 246.15587.
13C NMR (75 MHz, D2O/TPS): d = 26.6 (g-CH2), 29.7 (b-CH2),
43.1 (NCH2), 43.8 (NwCH2), 55.4 (a-CH), 60.9 (OCH3), 73.1
(OCH2), 159.0 (guanidine-C), 174.7 (CO2H).
Nd-[(Aminomorpholino)methylidene]-L-ornithine Bis(trifluo-
roacetate) (6g)
Yield: 184 mg (97%); colorless oil; Rf = 0.38 (i-PrOH–H2O–AcOH,
6:3:1).
HRMS (ESI): m/z calcd for C9H21N4O3 [M + H]+: 233.16082;
found: 233.16073.
1H NMR (300 MHz, D2O): d = 1.70 (m, 2 H, g-CH2), 1.96 (m, 2 H,
b-CH2), 3.30 (t, 2 H, 3J = 6.8 Hz, NCH2), 3.44 (t, 3J = 4.9 Hz, 4 H,
2 × NwCH2), 3.76 (t, 3J = 4.9 Hz, 4 H, 2 × OCH2), 4.09 (t, 3J = 6.2
Hz, 1 H, a-CH).
13C NMR (75 MHz, D2O/TPS): d = 26.5 (g-CH2), 29.6 (b-CH2),
43.9 (NCH2), 48.5 (NwCH2), 55.1 (a-CH), 68.3 (OCH2), 159.1
(guanidine-C), 174.4 (CO2H).
Nw-Allyl-L-arginine Bis(trifluoroacetate) (6c)24
Yield: 173 mg (98%); colorless oil; Rf = 0.54 (i-PrOH–H2O–AcOH,
6:3:1). The spectroscopic data were in good agreement with those
reported.
1H NMR (300 MHz, D2O): d = 1.74 (m, 2 H, g-CH2), 2.00 (m, 2 H,
b-CH2), 3.30 (t, 2 H, 3J = 6.7 Hz, NCH2), 3.87 (d, 3J = 4.6 Hz, 2 H,
NwCH2), 4.11 (t, 3J = 6.3 Hz, 1 H, a-CH), 5.25 (m, 1 H, CH=CH2),
5.29 (m, 1 H, CH=CH2), 5.90 (ddt, J = 17.3, 10.4, 4.8 Hz, 1 H,
CH=CH2).
HRMS (ESI): m/z calcd for C10H21N4O3 [M + H]+: 245.16082;
found: 245.16070.
Nd-[(Amino-2,5-dihydro-1H-pyrrol-1-yl)methylidene]-L-orni-
thine Bis(trifluoroacetate) (6h)
Yield: 175 mg (96%); light yellow oil; Rf = 0.44 (i-PrOH–H2O–
13C NMR (75 MHz, D2O/TPS): d = 26.5 (g-CH2), 29.6 (b-CH2),
43.0 (NCH2), 45.7 (NwCH2), 55.3 (a-CH), 119.0 (CH=CH2), 135.1
(CH=CH2), 158.6 (guanidine-C), 174.5 (CO2H).
AcOH, 6:3:1).
HRMS (ESI): m/z calcd for C9H19N4O2 [M + H]+: 215.15025;
found: 215.15029.
1H NMR (300 MHz, D2O): d = 1.75 (m, 2 H, g-CH2), 1.98 (m, 2 H,
b-CH2), 3.33 (t, 3J = 6.5 Hz, 2 H, NCH2), 4.10 (t, 3J = 6.2 Hz, 1 H,
a-CH), 4.20 (s, 4 H, NwCH2), 5.90 (s, 2 H, CH2CH=).
13C NMR (75 MHz, D2O/TPS): d = 26.7 (g-CH2), 29.6 (b-CH2),
43.6 (NCH2), 55.3 (a-CH), 56.4 (CH2CH=), 127.4 (CH2CH), 155.9
(guanidine-C), 174.5 (CO2H).
Nw-(But-3-enyl)-L-arginine Bis(trifluoroacetate) (6d)
Yield: 181 mg (99%); colorless oil; Rf = 0.61 (i-PrOH–H2O–AcOH,
6:3:1).
1H NMR (300 MHz, D2O): d = 1.76 (m, 2 H, g-CH2), 1.98 (m, 2 H,
b-CH2), 2.34 (br pseudo q, 2 H, CH2CH=CH), 3.27 (t, 3J = 6.7 Hz,
2 H, NCH2), 3.29 (t, 3J = 6.7 Hz, 2 H, NCH2), 4.11 (t, 3J = 6.3 Hz,
1 H, a-CH), 5.16 (m, 2 H, CH=CH2), 5.84 (ddt, J = 17.2, 10.3, 6.8
Hz, CH=CH2).
13C NMR (75 MHz, D2O/TPS): d = 26.6 (g-CH2), 29.6 (b-CH2),
35.1 (CH2CH=), 42.98 (NCH2), 43.0 (N-CH2), 55.3 (a-CH), 120.2
(CH=CH2), 137.6 (CH=CH2), 158.5 (guanidine-C), 174.4 (CO2H).
HRMS (ESI): m/z calcd for C10H21N4O2 [M + H]+: 229.16590;
found: 229.16596.
HRMS (ESI): m/z calcd for C10H19N4O2 [M + H]+: 227.15025;
found: 227.15026.
Nw,w-(2,2,2-Trifluoroethyl)-L-arginine Bis(trifluoroacetate) (6i)
Yield: 192 mg (99%); colorless oil; Rf = 0.70 (i-PrOH–H2O–AcOH,
6:3:1).
1H NMR (300 MHz, D2O): d = 1.77 (m, 2 H, g-CH2), 1.91 (m, 2 H,
b-CH2), 3.31 (m, 2 H, NCH2), 4.0 (q, 3J = 8.8 Hz, 2 H, NwCH2), 4.09
(m, 1 H, a-CH).
13C NMR (75 MHz, D2O/TPS): d = 26.4 (g-CH2), 29.6 (b-CH2),
45.1 (q, 3JC,F = 34.9 Hz, CH2CF3), 43.3 (NCH2), 55.2 (a-CH), 126.5
(q, JC,F = 278.8 Hz, CF3), 158.9 (guanidine-C), 174.4 (CO2H).
Nw-Propargyl-L-arginine Bis(trifluoroacetate) (6e)7
Yield: 173 mg (98%); colorless oil; Rf = 0.57 (i-PrOH–H2O–AcOH,
6:3:1). The spectroscopic data were not in full agreement with those
reported.
HRMS (ESI): m/z calcd for C8H16F3N4O2 [M + H]+: 257.12199;
found: 257.12193.
1H NMR (300 MHz, D2O): d = 1.75 (m, 2 H, g-CH2), 1.99 (m, 2 H,
b-CH2), 2.76 (m, 1 H, C≡CH), 3.28 (t, 3J = 6.8 Hz, 2 H, NCH2), 4.03
(d, 4J = 2.3 Hz, 2 H, NCH2C≡CH), 4.08 (t, 3J = 6.2 Hz, 1 H, a-CH).
13C NMR (75 MHz, D2O/TPS): d = 26.5 (g-CH2), 29.6 (b-CH2),
33.3 (NCH2C≡CH), 43.2 (NCH2), 55.3 (a-CH), 76.4 (C≡CH), 80.6
(C≡CH), 158.4 (guanidine-C), 174.6 (CO2H).
Acknowledgment
We thank Dr. Ulrich Girreser for performing NMR and MS experi-
ments and gratefully acknowledge the excellent technical assistance
of Mrs. Melissa Zietz.
HRMS (ESI): m/z calcd for C9H17N4O2 [M + H]+: 213.1346; found:
213.13448.
Synthesis 2008, No. 15, 2391–2397 © Thieme Stuttgart · New York

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Synthesis of Optically Pure Nw-Alkylated L-Arginines
2397
(10) Cardounel, A. J.; Cui, H.; Samouilov, A.; Johnson, W.;
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Synthesis 2008, No. 15, 2391–2397 © Thieme Stuttgart · New York