An Efficient Synthesis of Optically Pure N δ-Monomethylated l -Arginine and l -Ornithine

Article abstract of DOI:10.1055/s-0035-1561303

N ^ω-Methylated l-arginines such as asymmetric dimethyl-l-arginine (ADMA) and monomethyl-l-arginine (NMMA) are well-known endogenous modulators of the nitric oxide (NO) generating system. To understand the (patho)physiological role and impact of N ^δ-methylation of l-arginine and l-ornithine an efficient synthesis of the pure enantiomers was needed. A synthetic approach that furnished both the desired amino acids in 8-10 steps from commercially available N-Boc-l-ornithine in good overall yields (20-21%) and with high optical purity (>99% ee) is reported.

Full text of DOI:10.1055/s-0035-1561303

SYNTHESIS
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© Georg Thieme Verlag Stuttgart · New York
2016, 48, 723–729
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An Efficient Synthesis of Optically Pure Nδ-Monomethylated
L-Arginine and L-Ornithine
Felix-A. Littya
Ulrich Girresera
Bernd Clementa
Dennis Schade*b
a Department of Pharmaceutical and Medicinal Chemistry,
Pharmaceutical Institute, Christian-Albrechts-University of
Kiel, Gutenbergstraße 76, 24118 Kiel, Germany
b TU Dortmund, Department of Chemistry & Chemical Biology,
Otto-Hahn-Straße 6, 44227 Dortmund, Germany
dennis.schade@tu-dortmund.de
Received: 07.10.2015
Accepted after revision: 02.12.2015
Published online: 05.01.2016
modulation.5 Therefore, not surprisingly, dysregulations of
the NO modulating system are associated with the pathol-
ogy of numerous diseases.6
DOI: 10.1055/s-0035-1561303; Art ID: ss-2015-z0587-op
Post-translational methylation of arginine residues in
proteins can affect their physiological activity.1 For exam-
ple, in histones, arginine methylation can promote or pre-
vent the docking of key effector molecules and thus regu-
lates the transcription of specific genes (epigenetic regula-
tions).7 Furthermore, arginine methylation in non-histone
proteins plays a crucial role in influencing distinct cellular
functions as cellular development and tumorigenesis.8
While much is known about the above outlined Nω-
methyl-L-arginines, the (patho)physiological role of Nδ-
methylation is not well understood. Nδ-Methyl-L-arginine
(11, Schemes 1 and 2) was discovered in 1998 as a post-
translational modification of arginine residues in yeast cells
and is studied in our group regarding possible functions in
the NO modulating system.9 Moreover, Nδ-methyl-L-orni-
thine (10, Scheme 2) was identified as a degradation prod-
uct of base-treated Nδ-methyl-L-arginine (11).10 Strikingly,
latest findings suggest a physiological relevance in humans
since 11 has been detected and quantified in human plas-
ma.11
Abstract Nω-Methylated L-arginines such as asymmetric dimethyl-L-ar-
ginine (ADMA) and monomethyl-L-arginine (NMMA) are well-known
endogenous modulators of the nitric oxide (NO) generating system. To
understand the (patho)physiological role and impact of Nδ-methylation
of L-arginine and L-ornithine an efficient synthesis of the pure enantio-
mers was needed. A synthetic approach that furnished both the desired
amino acids in 8–10 steps from commercially available N-Boc-L-orni-
thine in good overall yields (20–21%) and with high optical purity (>99%
ee) is reported.
Key words alkylation, amino acids, bioorganic chemistry, chiral pool,
chiral resolution, protecting groups
Methylated arginines such as ADMA (asymmetric Nω,Nω-
dimethyl-L-arginine), NMMA (Nω-monomethyl-L-arginine),
and SDMA (symmetric Nω,Nω′-dimethyl-L-arginine) are
formed by post-translational methylation of arginine resi-
dues in proteins (Scheme 1).1,2 These modifications are cat-
alyzed by protein arginine methyltransferases (PRMTs).1
Within the proteolysis of these proteins, methylated argi-
nines are liberated and metabolized by the dimethylargi-
nine dimethylaminohydrolase (DDAH). They can act as
physiological modulators of the nitric oxide (NO) generat-
ing system by inhibition of nitric oxide synthases (NOSs).3
The degradation of L-arginine to L-ornithine and urea is cat-
alyzed by arginases, which represents the final step of the
urea cycle.4 Nitric oxide is involved in a plethora of physio-
logical processes, with the regulation of vascular tone and
tissue blood flow as one of its many functions. The anti-
atherosclerotic potential of NO is largely due to inhibition of
platelet aggregation and leukocyte adhesion on the endo-
thelial surface. In addition, NO is a neuromediator with
many physiological functions, including memory and pain
Therefore, to further study Nδ-methylated arginine 11
and its putative metabolite 10, an efficient synthetic proto-
col is needed. However, selective methylation of the Nδ-ni-
trogen of arginine is a challenge and can only be tackled us-
ing a suitable protecting group strategy.
The first synthesis of 10 and 11 was reported in 1983 by
the Steglich group, who obtained the compounds in race-
mic form.12 Since NOS, DDAH, and arginase are highly sub-
strate-specific for naturally occurring L-amino acids, the es-
tablishment of a synthetic concept for optically pure 10 and
11 would thus be desirable.13 We and others have described
a synthetic approach for a series of Nδ-methylated L-argi-
nine derivatives with already high enantiopurity (>98%
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ee).14,15 However, this strategy employed distinct boroxazo-
lidinones as an orthogonal amino acid protecting group,
which was highly dependent on the availability of optically
pure 10. Here, we present a conceptually different approach
that avoids these shortcomings by furnishing 7, a suitably
protected form of 10, in high yields and enantiopurity.
Previous work on the synthesis of 10 involved protect-
ing group (PG) strategies that were associated with a high
risk of racemization, such as the tosyl PG that allowed
monomethylation of the Nδ-nitrogen in ornithine.12,14,15
Cleavage of this PG can only be performed under rather
harsh reaction conditions, namely treatment with HBr in
glacial acetic acid and higher temperatures.14 These acidic
conditions cause rapid racemization of amino acids.16 In
fact, we determined that reaction times should not exceed
1.5 hours, and that HBr should be quickly removed under
high vacuum (<1 mbar) at low temperature, not higher than
50–60 °C.15
under mild conditions. 2-Nitrobenzenesulfonamides fulfill
these requirements as they have been reported to be
cleaved under mild conditions by treatment with nucleo-
philic agents like thiophenol.17 However, before introducing
the 2-nitrobenzenesulfonyl group, the first challenge was to
efficiently protect the amino acid moiety from methylation.
Commercially available Nα-Boc-protected L-ornithine 1 was
treated with phthalic anhydride to protect the reactive Nδ-
position and furnished
2 in good yields (60–70%)
(Scheme 2).18 Next, esterification of the α-carboxylic moi-
ety was performed with tert-butyl bromide according to a
literature-described protocol.19 To avoid later methylation
at the Boc-protected Nα-position, a second Boc group was
introduced by treatment of 3 with di-tert-butyl dicarbonate
(Boc2O) in anhydrous acetonitrile and DMAP as a catalyst to
furnish the fully protected ornithine 4.20 By treatment of 4
with hydrazine, the phthalimide PG could be efficiently re-
moved, delivering 5 in very good yield (90%). This amino
acid protected ornithine 5 served as an ideal building block
for the subsequent synthetic sequence. Compound 5 was
Hence, we aimed at utilizing a tosyl-like PG that similar-
ly enables selective monomethylation and can be removed
O
OH
O
OH
NH2
NH2
+
ADMA
N
NH
HN
N
NMMA
SDMA
proteolysis
PRMTs
CH3
(H)H3C
HN
NH2
Nδ-methyl-L-arginine
CH3(H)
physiological role and function?
DDAH
protein
synthesis
NH(CH3)2
NH2CH3
O
OH
O
OH
NOS
arginine
HN
NH2
NH2
+
NO
NH
NH2
O
NH
NH2
arginase
citrulline
O
OH
NH2
+
urea
ornithine
NH2
Scheme 1 Biochemical pathways of L-arginine and its methylated analogues. PRMTs: protein arginine methyltransferases; ADMA: asymmetric Nω,Nω-
dimethyl-L-arginine; SDMA: symmetric Nω,Nω′-dimethyl-L-arginine; NMMA: Nω-monomethyl-L-arginine, NO: nitric oxide; NOS: nitric oxide synthase (EC
1.14.13.39); DDAH: dimethylarginine dimethylaminohydrolase (EC 3.5.3.18); arginase (EC 3.5.3.1).
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F.-A. Litty et al.
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reacted with 2-nitrobenzenesulfonyl chloride and triethyl-
amine in CH2Cl2 to afford the sulfonamide 6, followed by
methylation with MeI. Removal of the 2-nitrobenzenesulfo-
nyl group was accomplished by reacting 7 with thiophenol
in the presence of K2CO3 as a base. The reaction went to
completion after 2 hours at room temperature to provide
Nδ-methylated ornithine 8 in form of the free amine in very
good yield (89%). N,N′-Bis-(tert-butyloxycarbonyl)thiourea
was used as a guanylation reagent. This reaction was car-
ried out with DIPEA for proton trapping and EDCI as the de-
sulfuration agent, instead of the more toxic (and oftentimes
less efficient) HgCl2. Finally, for both 8 and 9, protecting
groups were carefully removed with gaseous HCl under
moisture-free conditions in absolute diethyl ether at low
temperatures to minimize risks for acid-catalyzed racem-
ization. Under these conditions, the dihydrochloride salt (as
confirmed by NMR spectroscopy, see Supporting Informa-
tion) of Nδ-methyl-L-arginine precipitated and was further
purified by flash chromatography on reversed-phase (RP-
18) silica gel. In contrast to 11, the dihydrochloride of Nδ-
methyl-L-ornithine (10) did not readily precipitate but was
isolated as a highly hygroscopic white solid after chromato-
graphic workup.
O
O
Br
O
O
O
O
O
O
H2N
OH
NHBoc
N
Ot-Bu
NHBoc
N
OH
C2Cl4,
BTEAC, K2CO3,
DMAC,
55 °C, 5 h
NHBoc
100 °C, 6 h
O
O
1
3, 93%
2, 60–70%
O
O
S
Cl
O
O
O
NO2
Boc2O
N2H4*H2O
N
Ot-Bu
Boc
H2N
Ot-Bu
Boc
Et3N, CH2Cl2,
r.t., 2 h
MeCN, DMAP,
Ar atm,
r.t., 24–48 h
CH2Cl2/MeOH
(1:1),
r.t., 24 h
N
N
Boc
Boc
O
4, 77%
5, 90%
SH
O2N
O
O2N
O
O
O
O
O
MeI
S
S
N
Ot-Bu
N
Ot-Bu
H
K2CO3, DMF,
r.t., 6 h
K2CO3, DMF,
r.t., 2 h
N
CH3
N
Boc
Boc
Boc
Boc
6, 78%
7, 82%
S
Boc
O
N
O
BocHN
NHBoc
H3C
N
Ot-Bu
Boc
BocHN
N
CH3
Ot-Bu
Boc
H
DIPEA, EDCI,
CH2Cl2, r.t., 2 h
N
N
Boc
8, 89%
Boc
9, 98%
HCl(g), anhyd Et2O,
2 atm,
0 °C, 45 min
HCl(g), anhyd Et2O,
2 atm,
0 °C, 20 min
N
N
H
H
N+ Cl–
O
O
H Cl–
N+
H
H2N
N
OH
OH
+
NH3 Cl–
+
CH3
NH3 Cl–
CH3
10, 89% (21% overall yield, 8 steps)
11, 84% (20% overall yield, 10 steps)
Scheme 2 Linear synthesis of Nδ-methyl-L-arginine (11) and Nδ-methyl-L-ornithine (10). BTEAC: benzyltriethylammonium chloride; DMAC: N,N-di-
methylacetamide.
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There are different chromatographic (HPLC) methods
for the discrimination of amino acid enantiomers. A con-
ventional method is ligand exchange chromatography with
chiral eluents on common RP-18 columns. One of these
procedures was described by Gil-Av et al. and adapted to
the herein reported compound of interest, Nδ-methyl-L-
arginine (11).21 L-Proline in combination with copper ace-
tate forms stable diastereomeric complexes with Nδ-methyl-
arginine enantiomers resulting in distinct retention times.
Using this system, injection of 11 showed a single peak,
which corresponds to the second eluting peak (12.9 min,
see Figure 1) from racemic Nδ-methylarginine that we ob-
tained from the Steglich group.12 Considering that the here-
in presented synthetic sequence started with enantiopure
commercial material (1) with low risk for racemization, it
can be assumed that the peak at 12.9 minutes corresponds
to the L-enantiomer. To reinforce this assumption, we also
analyzed D- and L-arginine and could confirm that the L-en-
antiomer eluted second (see Supporting Information). The
limit of detection, determined for the D-enantiomer of race-
mic Nδ-methylarginine (10 mM), was 50 μM, based on a
signal-to-noise-ratio of more than 3:1. Thus, the enantio-
meric purity of 11 can be stated as ≥99% ee. Moreover, since
11 has been synthesized from 8, it can also be concluded
that Nδ-methyl-L-ornithine 10 exhibits >99% ee.
enantiomeric purity, we have developed a straightforward
synthetic strategy, utilizing a protecting group concept (i.e.,
2-nitrobenzenesulfonyl) that allowed installing the desired
Nδ-methyl group under mild reaction conditions and mini-
mal risk for racemization. We could ultimately verify opti-
cal purity by a reliable HPLC-based analytical method that
employed a chiral eluent on a conventional reversed-phase
column.
Melting points are uncorrected. 1H (300 MHz), 13C (75 MHz), and 35Cl
(29 MHz) NMR spectra were recorded on a Bruker Avance III 300
spectrometer at 298 K. Chemical shifts (δ values) were quoted in ppm
relative to TMS as an internal standard, 3-(trimethylsilyl)-1-propane-
sulfonic acid sodium salt (TPS) as an external standard or alternative-
ly, relative to the solvent residual signal. Spectra interpretation was
performed by first order analysis. Signal assignment was done via 1H,
1H-COSY and 13C-HSQC and -HMBC. Quantification of Cl– by 35Cl NMR
was verified by a three-point calibration with NaCl in D2O. Low-reso-
lution mass spectra were recorded using a Bruker amazon SL system
with LC coupling, electrospray ionization, in the positive mode. Re-
cordings of exact mass spectra were performed at the Department of
Physical Chemistry, Christian-Albrechts-University of Kiel, on a 7.05
Tesla Bruker APEX III FT-ICR mass spectrometer in ESI-positive mode;
samples were dissolved in EtOH and diluted with a solvent mixture
containing MeOH, H2O, and formic acid (49.9:49.9:0.2) to a concen-
tration of about 100 pmol/μL. Elemental analyses were performed on
a CHNS analyzer (HEKAtech GmbH) at the Department of Inorganic
Chemistry, Christian-Albrechts-University of Kiel. IR spectra were re-
corded on a Shimadzu IRAffinity-1S FTIR spectrometer equipped
with MIRacle 10 Single Reflection ATR Accessory. Optical rotations
were measured on a PerkinElmer 241 polarimeter at 20 °C. Reactions
were monitored by TLC on precoated silica gel plates (SiO2, 60, F254).
All compounds could be detected by either UV detection or by ninhy-
drin staining. Purification of synthesized compounds was carried out
on a Combi Flash Rf, version 1.8.2, flash chromatography apparatus
using Interchim PF-30SIHP-JP/12G or 40G or PF-15SIHP/12G silica gel
columns. Reverse phase chromatography was performed by column
chromatography using silica gel 60 silanized (0.063–0.200 mm,
Merck). All starting materials were commercially available and used
without further purification. Nα-(tert-Butyloxycarbonyl)-L-ornithine
(1) was purchased from Bachem. N,N′-Bis-(tert-butyloxycarbon-
yl)thiourea was purchased from Sigma Aldrich. All solvents were dis-
tilled and dried according to standard procedures.
Nα-(tert-Butyloxycarbonyl)-Nδ-phthalimido-L-ornithine (2)19
Nα-(tert-Butyloxycarbonyl)-L-ornithine (1; 1.94 g, 8.4 mmol) and
phthalic anhydride (2.22 g, 15.0 mmol) were suspended in CHCl3 (20
mL) and tetrachloroethylene (150 mL). This suspension was heated
for 2 h at 60 °C and an additional 6 h at 100 °C. The clear reaction
mixture was concentrated in vacuo to afford the crude product. Puri-
fication was carried out by flash column chromatography (silica gel,
CH2Cl2–MeOH, 0–5%); yield: 1.9 g (61%); white crystalline solid; mp
126 °C; Rf = 0.54 (CH2Cl2–MeOH, 9:1). The spectroscopic data were in
agreement with those reported.19
Figure 1 Representative chromatograms of racemic Nδ-methylargi-
nine (black) and optically pure Nδ-methyl-L-arginine 11 (turquoise) on a
VDS Optilab Nucleosil 100 C18 column with a chiral eluent composed
of L-proline and aqueous Cu(OAc)2 in ddH2O (pH 4.5) and post-column
o-PA derivatization (for details see the experimental section).
In summary, the establishment of an efficient synthesis
for optically pure Nδ-methyl-L-arginine and -ornithine rep-
resents an important advance in arginine chemistry and
will enable various studies of biological as well as pharma-
cological significance. Motivated by the necessity of high
IR (ATR): 2976, 1771, 1697, 1514, 1396, 1366, 1159, 1047, 718 cm–1
.
1H NMR (300 MHz, CDCl3): δ = 9.36 (br s, 1 H, OH), 7.80–7.86 (m, 2 H,
ArH), 7.68–7.75 (m, 2 H, ArH), 5.16 (d, 3J = 8.2 Hz, 1 H, NH), 4.14–4.37
(m, 1 H, α-CH), 3.72 (t, 3J = 6.8 Hz, 2 H, δ-CH2), 1.65–2.00 (m, 4 H, β,γ-
CH2), 1.43 [s, 9 H, C(CH3)3].
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13C NMR (75 MHz, CDCl3): δ = 176.5 (CO2H), 168.4 (2 × CO-Pht), 155.6
(CO-Boc), 134.0 (2 × ArCH), 132.0 (2 × ArC), 123.3 (2 × ArCH), 80.3
[C(CH3)3], 53.0 (α-CH), 37.4 (δ-CH2), 29.8 (β-CH2), 28.3 [C(CH3)3], 24.8
(γ-CH2).
MS (ESI): m/z = 541 [M + Na]+, 419 [M – C4H8 – CO2 + H]+, 319 [M – 2 ×
C4H8 – 2 × CO2 + H]+ 263 [M – 3 × C4H8 – 2 × CO2 + H]+.
Anal. Calcd for C27H38N2O8 (518.61): C, 62.53; H, 7.39; N, 5.40. Found:
C, 62.00; H, 7.34; N, 5.49.
MS (ESI): m/z = 725 [2 × M + H]+, 625 [2 × M – C4H8 – CO2 + H]+, 363 [M
+ H]+, 263 [M – C4H8 – CO2 + H]+.
Nα,Nα-Bis(tert-butyloxycarbonyl)-L-ornithine tert-Butyl Ester (5)
Anal. Calcd for C18H22N2O6 (362.38): C, 59.66; H, 6.12; N, 7.73. Found:
C, 59.21; H, 6.15; N, 7.58.
Compound 4 (1.88 g, 3.6 mmol) was dissolved in a mixture of CH2Cl2
and MeOH (1:1, 40 mL). Hydrazine hydrate (2.7 mL, 54 mmol, 15
equiv) was added and the reaction mixture was stirred magnetically
at r.t. After 24 h, the mixture was filtered to remove the precipitated
phthalhydrazide and quenched by adding sat. aq NaHCO3 (30 mL). The
organic phase was separated and the aqueous layer was extracted
with CH2Cl2 (3 × 20 mL). The combined organic phases were washed
with brine (30 mL), dried (Na2SO4), and concentrated under reduced
pressure. Compound 5 was at this point already pure (TLC) and used
in the following reaction without any purification; yield: 1.27 g
(90%); colorless oil; Rf = 0.37 (CH2Cl2–MeOH–aq NH3, 8.5:1.5:0.5).
1H NMR (300 MHz, CDCl3): δ = 4.72 (dd, 3J = 9.6, 5.2 Hz, 1 H, α-CH),
2.64–2.79 (m, 2 H, δ-CH2), 2.03–2.15 (m, 1 H, β-CH2), 1.80–1.94 (m, 1
H, β-CH2), 1.40–1.56 (m, 2 H, γ-CH2), 1.51 [2 s, 18 H, C(CH3)3], 1.45 [s,
9 H, C(CH3)3], 1.21 (br s, 2 H, NH2).
13C NMR (75 MHz, CDCl3): δ = 169.9 (CO2t-Bu), 152.5 (2 × CO-Boc),
82.7 [2 × C(CH3)3], 81.2 [C(CH3)3], 58.8 (α-CH), 42.0 (δ-CH2), 30.7 (β-
CH2), 28.1 [2 × C(CH3)3], 28.0 [C(CH3)3], 26.6 (γ-CH2).
Nα-(tert-Butyloxycarbonyl)-Nδ-phthalimido-L-ornithinetert-Butyl
Ester (3)19
Compound 2 (1.87 g, 5.2 mmol), benzyltriethylammonium chloride
(1.17 g, 5.2 mmol, 1 equiv), tert-butyl bromide (27 mL, 239 mmol, 46
equiv), and K2CO3 (19 g, 135 mmol, 26 equiv) were added to DMAC
(40 mL). This reaction mixture was heated for 5 h at 55 °C and cooled
to r.t. The salts were filtered and washed with EtOAc (3 × 25 mL). The
filtrate was washed with H2O (3 × 20 mL) and the combined aqueous
phases were extracted with EtOAc (1 × 25 mL). The combined organic
layers were dried (Na2SO4) and the solvent was removed under re-
duced pressure. The crude product was purified by flash column
chromatography (silica gel, cyclohexane–EtOAc, 0–20%); yield: 2.0 g
(93%); colorless oil; Rf = 0.48 (cyclohexane–EtOAc, 2:1). The spectro-
scopic data were in agreement with those reported.19
IR (ATR): 3399, 2978, 1771, 1740, 1694, 1508, 1393, 1150, 718 cm–1
.
1H NMR (300 MHz, CDCl3): δ = 7.81–7.88 (m, 2 H, ArH), 7.68–7.75 (m,
2 H, ArH), 5.06 (d, 3J = 8.3 Hz, 1 H, NH), 4.14–4.27 (m, 1 H, α-CH), 3.71
(t, 3J = 6.9 Hz, 2 H, δ-CH2), 1.62–1.91 (m, 4 H, β,γ-CH2), 1.45 [s, 9 H,
C(CH3)3], 1.43 [s, 9 H, C(CH3)3].
MS (ESI): m/z = 777 [2 × M + H]+, 389 [M + H]+, 289 [M – C4H8 – CO2 +
H]+.
Anal. Calcd for C19H36N2O6 (388.51): C, 58.74; H, 9.34; N, 7.21. Found:
C, 58.49; H, 9.61; N, 6.91.
13C NMR (75 MHz, CDCl3): δ = 171.6 (CO2t-Bu), 168.3 (2 × CO-Pht),
155.3 (CO-Boc), 133.9 (2 × ArCH), 132.1 (2 × ArC), 123.3 (2 × ArCH),
79.7, 80.3 [2 × C(CH3)3], 53.6 (α-CH), 37.6 (δ-CH2), 30.3 (β-CH2), 28.3
[C(CH3)3], 28.0 [C(CH3)3], 24.5 (γ-CH2).
Nα,Nα-Bis(tert-butyloxycarbonyl)-Nδ-(2-nitrophenylsulfonyl)-L-or-
nithine tert-Butyl Ester (6)
Compound 5 (1.27 g, 3.3 mmol) and 2-nitrosulfonyl chloride (0.95 g,
4.3 mmol, 1.3 equiv) were dissolved in anhydrous CH2Cl2 (60 mL).
Et3N (589 μL, 4.3 mmol, 1.3 equiv) was added and the reaction mix-
ture was stirred magnetically at 0 °C for 30 min and at r.t. for an addi-
tional 2 h. The solvent was removed in vacuo to afford the crude
product. Purification was performed by flash column chromatogra-
phy (silica gel, cyclohexane–EtOAc, 0–25%); yield: 1.46 g (78%); color-
less oil; Rf = 0.69 (cyclohexane–EtOAc, 1:1).
MS (ESI): m/z = 419 [M + H]+, 319 [M – C4H8 – CO2 + H]+, 263 [M – 2 ×
C4H8 – CO2 + H]+.
Anal. Calcd for C22H30N2O6 (418.49): C, 63.14; H, 7.23; N, 6.69. Found:
C, 62.70; H, 7.56; N, 7.12.
Nα,Nα-Bis(tert-Butyloxycarbonyl)-Nδ-phthalimido-L-ornithine
tert-Butyl Ester (4)
IR (ATR): 2978, 1734, 1697, 1554, 1366, 1155, 1130, 851 cm–1
.
Compound 3 (2.0 g, 4.8 mmol), DMAP (61 mg, 0.5 mmol), and di-tert-
butyl dicarbonate (10.9 g, 48 mmol, 10 equiv) were dissolved in anhy-
drous MeCN (25 mL) under an argon atmosphere. This reaction mix-
ture was stirred magnetically until the reaction was completed (TLC).
The mixture was diluted with CH2Cl2 (25 mL) and washed with sat. aq
NaHCO3 (20 mL), H2O (20 mL), and brine (20 mL). The organic phase
was dried (Na2SO4) and concentrated in vacuo. Purification was car-
ried out by flash column chromatography (silica gel, cyclohexane–
EtOAc, 0–20%); yield: 1.9 g (77%); colorless oil; Rf = 0.56 (cyclohex-
ane–EtOAc, 2:1).
1H NMR (300 MHz, CDCl3): δ = 8.10–8.16 (m, 1 H, ArH), 7.82–7.89 (m,
1 H, ArH), 7.69–7.77 (m, 2 H, ArH), 5.35 (t, 3J = 6.2 Hz, 1 H, NH), 4.63
(dd, 3J = 9.0, 5.5 Hz, 1 H, α-CH), 3.12 (q, 3J = 6.7 Hz, 2 H, δ-CH2), 2.02–
2.14 (m, 1 H, β-CH2), 1.78–1.91 (m, 1 H, β-CH2), 1.54–1.67 (m, 2 H, γ-
CH2), 1.49 [2 s, 18 H, C(CH3)3], 1.43 [s, 9 H, C(CH3)3].
13C NMR (75 MHz, CDCl3): δ = 169.4 (CO2t-Bu), 152.5 (2 × CO-Boc),
148.1 (ArC–N), 133.8 (ArC–S), 125.4, 131.1, 132.8, 133.5 (ArCH), 83.0
[2 × C(CH3)3], 81.5 [C(CH3)3], 58.2 (α-CH), 43.5 (δ-CH2), 28.0 [2 ×
C(CH3)3], 27.9 [C(CH3)3], 26.8 (β-CH2), 26.5 (γ-CH2).
IR (ATR): 2978, 1773, 1736, 1711, 1366, 1126, 1026, 719 cm–1
.
MS (ESI): m/z = 596 [M + Na]+, 362 [M – 3 × C4H8 – CO2 + H]+, 318 [M –
1H NMR (300 MHz, CDCl3): δ = 7.82–7.84 (m, 2 H, ArH), 7.69–7.73 (m,
2 H, ArH), 4.78 (dd, 3J = 9.3, 5.1 Hz, 1 H, α-CH), 3.72 (dt, 3J =6.9 Hz, 4J =
1.9 Hz, 2 H, δ-CH2), 2.04–2.16 (m, 1 H, β-CH2), 1.83–1.97 (m, 1 H, β-
CH2), 1.68–1.79 (m, 2 H, γ-CH2), 1.49 [2 s, 18 H, C(CH3)3], 1.43 [s, 9 H,
C(CH3)3].
13C NMR (75 MHz, CDCl3): δ = 169.6 (CO2t-Bu), 168.3 (2 × CO-Pht),
152.4 (2 × CO-Boc), 133.8 (2 × ArCH), 132.2 (2 × ArC), 123.2 (2 ×
ArCH), 82.9 [2 × C(CH3)3], 81.3 [C(CH3)3], 58.4 (α-CH), 37.5 (δ-CH2),
28.0 [2 × C(CH3)3], 27.9 [C(CH3)3], 26.5 (β-CH2), 25.6 (γ-CH2).
3 × C4H8 – 2 × CO2 + H]+.
Nα,Nα-Bis(tert-butyloxycarbonyl)-Nδ-methyl-Nδ-(2-nitrophenyl-
sulfonyl)-L-ornithine tert-Butyl Ester (7)
To a solution of 6 (1.45 g, 2.5 mmol) in DMF (15 mL) were added
K2CO3 (1.05 g, 7.6 mmol, 3 equiv) and MeI (475 μL, 7.6 mmol, 3 equiv).
The reaction mixture was stirred magnetically at r.t. for 6 h. The mix-
ture was quenched by adding 10% aq ammonia (10 mL) and extracted
with CH2Cl2 (3 × 10 mL). The combined organic phases were dried
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3
(Na2SO4) and concentrated under reduced pressure. The resulting oil
was purified by flash column chromatography (silica gel, cyclohex-
ane–EtOAc, 0–30%); yield: 1.22 g (82%); colorless oil; Rf = 0.67 (tolu-
ene–EtOAc, 7:3).
1H NMR (300 MHz, CDCl3): δ = 7.95–8.01 (m, 1 H, ArH), 7.63–7.71 (m,
2 H, ArH), 7.57–7.63 (m, 1 H, ArH), 4.70 (dd, 3J = 9.1, 5.5 Hz, 1 H, α-
CH), 3.11–3.34 (m, 2 H, δ-CH2), 2.89 (s, 3 H, NCH3), 2.02–2.14 (m, 1 H,
β-CH2), 1.79–1.92 (m, 1 H, β-CH2), 1.58–1.74 (m, 2 H, γ-CH2), 1.50 [2 s,
18 H, C(CH3)3], 1.44 [s, 9 H, C(CH3)3].
1H NMR (300 MHz, CDCl3): δ = 10.6 (br s, 1 H, NH), 4.72 (dd, J = 9.0,
5.4 Hz, 1 H, α-CH), 3.35–3.63 (m, 2 H, δ-CH2), 3.00 (s, 3 H, NCH3),
1.99–2.12 (m, 1 H, β-CH2), 1.77–1.92 (m, 1 H, β-CH2), 1.60–1.74 (m, 2
H, γ-CH2), 1.51 [2 s, 18 H, C(CH3)3], 1.48 [2 s, 18 H, C(CH3)3], 1.45 [s, 9
H, C(CH3)3].
13C NMR (75 MHz, CDCl3): δ = 169.6 (CO2t-Bu), 155.8 (C=N), 152.5 (4 ×
CO-Boc), 82.9 [4 × C(CH3)3], 81.3 [C(CH3)3], 58.5 (α-CH), 50.6 (δ-CH2),
36.8 (NCH3), 28.2 [2 × C(CH3)3], 28.1 [2 × C(CH3)3], 28.0 [C(CH3)3], 26.6
(β-CH2), 24.3 (γ-CH2).
13C NMR (75 MHz, CDCl3): δ = 169.5 (CO2t-Bu), 152.5 (2 × CO-Boc),
148.0 (ArC–N), 132.5 (ArC–S), 124.0, 130.9, 131.5, 133.4 (ArCH), 83.0
[2 × C(CH3)3], 81.4 [C(CH3)3], 58.3 (α-CH), 49.7 (δ-CH2), 34.4 (NCH3),
28.0 [2 × C(CH3)3], 27.9 [C(CH3)3], 26.4 (β-CH2), 24.8 (γ-CH2).
MS (ESI): m/z = 645 [M + H]+.
HRMS (ESI): m/z [M + H]+ calcd for C31H56N4O10: 645.4069; found:
645.4072.
Anal. Calcd for C31H56N4O10 (644.81): C, 57.74; H, 8.75; N, 8.69. Found:
C, 57.72; H, 9.01; N, 8.60.
MS (ESI): m/z = 610 [M + Na]+, 388 [M – 2 × C4H8 – 2 × CO2 + H]+, 376
[M – 3 × C4H8 – CO2 + H]+, 332 [M – 3 × C4H8 – 2 × CO2 + H]+.
Nδ-Methyl-L-ornithine Dihydrochloride (10)
Anal. Calcd for C26H41N3O10S·0.4 H2O (594.90): C, 52.49; H, 7.08; N,
7.06. Found: C, 52.74; H, 7.67; N, 6.85.
Compound 8 (0.22 g, 0.54 mmol) was dissolved in absolute Et2O (20
mL) under a N2 atmosphere and stirred magnetically at 0 °C for 30
min. Gaseous HCl was carefully bubbled through the solution for 20
min and the mixture was stirred at 0 °C for an additional 2 h. The re-
action mixture was left in the refrigerator for 24 h and then concen-
trated under vacuum. The white solid was taken up in double dis-
tilled H2O (2 × 1 mL) and purified by RP-18 column chromatography
with double distilled H2O as eluent. The product containing fractions
were combined and concentrated under reduced pressure; yield: 105
mg (89%); white, crystalline and very hygroscopic solid (mp not mea-
Nα,Nα-Bis(tert-butyloxycarbonyl)-Nδ-methyl-L-ornithine tert-Bu-
tyl Ester (8)
To a solution of 7 (1.21 g, 2.1 mmol) in DMF (15 mL) were added
K2CO3 (0.85 g, 6.2 mmol, 3 equiv) and thiophenol (509 μL, 4.1 mmol, 2
equiv). The reaction mixture was stirred magnetically at r.t. for 2 h
and washed with 10% aq ammonia (10 mL). The mixture was extract-
ed with CH2Cl2 (3 × 10 mL) and the combined organic phases were
dried (Na2SO4). The solvent was removed under reduced pressure and
purification was carried out by flash column chromatography (silica
gel, CH2Cl2–MeOH, 0–30%); yield: 0.74 g (89%); colorless oil; Rf = 0.52
(CH2Cl2–MeOH–aq NH3, 8.5:1.5:0.5).
20
surable); [α]D +11.5 (c 2.00, H2O); Rf = 0.37 (i-PrOH–H2O–AcOH,
6:3:1).
1H NMR (300 MHz, D2O/TPS): δ = 4.02 (t, 3J = 6.1 Hz, 1 H, α-CH), 3.06
(t, 3J = 7.5 Hz, 2 H, δ-CH2), 2.69 (s, 3 H, NCH3), 1.70–2.08 (m, 4 H, β,γ-
CH2).
13C NMR (75 MHz, D2O/TPS): δ = 173.8 (CO2H), 54.4 (α-CH), 49.7 (δ-
CH2), 34.6 (NCH3), 28.7 (β-CH2), 23.3 (γ-CH2).
MS (ESI): m/z = 461 [3 × M + Na]+, 293 [2 × M + H]+, 147 [M + H]+.
HRMS (ESI): m/z [M + H]+ calcd for C6H14N2O2: 147.1128; found:
IR (ATR): 2978, 1734, 1697, 1366, 1130, 849 cm–1
.
1H NMR (300 MHz, CDCl3): δ = 4.72 (dd, 3J = 9.5, 5.3 Hz, 1 H, α-CH),
2.97 (br s, 1 H, NH), 2.61–2.70 (m, 2 H, δ-CH2), 2.46 (s, 3 H, NCH3),
2.04–2.16 (m, 1 H, β-CH2), 1.82–1.94 (m, 1 H, β-CH2), 1.53–1.66 (m, 2
H, γ-CH2), 1.50 [2 s, 18 H, C(CH3)3], 1.44 [s, 9 H, C(CH3)3].
13C NMR (75 MHz, CDCl3): δ = 169.8 (CO2t-Bu), 152.5 (2 × CO-Boc),
82.8 [2 × C(CH3)3], 81.2 [C(CH3)3], 58.7 (α-CH), 51.1 (δ-CH2), 35.8
(NCH3), 28.0 [2 × C(CH3)3], 27.9 [C(CH3)3], 26.9 (β-CH2), 26.0 (γ-CH2).
147.1127.
MS (ESI): m/z = 425 [M + Na]+, 303 [M – C4H8 – CO2 + H]+.
HRMS (ESI): m/z [M + H]+ calcd for C20H38N2O6: 403.2803; found:
Nδ-Methyl-L-arginine Dihydrochloride (11)
Compound 9 (1.02 g, 1.58 mmol) was dissolved in absolute Et2O (80
mL) under a N2 atmosphere and stirred magnetically at 0 °C for 30
min. Gaseous HCl was carefully bubbled through the solution for
45 min and the reaction mixture was stirred at 0 °C for an additional
6 h. The product 11 precipitated during storage in the refrigerator and
was isolated after 24 h by careful solvent evaporation. The white solid
was taken up in double distilled H2O (2 × 1 mL) and purified by RP-18
column chromatography with double distilled H2O as eluent. The
product containing fractions were combined and concentrated under
vacuum; yield: 369 mg (84%); white solid; mp 210 °C (Lit.12 mp 210–
403.2798.
Anal. Calcd for C20H38N2O6 (402.53): C, 59.68; H, 9.52; N, 6.96. Found:
C, 59.31; H, 9.89; N, 6.63.
Nω,Nω′-Bis(tert-butyloxycarbonyl)-Nα,Nα-bis(tert-butyloxycarbon-
yl)-Nδ-methyl-L-arginine tert-Butyl Ester (9)
To a solution of compound 8 (0.71 g, 1.88 mmol) in anhydrous CH2Cl2
(20 mL) was added N,N′-bis-(tert-butyloxycarbonyl)thiourea (0.59 g,
2.13 mmol, 1.2 equiv) and DIPEA (611 μL, 3.6 mmol, 2 equiv). The re-
action mixture was stirred magnetically at 0 °C for 30 min; EDCI was
then added (0.69 g, 3.6 mmol, 2 equiv) and the mixture was stirred
for an additional 2 h at r.t. The solvent was removed in vacuo and pu-
rification was performed by flash column chromatography (silica gel,
cyclohexane–EtOAc, 0–30%); yield: 1.12 g (98%); colorless oil; Rf =
0.51 (cyclohexane–EtOAc, 1:1).
20
212 °C for DL-compound); [α]D +11.0 (c 2.00, H2O); Rf = 0.51 (i-
PrOH–H2O–AcOH, 6:3:1).
IR (ATR): 3361, 3167, 2947, 1720, 1622, 1504, 1427, 1231, 752 cm–1
1H NMR (300 MHz, D2O/TPS): δ = 4.04 (t, 3J = 6.1 Hz, 1 H, α-CH), 3.37
(t, 3J = 7.3 Hz, 2 H, δ-CH2), 2.99 (s, 3 H, NCH3), 1.62–2.02 (m, 4 H, β,γ-
CH2).
.
IR (ATR): 2978, 1740, 1699, 1608, 1366, 1231, 1047, 847 cm–1
.
13C NMR (75 MHz, D2O/TPS): δ = 173.8 (CO2H), 158.3 (C=N), 54.6 (α-
CH), 51.3 (δ-CH2), 37.7 (NCH3), 28.5 (β-CH2), 24.0 (γ-CH2).
35Cl NMR (29 MHz, D2O, NaCl): Calcd for C7H18Cl2N4O2: Cl, 27.15;
found: Cl, 24.66 (91%).
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Synthesis
MS (ESI): m/z = 753 [4 × M + H]+, 565 [3 × M + H]+, 377 [2 × M + H]+,
(2) (a) Krause, C. D.; Yang, Z.-H.; Kim, Y.-S.; Lee, J.-H.; Cook, J. R.;
Pestka, S. Pharmacol. Ther. 2007, 113, 50. (b) Paik, W. K.; Kim, S.
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189 [M + H]+.
HRMS (ESI): m/z [M + H]+ calcd for C7H16N4O2: 189.1346; found:
189.1348.
HPLC-Based Chiral Separation of Nδ-Methyl-D/L-arginine
Discrimination of the enantiomers was carried out on a Waters HPLC
system equipped with Waters W600 pump, Waters W474 fluores-
cence detector, and Waters 717 Plus autosampler; stationary phase:
VDS Optilab Nucleosil 100 C18, 300 × 4.0 mm, 10 μm column. Eluent
consisted of 17 mM L-proline and 4 mM Cu(OAc)2 in double distilled
H2O (pH 4.5) with a flow rate of 0.5 mL/min. For detection of Nδ-
methylarginine an online post-column derivatization equipped with
a 2.5 m Teflon reaction coil was installed. Derivatization reagent con-
tained 120 mL of o-phthalaldehyde (dissolved in 2 mL of MeOH), 0.3
mL of 2-mercaptoethanol, 0.1 M H3BO3, and 3 mM EDTA adjusted to
pH 9.25 with NaOH. The flow rate for post-column derivatization was
set to 1.0 mL/min. Detection of derivatized amino acids was carried
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out by a Waters W474 fluorescence detector, set to λex: 340 nm, λem
:
455 nm. Chromatography was performed isocratically at r.t. Repro-
ducibility of peak areas of three sequential injections was assessed
with a relative standard deviation of less than 1.2% for both enantio-
mers.
Acknowledgment
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3934.
(15) Schade, D.; Töpker-Lehmann, K.; Kotthaus, J.; Clement, B. J. Org.
Chem. 2008, 73, 1025.
We gratefully thank Mr. Martin Clemen for performing HRMS experi-
ments and Mrs. Melissa Zietz and Mr. Sven Wichmann for the excel-
lent technical assistance.
(16) Bada, J. L. Racemization of Amino Acids, In Chemistry and Bio-
chemistry of the Amino Acids; Barrett, G. C., Ed.; Springer: Dor-
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Supporting Information
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36, 6373.
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1561303.
S
u
p
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ortiInfogrmoaitn
S
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p
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ortioInfgrmoaitn
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© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 723–729