A flexible approach for the synthesis of selectively labelled L-arginine

Article abstract of DOI:10.1016/j.tetlet.2004.05.096

A simple and efficient synthesis of L-arginine has been achieved in 12 steps and 24% overall yield. Regioselective reduction and functional group manipulation of the β-side chain of aspartic acid allowed the preparation of an ornithine derivative, which was then guanylated with a bis-protected 1-guanyl-pyrazole and deprotected to give L-arginine. This approach allows the flexible incorporation of stable isotopes and this is demonstrated using potassium ¹³C-cyanide, which has resulted in the preparation of 5-¹³C-L-arginine.

Full text of DOI:10.1016/j.tetlet.2004.05.096

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 5739–5741
A flexible approach for the synthesis of selectively
labelled -arginine
L
Deborah J. Hamilton and Andrew Sutherland*
Department of Chemistry, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK
Received 6 April 2004; revised 4 May 2004; accepted 14 May 2004
Available online 11 June 2004
Abstract—A simple and efficient synthesis of
reduction and functional group manipulation of the b-side chain of aspartic acid allowed the preparation of an ornithine derivative,
which was then guanylated with a bis-protected 1-guanyl-pyrazole and deprotected to give -arginine. This approach allows the
flexible incorporation of stable isotopes and this is demonstrated using potassium 13C-cyanide, which has resulted in the preparation
of 5-13C-
-arginine.
Ó 2004 Elsevier Ltd. All rights reserved.
L-arginine has been achieved in 12 steps and 24% overall yield. Regioselective
L
L
L
-Arginine 1 plays a key role in the function and
thesis of L-arginine and demonstrate the utility of this
structure of many proteins.1 In particular, this amino
acid is an important structural motif found conserved in
type II polyketide synthase acyl carrier proteins (ACP),
which are essential for the production of a specific
polyketide product.2 NMR studies of several type II
ACPs have provided the solution structure of these
proteins revealing a common topology consisting of a
distorted four a-helical bundle.2–7
approach for the preparation of selectively labelled
analogues by the incorporation of a 13C-label at the C-5
position.
Our retrosynthesis of L-arginine is outlined in Scheme 1.
It was proposed that 1 could be prepared by coupling
commercially available N,N-bis(tert-butoxycarbonyl)-
1H-pyrazole-1-carboxamidine 2 and a suitably protected
Elucidation of protein structures using NMR spectro-
scopy has been facilitated by the incorporation of amino
acids selectively labelled with stable isotopes.3;8 The
importance of these biological studies has led to the
development of many methods for the synthesis of iso-
topically labelled amino acids.9 To further elucidate the
structure and mechanisms of polyketide ACPs we were
NH
CO2H
H2N
N
H
NH2
1
NBoc
CO2Me
NHBoc
+
H2N
interested in developing a flexible synthesis of
L-argi-
BocHN
N
N
nine, which would allow the selective incorporation of
stable isotopes leading to a series of labelled arginine
analogues. Several methods for the synthesis of labelled
D,L
-arginine have been reported.10 However, these are
limited by both the lack of stereocontrol and the intro-
duction of the stable isotope early in the synthetic
pathway. We now report a 12 step, stereospecific syn-
2
3
I
CO2Me
NBoc2
NC
CO2Me
NBoc2
+
KCN
5
4
CO2H
NH2
HO2C
Keywords: L-Arginine; Stable isotopes; Stereospecific synthesis; Gua-
nylation.
6
* Corresponding author. Tel.: +44-141-330-5936; fax: +44-141-330-
4888; e-mail: andrews@chem.gla.ac.uk
Scheme 1.
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.05.096

5740
D. J. Hamilton, A. Sutherland / Tetrahedron Letters 45 (2004) 5739–5741
ornithine derivative 3. We intended to synthesise 3 from
-aspartic acid 6 by regioselective reduction of the
b-carboxylate group followed by functional group
manipulation to give the required ornithine derivative 3
via intermediates 5 and 4.
a
+
NC
CO2Me
CO2Me
NHBoc
H3N
L
Cl-
NHBoc
11
12
b
The first stage of the synthesis involved the preparation
of nitrile 10 (Scheme 2).
H
N
L
-Aspartic acid 6 was converted
-aspartic
O
in two steps to N,N-di-tert-butoxycarbonyl
L
acid dimethyl ester 7.11 As reported by Martın and co-
workers, bis-protection of the amino functional group
allows regioselective reduction of the b-methyl ester
using DIBAL-H to give the corresponding aldehyde.11
The aldehyde was then further reduced using sodium
borohydride to afford, after column chromatogra-
phy, alcohol 8 in 91% yield over the two steps. Direct
synthesis of alcohol 8 from 7 using DIBAL-H was
attempted but this led to mixtures of starting material,
regiomeric alcohols and also significant amounts of the
diol. Alcohol 8 was then activated as the mesylate and
subsequently reacted with sodium iodide to give inter-
mediate 5 in high yield. Nucleophilic displacement of the
iodide with potassium 13C-cyanide in DMF gave the
isotopically labelled nitrile 9 in 87% yield. Introduction
of the nitrile functional group could have been
attempted using the mesylate resulting in a slightly
shorter synthesis.12 However, it was felt that displace-
ment of the iodide leaving group rather than the mesyl-
ate with the labelled cyanide would permit more efficient
utilisation of the isotopic label. Finally, selective
removal of one of the Boc-protecting groups was
achieved using trifluoroacetic acid to give 10 in 88%
yield.13
ꢀ
NHBoc
13
Scheme 3. Reagents and conditions: (a) H2, PtO2, CHCl3, MeOH,
85%; (b) NaHCO3, EtOAc, 75%.
The next step required deprotonation of the amine
hydrochloride to release the amine for subsequent cou-
pling with urethane protected 1-guanylpyrazole 2.
Treatment of 12 with sodium hydrogen carbonate
however, resulted in the formation of the cyclic amide
13. This was surprising as a similar ornithine deriva-
tive has been shown to be stable under such condi-
tions.15
To prevent cyclisation occurring, a-methyl ester 10 was
hydrolysed to the corresponding carboxylic acid 14
using lithium hydroxide in essentially quantitative yield
(Scheme 4). Hydrogenation, again using platinum oxide
as the catalyst gave the amine hydrochloride 15 in good
yield. Direct treatment of 15 with N,N-di-isopropyleth-
ylamine (2 equiv) and N,N-bis(tert-butoxycarbonyl)-1H-
pyrazole-1-carboxamidine 2 gave the tri-Boc protected
arginine analogue 16 cleanly and in 75% yield.15;16
Finally cleavage of the three Boc-protecting groups in
trifluoroacetic acid followed by purification by ion
The next stage required the synthesis of the ornithine
analogue and this was initially investigated using un-
labelled material. Hydrogenation of 11 in the presence
of chloroform using platinum oxide as the catalyst gave
the amine hydrochloride 12 in good yield (Scheme 3).14
exchange chromatography gave 5-13C-
87% yield.17
L
-arginine 17 in
13
NC
13
NC
a
CO2Me
CO2H
a, b
CO2H
NH2
CO2Me
NBoc2
HO2C
MeO2C
NHBoc
10
NHBoc
14
6
7
b
c
13
c
+
CO2H
NHBoc
d, e
I
CO2Me
NBoc2
HO
CO2Me
NBoc2
H3N
Cl-
15
8
5
NBoc
13
CO2H
f
BocHN
N
H
d
NHBoc
13
NC
13
NC
g
16
CO2Me
CO2Me
NBoc2
NH
NHBoc
10
13
CO2H
NH2
9
H2N
N
H
Scheme 2. Reagents and conditions: (a) i. TMSCl, MeOH, ii. NEt3,
Boc2O, 85% over two steps; (b) Boc2O, DMAP, MeCN, 96%; (c) i.
DIBAL-H, Et2O, )78 °C; ii. NaBH4, THF, H2O, 91% over two steps;
(d) MsCl, NEt3, DMAP, CH2Cl2, 85%; (e) NaI, acetone, D, 89%; (f)
K13CN, DMF, 87%; (g) TFA, CH2Cl2, 88%.
17
Scheme 4. Reagents and conditions: (a) LiOH, MeOH, H2O, 99%; (b)
H2, PtO2, CHCl3, MeOH, 87%; (c) 2, DIPEA, MeOH, 75%; (d) TFA,
87%.

D. J. Hamilton, A. Sutherland / Tetrahedron Letters 45 (2004) 5739–5741
5741
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In conclusion, a simple and efficient, 12-step synthesis of
-arginine has been achieved giving the target molecule
L
in 24% overall yield. This route has been developed to
allow the selective incorporation of stable isotopes
during the later stages of the synthesis thereby maxi-
mising efficacy of the isotopic label. We have demon-
strated this, using the relatively inexpensive potassium
13C-cyanide for the preparation of 5-13C-
L-arginine.
Similarly, use of other stable isotopes of cyanide (e.g.,
KC15N or K13C15N) or isotopically labelled N,N-bis-
(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine
(synthesised in two steps from commercially available
isotopically labelled cyanamide)16;18 would allow the
preparation of further analogues of labelled L-arginine
using this approach. Further studies towards the syn-
thesis of these compounds are currently in progress.
10. Martinkus, K. J.; Tann, C.-H.; Gould, S. J. Tetrahedron
1983, 39, 3493–3505; Prabhakaran, P. C.; Woo, N.-T.;
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ꢀ
3741–3744.
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Acknowledgements
13. Attempted hydrogenation of the di-Boc protected nitrile 9
at 60 psi and at 25 °C after 24 h returned only starting
material. However, under similar conditions the mono-
Boc analogues 11 and 14 were reduced in good yield.
14. Secrist, J. A., III; Logue, M. W. J. Org. Chem. 1972, 37,
335–336.
The authors gratefully acknowledge the University of
Glasgow and Lancaster Synthesis Ltd for financial
support.
15. Aurelio, L.; Brownlee, R. T. C.; Hughes, A. B. Org. Lett.
2002, 4, 3767–3769.
References and notes
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17. Selected data for 5-13C-
L-arginine 17: IR (KBr) 3262,
3105, 2912, 1683, 1641, 1606, 1563, 1469, 1333 cmꢀ1; ½aŠ
D
+12.4 (c 1.0, H2O); 1H NMR (400 MHz, D2O) d 1.49 (2H,
m), 1.68 (2H, m), 3.07 (2H, dt, J ¼ 139:6, 6.8 Hz), 3.49
(1H, t, J ¼ 6:0 Hz); 13C NMR (100 MHz, D2O) d 24.4 (d,
J ¼ 35:7 Hz), 29.1, 41.0, 55.0, 157.1, 174.5; MS (CI) m=z
176 (MHþ, 65%).
4. Xu, G.-Y.; Tam, A.; Lin, L.; Hixon, J.; Fritz, C. C.;
Powers, R. Structure 2001, 9, 277–287.
18. Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. J. Org.
Chem. 1992, 57, 2497–2502.