Photochemical desulfurization of L-cysteine derivatives

Article abstract of DOI:10.1016/S0957-4166(98)00295-X

Thiol groups can be reductively eliminated at room temperature by a photochemical method which makes use of triethylboron, triethylphosphite and visible light. Thus, after treating L-Cys 1a, D-Pen 1b, L-Cys-OMe 1c and glutathione (γ-L-Glu-L-Cys-Gly) 3, the corresponding desulfurized compounds L-Ala 2a, D-Val 2b, L-Ala-OMe 2c and γ-L-Glu-L-Ala-Gly 4, respectively, are prepared in high yields and with retention of configuration.

Full text of DOI:10.1016/S0957-4166(98)00295-X

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 2761–2764
∗
Photochemical desulfurization of L-cysteine derivatives
Asensio González and Gregorio Valencia b
Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain
a
a
b
Unit for Glycoconjugate Chemistry, C.I.D.-C.S.I.C., Jordi Girona 18-26, 08034 Barcelona, Spain
Received 9 June 1998; accepted 24 July 1998
Abstract
Thiol groups can be reductively eliminated at room temperature by a photochemical method which makes use
of triethylboron, triethylphosphite and visible light. Thus, after treating L-Cys 1a, D-Pen 1b, L-Cys-OMe 1c and
glutathione (γ-L-Glu-L-Cys-Gly) 3, the corresponding desulfurized compounds L-Ala 2a, D-Val 2b, L-Ala-OMe
2
c and γ-L-Glu-L-Ala-Gly 4, respectively, are prepared in high yields and with retention of configuration. © 1998
Elsevier Science Ltd. All rights reserved.
1. Introduction
The selective chemical modification of amino acid side chains in proteins is a subject of current
1
interest. The presence of free thiols and disulfide bridges in many proteins is of paramount importance
2
in biological systems. In this connection, the reductive desulfurization of the thiol function of L-cysteine
in a protein, to convert the amino acid to L-alanine, is a chemical modification with many implications
from the biochemical and structural point of view.
Only a few methods are known for thiol group elimination. Early work by Hoffmann on the conversion
of alkanethiols to the corresponding alkanes used triethyl phosphite under thermal and photochemical
3
conditions. Improved procedures later proposed by Walling also relied on thermal treatment coupled to
4
radical chain reactions initiated by catalytic amounts of azobisisobutyronitrile (AIBN).
Owing to the low thermal stability of many biological substances, these harsh conditions are of little
value in, for example, the selective chemical modification of amino acid side chains such as free thiol
and disulfide bridges which play very important roles in maintaining the active conformation of many
proteins and peptides. With the aim of developing new chemical tools for the study of sulfur related
structural features of proteins, different attempts have been made at the reductive desulfurization of L-
cysteine derivatives and L-cysteine containing peptides and proteins. However, up to now, the reaction
∗
Dedicated to the memory of Dr. Francesc R. Trull.
0957-4166/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00295-X

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6
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conditions assayed: i.e. Raney nickel, palladium catalysis, phosphorous acid at 100°C for 8 h and
8
triethyl phosphite at 110°C for 8 h, do not guarantee the integrity of protein substrates since these
reagents may also promote, for example, unwanted side chain hydrogenations, cleavage of peptide bonds
and difficult separations of the modified proteins from metal based reagents.
2. Results and discussion
In trying to improve some of these methods, we have treated L-Cys 1a with triethylphosphite under
thermal conditions. However, L-Ala 2a was not found among the reaction mixture which was always a
phosphorus containing viscous oil that was not further characterized. At the same time, photochemical
experiments by means of a 300 W visible light bulb, were conducted on L-Cys 1a either in acetonitrile
(ACN) or water solutions rendering unchanged L-Cys 1a. Since these results could be partly explained
by the limited solubility of L-Cys in ACN, a protective group of L-Cys was sought to improve the
homogeneity of the reaction systems.
9
Organoboranes have received a great deal of attention. In particular, Zwanenburg has described the
use of different organoboranes for the simultaneous protection of α-amino and carboxyl groups of
10
amino acids. These derivatives allow the solubilization of amino acids in organic solvents and offer
a number of advantages such as very easy and mild deprotection procedures. Although their applications
11
are not widespread in amino acid chemistry, the potential of triethylboron as an initiator of radical
12
chain reactions was considered a very suitable feature for the purposes of this work. Accordingly, new
experiments were conducted by treating L-Cys 1a in ACN with a stoichiometric amount of triethylboron
for 1.5 h under an argon atmosphere to form the corresponding protected amino acid. After addition
of triethylphosphite and irradiation with a light bulb for 6.5 h, the reaction mixture was quenched with
6
M HCl affording L-Ala hydrochloride 2a·HCl in quantitative yield. When D-penicillamine 1b was
processed under identical conditions, D-Val·HCl 2b·HCl was obtained in 95% yield (Scheme 1).
3 3 3
Scheme 1. (i) Et B/CH CN; (ii) (EtO) P/hν
The crucial role of triethylboron as radical initiator is evident in accomplishing this reductive desul-
furization which is also supported by a number of recent kinetic mechanism studies of free-radical
chain reactions of alkylthiyl radicals with organoboron and organophosphorous substrates.13 A further
argument in favor of the free-radical initiator hypothesis, rather than the protecting group solubilizing
effect, was obtained by performing experiments with a readily soluble L-Cys derivative. Thus, when
L-Cys-OMe·HCl 1c·HCl containing triethylphosphite was irradiated as before in ACN, the starting
compound was recovered. However, when the same experiment was conducted in the presence of
catalytic amounts of triethylboron, L-Ala-OMe·HCl 2c·HCl was obtained in high yield (93%).

A. González, G. Valencia / Tetrahedron: Asymmetry 9 (1998) 2761–2764
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Finally, from similar experiments performed on L-Ser, this sulfur elimination method was probed to be
specific for thiol functions since L-Ser was always recovered as the exclusive reaction product. Moreover,
this reaction system was successfully applied to a simple peptide model glutathione (γ-L-Glu-L-Cys-Gly)
3
, which was fully converted to the tripeptide, γ-L-Glu-L-Ala-Gly 4,14 in an acetonitrile/water mixture
after 8 h of irradiation (Scheme 2).
3 3 3
Scheme 2. (i) Et B/CH CN; (ii) (EtO) P/hν
In conclusion, we propose a new method for the efficient removal of the thiol function from L-Cys
15
and related molecules. The procedure relies on a photochemical method that may follow a free-radical
chain reaction mechanism initiated by triethylboron. Features such as mild reaction conditions (room
temperature), high yields, operational simplicity, lack of side reactions (hydroxyl groups not affected)
and compatibility of the reagents with the presence of water, makes this method an ideal procedure for
the elimination of thiol groups from peptides. Studies aimed at the application of this process in protein
chemistry are currently underway in our laboratories.
Acknowledgements
Support for this research was partially provided by DGICYT (Spain) through Grant PB94-0858.
References
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3
15. Typical experiment: A 1 M solution of Et B in tetrahydrofuran (10.5 mL, 10.5 mmol) was added to L-Cys 1a (1.21
g, 10 mmol) in 20 mL of acetonitrile. The reaction mixture was stirred at room temperature under argon for 90 min.
Triethylphosphite (5.6 mL, 33 mmol) was added and the resulting mixture was irradiated with a 300 W visible light bulb
located about 20 cm from the flask for 6.5 h in an open system (Dimroth refrigerant). The organic solvent was evaporated
under reduced pressure. 6 M HCl (6 mL) was added, the mixture was stirred for 5 min and then was extracted with
CH
2
Cl
2
(3×2 mL). Evaporation of the acidic fraction under high vacuum gave L-alanine hydrochloride 2a·HCl (1.2 g,
22
D
9
6% yield) identical in all respects with the authentic product. Selected spectral data: 2a·HCl: [α]
=+14.2 (c=10, 6 N
O, 50 MHz) δ 175.3 (C_O), 51.4 (CH), 18.0 (CH
). 2b·HCl:
O, 50 MHz) δ 174.3 (C_O), 61.1
2
0
13
HCl), lit.: [α]
D
=+14.5 (c=10, 6 N HCl); C NMR (D
2
3
2
2
23
13
[
(
α]
D
=−27.1 (c=3.4, 6 N HCl), lit.: [α]
D
=−27.4 (c=3.4, 6 N HCl); C NMR (D
2
2
2
25
13
CH), 31.8 (CH), 20.2 (CH
3
), 19.7 (CH3). 2c·HCl: [α]
D
=+6.8 (c=1.6, CH
3
OH), lit.: [α]
D 3
=+7.0 (c=1.6, CH OH); C
NMR (D
2
3 3
O, 50 MHz) δ 174.1 (C_O), 56.4 (CH ), 51.6 (CH), 18.0 (CH ). Glutathione (γ-L-Glu-L-Cys-Gly) 3 treated
in acetonitrile/water with triethylboron for 2 min and irradiated with triethylphosphite for 8 h was converted into γ-
L-Glu-L-Ala-Gly 4.14 The reaction was checked by RP-HPLC and MALDI-TOF-MS. Glutathione 3, [M+H]=308.12,
1
3
[
1
M+Na]=331.11. Corresponding alanyl tripeptide 4, [M+H]=276.10. C NMR (D
74.1, 54.8 (CH), 52.5 (CH), 43.9 (CH ), 33.7 (CH ), 28.1 (CH ), 19.3 (CH ).
2
O, 50 MHz) δ 177.1, 175.6, 175.3,
2
2
2
3