Disulfide-bridge formation through solvent-free oxidation of thiol amino acids catalysed by peroxidase or hemin on mineral supports

Article abstract of DOI:10.1039/a907027c

Air oxidation of hydrophilic thiols to disulfides was performed in neutral conditions on mineral supports activated by hemin or peroxidase. The Royal Society of Chemistry 1999.

Full text of DOI:10.1039/a907027c

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Disulfide-bridge formation through solvent-free oxidation of thiol
amino acids catalysed by peroxidase or hemin on mineral supports
a
b
Eryka Guibé-Jampel and Michel Therisod*
a
b
UMR 8615 Réactivité et Synthèses Sélectives and Lab. Chimie Bioorganique
et Bioinorganique, I.C.M.O. - Bat. 410-420, Université Paris-Sud, F-91405 Orsay cedex.
E-mail: therisod@icmo.u-psud.fr; eriguibe@icmo.u-psud.fr
Received (in Cambridge, UK) 31st August 1999, Accepted 1st October 1999
Air oxidation of hydrophilic thiols to disulfides was
performed in neutral conditions on mineral supports
activated by hemin or peroxidase.
common hemeproteins, is known to catalyse the oxidation of
cysteine and dithiothreitol; it has recently been used to prepare
5
aryl and alkyl disulfides under similar conditions to hemin
6
catalysis. Consequently, we studied the performance of a

Oxidation of cysteine and related compounds has been exten-
sively studied. Despite its importance in biochemical processes,
the controlled formation of disulfide bridges remains a sig-
weakly basic mineral support (Hyflo Super Cel , Fluka: HSC),
activated by hemin or peroxidase (EC 1.11.1.7, Novo SP
676) to catalyse the oxidation by air of amino acid-thiols, as
exemplified below for N-acetylcysteine (Fig. 1).
1
nificant challenge. We have recently reported that aromatic
thiols dispersed on a weakly basic support could be easily and
selectively oxidised by air in solvent-free conditions. Likewise,
Freeze-dried peroxidase and the substrate were co-dispersed
on HSC; the coated support was hydrated by a small amount of
water, and shaken until a loose powder was obtained. Altern-
atively, the enzyme was dispersed on the support, and the
powder was impregnated by a methanolic solution of the sub-
strate, afterwards the mixture was dried. N-Acetylcysteine thus
gave 85% of N-acetylcystine at 75 ЊC after 2 hours if impreg-
nated by methanol, after 45 min if dispersed on hydrated HSC,
and less than 5% of disulfide after 2 hours at 75 ЊC if dispersed
on dry HSC (Fig. 2).
Hemin-activated HSC was prepared by impregnation with a
pyridine solution of hemin and was then dried. The substrate
was then added to the coated support, and the mixture was
hydrated with a small amount of water. Under these solvent-
free optimised conditions, complete oxidation of cysteine is
achieved in 15 min at 75 ЊC (Table 1); cysteine hydrochloride is
2

-cysteine dispersed on wet Hyflo Super Cel (HSC) is rapidly
converted in 30 min into a mixture of racemic and meso-cystine
by heating in air at 75 ЊC (Table 1, entry 1). However, several
compounds like cysteine hydrochloride, cysteine methyl ester
hydrochloride, N-acetylcysteine and glutathione remained
unchanged under these conditions. Thus, in order to perform
smooth oxidation of these thiols in neutral conditions, we
have foreseen the use of a support activated with an efficient
catalyst. Transition metal salts or complexes have been
employed for the catalytic or stoichiometric oxidation of
3
thiols into disulfides. Among them, hemin (ferriproto-
porphyrin() chloride) was reported to catalyse at a 0.2% molar
ratio the oxidation by oxygen of alkylthiols in alkaline aqueous
4
solution. Horseradish peroxidase (HRP), one of the most
Table 1 Oxidation of thiols to symmetrical disulfides
a
Ϫ3
Catalyst (3 × 10
Temperature/
ЊC
Conversion
(%)
b
Entry
Thiol (100 mg)
mol equiv.)
Time/h
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
-Cysteine
-Cysteine
-Cysteine
—
75
75
75
75
75
75
75
75
20
75
20
75
20
20
20
75
20
45
20
45
6
0.5
0.25
0.5
3
3
3
0.5
0.5
18
0.5
18
0.5
24
5
5
3
28
5
28
5
72
72
0.2
0.2
0.2
>98
Hemin
Peroxidase
—
Hemin
Peroxidase
—
96
100
<1.5
85
90
0
2.5
80
85
80
73
0
77
95
<3
84
>92
87
>98
>95
>95
30
-CysteineؒHCl
-CysteineؒHCl
-CysteineؒHCl
N-Ac--Cysteine
N-Ac--Cysteine
N-Ac--Cysteine
N-Ac--Cysteine
N-Ac--Cysteine
N-Ac--Cysteine
-CysteineOMeؒHCl
-CysteineOMeؒHCl
-CysteineOMeؒHCl
Glutathione
Glutathione
Glutathione
Glutathione
Glutathione
Penicillamine
Penicillamine
Penicillamine
Penicillamine
FeCl3
Hemin
Hemin
Peroxidase
Peroxidase
—
Hemin
Peroxidase
—
Hemin
Hemin
Peroxidase
Peroxidase
Hemin
Peroxidase
—
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
6
75
75
75
Hemin
Peroxidase
35
60
Penicillamine
a
c
b
The hemin content of peroxidase was determined spectrophotometrically as 1.5 mg of hemin per 100 mg of peroxidase. Determined by NMR.
0% decomposition.
4
J. Chem. Soc., Perkin Trans. 1, 1999, 3067–3068
This journal is © The Royal Society of Chemistry 1999
3067

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Fig. 1 Preparation of activated HSC and catalysed oxidation of
Ϫ1
N-acetylcysteine (2 h, 80 ЊC). i: hemin (pyridine), 1.5 mg g HSC;
Ϫ1
ii: drying; iii: freeze-dried peroxidase, 100 mg 500 mg HSC.
Fig. 3 Oxidation of -penicillamine dispersed on hydrated HSC
75 ЊC) activated by: (A): peroxidase; (B): hemin; (C): no catalyst.
(
mg of hemin (Fluka) dissolved in 10 ml of pyridine were added
to 10 g of HSC, and the mixture was dried on a rotatory evap-
orator (pyridine was not retained on HSC under these condi-
tions). The activated supports remains active for months if
stored in the refrigerator (peroxidase) or at room temperature
(
hemin).
Typical procedure for an oxidation reaction
Reaction mixtures were prepared by mechanical dispersion of
Ϫ1
the thiol (200 mg g ) on the activated support. Water (0.5 ml
g ) was added and the mixture was carefully mixed until an
Fig. 2 Dependence of the oxidation of N-acetylcysteine catalysed by
hemin-activated HSC on the amount of added water (2 h, 80 ЊC).
Ϫ1
homogeneous (hydrated) loose solid was obtained. This solid
was left at room temperature or heated in an open vessel for the
indicated time. Products were eluted from the support either
with diluted hydrochloric acid (cystine, oxidised glutathione,
penicylamine) or methanol (N-acetylcystine, cystine methyl
ester). The solution was decolorised by addition of activated
carbon, filtered and evaporated.
oxidised within 3 hours, N-acetylcysteine within 60 min at 75 ЊC
or 24 h at 20 ЊC. Cysteine methyl ester hydrochloride, which
decomposes rapidly in alkaline solutions, can be quantitatively
oxidised under neutral conditions at 20 ЊC.†
We have tested the method on the peptide model, reduced
glutathione. Like N-acetylcysteine, glutathione is not oxidised
in the absence of a catalyst. On heating or after a long storage
in air on HSC, some decomposition (up to 40%) occurs. On
hemin or peroxidase activated HSC, clean and complete oxid-
ation can be achieved at 20 or 45 ЊC. The rate of oxidation of
N-acetylcysteine or glutathione is similar if catalysed by hemin
or peroxidase. Nevertheless, an effect of the protein environ-
ment can be observed for the oxidation of tertiary thiols like
penicylamine (dimethylcysteine) (Fig. 3). Neither overoxidation
nor other side reactions could be observed with any substrates
under study. All products were quantitatively recovered as
described below in the Experimental section.
To summarise, disulfide bridges can be successfully formed
by oxidation of amino acid-thiols catalysed by hemin or per-
oxidase immobilised on a weakly basic mineral support without
solvent. This environmentally friendly method offers consider-
able synthetic advantages in terms of yield, selectivity and
simplicity of reaction and work-up procedures.
Acknowledgements
We are grateful to Novo Nordisk for the kind supply of
peroxidase.
Notes and references
† Hemin on Celite, neutral and basic alumina or silica gel instead of
HSC can be used under similar conditions.
1
2
3
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Comprehensive Organic Synthesis, ed. B. M. Trost and I. Fleming,
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Wilkes and D. Reynolds, in Comprehensive Organic Functional Group
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1
Experimental
Preparation of catalysts
4
5
C. F. Cullis, J. D. Hopton, C. J. Swan and D. L. Trimm, J. Appl.
Chem., 1968, 18, 335.
Peroxidase: Novo SP 676 is a freeze-dried enzyme from Novo
Nordisk; specific activity: 2675 KPODU/g [one PODU con-
verts 1 µmol of hydrogen peroxide per min in a system where
J. Olsen, L. Davis and M. F. Tsan, Biochim. Biophys. Acta, 1976, 445,
3
1
24; L. S. Harman, C. Mottley and R. P. Mason, J. Biol. Chem.,
984, 259, 5606.
M. Sridhar, S. Kumara Vadivel and U. T. Bhalerao, Synth. Commun.,
1998, 28, 1499.
2
,2Ј-azinobis(3-ethylbenzothiazoline-6-sulfonate) is oxidised].
6
Peroxidase-activated support was prepared by mechanical
dispersion of the enzyme (200 mg g ) on HSC with a vortex
Ϫ1
mixer. Hemin-activated support was prepared as follows: 32.6
Communication 9/07027C
3
068
J. Chem. Soc., Perkin Trans. 1, 1999, 3067–3068