3
310
C. Bienvenu et al. / Tetrahedron Letters 51 (2010) 3309–3311
Scheme 1. (a) Coupling step: lipoic acid, DIC/HOBt, CH
CH Cl (2Â), washing with water (2Â), 0.1 M acetic
(5Â). (b) Reduction step: NaBH
acid (2Â), EtOH (2Â) then drying.
2
Cl
2
(2Â), filtration, washing
2
2
4
either on Sephadex, Sepharose,10,15 polyacrylamide,10 cellulose
1
0
1
0
resins (for reactions in aqueous media) or on a poly(4-vinylpyri-
1
4
dine) resin (for reactions in organic solvents). However, none of
these systems can be used in both hydrophilic and apolar solvents.
We describe herein a very simple one-pot, two-step synthesis of
a dihydrolipoyl-supported polymer starting from the commercially
Scheme 2. (a) DHLA-polymer supported reducing agent (15 equiv), formate buffer
pH 7.5; (b) filtration, washing and aporation.
available Aminomethyl-ChemMatrixÒ (Scheme 1). This new ‘di-
thiol’ material fulfil the required specifications, that is, (i) high
reducing abilities owing to the formation of a stable dithiolane
moiety upon oxidation, (ii) good swelling properties in a wide
range of solvent composition (aqueous or organic) for a high load-
ing capacity (e.g., up to 1 mmol/g such as conventional PS-based
resins; for comparison, Sephadex, Sepharose or cellulose resins
once chemically modified to exhibit aminoethyl residues, possess
lower capacities, which are in the 0.36–0.29 mequiv/g range), (iii)
easy work-up that allows complete recovery of highly valuable thi-
ols from the reduction of the corresponding disulfide substrates
without any additional purification steps and (iv) possibility to
regenerate quantitatively the di-thiol reducing polymer. The ease
of implementation of this procedure as well as its versatility should
render this strategy routinely applicable for most biochemists and
biologists.
2
disulfide ([MeOPEG-S] ; n = 45) were chosen (Scheme 2). The
whole process is described in Refs. 20 and 21.
The optimal conditions were established for the reduction of oxi-
dized glutathione. Freshly regenerated DHLA-polymer supported
reducing agent was used in all cases. Completion of the reaction
was performed using Ellman’s tests by monitoring the UV absor-
bance at 410 nm. Results showed that for 15 equiv of DHLA-polymer
supported reducing agent the reaction was completed in 50 min. To
check for an eventual re-oxidation, the thiol content of the sulfide
compound obtained was measured again after its recovering. In all
cases tested, the thiol content was higher than 98%. Recycled resin
1
6
4
obtained after subsequent NaBH reduction procedure as men-
tioned in Scheme 1 was tested and re-used in ten consecutive runs
without showing any obvious loss of activity.
2
Concerning the reduction of [MeOPEG-S] into MeOPEG-SH, it
could be done in DMF as well as in acetonitrile. In the latter case,
for completion, the DHLA matrix had to be used in larger excesses
2
. Preparation of the resin-supported lipoyl redox
The lipoyl polymer supported conjugate was prepared in a one-
(
30 equiv instead of 15 in DMF).
pot, two-step procedure (Scheme 1; coupling and reduction step,
see Ref. 17). The coupling step was performed by reacting lipoic
acid with the PEG Aminomethyl-ChemMatrixÒ resin using diiso-
propyl-carbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) as the
coupling agent.
This coupling step was repeated twice and completion of the
reaction was monitored with Kaiser’s test (negative).18 At this
stage, the lipoyl-grafted resin can be conserved indefinitely if kept
dry. Reduction of the 1,2-dithiolane ring to generate the potent di-
hydrolipoyl reductor was accomplished by running two subse-
In conclusion, an efficient, stable and 100% recyclable PEG ma-
trix supporting the dihydrolipoyl residue as the reducing agent
was synthesized in a one-pot, two-step procedure. Reduction of
disulfide substrates proceeds quantitatively affording the targeted
thiols without any purification step. This reduction can be per-
formed either in 100% aqueous volatile buffers or in 100% organic
solvents. This very simple protocol can be utilized by researchers
that routinely work with cystine containing proteins or organic
disulfide and seek for the total recovery of the targeted reduced
thiols without setting up heavy strategies.
4
quent reduction steps with NaBH . To prevent from re-oxidation,
the resin was acidified with 0.1 M acetic acid. The thiol content
determined by Ellman’s test was shown to be quantitative (UV
titration at 410 nm, data not shown).19 The reduced di-thiol form
of the resin can be conserved for months after EtOH washing and
if kept dried in an appropriate dioxygen-free atmosphere. How-
ever, reduction should be preferably performed just prior use for
better results.
References and notes
1. Jocelyn, P. C.Biochemistry of the SH Group The Occurrence, Chemical Properties,
Metabolism, and Biological Function of Thiols and Disulfides; Academic Press:
London, 1972.
2.
(a) Oae, S. Organic Sulfur Chemistry: Structure and Mechanism; CRC Press: Boca
Raton, FL, 1991; (b) Patai, S. The Chemistry of the Thiol Group; S. Patai, Ed.; J.
Wiley and Sons Ltd: London, 1974.
3.
Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, G. R.; Whitesides, G. M. Chem. Rev.
2005, 105, 1103–1170.
4
5
.
.
Saito, G.; Swanson, J. A.; Lee, K. D. Adv. Drug Deliv. Rev. 2003, 55, 199–215.
Zuber, G.; Dauty, E.; Nothisen, M.; Belguise, P.; Behr, J.-P. Adv. Drug Deliv. Rev.
3
. Reduction potential of the supported dihydrolipoyl reducing
agent
2001, 52, 245–253.
6
7
.
.
Balakirev, M.; Schoehn, G.; Chroboczek, J. Chem. Biol. 2000, 7, 813–819.
(a) Le Gourrierec, L.; Di Giorgio, C.; Greiner, J.; Vierling, P. Tetrahedron 2008, 64,
To set up a protocol, as simple as possible, the possibility of
2233–2240; (b) Le Gourrierec, L.; Di Giorgio, C.; Greiner, J.; Vierling, P. New J.
using highly volatile buffers was investigated in order to perform
the total disulfide reduction that could result into a simple filtra-
tion-evaporation step procedure. We thus established two proce-
dures, one for a 100% aqueous environment with ammonium
formate buffer and the other running in a 100% organic solvent
supplemented with diethylamine. Disulfides to reduce were cho-
sen regarding practical interest for our group but also and more
generally for chemists and biochemists. Thus as examples, a small
peptide (oxidized glutathione) and methoxypolyethylene glycol
Chem. 2008, 32, 2027–2042.
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1
0. (a) Gorecki, M.; Patchornik, A. Biochim. Biophys. Acta, Protein Struct. 1973, 303,
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1
1
1
1
3. Amos, R. A.; Fawcett, S. M. J. Org. Chem. 1984, 49, 2637–2639.
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