Full text of DOI:10.1016/S0040-4039(99)02045-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 165–168
A novel method for sequence independent incorporation of
activated/protected cysteine in Fmoc solid phase peptide
synthesis
Ajit K. Ghosh and Erkang Fan ∗
Department of Biological Structure and Biomolecular Structure Center, University of Washington, Box-357742, Seattle,
Washington 98195-7742, USA
Received 6 August 1999; revised 20 October 1999; accepted 22 October 1999
Abstract
In conventional stepwise Fmoc solid phase peptide synthesis, incorporation of cysteine with nitro-pyridine
sulfenyl (Npys) or pyridine sulfenyl (Pys) groups which act as an activation/protection of the thiol function has
been accomplished in a sequence independent manner. © 1999 Elsevier Science Ltd. All rights reserved.
Peptides are attractive targets for drug discovery because they have been shown to be diagnostically
and therapeutically important in many areas of biomedical research.1 With the rapid advancement of
combinatorial chemistry and high throughput screening the lead drug discovery process has undergone
dramatic changes. Consequently, in recent times, interest in the solid phase peptide synthesis has
increased significantly. In this connection the protection/activation of the thiol function in cysteine with
nitro-pyridine sulfenyl (Npys) or pyridine sulfenyl (Pys) group has found increasing use in unsymmetrical
disulfide formation,2 protein–peptide covalent linkage3 and for the synthesis of cyclic peptides.4
Both Npys and Pys groups are base labile and do not survive under Fmoc removal condition in stepwise
solid phase peptide synthesis.2,5 Therefore, one cannot have Cys(Npys) or Cys(Pys) at non N-terminal
position in a peptide while performing Fmoc chemistry. However, at the N-terminal position, Boc-
Cys(Npys) could be coupled to a peptide sequence which has been assembled by using Fmoc strategy.
The only option left to introduce Cys(Npys) into non N-terminal position of a resin bound fully assembled
peptide made by Fmoc chemistry is to replace thioether type protecting groups by Npys. This needs to
react resin bound peptide with Npys-Cl. Unfortunately this method has several serious limitations.2,4 It
depends on the type of resin used, and works only for very small peptides. In addition, Npys-Cl is highly
hygroscopic and the reaction is very slow. Any attempt to increase the reaction rate by using higher
proportion of Npys-Cl leads to unidentified side product.2,6
∗
Corresponding author. Fax: (206)-685-7002; e-mail: erkang@u.washington.edu (E. Fan)
0040-4039/00/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(99)02045-6
tetl 15973

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In this communication, we wish to report a very convenient and high yielding method to introduce
activated/protected cysteine at either an N-terminal or a non N-terminal position in a peptide sequence
assembled by Fmoc strategy. In our approach, we first synthesize the peptide sequence on resin.
While cleaving the peptide from resin, we use 4–5 equivalents of 2,20-dithio-bis-(pyridine) (or the 5-
nitro substituted analogue) in the cleavage mixture (Scheme 1), since disulfide bond exchange can be
conducted under acidic conditions.7 We have designed four peptide sequences (1–4 in Scheme 1 and
Table 1) to test the generality of our procedure. These peptide sequences cover all common protected
side chains in Fmoc peptide synthesis. This is to ensure that our procedure is not only compatible with
those protection groups, but also compatible to each of those reactive side chains. In addition, sequences
1 and 2 test the sequence independence of our procedure, with cysteine at either the middle or the N-
terminal of the peptide chain. Sequence 3 demonstrates the ability to modify the N-terminal of a peptide
chain in our approach, which is not achievable by using Npys/Pys protected cysteine in a conventional
approach. Sequence 4 tests our procedure in the presence of both cysteine and methionine.
Scheme 1. *Fmoc amino acids: Glu(OtBu)OH, Gln(Trt)OH, Tyr(OtBu)OH, Arg(Pbf)OH, Lys(BOC)OH, Cys(Trt)OH,
His(Trt)OH, Trp(BOC)OH, Ser(OtBu)OH, MetOH. Only Cys was coupled as symmetrical anhydride and all others with 3
equiv. HOBt, 3 equiv. PyBOP, 6 equiv. DIPEA in DMA for 40 to 60 mins
The actual synthesis was straightforward and the results were excellent with our procedure. In a
typical experiment, for example, after the synthesis of peptide sequence 1 on Wang resin, the peptide
was cleaved with a mixture of TFA:H2O:TIS (95:2.5:2.5., TIS: triisopropylsilane) which contains 5
equivalents of 2,20-dithio-bis-(pyridine). After the removal of resin and most of the solvent, the crude
peptide 1A was obtained by ether precipitation. Analysis by electrospray mass spectrometry (ES-MS) and
analytical HPLC of this crude sample shows mostly one compound (Scheme 2). In this crude mixture, no
unmodified peptide with free cysteine was detected (by ES-MS or HPLC), indicating that the formation
of Cys-(Pys) was quantitative. Preparative HPLC purification of this ether precipitated crude product
afforded 66% of pure peptide 1A (Table 1). Subsequent experiments using 2,20-dithio-bis-(5-nitro-
pyridine) in cleavage mixture instead of 2,20-dithio-bis-(pyridine) produced similar results (1B and 2B in
Table 1). According to our procedure, one should be able to freely modify the N-terminal amino group.
We have carried out acetylation to give an excellent yield of 3. We also tested our procedure on a peptide
sequence containing more than one cysteine. A mixture of products with intramolecular disulfide bond
and bis-Npys modification were obtained. We are currently investigating modified procedures which
would lead to selective formation of either the cyclic product or the bis-Npys (bis-Pys) modified peptide.
Methionine suffers easy acid catalyzed oxidation of thioether during cleavage. In order to prevent
oxidation ethyl methyl sulfide or thioanisole has been routinely used in cleavage mixture. To prove the

167
Table 1
generality of our method we applied it (under nitrogen, with 2,20-dithio-bis-(5-nitro-pyridine) but without
the addition of other thio-scavengers) to a resin bound peptide 4, containing both cysteine and methionine.
Analysis of crude sample after ether precipitation by ES-MS showed no evidence of oxidized product.
Methionine oxidation may have been prevented by the combined effects of a nitrogen atmosphere and the
presence of 2-thio-5-nitropyridine, a by-product formed during the cleavage reaction. After preparative
HPLC we obtained more than 60% yield of pure peptide 4.
In conclusion, a novel method for sequence independent incorporation of Npys and Pys activa-
ted/protected cysteine in Fmoc solid phase peptide synthesis has been developed. Some unique merits of
our method include: operational simplicity, no need to consider dry reaction condition, fast and efficient
reaction, and the use of commercially available and non-moisture sensitive reagents. Modification of N-
terminal amino group could be done if required. We believe our procedure would be a beneficial addition
to the peptide chemistry.
Acknowledgements
This work is supported by the School of Medicine and the Department of Biological Structure,
University of Washington.

168
Scheme 2. A: HPLC profile of the crude 1A from ether precipitation shows mainly the product at 26.5 min. B: Electro-
spray mass spectrum of crude 1A with the following main peaks: m/z 468.3 ((1A+3H)3+), 701.9 ((1A+2H)2+), and 221
(2,20-dithio-bis-(pyridine)). The peaks for unmodified peptide (m/z 1294 or its multiple charged forms at 647.5 or 432.2) were
undetected. The internal standard showed a down-shift of 0.3 mass units during measurement. Therefore, the observed mass for
1A is 1402.6, an average of the triply charged peak at 468.6 and the doubly charged peak at 702.2
References
1. (a) Gordon, E. M.; Barrett, R. W.; Dower, W. J.; Fodor, S. P.; Gallop, M. A. J. Med. Chem. 1994, 37, 1385; (b) Hocart, S. J.;
Jain, R.; Murphy, W. A.; Taylor, J. E.; Coy, D. H. J. Med. Chem. 1999, 42, 1863–1871.
2. Simmonds, R. G.; Tupper, D. E.; Harris, J. R. Int. J. Pept. Protein Res. 1994, 43, 363–366.
3. Drijfhout, J. W.; Perdijk, E. W.; Weijer, W. J.; Bloemhoff, W. Int. J. Pept. Protein Res. 1988, 32, 161.
4. Matsueda, R.; Higashida, S.; Ridge, J. R.; Matsueda, G. Chem. Lett. 1982, 921–924.
5. Ploux, O.; Chassing, G.; Marquet, A. Int. J. Pept. Protein Res. 1987, 29, 62–169.
6. Ghosh, A. K.; Fan, E., unpublished results.
7. Carlsson, J.; Drevin, H.; Axen, R. J. Biochemistry 1978, 173, 723–737.