Synthesis of a selenocysteine-containing peptide by native chemical ligation

Article abstract of DOI:10.1021/ol015712o

(equation presented) A new method for the synthesis of selenocysteine derivatives and selenocysteine-containing peptides is described. Fmoc-Se-p-methoxybenzylselenocysteine (1) was prepared and used for solid-phase synthesis of peptides with an N-terminal unprotected selenocysteine. Subsequent native chemical ligation with a peptide thioester provided a 17-mer that corresponds to the C-terminus of ribonucleotide reductase with selenocysteine in place of cysteine.

Full text of DOI:10.1021/ol015712o

ORGANIC
LETTERS
2001
Vol. 3, No. 9
1331-1334
Synthesis of a
Selenocysteine-Containing Peptide by
Native Chemical Ligation
Matt D. Gieselman, Lili Xie, and Wilfred A. van der Donk*
Department of Chemistry, UniVersity of Illinois at Urbana-Champaign,
600 S. Mathews AVe., Urbana, Illinois 61801
Vddonk@uiuc.edu
Received February 14, 2001
ABSTRACT
A new method for the synthesis of selenocysteine derivatives and selenocysteine-containing peptides is described. Fmoc-Se-p-
methoxybenzylselenocysteine (1) was prepared and used for solid-phase synthesis of peptides with an N-terminal unprotected selenocysteine.
Subsequent native chemical ligation with a peptide thioester provided a 17-mer that corresponds to the C-terminus of ribonucleotide reductase
with selenocysteine in place of cysteine.
The synthesis of peptides containing selenocysteine (Sec) is
rapidly gaining interest with the discovery of an increasing
number of proteins containing this amino acid.1 In addition,
selenocysteine derivatives can serve as convenient precursors
to dehydroamino acids,2 which are useful electrophilic
handles for the chemoselective preparation of peptide
conjugates.3 The Fmoc-protected amino acids 1 and 2 have
been used as precursors for solid-phase peptide synthesis
(SPPS) of selenocysteine-containing peptides.2a,4 Compound
1 allows the incorporation of free selenocysteine into peptides
or the selenocystine derivatives thereof,4 whereas 2 is used
for the mild oxidative introduction of dehydroalanines.2a
Unfortunately, the reported syntheses of both compounds are
not very suitable for gram-scale preparations. Se-p-Meth-
oxybenzyl-protected selenocysteine, 1, has been previously
prepared from Boc-serine in eight steps in 24% overall
yield.4a,5 Fmoc-Se-phenylselenocysteine 2 was obtained from
Boc-protected serine â-lactone,2a,c the preparation of which
is well-known to suffer in yield upon scale-up.6 We describe
here two new synthetic routes to these compounds that can
be carried out in multigram quantities. Notably, 2 has been
prepared on a 15-g scale without the need for chromato-
graphic purification steps. We also show the utility of 1 for
the synthesis of selenocysteine-containing peptides and their
use in native chemical ligations.7 Specifically, we have used
(1) (a) Stadtman, T. C. J. Biol. Chem. 1991, 266, 16257-60. (b)
Stadtman, T. C. Annu. ReV. Biochem. 1996, 65, 83-100.
(2) (a) Okeley, N. M.; Zhu, Y.; van der Donk, W. A. Org. Lett. 2000, 2,
3603-3606. (b) Hashimoto, K.; Sakai, M.; Okuno, T.; Shirahama, H. Chem.
Commun. 1996, 1139-1140. (c) Sakai, M.; Hashimoto, K.; Shirahama, H.
Heterocycles 1997, 44, 319-324.
(3) Zhu, Y.; van der Donk, W. A. Org Lett. 2001, 3, in press.
(4) (a) Koide, T.; Itoh, H.; Otaka, A.; Yasui, H.; Kuroda, M. Chem.
Pharm. Bull. 1993, 41, 502-506. (b) Koide, T.; Itoh, H.; Otaka, A.; Furaya,
M.; Kitajima, Y.; Fuji, N. Chem. Pharm. Bull. 1993, 41, 1596-1600 (c)
Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-Bohner, S.; Siedler, F.
Biopolymers 1996, 40, 207-234.
(5) Stocking, E. M.; Schwarz, J. N.; Senn, H.; Salzmann, M.; Silks, L.
A. J. Chem. Soc., Perkin Trans. 1 1997, 2443-2447.
(6) Pansare, S. V.; Arnold, L. D.; Vederas, J. C. Org. Synth. 1991, 70,
10-17.
10.1021/ol015712o CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/11/2001

this technique for the preparation of a selenocysteine-
containing analogue of the C-terminal peptide of ribonucleo-
tide reductase.
alities that allowed their independent manipulation. The free
carboxylate of Fmoc-Ser was converted to the allyl ester,
followed by activation of the alcohol with p-toluenesulfonyl
chloride to provide 7. p-Methoxybenzyldiselenide (PMBSe)2,
obtained by treating selenium powder with super hydride
followed by PMBCl, was reduced to the selenol with
hypophosphoric acid. Without further purification, the result-
ing selenol was added to 7 under basic conditions to yield
the fully protected selenocysteine derivative. Selective depro-
tection of the carboxylate with catalytic palladium and
morpholine gave 1 in 61% yield over four steps, a marked
improvement over the existing route. The optical rotation
of the final product after recrystallization compared well with
the previously reported value4a and that obtained for the same
compound produced via Scheme 1. This suggests that the
introduction of the selenide occurred without any extensive
racemization in agreement with a previous account of a
similar reaction.8 The preparation of 2 followed a similar
route except that the carboxylate protecting group was
changed to a diphenylmethyl ester (Scheme 3). The four-
Our initial route to 1 focused on modifications to literature
preparations of selenocystine5 and Se-PMB-Sec.4a Boc-
protected serine was converted into bromoalanine methyl
ester 3, which was transformed with dilithium diselenide to
the selenocystine derivative 4. Deprotection of both the
amino and carboxylate functionalities under acidic conditions,
followed by recrystallization and cation exchange chroma-
tography, provided selenocystine 5. Reduction of the di-
selenide and protection of the selenol with p-methoxybenzyl
chloride gave 6, which was converted into the desired product
in 55% unoptimized yield. Although literature yields for
some of the individual steps in Scheme 1 are somewhat
Scheme 1
Scheme 3
higher than in our hands, the route is lengthy and initial
attempts toward scale-up did not look promising. In light of
these results, we decided to evaluate an alternative synthetic
route to 1 that would require fewer steps and be amenable
to scale-up. Scheme 2 depicts the strategy that we ultimately
step sequence provided 2 in 37% overall yield on a 15-g
scale, which would not have been achievable with our
previously reported procedure.2a
Selenocysteine is often referred to as the 21st natural amino
acid as it is ribosomally incorporated into several important
redox enzymes.1,9 Replacement of cysteine by selenocysteine
in proteins and peptides can impose interesting properties10,11
because of the higher acidity of the selenol,4a,12 the lower
redox potential,11,13 and the greater nucleophilicity of the
Scheme 2
(7) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science
1994, 266, 776-779. (b) Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem.
2000, 69, 923-960.
(8) Theodoropoulos, D.; Schwartz, I. L.; Walter, R. Biochemistry 1967,
6, 3927-3932.
(9) (a) Leinfelder, W.; Zehelein, E.; Mandrand-Berthelot, M. A.; Bo¨ck,
A. Nature 1988, 331, 723-5. (b) Leinfelder, W.; Forchhammer, K.; Veprek,
B.; Zehelein, E.; Bo¨ck, A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 543-
547 (c) Low, S. C.; Berry, M. J. TIBS 1996, 203-208.
(10) Selenosubtilisin is an interesting example of altered properties of
an enzyme by selenocysteine substitution; see (a) Wu, Z. P.; Hilvert, D. J.
Am. Chem. Soc. 1990, 112, 5647-8. (b) Syed, R.; Wu, Z. P.; Hogle, J. M.;
Hilvert, D. Biochemistry 1993, 32, 6157-64.
(11) Besse, D.; Siedler, F.; Diercks, T.; Kessler, H.; Moroder, L. Angew.
Chem., Int. Ed. Engl. 1997, 36, 883-885.
(12) (a) Nygard, B. Ark. Kemi 1967, 27, 341-361. (b) Arnold, A. P.;
Tan, K. S.; Rabenstein, D. L. Inorg. Chem. 1986, 25, 2433-2437.
(13) Mu¨ller, S.; Senn, H.; Gsell, B.; Vetter, W.; Baron, C.; Bo¨ck, A.
Biochemistry 1994, 33, 3404-3412.
used for this purpose. Three orthogonal protecting groups
were used for the amino, carboxylate, and selenol function-
1332
Org. Lett., Vol. 3, No. 9, 2001

selenolate compared to the thiolate.14 Unfortunately, the
conditions necessary for selenocysteine incorporation by the
cell translational machinery are very specific. As a result,
preparation of non-natural selenocysteine-containing peptides
and proteins by molecular biology methods is difficult,
although some recent successful approaches have been
documented.15 We envisioned that the practical synthesis of
1 in combination with the recently developed native chemical
ligation technique7 could overcome this limitation. In prin-
ciple, two different approaches can be used to substitute a
cysteine with a selenocysteine in a protein or peptide using
the native ligation strategy. One could either position the
selenocysteine in the interior of one of the ligation partners
or at the site of ligation itself. In the former method, an
additional cysteine is required at the junction site,16 limiting
the applicability. The latter method would be more powerful
as it does not require a second nearby cysteine. To the best
of our knowledge, native chemical ligations using seleno-
cysteine have not been reported previously. Therefore, we
selected a test peptide to investigate the feasibility of the
approach.
resin. It contains the protected selenocysteine in place of
Cys754 at its N-terminus and a tBu disulfide protected
cysteine at position 759 (Sec(PMB)ESGAC(S-tBu)KI-OH)
(Scheme 4). After cleavage from the resin, performed under
Scheme 4
The C-terminus of class Ia ribonucleotide reductase (RNR)
contains two cysteine residues (Cys754 and Cys759, E. coli
numbering) that are involved in shuttling reducing equiva-
lents into the active site of the protein.17 During ribonucleo-
tide reduction a disulfide linkage is formed by two cysteines
in the active site. This disulfide is subsequently reduced via
a dithiol-disulfide interchange with the two C-terminal
cysteines (C754 and 759). The disulfide so formed at the
C-terminus is reduced in turn by thioredoxin, preparing RNR
for another turnover. Substitution of one or both of the
cysteines at the C-terminus with selenocysteines would
introduce altered redox properties11 that may be used to
investigate the two dithiol-disulfide interchange reactions.18
Two peptide segments were synthesized that correspond to
residues 745-753 and 754-761 of the C-terminus of RNR.
Peptide 10 was synthesized by Fmoc-based SPPS on Wang
conditions that maintained the PMB protecting group, and
HPLC purification the desired peptide was obtained in 58%
yield.
Oxidative deprotection of the selenocysteine derivative
yielded two different products depending on the amount of
iodine used (Scheme 4). Treatment of 10 with 15 equiv of
I2 in AcOH/MeCN/H2O4c,19 gave the diselenide 11 in 61%
yield, in which the cysteine retained the tert-butyl disulfide
protecting group. Alternatively, when only 1 equiv of I2 was
used, 11 and 12 were recovered in 86% combined yield.
Product 12 is presumably formed when the free selenol
liberated at the N-terminus reacts with the disulfide at
Cys759.20 Peptide 13 (Ac-LVPSIQDDG-SBn, residues 745-
753) containing a C-terminal benzyl thioester was synthe-
sized using a safety catch resin21 in 61% yield.
(14) Huber, R. E.; Criddle, R. S. Arch. Biochem. Biophys. 1967, 122,
164-173.
(15) (a) Boschi-Muller, S.; Muller, S.; Van Dorsselaer, A.; Bo¨ck, A.;
Branlant, G. FEBS Lett. 1998, 439, 241-245. (b) Arner, E. S.; Sarioglu,
H.; Lottspeich, F.; Holmgren, A.; Bo¨ck, A. J. Mol. Biol. 1999, 292, 1003-
1016 (c) Sandman, K. E.; Benner, J. S.; Noren, C. J. J. Am. Chem. Soc.
2000, 122, 960-961.
(16) More recent techniques have been developed where the presence
of a cysteine at ligation sites is not required; see (a) Canne, L. E.; Bark, S.
J.; Kent, S. B. H. J. Am. Chem. Soc. 1996, 118, 5891-5896. (b) Beligere,
G. S.; Dawson, P. E. J. Am. Chem. Soc. 1999, 121, 6332-6333. (c) Saxon,
E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2, 2141-2143. (d)
Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2, 1939-
1941. (e) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2001, 3,
9-12. (f) Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526-
533.
(17) (a) Mao, S. S.; Holler, T. P.; Yu, G. X.; Bollinger, J. M.; Booker,
S.; Johnston, M. I.; Stubbe, J. Biochemistry 1992, 31, 9733-9743. (b)
Stubbe, J.; van der Donk, W. A. Chem. Biol. 1995, 2, 793-801.
(18) A redox active cysteine-selenocysteine pair is actually present in
mammalian thioredoxin reductase. This pair cycles between the free amino
acids and the selenosulfide oxidation states; see (a) Lee, S. R.; Bar-Noy,
S.; Kwon, J.; Levine, R. L.; Stadtman, T. C.; Rhee, S. G. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 2521-2526. (b) Zhong, L.; Arne´r, E. S.; Holmgren,
A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5854-5859.
(19) Kamber, B.; Hartmann, A.; Eisler, K.; Riniker, B.; Rink, H.; Sieber,
P.; Rittel, W. HelV. Chim. Acta 1980, 63, 899-915.
(20) Thermodynamically, selenosulfide 12 is expected to be less stable
than diselenide 11; see ref 11.
In parallel reactions, peptides 11 and 12 were ligated with
the thioester 13 in sodium phosphate buffer (0.1 M)
(21) (a) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem.
Soc., Chem. Commun. 1971, 636-637. (b) Shin, Y.; Winans, K. A.; Backes,
B. J.; Kent, S. B. H.; Ellman, J. A.; Bertozzi, C. R. J. Am. Chem. Soc.
1999, 121, 11684-11689 (c) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi,
A. J. Am. Chem. Soc. 1999, 121, 11369-11374.
(22) All yields are after purification by preparative RP-HPLC and are
based on thioester starting material.
(23) See, for instance, (a) Dawson, P. E.; Churchill, M. J.; Ghadiri, M.
R.; Kent, S. B. H. J. Am. Chem. Soc. 1997, 119, 4325-4329. (b) Miranda,
L. P.; Alewood, P. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1181-1186.
(c) Kochendoerfer, G. G.; Salom, D.; Lear, J. D.; Wilk-Orescan, R.; Kent,
S. B.; DeGrado, W. F. Biochemistry 1999, 38, 11905-11913.
(24) Singh, R.; Whitesides, G. M. J. Org. Chem. 1991, 56, 2332-7.
Thiophenol may generate low but sufficient concentrations of the free selenol
for the ligation reaction to proceed. Selenosulfides such as 12 are reported
to be more easily reduced than diselenides (ref 11).
(25) Vedejs, E.; Diver, S. T. J. Am. Chem. Soc. 1993, 115, 5.
(26) A dehydroalanine-containing ligation product would have identical
mass as 15.
(27) (a) Harpp, D. N.; Gleason, J. G.; Snyder, J. P. J. Am. Chem. Soc.
1968, 90, 4181-4182. (b) Harpp, D. N.; Gleason, J. G. J. Org. Chem. 1971,
36, 73-80. (c) Harpp, D. N.; Gleason, J. G. J. Am. Chem. Soc. 1971, 93,
2437-2445. (d) Wakamiya, T.; Shimbo, K.; Sano, A.; Fukase, K.; Shiba,
T. Bull. Chem. Soc. Jpn. 1983, 56, 2044-2049. (e) Lai, Y. H.; Soo, T. B.
Org. Lett., Vol. 3, No. 9, 2001
1333

containing 6 M Gn‚HCl (pH 7.5) and thiophenol (4%)
(Scheme 5). The ligation product 14 was isolated as the
(Et2N)3P has been used previously for desulfurization of
disulfides to provide dialkylthioethers.27 A similar reaction
may have been induced by TCEP with the selenosulfide 12,
producing a lanthionine. The formation of either an N-
terminal dehydroalanine or a lanthionine would also explain
the very slow formation of a ligated product in poor yield,
as it would not be assisted by initial attack of a selenolate
on the thioester of 13. Instead, 15 might have been formed
by direct attack of the N-terminal amine of the lanthionine
derivative of 12. Regardless of the exact structure of the
product and the pathway(s) of its formation, it appears that
TCEP is not a reagent of choice for native chemical ligations
with selenocysteine if this residue can form a selenosulfide
with a nearby cysteine.
Scheme 5
The deselenation suggests a potential application for
ligation products containing selenocysteine. Dawson recently
reported on native chemical ligations combined with sub-
sequent reductive desulfurization to provide alanines at the
site of ligation.16f A limitation of this interesting extension
of the ligation repertoire lies in the inability to control
regioselective desulfurizations if other cysteines are present
in the peptide or protein.16f We are currently exploring
whether selenocysteines can be chemoselectively deselenated
in the presence of cysteines.
In summary, we demonstrate high yielding short syntheses
of FmocSec(PMB) (1) and FmocSec(Ph) (2). The p-meth-
oxybenzyl group of the N-terminal selenocysteine can be
removed with concomitant selenosulfide or diselenide forma-
tion by treatment with I2. To demonstrate the utility of 1, a
C-terminal peptide of ribonucleotide reductase was synthe-
sized by employing the first reported example of native
chemical ligation with selenocysteine. We are currently
investigating extension of this methodology to expressed
protein ligations.28
mixed selenosulfide in 56% yield when starting with 11 and
60% yield when starting with 12.22 Compared to typical
literature procedures for native chemical ligations using
peptides with N-terminal cysteines (∼1 mM, 8-24 h),23 the
ligations with selenocysteine derivatives 11 and 12 needed
a somewhat higher concentration (3 mM) for complete
conversion in 24 h. Given the significantly lower pKa of a
selenol compared to a thiol4a,12 and the higher nucleophilicity
of the resulting selenolate in comparison to the thiolate,14 it
may be surprising that the ligations did not proceed faster.
We speculate that the rate-limiting step in our reactions may
actually be the generation of the free selenol from diselenide
11 or selenosulfide 12 by reduction with thiophenol, since
diselenides are not readily reduced by monothiols.11,24
Interestingly, when triscarboxyethylphosphine (TCEP) was
added to the ligation reaction with 12, no selenocysteine-
containing ligation product was obtained. The addition of
phosphine was prompted by the possibility that it might
facilitate the reduction of the selenosulfide and perhaps also
act as an acyl transfer catalyst.16d,25 However, the reaction
proceeded very slowly and produced a ligation product after
7 days in poor yield (25%) that showed a molecular ion
corresponding to loss of a selenium atom. The typical
isotopic distribution characteristic for selenium was also
absent from the molecular ion. Either this peptide may be
an elimination product giving a dehydroalanine derivative,
or it may consist of a lanthionine-containing peptide 15.26
Acknowledgment. This work was supported by grants
from the Burroughs-Welcome Fund (APP 1920), the NIH
(GM58822), the Beckman Foundation (BYI award), and the
Research Corporation (R10187). L.X. acknowledges support
from a Zeneca Graduate Fellowship. NMR spectra were
obtained in the Varian Oxford Instrument Center for Excel-
lence, funded in part by the W.M. Keck Foundation, NIH
(PHS 1 S10 RR10444), and NSF (CHE 96-10502). Mass
spectra were recorded on a Voyager mass spectrometer
funded in part by the NIH (RR 11966).
Supporting Information Available: Experimental pro-
cedures for all transformations that produced previously
unknown compounds, as well as their full spectral charac-
terization, and a general procedure for the ligation reaction.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Heterocycles 1985, 23, 1205-1214. (f) Olsen, R. K.; Kini, G. D.; Hennen,
W. J. J. Org. Chem. 1985, 50, 4332-4336 (g) Fukase, K.; Kitazawa, M.;
Sano, A.; Shimbo, K.; Horimoto, S.; Fujita, H.; Kubo, A.; Wakamiya, T.;
Shiba, T. Bull. Chem. Soc. Jpn. 1992, 65, 2227-2240.
(28) (a) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 6705-6710. (b) Evans, T. C.; Benner, J.; Xu, M. Q. Protein Sci.
1998, 7, 2256-2264. (c) Cotton, G. J.; Muir, T. W. Chem. Biol. 1999, 6,
R247-R256. (d) Noren, C. J.; Wang, J. M.; Perler, F. B. Angew. Chem.,
Int. Ed. 2000, 39, 451-466.
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