Facile chemoselective synthesis of dehydroalanine-containing peptides.

Article abstract of DOI:10.1021/ol006485d

Useful methodology is described for the synthesis of dehydroalanine residues (II) within peptides. The unnatural amino acid (Se)-phenylselenocysteine (I) can be incorporated into growing peptide chains via standard peptide synthesis procedures. Subsequent

Full text of DOI:10.1021/ol006485d

ORGANIC
LETTERS
2000
Vol. 2, No. 23
3603-3606
Facile Chemoselective Synthesis of
Dehydroalanine-Containing Peptides†
Nicole M. Okeley,‡ Yantao Zhu,‡ and Wilfred A. van der Donk*
Department of Chemistry, UniVersity of Illinois, Urbana, Illinois 61801
Vddonk@aries.scs.uiuc.edu
Received August 19, 2000
ABSTRACT
Useful methodology is described for the synthesis of dehydroalanine residues (II) within peptides. The unnatural amino acid (Se)-
phenylselenocysteine (I) can be incorporated into growing peptide chains via standard peptide synthesis procedures. Subsequent oxidative
elimination affords a dehydroalanine at the desired position. The oxidation conditions are mild and tolerate functionalities commonly found
in peptides, including variously protected cysteine residues. To illustrate its utility, cyclic lanthionines have been synthesized by this method.
The R,â-unsaturated amino acids dehydroalanine (1) and
dehydrobutyrine (2) are found in a variety of biological
containing peptides (dehydropeptides) has previously been
accomplished by a number of different methods.3 The most
common approach involves incorporation of a masked
residue into the peptide and its subsequent conversion to the
dehydroamino acid. An abundance of precursors have been
used in this fashion to generate 1 and 2 within peptides, such
as the activation and elimination of serine or threonine
derivatives,4 Hoffmann elimination from 2,3-diaminopropi-
onic acid or asparagine residues,5 and oxidative elimination
of S-alkyl or S-aryl cysteines.6 Although these methodologies
have allowed the synthesis of a number of natural products
containing dehydroamino acids, most procedures are not
polypeptides and natural products.1 In nature they impart
interesting biological activities, while synthetically they can
be versatile precursors to unnatural amino acids2 and have
been used to alter the biological and structural properties of
peptides and proteins. The synthesis of dehydroamino acid
(3) (a) For a review, see: Schmidt, U.; Lieberknecht, A.; Wild, J.
Synthesis 1988, 3, 159-172. (b) Rich, D. H.; Tam, J. P. Tetrahedron Lett.
1975, 211-212. (c) Srinivasan, A.; Stephenson, R. W.; Olsen, R. K. J.
Org. Chem. 1977, 42, 2253-2256. (d) Somekh, L.; Shanzer, A. J. Org.
Chem. 1983, 48, 907-908. (e) Paquet, A. Tetrahedron Lett. 1990, 31, 5269-
5272. (f) Zetterstrom, M.; Trogen, L.; Hammarstrom, L. G.; Juhlin, L.;
Nilsson, B.; Damberg, C.; Bartfai, T.; Langel, U¨ . Acta Chem. Scand. 1995,
49, 696-700. (g) Yamada, M.; Miyajima, T.; Horikawa, H. Tetrahedron
Lett. 1998, 39, 289-292. (h) Coghlan, P. A.; Easton, C. J. Tetrahedron
Lett. 1999, 40, 4745-4748. (i) Stohlmeyer, M. M.; Tanaka, H.; Wandless,
T. J. J. Am. Chem. Soc. 1999, 121, 6100-6101.
(4) (a) Srinivasan, A.; Stephenson, R. W.; Olsen, R. K. J. Org. Chem.
1977, 42, 2256-2260. (b) Andruszkiewicz, R.; Czerwinski, A. Synthesis
1982, 968-969. (c) Balsamini, C.; Duranti, E.; Mariani, L.; Salvatori, A.;
Spadoni, G. Synthesis 1990, 9, 779-781. (d) Berti, F.; Ebert, C.; Gardossi,
L. Tetrahedron Lett. 1992, 33, 8145-8148. (e) Ranganathan, D.; Shah,
K.; Vaish, N. J. Chem. Soc., Chem. Commun. 1992, 16, 1145-1147. (f)
Shin, C.; Okumura, K.; Ito, A.; Nakamura, Y. Chem. Lett. 1994, 1301-
1304.
† Abbreviations: Boc, tert-butoxycarbonyl; Fmoc, N-9-fluorenylmethoxy-
carbonyl; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate; NMM, N-methylmorpholine.
‡ These authors contributed equally to this work.
(1) (a) Bodanszky, M.; Scozzie, J. A.; Muramatsu, I. J. Antibiot. (Tokyo)
1970, 23, 9-12. (b) Berdy, J. AdV. Appl. Microbiol. 1974, 18, 309-406.
(c) Tori, K.; Tokura, K.; Okabe, K.; Ebata, M.; Otsuka, H.; Lukacs, G.
Tetrahedron Lett. 1976, 3, 185-188 (d) Pascard, C.; Ducruix, A.; Lunel,
J.; Prange´, T. J. Am. Chem. Soc. 1977, 99, 6418-6423. (e) Pearce, C. J.;
Rinehart, K. L., Jr. J. Am. Chem. Soc. 1979, 101, 5069-5070. (f) Pedras,
M. S. C.; Taylor, J. T.; Nakashima, T. T. J. Org. Chem. 1993, 58, 4778-
4780. (g) Hamann, M. T.; Scheuer, P. J. J. Am. Chem. Soc. 1993, 115,
5825-5826. (h) Lau, R. C.; Rinehart, K. L., Jr. J. Antibiot. (Tokyo) 1994,
47, 1466-1472. (i) Sahl, H.-G.; Jack, R. W.; Bierbaum, G. Eur. J. Biochem.
1995, 230, 827-853. (j) Strohl, W. R.; Floss, H. G. Biotechnology 1995,
28, 223-238. (k) Dawson, J. F.; Holmes, C. F. Front. Biosci. 1999, 4,
D646-D658.
(2) Ferreira, P. M. T.; Maia, H. L. S.; Monteiro, L. S. Tetrahedron Lett.
1999, 40, 4099-4102.
(5) (a) Nomoto, S.; Sano, A.; Shiba, T. Tetrahedron Lett. 1979, 6, 521-
522. (b) Blettner, C.; Bradley, M. Tetrahedron Lett. 1994, 35, 467-470.
10.1021/ol006485d CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/24/2000

Table 1. Oxidation of Model Dehydropeptides
entry
peptide
dehydropeptidea,b
solvent
yield (%)c
1
2
3
4
5
Fmoc-Cys(SEt)Sec(Ph)-ODPM
[Boc-Sec(Ph)Cys-OMe]2
Boc-Sec(Ph)Cys(Trt)-OMe
Fmoc-TrpSec(Ph)-ODPM
AlaMetSec(Ph)Ala
Fmoc-Cys(SEt)Dha-ODPM
[Boc-DhaCys-OMe]2
Boc-DhaCys(Trt)-OMe
Fmoc-TrpDha-ODPM
AlaMetDhaAla
CH2Cl2/MeOH
MeOH
THF
CH2Cl2/MeOH
MeCN/H2O
70
75
83
64d
62
a Conditions for entries 1-4: aq NaIO4 (4 equiv) was added at 25 °C to solutions of the peptides (final peptide concentrations 7-30 mM). For entry 3,
NaIO4 was added at 0 °C and warmed to rt. For entry 5, 1.1 equiv of NaIO4 and 1 equiv of NEt3 were used. b Sec, selenocysteine; Dha, dehydroalanine;
DPM, diphenylmethyl. c Yields are for products after purification by flash chromatography (entries 1-4) or HPLC (entry 5). d The crude yield of this
1
reaction was 98% (95% pure material by H NMR).
selective or are incompatible with preexisting amino acid
residues in the peptide, thus requiring cumbersome protecting
strategies. A facile, site-specific, and chemoselective method
of introduction has not been previously described.7 Our
search for a mild method of introducing these amino acids
in a manner compatible with standard solid-phase peptide
synthesis (SPPS), led us to a previously reported yet
unexploited method for the generation of dehydroalanine via
oxidative elimination from the unnatural amino acid (Se)-
phenylselenocysteine (Sec(Ph)).8,9 In this communication, we
report our initial results on the further development and
modification of this chemistry to allow the synthesis of
dehydropeptides via either solution- or solid-phase peptide
synthesis.
peptide synthesis, the Boc protected amino acid was con-
verted to its Fmoc derivative via standard methods. The
resulting Boc or Fmoc amino acids were incorporated into
growing chains via standard solid- or solution-phase peptide
chemistry.
The short dehydropeptides listed in Table 1 were used to
optimize the desired conditions for chemoselective oxidative
elimination. These peptides were designed to test the
tolerance of oxidation sensitive amino acids, including
various protected cysteine residues, methionine and tryp-
tophan, unprotected at the indole moiety. The oxidative
eliminations were accomplished with sodium periodate in
less than 2 h, except in the case of an N-terminal Sec(Ph),
which required 12 h (Table 1, entries 2 and 3), reflecting
the lower reactivity of a carbamate versus an amide.13
Cysteine protecting groups that proved to be compatible
include mixed (Table 1, entry 1) and symmetrical (entry 2)
disulfides, as well as trityl (entry 3). All reaction products
were obtained in moderate to good yields after purification
without side-chain modification of tryptophan or the pro-
tected cysteines.14 When methionine was present in the
peptide, the reaction was carried out with 1.1 equiv of oxidant
To limit the number of possible diastereomeric peptides
containing phenylselenocysteine, we desired the optically
pure amino acid. The synthesis of (R)-FmocSec(Ph) was
modified from existing literature reports (Scheme 1). Con-
Scheme 1
(7) For example, methods based on activation and elimination of serine,
threonine or 2,3-diaminopropionic acid require either that no other Ser, Thr,
or Lys residues are present in the peptides or that they are orthogonally
protected compared to the residues to be eliminated. Bradley and co-workers
recently reported methodology involving incorporation of S-methylcysteine
into peptides followed by oxidation to the sulfoxide and either pyrolytic or
basic (DBU) elimination, which constitutes the most versatile approach to
date.6e However, this method is not compatible with protected cysteine
residues.6d Therefore, the authors resorted to segment couplings to prepare
peptides containing both cysteine and dehydroalanine (Dha).6e
(8) Selenocysteine occurs naturally and is incorporated into proteins via
the ribosomal machinery. Hence it has been termed the 21st physiological
amino acid and has been given the three-letter abbreviation Sec. The
oxidative elimination of Sec(Ph) has been utilized previously by Shirahama
and co-workers9b,c for the solution phase synthesis of Alternariolide. This
4-residue cyclic peptide does not contain reactive amino acid side chains,
necessitating the studies reported in this communication.
(9) (a) Walter, R.; Roy, J. J. Org. Chem. 1971, 36, 2561-2563. (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.
(10) Pansare, S. V.; Arnold, L. D.; Vederas, J. C. Org. Synth. 1991, 70,
10-17.
(11) Liotta, D.; Markiewicz, W.; Santiesteban, H. Tetrahedron Lett. 1977,
50, 4365-4368.
version of Boc-L-serine to its â-lactone,10 followed by ring
opening with phenylselenide anion11 generated by the reac-
tion of diphenyl diselenide with sodium trimethoxyborohy-
dride,12 provided BocSec(Ph) in 93% yield. For use in Fmoc
(6) (a) Rich, D. H.; Tam, J.; Mathiaparanam, P.; Grant, J. A.; Mabuni,
C. J. Chem. Soc., Chem. Commun. 1974, 21, 897-898. (b) Rich, D. H.;
Tam, J. P. J. Org. Chem. 1977, 42, 3815-3820. (c) Rich, D. H.; Bhatnager,
P.; Mathiaparanam, P.; Grant, J. A.; Tam, J. P. J. Org. Chem. 1978, 43,
296-302. (d) Burrage, S. A.; Raynham, T.; Bradley, M. Tetrahedron Lett.
1998, 39, 2831-2834. (e) Burrage, S.; Raynham, T.; Williams, G.; Essex,
J. W.; Allen, C.; Cardno, M.; Swali, V.; Bradley, M. Chem.sEur. J. 2000,
6, 1455-1466.
(12) (a) Berkowitz, D. B.; Smith, M. K. Synthesis 1996, 39. (b) Pedersen,
M. L.; Berkowitz, D. B. J. Org. Chem. 1993, 58, 6966-6975.
(13) (a) We and others13b have noted that selenoxides in amino acids
and peptides can be unusually stable. In certain cases they can be isolated
and purified and require elevated temperatures to undergo elimination. (b)
Woiwode, T. F.; Wandless, T. J. J. Org. Chem. 1999, 64, 7670-7674.
3604
Org. Lett., Vol. 2, No. 23, 2000

Table 2. Synthesis of Dehydropeptides
entry
peptidea,b
yield (%)c
dehydropeptided
oxidant
yield (%)
1
2
3
4
5
6
7
8
Fmoc-GLPU(Ph)VIA
Fmoc-ISVU(Ph)RSTS
Ac-GLPU(Ph)VIA
Ac-ISVU(Ph)RSTS
Ac-GGC(StBu)PU(Ph)VIA
LU(Ph)PGC(Trt)VG
[LU(Ph)ANCKI]2
44
27
40
34
33
15
30e
47
Fmoc-GLPDhaVIA
Fmoc-ISVDhaRSTS
Ac-GLPDhaVIA
NaIO4
NaIO4
H2O2
NaIO4
NaIO4
NaIO4
NaIO4
NaIO4
72
67
82
82
84
80
33
72
Ac-ISVDhaRSTS
Ac-GGC(StBu)PDhaVIA
LDhaPGC(Trt)VG (3)
[LDhaAECKI]2 (4)
RIADhaIALC(StBu)K
RIAU(Ph)IALC(StBu)K
a U, selenocysteine; U(Ph), (Se)-phenyl selenocysteine. b Conditions: Wang resins preloaded with the C-terminal amino acids were used. Piperidine was
used as deprotectant, HBTU and NMM were used as activators, and DMF was the solvent. c Yields are for HPLC purified products after SPPS and are based
on the supplier-specified loading of resin. d Conditions: the oxidant (4 equiv) was added at 25 °C to solutions of the peptides (final peptide concentrations
1-6 mM). Solvents were MeOH (entries 1, 3, 4) or H2O/MeCN (entries 2 and 5-8). e The symmetrical disulfide was formed by oxidation of the purified
free thiopeptide with I2. The yield is for both SPPS and oxidation.
as partial oxidation of the methionine to the sulfoxide was
otherwise observed.14
using tris(carboxyethyl)phosphine (TCEP) as monitored by
HPLC and mass spectrometry.
In an alternative approach, trityl-protected or free cysteine
residues were chemoselectively oxidized with I2 to the
corresponding symmetrical disulfides without modification
of the phenylselenide (Scheme 2). The dehydroalanine was
Beyond its utility for the synthesis of dehydropeptides,
this methodology is well suited for the synthesis of a class
of cyclic peptide thioethers known as lanthionines. Lanthion-
ines are structures that are almost exclusively found in
lantibiotics, a class of posttranslationally modified antibiotics.1i
Among these is the promising antibacterial agent nisin, which
interacts with lipid II, the physiological target of vancomy-
cin.15 To illustrate the utility of the current methodology for
lanthionine synthesis, we prepared two cyclic thioethers 5
and 6 (Scheme 3). After introduction of dehydroalanines into
Scheme 2
Scheme 3
then unmasked chemoselectively in the presence of the new
cystine residue (Table 1, entry 2). In general, H2O2 could
also be used as a suitable oxidant for the reactions in Tables
1 and 2, however m-CPBA provided a complex mixture of
products.
The versatility of this chemoselective and site-specific
method was further demonstrated with longer peptide
sequences synthesized by standard SPPS procedures (Table
2). The oxidations were monitored by RP-HPLC, and
generally quantitative conversion was observed in as little
as 0.5 h and as long as 2 h. Typical functionalities found in
peptides such as amines, amides, guanidines, alcohols, and
acids did not interfere when left unprotected during the
oxidation (Table 2).14 Cysteine residues protected as sym-
metrical disulfides were subsequently quantitatively reduced
peptides 3 and 4 (Table 2), a clean intramolecular Michael
addition of a cysteine onto the dehydroalanines was ac-
complished by deprotection of the cysteines followed by
adjustment of the pH of the reaction. One new species was
produced in both cases as monitored by HPLC, consistent
(14) The transformations in Tables 1 and 2 generally showed spot-to-
spot TLC or peak-to-peak HPLC conversions. In one case of a methionine-
containing peptide we did observe a byproduct containing both a dehy-
droamino acid and the sulfoxide of methionine. Addition of Et3N suppressed
formation of this side product to 13%. Since no other products were isolated
in significant quantities, we believe that the moderate yields obtained in
some cases in Tables 1 and 2 reflect losses during purification.
(15) Breukink, E.; Wiedemann, I.; van Kraaij, C.; Kuipers, O. P.; Sahl,
H.-G.; de Kruijff, B. Science 1999, 286, 2361-2364.
Org. Lett., Vol. 2, No. 23, 2000
3605

with similar observations recently reported by Toogood16 and
Bradley and co-workers.6e The single diastereomers were
assigned by NMR to the natural (2S,6R)-lanthionine ring
systems (“meso”-lanthionines) in the latter studies.
Acknowledgment. This work was supported by grants
from the Burroughs-Wellcome Fund (APP 1920), the Na-
tional Institutes of Health (GM58822), and the Research
Corporation (R10187). N.M.O. acknowledges support from
an Abbott Laboratories Graduate Fellowship. We also thank
Ms. Hao Zhou for assistance in preparing BocSec(Ph) and
Mr. Matt Gieselman for assistance in preparing FmocSec-
(Ph). NMR spectra were obtained in the Varian Oxford
Instrument Center for Excellence, 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 National Institutes
of Health (RR 11966).
In conclusion, a facile, site-specific, and chemoselective
method of incorporating dehydroamino acids into peptides
has been developed. This method allows unmasking of the
dehydroalanine after global deprotection in the ultimate step
of the synthesis of dehydropeptides, minimizing possible side
reactions. The method is fully compatible with all physi-
ological amino acids including appropriately protected cys-
teines, and the peptides can be assembled using standard
Fmoc SPPS. Its utility is demonstrated by the synthesis of a
variety of peptides including the intriguing cyclic lanthion-
ines. Given the high efficiency and yield of the oxidation
reaction, we are currently investigating the possibility of on-
resin oxidative eliminations to form resin-bound dehydro-
peptides.
Supporting Information Available: A general procedure
for the oxidation reactions is described, as well as charac-
terization of the peptides. This material is available free of
charge via the Internet at http://pubs.acs.org.
(16) Toogood, P. L. Tetrahedron Lett. 1993, 34, 7833-7836.
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