Site-selective conjugation of thiols with aziridine-2-carboxylic acid-containing peptides

Article abstract of DOI:10.1021/ja046793x

The synthesis and convergent site-selective conjugation of aziridine-2-carboxylic acid-containing peptides with thiols, both in solution and on solid support, are described. The synthesis and use of FmocAzyOH in this capacity demonstrate both the efficient incorporation and tolerance of the Azy moiety in multistep Fmoc solid-phase peptide synthesis (SPPS), as well as the competence of solution and on-bead ligation through a highly regioselective base-promoted aziridine ring-opening process. Copyright

Full text of DOI:10.1021/ja046793x

Published on Web 09/15/2004
Site-Selective Conjugation of Thiols with Aziridine-2-Carboxylic
Acid-Containing Peptides
Danica P. Galonic´, Wilfred A. van der Donk,* and David Y. Gin*
Department of Chemistry, UniVersity of Illinois, Urbana, Illinois 61801
Received May 31, 2004; E-mail: vddonk@scs.uiuc.edu; gin@scs.uiuc.edu
Table 1. Azy-Tripeptide Conjugation with Thiols
Biological roles of proteins are often determined by their
posttranslational modifications that result in the introduction of
various functionalities such as carbohydrates and lipids. The
resulting conjugates play pivotal roles in numerous processes,
including cell adhesion, signal transduction, and immune response.1
As such, the development of methods for site-selective peptide and
protein modifications has been a focus of extensive research.2
Herein, we report a strategy for the site- and stereoselective
conjugation of electrophilic aziridine-carboxylic acid-containing
peptides with thiols promoted by DBU. The methodology is also
adapted to solid-phase peptide synthesis (SPPS), in which incor-
poration of the aziridine-carboxylic acid residue into peptides and
their ligation with thiols are effected on a solid support.
Aziridine-2-carboxylic acid (Azy)-containing peptides have the
potential to function as useful precursors to modified peptide
derivatives.3 However, only short (di- and tri-) peptides of this type
have been prepared, presumably as a result of the lability4 of the
Azy-containing peptide backbone toward consecutive amino acid
couplings. Moreover, nucleophilic ring openings of the aziridine
in these short peptides have employed only Lewis or protic acid
promoters,3b,d which are likely to be rendered ineffective by the
multiple Lewis basic functionalities in larger peptides.
Scheme 2 a
To evaluate nonacidic conditions for the reaction of aziridine-
containing peptides with thiol nucleophiles, N-acetylaziridine-2-
carboxamide 1 (Scheme 1) was reacted with ethanethiol in the
Scheme 1
a Reagents and conditions: (a) FmocAzyOH, HBTU, NMM; DBU; (b)
FmocPheOH, HBTU, NMM; DBU; (c) FmocGlyGlyOH, HBTU, NMM;
DBU; (d) Ac2O, pyridine; (e) FmocGlu(tBu)GlyOH, HBTU, NMM; DBU;
(f) FmocSer(tBu)OH, HBTU, NMM; DBU; (g) FmocLys(Boc)OH, HBTU,
NMM; DBU.
presence of DBU as a catalyst (0.1 equiv). Aziridine opening
proceeded with high regioselectivity, providing cysteine derivative
2 (61%), accompanied by a small amount of the â2-amino acid
derivative 3 (4%).5
generated cysteine derivative, providing S-mucin isosteres from the
coupling of preformed Azy-peptides with carbohydrates.7
Further expansion of the versatility of the aziridine-peptide
conjugation was realized through the rapid construction of Azy-
containing peptides via SPPS. Due to the known acid sensitivity
of acylated aziridines,3a Fmoc-based solid-phase synthesis was
chosen over Boc-based SPPS. Likewise, polystyrene with a
2-chlorotrityl linker was selected as the polymer support as it can
be cleaved under weakly acidic conditions.8 Adhering to these
criteria, several peptides were prepared on solid support using
FmocAzyOH as a novel amino acid monomer,9 HBTU as the
coupling agent, and 1% DBU as the Fmoc deblocking agent
(Scheme 2).10 Thus, the solid-supported Azy-pentapeptides 10 and
11 (Scheme 2A), as well as the Azy-heptapeptide 12 (Scheme 2B),
incorporating additional functionalized residues such as glutamic
acid, serine, and lysine, were readily accessed. It is important to
note that attempts to extend Azy-containing peptides by sequential
The application of this aziridine opening protocol to peptide
substrates was established with the reaction of a series of tripeptides
containing a central Azy moiety. Each of the peptides 4a-d (Table
1) was prepared using a modification of Okawa’s procedure.3a
Reaction of either ethanethiol or the sterically demanding tert-butyl
thiol with BocAlaAzyAlaOBn (4a) in the presence of DBU (0.1
equiv) proceeded smoothly at 23 °C to provide tripeptide-thiol
conjugates in good yield, strongly favoring the R-amino acid product
5 over its â2-isomer 6 (ratio g 20:1, entries 1 and 2). The procedure
is also amenable to carbohydrate thiols, exemplified by the coupling
of the C1 thio analogue of the TN-antigen 7 (Table 1, entries 3-6).6
Importantly, these reactions proceed with retention of both the
R-anomeric configuration of 7 and the L-configuration of the newly
9
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J. AM. CHEM. SOC. 2004, 126, 12712-12713
10.1021/ja046793x CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Table 2. On-Bead Conjugation of Azy-Tripeptides with Thiolsa
glycoconjugates 15 and 17 (19% over 14 steps and 43% over 10
steps, respectively) from 8 (g88%/step).
This work describes the synthesis and convergent site-selective
conjugation of aziridine-2-carboxylic acid-containing peptides with
thiols, both in solution and on solid support. The synthesis and use
of FmocAzyOH in this capacity demonstrate both the efficient
incorporation and tolerance of the Azy moiety in multistep Fmoc-
SPPS, as well as the competence of solution and on-bead ligation
through a highly regioselective base-promoted aziridine ring-
opening process. The strategy presents prospects not only for the
convergent preparation of complex oligosaccharyl-peptide con-
jugates and multivalent glycopeptide displays but also for the rapid
assembly of Azy-containing peptides for functional proteomics
studies.12
peptide conjugate;
time (h),
temp ( C)
yield from 8 or 9, %
entry
peptide
R2SH
°
(yield/step, %)
1
2
3
4
5
10
10
11
10
11
EtSH
tBuSH
EtSH
7
16, 23
28, 23
22, 23
22, 60
22, 60
13a; 52% (93%)a
13b; 40% (90%)a
14a; 35% (89%)b
13c; 21% (86%)a,c
14b; 17% (84%)b,c
7
a Reagents and conditions: (a) resin cleavage: (CF3)2CHOH:CH2Cl2
(20% v/v). b Resin and tBu-ester removal: TFA:CH2Cl2:iPr3SiH:HS(CH2)2SH
) 10:10:1:1. c Acetate removal: NaOMe, MeOH, pH 8.5.
Acknowledgment. This research was supported by the NIH
(GM58833 and GM58822). A Procter and Gamble Fellowship to
D.P.G. is acknowledged. We thank Jennifer Showerman for useful
suggestions on SPPS, and Kate Lindley for initial exploratory work
in base-promoted aziridine thiolysis.
Scheme 3 a
Supporting Information Available: Experimental details . This
information is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) (a) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A.
Science 2001, 291, 2370-2376. (b) Bertozzi, C. R.; Kiessling, L. L.
Science 2001, 291, 2357-2364. (c) Seitz, O.; Heinemann, I.; Mattes, A.;
Waldmann, H. Tetrahedron 2001, 57, 2247-2277.
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a Reagents and conditions: (a) DBU, DMF, 60 °C; (b) TFA:CH2Cl2:
iPr3SiH:HS(CH2)2SH ) 10:10:1:1; (c) NaOMe, MeOH, pH 8.5; (d) DBU,
DMF, 23 °C; (e) (CF3)2CHOH:CH2Cl2 (20% v/v).
(3) (a) Okawa, K.; Nakajima, K. Biopolymers 1981, 20, 1811-1821. (b)
Wakamiya, T.; Shimbo, K.; Shiba, T.; Nakajima, K.; Neya, M.; Okawa,
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coupling of single Fmoc-amino acids were unsuccessful, presumably
due to intramolecular aziridine N-deacylation by the liberated amine
upon Fmoc removal at the [Azy+2] position (Scheme 2). Fortu-
nately, employing a dipeptide building block for simultaneous
incorporation of the [Azy+2] and [Azy+3] residues avoided
aziridine deacylation, allowing for further sequential peptide
elongation.11
After successful incorporation of the Azy into peptides via SPPS,
each was used for on-bead ligation with thiols (Table 2). The
polymer-supported peptides were treated with the thiols (5-10
equiv) in the presence of DBU (1 equiv) in DMF. After 16-28 h,
the products were removed from the solid support with concomitant
peptide deprotection ((CF3)2CHOH or TFA). Preparative RP-HPLC
purification provided the deprotected peptide conjugates in good
overall isolated yields (calculated from 8 and 9). For example,
preparation of pentapeptides 10 and 11 and ligation with EtSH or
tBuSH provided peptide conjugates 13a-b and 14a, each in a nine-
step sequence from 8 or 9 with an average yield of at least 89%
per step (Table 2, entries 1-3).
In like manner, the C1 thio analogue of the TN-antigen 7 was
reacted with polymer-supported pentapeptides 10 and 11 (Table 2,
entries 4-5). Release of thioglycopeptides from the resin, followed
by acetate removal provided, after RP-HPLC purification, the
thioglycopeptides 13c and 14b in 21 and 17% yield, respectively,
each over a 10-step sequence (g84%/step). Finally, on-bead
synthesis and ligation of heptapeptide 12 with thiol 7 (Scheme 3A),
as well as peptide 10 with the more complex thio-STN-antigen 167h
(Scheme 3B), proceeded with equally high efficiency providing,
after release from solid support, deprotection and RP-HPLC,
(5) For aminolysis of N-Ns-Azy-OtBu with benzylamine and Et3N (0.2 equiv),
see: Turner, J. J.; Sikkema, F. D.; Filippov, D. V.; van der Marel, G. A.;
van Boom, J. H. Synlett 2001, 1727-1730.
(6) In each case, the minor â2 product was obtained as a single diastereomer,
assumed to result from inversion (absolute configuration not determined).
(7) For approaches to the preparation of glycopeptide mimetics, see: (a)
Recent review: Peri, F.; Nicotra, F. Chem. Commun. 2004, 623-627.
(b) Zhao, Y.; Kent, S. B. H.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 1629-1633. (c) Rodriguez, E. C.; Winans, K. A.; King, D. S.;
Bertozzi, C. R. J. Am. Chem. Soc. 1997, 119, 9905-9906. (d) Zhu, Y.,
van der Donk, W. A. Org. Lett. 2001, 3, 1189-1192. (e) Cohen, S. B.;
Halcomb, R. L. J. Am. Chem. Soc. 2002, 124, 2534-2543. (f) Liu, H.;
Wang, L.; Brock, A.; Wong, C.-H.; Schultz, P. G. J. Am. Chem. Soc.
2003, 125, 1702-1703. (g) Watt, G. M.; Lund, J.; Levens, M.; Kolli, V.
S. K.; Jefferis, R.; Boons, G.-J. Chem. Biol. 2003, 10, 807-814. (h)
Galonic´, D. P.; van der Donk, W. A.; Gin, D. Y. Chem. Eur. J. 2003, 9,
5997-6006. (i) Zhu, X.; Schmidt, R. R. Chem. Eur. J. 2004, 10, 875-
887. (j) Gamblin, D. P.; Garnier, P.; van Kasteren, S.; Oldham, N. J.;
Fairbanks, A. J.; Davis, B. G. Angew. Chem., Int. Ed. 2004, 43, 828-
833. For ligation approaches to the preparation of native linkages, see,
for example: (k) Miller, J. S.; Dudkin, V. Y.; Lyon, G. J.; Muir, T. W.;
Danishefsky, S. J. Angew. Chem., Int. Ed. 2003, 42, 431-434.
(8) Bollhagen, R.; Schmiedberger, M.; Barlos, K.; Grell, E. J. Chem. Soc.,
Chem. Commun. 1994, 2559-2560.
(9) For the preparation of FmocAzyOH, see Supporting Information.
(10) Wade, J. D.; Bedford, J.; Sheppard, R. C.; Tregear, G. W. Pept. Res.
1991, 4, 194-199.
(11) Analytical samples of Azy-peptides from SPPS were assayed ((CF3)2-
CHOH cleavage; ESI-MS, 1H NMR) to be 80-90% pure prior to ligation.
(12) Moroder, L.; Musiol, H.-J.; Scharf, R. FEBS Lett. 1992, 299, 51-53.
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