Chemoselective peptidomimetic ligation using thioacid peptides and aziridine templates

Article abstract of DOI:10.1021/ja104488d

Chemoselective peptidomimetic ligation has been made possible using thioacid peptides and NH aziridine-terminated amino acids and peptides. In the course of this reaction, a reduced amide bond is incorporated into the backbone of a peptide. This process e

Full text of DOI:10.1021/ja104488d

Published on Web 07/28/2010
Chemoselective Peptidomimetic Ligation Using Thioacid Peptides and
Aziridine Templates
Naila Assem, Aditya Natarajan, and Andrei K. Yudin*
DaVenport Research Laboratories, Department of Chemistry, UniVersity of Toronto, 80 St. George Street, Toronto,
Ontario, Canada M5S 3H6
Received May 24, 2010; E-mail: ayudin@chem.utoronto.ca
Abstract: Chemoselective peptidomimetic ligation has been
made possible using thioacid peptides and NH aziridine-
terminated amino acids and peptides. In the course of this
reaction, a reduced amide bond is incorporated into the backbone
of a peptide. This process enables incorporation of reduced
cysteine, reduced substituted cysteine, reduced phenylalanine,
and reduced alanine. Our method should be adaptable to other
unnatural amino acid residues at the ligation site. Experiments
aimed at evaluating the chemoselectivity of this process in the
presence of competing thiol nucleophiles suggest high specificity
at micromolar concentrations. This holds even in the presence
of glutathione, which neutralizes xenobiotic electrophiles in cells.
Figure 1. Strained peptide diamines used in this study.
Structural modification of peptide backbones has been widely
used in the study of biological processes.1,2 Reduced peptide bonds
are present in a number of potent protease inhibitors. These linkages
display interesting conformational preferences, inducing folds that
are similar to γ- and ꢀ-turns. The secondary amine of the reduced
amide bond is protonated at physiological pH, which enables
formation of a hydrogen bond between the protonated nitrogen
Figure 2. Preparation of thioacid starting materials.
center and carbonyl oxygen, similar to (i + 2) or (i + 3) interactions
in peptides.3 Chemoselective strategies for linking two peptide
fragments by a peptide-bond mimetic hold significant potential for
discovery of novel peptide and protein scaffolds. Our contribution
describes efforts to develop peptidomimetic ligation during which
a reduced amide bond is incorporated into the backbone of a peptide.
This process enables incorporation of reduced cysteine, reduced
substituted cysteine, reduced phenylalanine, and reduced alanine
and relies on unprotected aziridine aldehydes.4 Side-chain variation
at the ligation site is a function of substituents present in the
aziridine aldehyde reagent. Experiments aimed at evaluating the
chemoselectivity of this reaction in the presence of competing thiol
nucleophiles suggest high specificity. This holds even in the
presence of glutathione, which neutralizes xenobiotic electrophiles
in cells.
In order to demonstrate peptidomimetic ligation, we employed
the recently developed unprotected aziridine aldehydes.4 The peptide
aziridines 1-8 (Figure 1) were prepared via reductive amination
between aziridine aldehyde dimers and peptides. We tested the
feasibility of peptidomimetic ligation between templates 1-8 and
thioacid-terminated peptides prepared using sodium sulfide and
carbonyldiimidazole (CDI) (Figure 2).5 Mechanistically, the aziri-
dine ring opening was projected to trigger S-to-N acyl group
transfer. We found that thioamino acids and the corresponding
COSH-containing peptides underwent clean conjugation with
templates 1-8, delivering a single addition/rearrangement product
in every attempted reaction (Scheme 1 and Table 1). Reduced
cysteine and its substituted derivatives are readily accessible using
this method.6 Subsequent treatment with Raney Ni produces the
corresponding desulfurized residues, opening the possibility of
introducing a desired side chain next to the reduced amide bond
by choosing an appropriate aziridine aldehyde reagent.7
Several control elements are responsible for the observed
chemoselectivity. One of the factors is the attenuated reactivity of
the thioamino acids, which contrasts with the double acylation
product observed with the more reactive thiobenzoic acid.8 We were
particularly pleased to note that the reactions with thioacids remain
effective at low molarity. A thousandfold dilution did not diminish
the efficiency of the process.9 Mechanistically, the thioester
generated upon aziridine ring opening is trapped by the primary
amine. The acyl transfer proceeds via a five-membered cyclic
intermediate.10 A balance of relative rates results in the peptido-
mimetic rather than peptidic backbone (Scheme 1).11 We did not
observe competition from the aza-Payne rearrangement.12 The
reaction was also found to proceed without epimerization. The
aziridine ring opening was regioselective. We posit that this type
of regioselectivity is unique to the unsubstituted NH aziridine-
containing templates that do not have electron-withdrawing sub-
stituents at the carbon adjacent to the three-membered ring.13 The
ring opening was also regioselective for the phenyl-substituted
aziridine ring (see the Supporting Information). We were also happy
to note that unprotected acid-terminated peptides readily participate
in this chemistry. We ran experiments aimed at further evaluating
the chemoselectivity of our protocol. Because of the ubiquity of
thiol nucleophiles in biological systems, it was important to perform
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10986 J. AM. CHEM. SOC. 2010, 132, 10986–10987
10.1021/ja104488d  2010 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Scope of Peptidomimetic Ligationa
a The peptide diamine concentration was 0.1 M in ethanol, and 1 equiv of thioamino acid was used in each case. b Isolated yields. c Desulfurization
reaction run with Raney Ni on entry 13. d Desulfurization reaction run with Raney Ni on entry 16. e 1:1 mixture of diastereoisomers.
protocols for long-chain thioacid synthesis,15 it should be possible
Scheme 1. Mechanism of Peptidomimetic Ligation
to extend this methodology to the solid phase. Equally attractive is
the possibility of interfacing this reaction with the subtiligase-
catalyzed thioacid synthesis.16 We also note that the sequence of
bonds that are installed as a result of our method matches the
repeating structure present in protein nucleic acids (PNAs), which
should facilitate construction of substituted PNAs under mild
conditions.
Acknowledgment. We thank NSERC for financial support. N.A.
thanks NSERC for a CGS-D Fellowship.
Supporting Information Available: Complete experimental details
and characterization data for new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Muir, T. W. Annu. ReV. Biochem. 2003, 72, 249–289.
(2) (a) Cheng, R. P.; Gellman, S. H.; Degrado, W. F. Chem. ReV. 2001, 101,
3219–3232. (b) Seebach, D.; Beck, A. C.; Bierbaum, D. J. Chem.
BiodiVersity 2004, 1, 1111–1239.
(3) For NMR and X-ray characterization, see: Grand, V.; Aubry, A.; Dupont,
V.; Vicherat, A.; Marraud, M. J. Pept. Sci. 1996, 2, 381–391.
(4) Hili, R.; Yudin, A. K. J. Am. Chem. Soc. 2006, 128, 14772–14773.
(5) The thioamino acids were prepared through activation of the carboxylic
acid with CDI followed by addition of sodium sulfide. No purification was
found to be necessary after workup with hydrochloric acid.
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control experiments with glutathione present in the reaction mixture.
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Pleasingly, the ligation remained thioacid-specific even under these
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see: Dirksen, A.; Dawson, P. E. Bioconjugate Chem. 2008, 19, 2543–2548.
(10) For S-to-N acyl transfer during native chemical ligation, see: Dawson, P. E.;
Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science 1994, 266, 776–779.
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conditions.14
In summary, the first example of chemoselective peptidomimetic
ligation has been made possible using thioamino acids and NH
aziridine-terminated amino acids and peptides. This method enables
incorporation of a peptidomimetic linkage at the site of ligation.
The technique is not accompanied by epimerization and appears
to perform chemoselectively in the presence of competing thiol
nucleophiles known to react with aziridines. In view of the
accessibility of aziridine aldehydes, it should be possible to generate
a wide range of peptidomimetics and backbone-modified proteins
under mild conditions. With the recently developed solid-phase
(12) Ibyka, T. Chem. Soc. ReV. 1998, 27, 145–154.
(13) Tam, J. P.; Lu, Y. A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 12485–12489.
(14) See the Supporting Information.
(15) Crich, D.; Kasinath, S. J. Org. Chem. 2009, 74, 7383–7388.
(16) Tan, X. H.; Zhang, X.; Yang, R.; Liu, C. F. ChemBioChem 2008, 9,
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