Aziridine-mediated ligation and site-specific modification of unprotected peptides

Article abstract of DOI:10.1021/ja207133t

A synthesis of aziridine-containing peptides via the Cu(II)-promoted coupling of unprotected peptide thioacids and N-H aziridine-2-carbonyl peptides is reported. The unique reactivity of the resulting N-acylated aziridine-2-carbonyl peptides facilitates t

Full text of DOI:10.1021/ja207133t

COMMUNICATION
pubs.acs.org/JACS
Aziridine-Mediated Ligation and Site-Specific Modification of
Unprotected Peptides
Frank Brock Dyer, Chung-Min Park, Ryan Joseph, and Philip Garner*
Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, United States
S Supporting Information
b
Scheme 1. Aziridine-Mediated Peptide Ligation Concept
ABSTRACT: A synthesis of aziridine-containing peptides
via the Cu(II)-promoted coupling of unprotected peptide
thioacids and NÀH aziridine-2-carbonyl peptides is re-
ported. The unique reactivity of the resulting N-acylated
aziridine-2-carbonyl peptides facilitates their subsequent
regioselective and stereoselective nucleophilic ring-opening
to give unprotected peptides that are specifically modified
at the ligation site. The aziridine-mediated peptide ligation
concept is exemplified using H2O as the nucleophile, pro-
ducing a XaaÀThr linkage (where Xaa can be an epimeriz-
able and hindered amino acid). The overall process is
compatible with a variety of unprotected amino acid func-
tionality, most notably the N-terminal and Lys side chain
amines.
Scheme 2. Initial Coupling Reaction Optimization
ative chemical ligation (NCL)1 enables the convergent
N
synthesis of peptides and proteins under mild reaction
conditions without the need for protecting groups. The NCL
process is characterized by the chemoselective coupling of
unprotected peptide thioesters (peptide1-Xaa-SR) and unpro-
tected cysteinyl peptides (H-Cys-peptide2) to give ligation
products peptide1-Xaa-Cys-peptide2. Since an N-terminal Cys
residue is required,2 the general application of NCL to peptide/
protein synthesis is limited to ligation at peptide linkages XaaÀ
Cys, where Xaa is preferably an unhindered amino acid. It follows
that the incorporation of post-translationally modified or un-
natural amino acids at the ligation site is not feasible with NCL.
We now disclose a ligation protocol that combines the con-
vergent synthesis of unprotected aziridine-2-carbonyl containing
peptides with their controlled site-specific chemical modification.
The key reaction (Scheme 1) involves chemoselective Cu(II)-
promoted coupling of a peptide thioacid 13 with an aziridine-2-
carbonyl (Azy) peptide 2 to give the initial ligation product 3
under native conditions. The unique properties of the chemical
species involved, a moderately acidic thioacid combined with a
moderately basic aziridine, enable the aziridine-mediated peptide
ligation to be performed without peptide protecting groups. The
Azy-containing peptide 3 may be converted to a site-specifically
modified peptide 4 through regioselective opening of the aziridine
ring by a nucleophile. Modifications may be introduced via the
aziridine substituent (“R”) and/or the nucleophilic species (“Nu”).
The potential utility of using an aziridine embedded in the
backboneof a peptide asan electrophilic handle for the site-specific
introduction of modifications has been recognized for some
time.4À6 The structural uniqueness of this unit has also been noted.7
However, the difficulty associated with the synthesis and manipula-
tion of unprotected Azy-containing peptides has limited the full
exploration and exploitation of their properties. In this communica-
tion, the aziridine-mediated peptide ligation concept is illustrated
with a methyl-substituted aziridine-2-carbonyl moiety using water as
the nucleophile, with the net result being ligation at a threonine site.
Our study began with thioacetic acid (5) serving as a model for 1
and the known6 aziridine H-Azy(Me)-NHBn (6, Azy(Me) =(2S,3S)
3-methylaziridine-2-carbonyl) as a model for 2. In the presence of
Cu(OAc)2 H2O, thioacid 5 reacted rapidly with aziridine 6 in
3
mixtures of DMF and phosphate-citrate buffer (pH range 4.2À7.2)
to give 7(Scheme2).StoichiometricCu(II) wasfoundtobeessential
for clean and efficient coupling. In its absence, a complex mixture of
products was observed, emanating from nonregioselective ring-open-
ing of 6 by 5 and, presumably, S- to N-acyl transfer.8,9 The use of
AgOAc instead of Cu(OAc)2 H2O resulted in a much slower
3
reaction.10 The subpar performance with K3Fe(CN)6 suggested that
the Cu(II)-mediated reaction does not simply involve an oxidative
mechanism.11 These experiments established the critical role that
Cu(II) plays in the aziridine-mediated coupling process as well as the
reaction’s compatibility with aqueous conditions.
Received: July 29, 2011
Published: November 23, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ja207133t J. Am. Chem. Soc. 2011, 133, 20033–20035
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COMMUNICATION
The next stage of reaction optimization focused on assessing
the level of epimerization during the coupling of Ac-Phe-SH
(8)12 to 6 and defining conditions to minimize it (Table 1).
This coupling reaction proceeded rapidly and cleanly in DMF-
aqueous buffers to produce Ac-Phe-Azy(Me)-NHBn (9). How-
ever, 13À14% of the epimeric product Ac-phe-Azy(Me)-NHBn
(epi-9) was also observed in these reactions (entries 1À3). The
level of epimerization could be reduced to 6% when the reaction
was performed in DMF alone but the crude yield was lowered
(entry 4). The inclusion of 1-hydroxybenzotriazole (HOBt) in
the reaction mixture was found to reduce the level of epimeriza-
tion to 10% in DMF-aqueous buffer and 5% in DMF. HOBt also
increased the yield of the ligation reaction. The optimal coupling
hydrolysis product Ac-Phe-Thr-NHBn (14) was isolated in good
overall yield after standard workup and purification (entry 1).13
The formation of 14 is consistent with regioselective and stereo-
selective nucleophilic opening of the aziridine ring at C3 by H2O.
The reaction of diastereomerically pure dipeptide thioacid Fmoc-
Phe-Ala-SH (15) and aziridine 6 produced Fmoc-Phe-Ala-Thr-
NHBn (16) (entry 2).14 Comparison with an authentic sample
of Fmoc-Phe-ala-Thr-NHBn (epi-16) established the level of
epimerization at 5%.
The coupling/ring-opening sequence was then performed
with H-Lys-Tyr-Thr-SH (17)15 and 6 to produce H-Lys-Tyr-
Thr-Thr-NHBn (18) in good yield (entry 3). An analogous
experiment using the peptide thioacid H-Glu-Tyr-Thr-SH (19)
produced H-Glu-Tyr-Thr-Thr-NHBn (20) (entry 4). Compat-
ibility of the coupling reaction with aqueous buffer and the
denaturant urea was established (entries 5 and 6). The coupling/
ring-opening protocol was also applied to a Cys-containing
peptide thioacid 21 to afford a mixture of peptide 22 and its
disulfide 23 (entry 7).16 Finally, we extended the ligation to the
union of thioacids 17 and 19 with the aziridine-containing
tripeptide H-Azy(Me)-Phe-Gly-NH2 (24)17 to give hexapep-
tides 25 and 26, respectively (entries 8 and 9). To summarize, the
aziridine-mediated ligation is compatible with free NH2, CO2H,
OH (aliphatic and aromatic), and SH functional groups (by virtue
of in situ protection as a disulfide). The facility of ligation employ-
ing an equimolar quantity of relatively hindered thioacid (reaction
complete within 1À2 h) is also noteworthy.
conditions were thus defined as 1 equiv of Cu(OAc)2 H2O and
3
2 equiv of HOBt in DMF.
We were now ready to combine the coupling reaction with an
aziridine ring-opening reaction and address the chemoselectivity
issue (Table 2). It was decided to use H2O as the nucleophile
converting the unprotected Azy(Me)-containing peptide to a
Thr-containing peptide (10 + 11 f [12] f 13). First, the
coupling of 8 and 6 was repeated but, rather than isolate 9, the
reaction mixture was treated directly with 10% TFA/H2O. The
Table 1. Optimization of Coupling Reaction Conditions
While much work remains to be done to develop the aziridine-
mediated peptide ligation concept, these preliminary results
are encouraging. The method disclosed herein enables one to
synthesize unprotected aziridine-containing peptides and regio-
selectively hydrolyze the embedded aziridine moiety to give
products corresponding to ligation at XaaÀThr linkages.18 It is
anticipated that the aziridine ring-opening reaction will not be
limited to the use of water as a nucleophile.4,6
HOBt
9 + epi-9
epi-9
entry
1
solvent
(equiv)
(% yield)
(mol %)
1:1 DMF-buffer
(pH 7.2)
0
0
0
68
82
87
13
13
14
2
3
1:1 DMF-buffer
(pH 6.2)
1:1 DMF-buffer
(pH 5.2)
’ ASSOCIATED CONTENT
4
5
DMF
0
2
53
88
6
S
Supporting Information. Experimental procedures and
b
1:1 DMF-buffer
(pH 7.2)
10
characterization data for all new compounds is provided. This
material is available free of charge via the Internet at http://pubs.
acs.org.
6
DMF
2
89
5
Table 2. Aziridine-Mediated Peptide Ligation at XaaÀThr Sites
entry
thioacid (1.1 equiv)
aziridine
solvent
product
% yield
1
2
3
4
5
6
7
Ac-Phe-SH (8)
H-Azy(Me)-NHBn (6)
H-Azy(Me)-NHBn (6)
H-Azy(Me)-NHBn (6)
H-Azy(Me)-NHBn (6)
H-Azy(Me)-NHBn (6)
H-Azy(Me)-NHBn (6)
H-Azy(Me)-NHBn (6)
DMF
DMF
DMF
DMF
Ac-Phe-Thr-NHBn (14)
69
72
80
69
71
88
43
40
78
77
Fmoc-Phe-Ala-SH (15)
H-Lys-Tyr-Thr-SH (17)
H-Glu-Tyr-Thr-SH (19)
H-Glu-Tyr-Thr-SH (19)
H-Glu-Tyr-Thr-SH (19)
H-Cys-Tyr-Ala-SH (21)
Fmoc-Phe-Ala-Thr-NHBn (16)
H-Lys-Tyr-Thr-Thr-NHBn (18)
H-Glu-Tyr-Thr-Thr-NHBn (20)
H-Glu-Tyr-Thr-Thr-NHBn (20)
H-Glu-Tyr-Thr-Thr-NHBn (20)
H-Cys-Tyr-Ala-Thr-NHBn (22)
(H-Cys-Tyr-Ala-Thr-NHBn)2 (23)
H-Lys-Tyr-Thr-Thr-Phe-Gly-NH2 (25)
H-Glu-Tyr-Thr-Thr-Phe-Gly-NH2 (26)
0.2 M Pi-citrate buffer, pH 6.9
8 M urea in 0.1 M NaPi, pH 7.5
DMF
8
9
H-Lys-Tyr-Thr-SH (17)
H-Glu-Tyr-Thr-SH (19)
H-Azy(Me)-Phe-Gly-NH2 (24)
H-Azy(Me)-Phe-Gly-NH2 (24)
DMF
8 M urea in 0.1 M NaPi, pH 7.5
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COMMUNICATION
’ AUTHOR INFORMATION
(16) MS analysis of this coupling reaction indicated predominant
formation of a disulfide corresponding to intermediate 12, which implies
that the free thiol may be undergoing an in situ protection. Reductive
disulfide cleavage likely occurs during the workup with aqueous NaSH,
which can act as a reducing agent. Minor products emanating from
perthioester intermediates were also detected. See: Liu, C. F.; Rao, C.;
Tam, J. P. Tetrahedron Lett. 1996, 37, 933–936.
Corresponding Author
ppg@wsu.edu
’ REFERENCES
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rt, 48% yield) followed by deprotection (TFA, (1:1) CHCl3-MeOH,
0 °C, 61% yield).
(18) A protocol for ligation at Thr via chemical ligation of a γ-thiol-
substituted N-terminal Thr peptide followed by postligation desulfur-
ization was recently reported. See ref 2m.
(2) Efforts to overcome this requirement include: (a) Tam, J. P.; Yu,
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(3) Danishefsky has reported the HOBt-mediated oxidative cou-
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(12) Thioacid 8 was prepared from commercially available Ac-Phe-
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(13) The structure of 14 was confirmed through comparison with an
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(14) The Fmoc protecting group was retained in this example to
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(15) Peptide thioacids 17, 19, and 21 were prepared by deprotection
(TFA, DCM, Et3SiH, 0 °C) of their STmb thioester precursors in 73, 53,
and 45% yields.
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dx.doi.org/10.1021/ja207133t |J. Am. Chem. Soc. 2011, 133, 20033–20035