Aziridine-2-carboxylic acid-containing peptides: Application to solution- and solid-phase convergent site-selective peptide modification

Article abstract of DOI:10.1021/ja050304r

The development of a method for site- and stereoselective peptide modification using aziridine-2-carboxylic acid-containing peptides is described. A solid-phase peptide synthesis methodology that allows for the rapid generation of peptides incorporating the aziridine residue has been developed. The unique electrophilic nature of this nonproteinogenic amino acid allows for site-selective conjugation with various thiol nucleophiles, such as anomeric carbohydrate thiols, farnesyl thiol, and biochemical tags, both in solution and on solid support. This strategy, combined with native chemical ligation, provides convergent and rapid access to complex thioglycoconjugates.

Full text of DOI:10.1021/ja050304r

Published on Web 05/03/2005
Aziridine-2-carboxylic Acid-Containing Peptides: Application
to Solution- and Solid-Phase Convergent Site-Selective
Peptide Modification
Danica P. Galonic´, Nathan D. Ide, Wilfred A. van der Donk,* and David Y. Gin*
Contribution from the Department of Chemistry, UniVersity of Illinois, Urbana, Illinois 61801
Received January 17, 2005; E-mail: vddonk@scs.uiuc.edu; gin@scs.uiuc.edu
Abstract: The development of a method for site- and stereoselective peptide modification using aziridine-
2-carboxylic acid-containing peptides is described. A solid-phase peptide synthesis methodology that allows
for the rapid generation of peptides incorporating the aziridine residue has been developed. The unique
electrophilic nature of this nonproteinogenic amino acid allows for site-selective conjugation with various
thiol nucleophiles, such as anomeric carbohydrate thiols, farnesyl thiol, and biochemical tags, both in solution
and on solid support. This strategy, combined with native chemical ligation, provides convergent and rapid
access to complex thioglycoconjugates.
Introduction
of these methods in the rapid preparation of various modified
peptides and proteins. To overcome these limitations, alternate
Carbohydrate and lipid moieties introduced via the post-
translational modification of proteins are key determinants of
their structure and biological activity.1 Numerous biological
processes, such as immunity and cell signaling, are controlled
by complex glycoconjugates, generated by the addition of
carbohydrate moieties to side chains of serine, threonine, and
asparagine residues.2 Lipoproteins, exemplified by the Ras
proteins, are key regulators of eukaryotic cell growth.3 As a
result of their biological and therapeutic significance, determi-
nation of the functional roles of these post-translationally
introduced functionalities has been a focus of extensive
research.1d,4 Obtaining homogeneous samples of conjugates
necessary for such studies has been a major challenge. For
example, natural glycoproteins are often expressed in many
different glycoforms;2 similarly, lipidated proteins cannot be
obtained from yeast and bacterial expression systems.1a,d
Therefore, efficient methods for the preparation of homogeneous
modified proteins and peptides are needed.
convergent ligation approaches have been developed. These
strategies rely on the late-stage coupling of preformed peptide/
protein and carbohydrate components, where chemoselectivity
results from the conjugation of carbohydrate nucleophiles with
unique electrophilic sites incorporated in peptide and protein
substrates, providing both native glycopeptides and their mi-
metics.6 Peptide electrophiles could be generated either via
modification of existing amino acid residues (e.g., Cys6i,j and
Asp6h,l), or via incorporation of non-proteinogenic amino acids
(e.g., dehydroalanine,6c,f ketones,6a,g cyclic sulfamidates,6d and
halogenated residues6m). Alternatively, chemoselectivity in
carbohydrate-peptide conjugations has been realized through
the application of a copper(I)-catalyzed [3 + 2] cycloaddition
between azide and alkyne functionalities.7 In analogy to
glycopeptides, lipopeptides have been prepared both by incor-
poration of preformed cysteine derivatives into a growing
(5) (a) Sames, D.; Chen, X.-T.; Danishefsky, S. J. Nature 1997, 389, 587-
591. (b) Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed. 2000, 39,
836-863. (c) Herzner, H.; Reipen, T.; Schultz, M.; Kunz, H. Chem. ReV.
2000, 100, 4495-4537. (d) Keil, S.; Claus, C.; Dippold, W.; Kunz, H.
Angew. Chem., Int. Ed. 2001, 40, 366-369.
Traditional synthetic approaches for the generation of ho-
mogeneous glycopeptides rely on the formation of fully
protected glycosylated amino acid building blocks and their
subsequent incorporation into peptides, mainly via solid-phase
peptide synthesis.5 These methods suffer from loss of valuable
glycosylated intermediates in each iteration of the solid-phase
synthesis. Furthermore, lack of convergence limits application
(6) (a) Marcaurelle, L. A.; Bertozzi, C. R. Chem. Eur. J. 1999, 5, 1384-1390.
(b) Hang, H. C.; Bertozzi, C. R. Acc. Chem. Res. 2001, 34, 727-736. (c)
Zhu, Y.; van der Donk, W. A. Org. Lett. 2001, 3, 1189-1192. (d) Cohen,
S. B.; Halcomb, R. L. J. Am. Chem. Soc. 2002, 124, 2534-2543. (e) Davis,
B. G. Chem. ReV. 2002, 102, 579-602. (f) Galonic´, D. P.; van der Donk,
W. A.; Gin, D. Y. Chem. Eur. J. 2003, 9, 5997-6006. (g) Liu, H.; Wang,
L.; Brock, A.; Wong, C.-H.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125,
1702-1703. (h) Miller, J. S.; Dudkin, V. Y.; Lyon, G. J.; Muir, T. W.;
Danishefsky, S. J. Angew. Chem., Int. Ed. 2003, 42, 431-434. (i) Watt,
G. M.; Lund, J.; Levens, M.; Kolli, V. S. K.; Jefferis, R.; Boons, G.-J.
Chem. Biol. 2003, 10, 807-814. (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. (k) Peri, F.; Nicotra, F. Chem. Commun. 2004,
623-627. (l) Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J.
J. Am. Chem. Soc. 2004, 126, 6576-6578. (m) Zhu, X.; Schmidt, R. R.
Chem. Eur. J. 2004, 10, 875-887.
(1) (a) Kadereit, D.; Kuhlmann, J.; Waldmann, H. ChemBioChem. 2000, 1,
144-169. (b) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357-
2364. (c) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R.
A. Science 2001, 291, 2370-2376. (d) Seitz, O.; Heinemann, I.; Mattes,
A.; Waldmann, H. Tetrahedron 2001, 57, 2247-2277.
(2) (a) Seitz, O. ChemBioChem. 2000, 1, 214-246. (b) Grogan, M. J.; Pratt,
M. R.; Marcaurelle, L. A.; Bertozzi, C. R. Annu. ReV. Biochem. 2002, 71,
593-634.
(3) Vojtek, A. B.; Der, C. J. J. Biol. Chem. 1998, 273, 19925-19928.
(4) (a) Meldal, M.; St Hilaire, P. M. Curr. Opin. Chem. Biol. 1997, 1, 552-
563. (b) Slovin, S. F.; et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5710-
5715. (c) Wong, C.-H. Acc. Chem. Res. 1999, 32, 376-385.
(7) (a) Kuijpers, B. H. M.; Groothuys, S.; Keereweer, A. B. R.; Quaedflieg, P.
J. L. M.; Blaauw, R. H.; van Delft, F. L.; Rutjes, F. P. J. T. Org. Lett.
2004, 6, 3123-3126. (b) Lin, H.; Walsh, C. T. J. Am. Chem. Soc. 2004,
126, 13998-14003.
9
10.1021/ja050304r CCC: $30.25 © 2005 American Chemical Society
J. AM. CHEM. SOC. 2005, 127, 7359-7369
7359

A R T I C L E S
Scheme 1
Galonic et al.
Scheme 2 a
peptide chain8 and by site-selective alkylation of this nucleo-
philic residue with farnesyl halides as electrophiles.9
The electrophilic aziridine functionality represents a valuable
synthetic building block due to its ability to undergo ring-
opening reactions with a wide range of nucleophiles.10 This
reactivity profile of the aziridine moiety has prompted the
preparation of aziridine-2-carboxylic acid (Azy)11 as well as
short peptides incorporating this subunit.12 However, these
pioneering procedures suffer from low yields of the desired Azy-
containing peptides, as well as the incompatibility of the required
protecting groups with established solid-phase peptide synthesis
protocols. Furthermore, to date only di- and tripeptides incor-
porating the Azy moiety have been prepared. Clearly, a more
versatile and rapid route to aziridine-2-carboxylic acid containing
peptides would be valuable, given their potential to serve as
electrophiles in peptide conjugations with appropriate nucleo-
philes. Indeed, nucleophilic ring-opening of the Azy moiety with
simple thiol nucleophiles has been carried out in the presence
of a catalytic amount of boron trifluoride etherate13 or stoichio-
metric amounts of thiobenzoic acid.14 In the context of longer
peptides, these acidic promoters are likely to be inefficient given
the presence of multiple Lewis basic sites, and thus, these
methods are not applicable to the conjugation of elaborate Azy-
containing peptides.
Herein, we report details of our efforts to develop a new
approach to site- and stereoselective peptide modification using
aziridine-2-carboxylic acid-containing peptides 1 (Scheme 1).15
It was anticipated that, in the presence of a suitable catalyst,
thiol addition to the less sterically hindered C3-carbon of the
aziridine moiety would provide the desired cysteine adduct 2.
This approach allows for the late-stage coupling of preformed
aziridine-2-carboxylic acid-containing peptides with complex
thiol moieties such as lipids, carbohydrates, and biochemical
tags.
i
a Reagents and conditions: (a) BOP, Pr2NEt, CHCl3 (92%); (b) MsCl,
Et3N, CH2Cl2; (c) Et3N, THF (91% over two steps); (d) TFA, MeOH, CHCl3
(66% for 6); (e) BocAAOH, BOP, iPr2NEt, CHCl3 (88% for 7, 76% for 8,
81% for 9, 89% for 10); (f) BnNH2, EDC‚HCl, HOBt, CH2Cl2 (96%); (g)
i
Et3N, DME (79% for 12 over two steps); (h) BocGlyOH, BOP, Pr2NEt,
CHCl3 (86% over two steps); (i) BnBr, THF (80%); (j) H2, Pd/BaSO4, THF
(97% over three steps); (k) HAlaNHMe, BOP, Pr2NEt, CHCl3 (77%); (l)
BocAlaOH, BOP, Pr2NEt, CHCl3 (99%).
i
i
tion of short aziridine-containing peptides for exploratory
investigations of thiol conjugation followed a strategy based
on the early work of Okawa and Nakajima.12b Tripeptides
containing a central Azy moiety were prepared by initial
coupling of N-trityl-protected serine 3 with alanine benzyl ester
4 (Scheme 2A). The resulting dipeptide 5 was reacted with
methanesulfonyl chloride and subsequently treated with triethyl-
amine in THF to afford the corresponding N-tritylaziridine-
containing dipeptide. Trityl removal followed by acylation of
the free aziridine in 6 with various Boc-protected amino acids
led to the generation of tripeptides 7-10 that possess a central
aziridine moiety.
Similarly, two 3-MeAzy-containing peptides were prepared.
3-MeAzy-containing dipeptide 13 was synthesized starting from
N-tritylthreonine 11 (Scheme 2B). Following the initial protec-
tion of the carboxylic acid moiety in the form of a benzylamide,
formation of trityl-3-MeAzy was achieved via a two-step
sequence, consisting of conversion of the alcohol to mesylate
and closure of the aziridine ring with triethylamine to generate
trityl-protected aziridine 12. Trityl deprotection and acylation
with BocGlyOH allowed for the generation of 3-MeAzy-
containing dipeptide 13. For the synthesis of tripeptide 15
possessing a central 3-MeAzy moiety, Trt-3-MeAzyOH mono-
mer 14, prepared from N-tritylthreonine 11 in four steps,12c,16
was coupled with alanine N-methylamide (Scheme 2B). Re-
moval of the trityl group and subsequent acylation with
BocAlaOH provided the desired BocAla-3-MeAzy-AlaNHMe
tripeptide 15 in good overall yield.
Results and Discussion
Synthesis of Aziridine-2-carboxylic Acid-Containing Pep-
tides and Conjugation with Thiol Nucleophiles. The prepara-
(8) (a) Durek, T.; Alexandrov, K.; Goody, R. S.; Hildebrand, A.; Heinemann,
I.; Waldmann, H. J. Am. Chem. Soc. 2004, 126, 16368-16378. (b) Kragol,
G.; Lumbierres, M.; Palomo, J. M.; Waldmann, H. Angew. Chem., Int. Ed.
2004, 43, 5839-5842.
(9) (a) Ghomashchi, F.; Zhang, X.; Liu, L.; Gelb, M. H. Biochemistry 1995,
34, 11910-11918. (b) Koppitz, M.; Spellig, T.; Kahmann, R.; Kessler, H.
Int. J. Pept. Protein Res. 1996, 48, 377-390.
(10) (a) Tanner, D. Angew. Chem., Int. Ed. Engl. 1994, 33, 599-619. (b)
Sweeney, J. B. Chem. Soc. ReV. 2002, 31, 247-258. (c) Hu, X. E.
Tetrahedron 2004, 60, 2701-2743.
(11) Full description of each abbreviated term is given in the Supporting
Information.
(12) (a) Nakajima, K.; Tanaka, T.; Morita, K.; Okawa, K. Bull. Chem. Soc.
Jpn. 1980, 53, 283-284. (b) Okawa, K.; Nakajima, K. Biopolymers 1981,
20, 1811-1821. (c) Korn, A.; Rudolph-Bo¨hner, S.; Moroder, L. Tetrahedron
1994, 50, 1717-1730.
(13) (a) Kuyl-Yeheskiely, E.; Dreef-Tromp, C. M.; van der Marel, G. A.; van
Boom, J. H. Recl. TraV. Chim. Pays-Bas 1989, 108, 314-316. (b) Nakajima,
K.; Oda, H.; Okawa, K. Bull. Chem. Soc. Jpn. 1983, 56, 520-522. (c)
Kogami, Y.; Okawa, K. Bull. Chem. Soc. Jpn. 1987, 60, 2963-2965. (d)
Wipf, P.; Uto, Y. J. Org. Chem. 2000, 65, 1037-1049.
(15) For initial report, see Galonic´, D. P.; van der Donk, W. A.; Gin, D. Y. J.
Am. Chem. Soc. 2004, 126, 12712-12713.
(16) Tanaka, T.; Nakajima, K.; Okawa, K. Bull. Chem. Soc. Jpn. 1980, 53,
1352-1355.
(14) Wakamiya, T.; Shimbo, K.; Shiba, T.; Nakajima, K.; Neya, M.; Okawa,
K. Bull. Chem. Soc. Jpn. 1982, 55, 3878-3881.
9
7360 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005

Aziridine-2-carboxylic Acid-Containing Peptides
A R T I C L E S
Table 1. Conjugation of R-SH-GalNAc with Azy Tripeptide
of the ligation. Despite the high yield observed in the conjuga-
tion reaction with n-Bu3P as the catalyst, its oxidative instability
makes it a nonideal catalyst for this transformation.19 The initial
mechanistic rationale by Hou et al.7a as to the role of the
phosphine in the ring opening of N-tosyl aziridines 19 is that
of the formation of a phosphonium intermediate 20 (eq 1). This
entry
catalyst
solvent (time)
yield, % (17:18)
1
2
3
4
5
6
7
8
9
PBu3
PBu3
Et2PCH2CH2PEt2
Me2PhP
Et3N
CH3CN (20 h)
CH3CN (4 h)
CH3CN (8 h)
CH3CN (33 h)
CH3CN (24 h)
CH3CN (19 h)
CH3CN (4 h)
DMF (10 h)
81 (4.2:1.0)
78 (4.5:1.0)
68 (4.3:1.0)
44 (3.7:1.0)
66 (3.7:1.0)
37 (3.3:1.0)
78 (4.5:1.0)
80 (3.1:1.0)
79 (4.6:1.0)
83 (6.3:1.0)
a
intermediate serves as base initiator that leads to the generation
of thiolate, capable of aziridine thiolysis. If the phosphine simply
initiated a base-catalyzed process, it was anticipated that a
similar outcome could be obtained with tertiary amine catalysts.
To test this hypothesis, the conjugation reaction was performed
in the presence of a substoichiometric amount of triethylamine
(entry 5),20 which did promote the transformation, albeit only
in moderate yield and with diminished regioselectivity. Further
screening of tertiary amine bases revealed that DABCO (entry
6) is not an efficient catalyst in this reaction, as significant
decomposition of the carbohydrate coupling partner was ob-
served and the conjugated products were isolated in a low 37%
yield. However, an increase in both yield and selectivity was
obtained with DBU. In the presence of 0.1 equiv of DBU in
acetonitrile, conjugated products were obtained in 78% yield,
favoring the desired cysteine conjugate over the undesired
â-homoglycine adduct in a ratio of 4.5:1, in a kinetically
controlled ligation process (entry 7).21 Variations in solvent
revealed that chloroform provides the optimal medium for this
transformation (entry 10), affording conjugated products in a
combined yield of 83%, further favoring the cysteine adduct
(ratio 6.3:1.0). In all of these cases, ligation proceeds with
complete retention of the R-anomeric configuration of the
carbohydrate nucleophile, and the (R) configuration at the
R-carbon of the newly formed L-cysteine derivative is secured.
Efficient regio- and stereoselective conjugation of R-thioGalNAc
16 with aziridine-containing peptide 7 demonstrates the ap-
plicability of this conjugation approach to the generation of thio-
linked glycopeptides. The importance of these thioglycopeptides
is highlighted by reports of their enhanced stability toward both
chemical and enzymatic degradation, as compared with their
O-linked counterparts.22
DABCO
DBU
DBU
DBU
DBU
DME (3 h)
CHCl3 (11 h)
10
a Reaction performed in the presence of 1 equiv of catalyst.
To evaluate nonacidic conditions for the conjugation of
aziridine-2-carboxylic acid-containing peptides with thiols with
an initial focus on the generation of glycopeptides, various
catalysts were screened for the coupling of the aziridine-
containing tripeptide BocAlaAzyAlaOBn (7) with the C1 thio
analogue of the tumor-associated TN antigen (16) (Table 1). It
was anticipated that the aziridine moiety positioned between
two amino acid residues in 7 would be a good mimic of the
Azy functionality within longer peptides. Furthermore, the
GalNAc-derived C1 R-thiol 16 is a core carbohydrate unit found
in various R-O-linked, mucin-related glycopeptides.2 To allow
for the generation of thioisosteres of these glycopeptides, it was
critical not only to find appropriate conjugation conditions
suitable for the addition of carbohydrate-thiol nucleophiles to
the less sterically hindered C3 carbon of the Azy moiety but
also to assess the stereochemical consequences at the anomeric
position of the carbohydrate coupling partner.
On the basis of reports by Hou and co-workers17 that
phosphines are efficient catalysts in the reaction of aziridines
derived from cyclohexene and styrene with simple thiol nu-
cleophiles (thiophenol, tert-butyl thiol, and p-methylbenzylthiol),
the thio-TN antigen 16 was reacted with the tripeptide 7 in the
presence of n-Bu3P (0.1 equiv; Table 1, entry 1) in acetonitrile
at 23 °C. The corresponding carbohydrate-peptide conjugates
were obtained in good yield, favoring adduct 17 (resulting from
the addition of thiol to the C3 position of the Azy moiety, Cys-
like adduct) over its constitutional isomer 18 (resulting from
the thiol adding to the C2 position, â2-HGly-like adduct).18
While addition of a stoichiometric amount of n-Bu3P resulted
in a faster reaction (entry 2), the ligated products were obtained
in similar yield and selectivity. Use of bisphosphines, such as
1,2-bis(diethylphosphino)ethane (entry 3), did not offer any
advantage in the conjugation reaction, as the products were
obtained in diminished yield. Finally, use of Me2PhP (entry 4)
led to a marked decrease in isolated yield, demonstrating that
introduction of an electron-withdrawing substituent, even in the
form of a single aryl group, significantly decreases the efficiency
The ligation protocol employing DBU as a catalyst was
applied to the conjugation of different thiol nucleophiles and
Azy-containing peptides (Table 2). Both ethanethiol and the
more sterically demanding tert-butyl thiol reacted efficiently
with BocAlaAzyAlaOBn (7) in the presence of DBU (0.1 equiv)
at 23 °C to afford tripeptide-thiol conjugates in high yield,
strongly favoring the product resulting from the addition of thiol
to the C3 position of the Azy moiety (Table 2, entries 1 and 2).
(19) Buckler, S. A. J. Am. Chem. Soc. 1962, 84, 3093-3097.
(20) Et3N (0.2 equiv) was previously used as a catalyst in aminolysis of N-Ns-
Azy-OtBu with benzylamine: Turner, J. J.; Sikkema, F. D.; Filippov, D.
V.; van der Marel, G. A.; van Boom, J. H. Synlett 2001, 1727-1730.
(21) The major product remained unchanged after resubjection to the reaction
conditions.
(17) (a) Hou, X.-L.; Fan, R.-H.; Dai, L.-X. J. Org. Chem. 2002, 67, 5295-
5300. (b) Fan, R.-H.; Hou, X.-L. J. Org. Chem. 2003, 68, 726-730.
(18) The minor â2 amino acid adduct was obtained as a single diastereomer,
which is presumably formed via invertive displacement at the aziridine
R-carbon.
(22) (a) Baran, E.; Drabarek, S. Pol. J. Chem. 1978, 52, 941-946. (b) Gerz,
M.; Matter, H.; Kessler, H. Angew. Chem., Int. Ed. Engl. 1993, 32, 269-
271. (c) Driguez, H. ChemBioChem 2001, 2, 311-318. (d) Jahn, M.; Marles,
J.; Warren, R. A. J.; Withers, S. G. Angew. Chem., Int. Ed. 2003, 42, 352-
354.
9
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A R T I C L E S
Galonic et al.
Table 2. Conjugation of Thiols with Aziridine-containing Peptides
reactivity toward thiol nucleophiles compared with Azy-
containing peptides, likely a result of the increased steric bulk
of the methyl substituent.
While these initial experiments established the feasibility of
base-catalyzed Azy thiolysis, further investigations into the
conjugation of thiol nucleophiles with more complex aziridine-
containing peptides were impeded by the lack of an efficient
method to prepare more elaborate peptide electrophiles. Present
solution-phase synthesis of Azy-containing peptides requires
introduction and multistep elaboration of TrtSerOH prior to
further peptide chain extension. Clearly, an improved method
was needed for the rapid generation of diverse Azy-containing
peptide electrophiles.
Solid-Phase Synthesis of Azy-Containing Peptides. Given
the encouraging results for the conjugation of short Azy-
containing peptides with thiol nucleophiles, extension of the
scope of this methodology to more diverse peptides was
investigated. An Fmoc-based solid-phase peptide synthesis
(SPPS) strategy was adopted for the preparation of longer Azy-
containing peptides, on the basis of the observation that the
acylated Azy moiety is highly unstable in the presence of acids,
thereby precluding the possibility of Boc-based SPPS.12b The
acid sensitivity of the Azy residue was also considered when
choosing the appropriate linker for the solid-phase synthesis.
For this purpose, polystyrene-attached 2-chlorotrityl linker was
selected as it can be cleaved under mildly acidic conditions (20%
hexafluoro-2-propanol in dichloromethane)25 found to be com-
patible with the acylated Azy moiety. This allowed for the
cleavage of aziridine-peptides from the resin and assessment
of coupling efficiencies during the course of SPPS. In addition
to the acid instability of the Azy moiety, this subunit is also
labile to standard basic Fmoc deprotection conditions (20%
piperidine in DMF), which were found to affect both aziridine
ring opening and deacylation. To avoid these side reactions,
1% DBU in DMF was used for Fmoc deprotection during the
iterative cycles of solid-phase synthesis.26
In accordance with these parameters, solid-phase synthesis
was carried out starting with 2-chlorotrityl polystyrene resin
preloaded with glycine (21), to which FmocPheOH was coupled
by use of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) as the activating agent and
N-methylmorpholine (NMM) as a base in DMF (Scheme 3).
After removal of Fmoc with 1% DBU in DMF, FmocAlaOH
was incorporated, followed successively by FmocAzyOH, and
FmocGlyOH to form pentapeptide 22 with high efficiency.
However, attempts to extend this Azy-containing peptide by
the coupling of FmocGlyOH at the [Azy + 2] site, followed
by Fmoc removal and attempted coupling of the AcGlyOH at
the [Azy + 3] position, were unsuccessful. Upon removal of
the resin, pentapeptide AcGlyAzyAlaPheGlyOH (25) was
obtained as the major product. Its formation could be rational-
ized by the intramolecular N-deacylation of the Azy moiety by
the amine liberated upon removal of Fmoc from the amino acid
in the [Azy + 2] position, followed by the acylation of the
resulting free aziridine 24 with AcGlyOH (Scheme 3). This
problem was particularly significant in those peptides where
an amino acid with a sterically undemanding side chain was
entry
RSH
peptide
solvent (time)
yield, % (C3:C2 adduct)
1
2
3
4
5
6
7
8
9
EtSH
tBuSH
16
16
16
EtSH
EtSH
16
7
7
8
CH3CN (4 h)
CH3CN (8 h)
CHCl3 (3 h)
CHCl3 (46 h)
CHCl3 (19 h)
CHCl3 (2 h)
CHCl3 (15 h)
CHCl3 (18 h)
CHCl3 (15 h)
85 (20:1)a
84 (C3 only)a
93 (5.3:1)a
9
89 (13:1)a
10
13
15
13
15
90 (16:1)a
36 (C3 only)b
61 (C3 only)c
65 (C3 only)b
43 (C3 only)b
16
a Reaction performed in the presence of 0.1 equiv of DBU. b Reaction
performed in the presence of 1 equiv of DBU. c Reaction performed in the
presence of 0.5 equiv of DBU.
Similarly, thio-TN antigen 16 was conjugated with the Azy-
containing peptides 8-10 in high yields (Table 2, entries 3-5).
In all cases, the constitutional isomer resulting from the addition
of the thiol to the less sterically hindered C3 carbon of the
aziridine was strongly favored.15
Encouraged by these results, we explored an extension of
this ligation methodology to threonine-derived methylaziridine-
carboxylic acid- (3-MeAzy-) containing peptides. Reaction of
these peptide electrophiles with carbohydrate thiol nucleophiles
would allow for the generation of thio analogues of threonine
glycosylation sites found in numerous mucin glycopeptides.2a
To test the possibility of conjugation of 3-MeAzy-containing
peptides with thiols, ethanethiol was reacted with BocGly-3-
MeAzyNHBn dipeptide (13) in the presence of a stoichiometric
amount of DBU (Table 2, entry 6).23 The conjugate resulting
from the addition of thiol to the C3 position of the 3-MeAzy
moiety was obtained in a low 36% yield, although no product
resulting from the addition of thiol to the C2 position of the
aziridine was observed. The major byproduct in this transforma-
tion, H-3-MeAzyNHBn, is a result of deacylation of the aziridine
via addition of thiolate to the acyl moiety of glycine.24 It was
presumed that this side reaction could be suppressed if a peptide
substrate with a more sterically demanding acyl substituent on
the aziridine nitrogen were used. Indeed, when tripeptide 15,
incorporating Ala instead of Gly on the aziridine group, was
reacted with ethanethiol in the presence of DBU (0.5 equiv,
entry 7), the desired conjugation product was isolated in 61%
yield. Reactions of the C1-thio analogue of TN antigen (16) with
3-MeAzy-containing peptides were also investigated (entries 8
and 9). In the presence of DBU (1 equiv), the dipeptide
glycoconjugate was obtained in 65% yield while the tripeptide-
carbohydrate adduct was prepared in a modest 43% yield. In
both instances, the peptide-carbohydrate coupling proceeded
with retention of the R-anomeric configuration of the carbo-
hydrate coupling partner, generating thio analogues of the
threonine-derived glycosylation sites as a single diastereoisomer
and constitutional isomer. Under these conjugation conditions,
the 3-MeAzy peptide electrophiles have significantly attenuated
(23) Low conversion to the desired product was observed after prolonged reaction
time when 0.1 equiv of DBU was used in this reaction.
(24) A similar mode of reactivity of acylated aziridines was previously observed
by Okawa in the reaction of acylated Azy-containing peptides with primary
amines (ref 12a).
(25) Bollhagen, R.; Schmiedberger, M.; Barlos, K.; Grell, E. J. Chem. Soc.,
Chem. Commun. 1994, 2559-2560.
(26) Wade, J. D.; Bedford, J.; Sheppard, R. C.; Tregear, G. W. Pept. Res. 1991,
4, 194-199.
9
7362 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005

Aziridine-2-carboxylic Acid-Containing Peptides
A R T I C L E S
Scheme 3 a
acid, serine, and lysine (29, 32, 33). Analytical samples of all
of the prepared peptides were obtained via cleavage from the
resin with hexafluoro-2-propanol in dichloromethane (20% v/v).
1H NMR and ESI-MS analysis showed the crude peptides to
be >80% pure.
Thiol Ligation on Solid Support. Successful incorporation
of the Azy moiety via SPPS was followed by investigation of
the ability of these compounds to undergo site-selective
conjugation with various thiols. The polymer-supported peptides
(Table 3) were treated with an excess of thiol (5-10 equiv) in
the presence of DBU (1 equiv) in DMF at 23 °C. Following
the ligation, detachment from the resin was accomplished with
either hexafluoro-2-propanol or TFA, which effected the
concomitant removal of the acid-labile amino acid side-chain
protecting groups. Each of the peptide conjugates was purified
by preparative RP-HPLC to afford a net yield of product as
calculated over the multistep sequence commencing with the
single amino acid preloaded resin.
Initial couplings were performed with simple alkanethiols.
Ethanethiol as well as the sterically demanding tert-butyl thiol
was efficiently conjugated with polymer-supported pentapeptides
27 and 29 to provide conjugates 38-40 (Table 3, entries 1-3),
each in a nine-step sequence starting with alanine- or aspartic
acid-preloaded resins 26 and 28. High yields and regio-
selectivities in these transformations established the feasibility
of the ligation on solid support as the average yield for these
couplings was at least 89% per step. Incorporation of more
complex alkanethiols included a biotin tag, commonly used to
probe peptide-receptor/antibody interactions.28 Thus aziridine-
containing pentapeptide 27 (entry 4), as well as the more
complex heptapeptide 33 (entry 5) were conjugated with the
biotin thiol 34 to provide the desired cysteine adducts in good
yields (94% and 89% per step, respectively, over 9- and 13-
step sequences, starting from 26). Moreover, the allylic thiol
moiety of farnesyl thiol 35 was also efficiently coupled with
the aziridine-containing peptides 27 and 31, affording lipopep-
tides 43 and 44 with an average yield of at least 90% per step,
starting with the single amino acid preloaded resin (entries 6
and 7). The importance of a convergent synthetic strategy for
the preparation of these peptides is highlighted by the involve-
ment of farnesylated proteins in gene expression, apoptosis, and
remodeling of the actin cytoskeleton.29 Compared to the
frequently used cysteine alkylation protocol with farnesyl
halides, this late-stage convergent coupling between the Azy
moiety and farnesyl thiol, in which the respective roles of
nucleophile and electrophile are reversed, surmounts the com-
monly encountered problems of cysteine oxidation as well as
overalkylation of peptide substrates.1a,d
a Reagents and conditions: (a) FmocPheOH, HBTU, NMM, DMF; (b)
DBU, DMF; (c) FmocAlaOH, HBTU, NMM, DMF; (d) FmocAzyOH,
HBTU, NMM, DMF; (e) FmocGlyOH, HBTU, NMM, DMF; (f) AcGlyOH,
HBTU, NMM, DMF; (g) (CF3)2CHOH:CH2Cl2 (20% v/v).
Scheme 4 a
a Reagents and conditions: (a) FmocAzyOH, HBTU, NMM, DMF; (b)
DBU, DMF; (c) FmocPheOH, HBTU, NMM, DMF; (d) FmocGlyGlyOH,
HBTU, NMM, DMF; (e) Ac2O, Pyr; (f) FmocValOH, HBTU, NMM, DMF;
(g) FmocPheGlyOH, HBTU, NMM, DMF; (h) FmocAlaOH, HBTU, NMM,
DMF; (i) FmocLys(Boc)OH, HBTU, NMM, DMF; (j) FmocGlu(tBu)-
GlyOH, HBTU, NMM, DMF; (k) FmocSer(tBu)OH, HBTU, NMM, DMF.
incorporated in the [Azy + 1] position. Indeed, the susceptibility
of aziridinyl amides toward deacylation is reinforced by an
elongated C(O)-N bond compared with non-aziridine-derived
amides due to reduced amide-imidate resonance.27 To circum-
vent this problem, a strategy involving the simultaneous
introduction of a dipeptide unit incorporating both the [Azy +
2] and [Azy + 3] residues was envisioned. Intramolecular
deacylation of the aziridine functionality would be disfavored
as this route would avoid a six-membered transition state.
Indeed, successful extensions of the Azy-containing peptide
backbone were achieved with this strategy (Scheme 4). Through
multiple applications of this solid-phase synthesis protocol, a
series of Azy-containing peptides were rapidly prepared, includ-
ing those possessing functionalized residues such as glutamic
The solid-supported aziridine ligation protocol is also useful
for the preparation of thioglycopeptides. The thio-TN antigen
16 was reacted with polymer-supported pentapeptides 27 and
29 to give, after release of thioglycopeptide from the resin,
acetate deprotection, and RP-HPLC purification, the thioglyco-
peptides 45 and 46 in 21% and 17% yield, respectively, each
over a 10-step sequence (entries 8 and 9, Table 3). Similar
efficiency was observed when heptapeptide 33 (entry 10) was
(28) Fu, P.; Layfield, S.; Ferraro, T.; Tomiyama, H.; Hutson, J.; Otvos Jr., L.;
Tregear, G. W.; Bathgate, R. A. D.; Wade, J. D. J. Peptide Res. 2004, 63,
91-98.
(29) (a) Barbacid, M. Annu. ReV. Biochem. 1987, 56, 779-827. (b) Bos, J. L.
Cancer Res. 1989, 49, 4682-4689.
(27) Shao, H.; Jiang, X.; Gantzel, P.; Goodman, M. Chem. Biol. 1994, 1, 231-
234.
9
J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005 7363

A R T I C L E S
Galonic et al.
Table 3. On-Bead Conjugation of Azy Peptides with Thiols
a DBU, DMF, 23 °C. b DBU, CHCl3, DMF, 23 °C. c DBU, DMF, 60 °C. d (CF3)2CHOH/CH2Cl2 (20% v/v). e TFA/CH2Cl2/iPr3SiH/HS(CH2)2SH, 10:10:
1:1. f NaOMe, MeOH, pH 8.5.
reacted with thiol 16 to provide glycoconjugate 47 (19% yield
over 14 steps, average of 88% per step).30 In addition, the thio
analogue of the mucin-related STN antigen 36, which incor-
porates a ketal glycosidic linkage between sialic acid and
galactosamine as well as a free carboxylic acid functionality,
reacted smoothly with the pentapeptide 27, affording the
disaccharide-pentapeptide conjugate 48 in 43% yield over a 10-
step sequence, an efficiency of 92% per step (entry 11). The
complete retention of the R-anomeric configuration in the
coupling of the thio-TN and STN carbohydrates punctuates the
potential of this approach to prepare and study glycosylated
S-mucin peptide fragments, especially in light of reports
highlighting the critical role of the core GalNAc R-anomeric
stereochemistry in properly organizing the peptide backbone
of the mucin glycopeptide motif.31 Finally, the anomeric thiol
of unprotected carbohydrates, exemplified by the â-thio-
glucopyranose 37, is also amenable to this conjugation approach.
Azy-containing pentapeptides 27 and 32, when subjected to
conjugation with this nucleophile, provided the desired R-amino
acid adducts in 32% and 33% yields, each over a 9-step
(31) (a) Liang, R.; Andreotti, A. H.; Kahne, D. J. Am. Chem. Soc. 1995, 117,
10395-10396. (b) Live, D. H.; Williams, L. J.; Kuduk, S. D.; Schwarz, J.
B.; Glunz, P. W.; Chen, X.-T.; Sames, D.; Kumar, R. A.; Danishefsky, S.
J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3489-3493. (c) Schuman, J.;
Qiu, D.; Koganty, R. R.; Longenecker, B. M.; Campbell, A. P. Glyco-
conjugate J. 2000, 17, 835-848. (d) Coltart, D. M.; Royyuru, A. K.;
Williams, L. J.; Glunz, P. W.; Sames, D.; Kuduk, S. D.; Schwarz, J. B.;
Chen, X.-T.; Danishefsky, S. J.; Live, D. H. J. Am. Chem. Soc. 2002, 124,
9833-9844. (e) Schuman, J.; Campbell, A. P.; Koganty, R. R.; Longe-
necker, B. M. J. Pept. Res. 2003, 61, 91-108.
(30) Reactions in entries 8-10 were carried out by heating the reaction mixture
to 60 °C to allow for faster reaction. A decrease in the yields of the isolated
products in these reactions may be attributed to the thermal instability of
the 2-chlorotrityl linker.
9
7364 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005

Aziridine-2-carboxylic Acid-Containing Peptides
A R T I C L E S
Table 4. On-Bead Conjugation of Azy Peptides with Thiols
appreciably different. While the reaction of EtSH (10 equiv)
with the polymer-supported AcValAzyAlaAlaGly pentapeptide
(51) (entry 1) reached completion in less than 24 h, reaction of
pentapeptide AcValAzyAlaProGly (53) with EtSH achieves only
75% conversion after 6 days (entry 5) under the same condi-
tions.35 A similar trend in ligation regioselectivity and reaction
rate was also evident in the conjugation of carbohydrate-derived
thiol 16 with the resin-attached Pro-containing peptide
AcValAzyAlaProGly (53) (entry 6, ∼60% conversion, 6 days;
C3:C2 adduct ) 3.7:1.0).36 These data imply that the nature of
the amino acid in the [Azy - 2] position may be a critical
determinant of the regioselectivity in the aziridine ring-opening
process. One obvious difference between Ala- and Pro- contain-
ing peptides is the absence of an NH group in the [Azy - 2]
position in the latter. Indeed, reaction of ethanethiol with the
polymer-supported pentapeptide AcValAzyAlaSarGly (54) (en-
try 7), which also possesses a tertiary amide group in the [Azy
- 2] position (Sar ) sarcosine ) N-methylglycine), provided
a mixture of conjugated products favoring the C3 adduct (C3:
C2 adduct ) 3.7:1.0), an outcome similar to that of the otherwise
identical proline-containing peptides. The potential significance
of the [Azy - 2] NH group in enhancing rate and facilitating
addition of nucleophile to the C2 position may be attributed to
an inductive activation of the R-carbon of the Azy residue
resulting from the involvement of its carbonyl moiety in a
hydrogen bond with the amide proton of the [Azy - 2] amino
acid in a γ-turn-like conformation.37 Alternatively, similar
carbonyl activation via interchain hydrogen bonding38 could
arise from high resin loadings.39 However, direct evidence for
these hypotheses has yet to be secured, as no significant change
in regioselectivity was observed when ligations were performed
in the presence of polar additives such as DMSO and ethylene-
carbonate.40 Alternatively, given that insertion of Pro and Sar
into peptides is known to inhibit peptide aggregation,41 the
regiochemical outcomes in Table 4 may arise from more subtle
conformational effects caused by the [Azy - 2] residue. Studies
are underway to further explore these intriguing aspects of the
reactivity of various Azy-containing peptide substrates, as well
as the influence that this residue has on the conformational
properties of the resulting peptides.
a (CF3)2CHOH/CH2Cl2 (20% v/v). b NaOMe, MeOH, pH 8.5.
sequence starting from 26 and 21, respectively (entries 12 and
13). During the course of the ligation, the â-anomeric config-
uration of the carbohydrate thiol remained unchanged. High
levels of chemoselectivity for the addition of thiol to the Azy
moiety in these transformations eliminate the need for protection
of carbohydrate peripheral hydroxyl functionalities. These
examples demonstrate not only the high efficiency of the Azy
incorporation during the course of SPPS but also the tolerance
of this residue to multiple rounds of amino acid couplings and
Fmoc deprotections during the course of SPPS.
Regioselectivity of Azy Opening as a Function of Peptide
Structure. During the course of investigation of on-bead ligation
with additional peptide substrates in which the Azy moiety was
further removed from the solid support, variations in regio-
selectivity were observed (Table 4). When ethanethiol was
reacted with resin-supported AcValAzyAlaAlaGly pentapeptide
51 (Table 4, entry 1), the ethylthio-â2-homoglycine-containing
peptide, resulting from thiol addition to C2, was observed as
the major product (C3:C2 adduct ) 1.0:5.6).32 Comparable
levels of regioselectivity favoring thiol addition to the more
sterically hindered C2 carbon of the aziridine moiety were
observed in the reaction of ethanethiol and farnesyl thiol (35)
with the resin-bound AcValAzySer(tBu)AlaGly (52) and
AcValAzyAlaAlaGly (51) pentapeptides as well (entries 2 and
3).33 A similar, although less marked, effect was observed in
the conjugation of peptide 52 with TN antigen 16, in which two
constitutional isomers were generated in the ratio 1.0:1.4, slightly
favoring the product resulting from thiol addition to the C2
carbon of the aziridine (entry 4).34 However, when ethanethiol
was reacted with AcValAzyAlaProGly pentapeptide 53 (entry
5), which differs from 51 only in the replacement of Ala with
Pro in the [Azy - 2] position, the C3 adduct was obtained as
the major product (C3:C2 adduct ) 5.8:1.0). Interestingly, the
reaction rates for these two ligations (entries 1 vs 5) were also
C-Terminal Extension of Glycopeptides via Native Chemi-
cal Ligation. Given the observed variations in regioselectivity
of aziridine opening in peptides with multiple residues C-
(35) A substantial decrease in the ligation rate for the peptides possessing N-alkyl
amino acids in the [Azy-2] position was observed for all of the tested
peptides.
(36) While the expected glycosylated cysteine adduct was the major component
of the crude mixture, two other minor carbohydrate-containing products
were observed after cleavage from the solid support. Upon removal of
O-acetates with sodium methoxide in methanol, a C3:C2 adduct ratio of
3.7:1.0 was obtained (HPLC-ESI).
(37) (a) Burgess, K.; Ho, K.-K.; Pettitt, B. M. J. Am. Chem. Soc. 1994, 116,
799-800. (b) Burgess, K.; Li, W.; Lim, D.; Moye-Sherman, D. Biopolymers
1997, 42, 439-453. (c) Vass, E.; Hollo´si, M.; Besson, F.; Buchet, R. Chem.
ReV. 2003, 103, 1917-1954. (d) Jime´nez, A. I.; Ballano, G.; Cativiela, C.
Angew. Chem., Int. Ed. 2005, 44, 396-399.
(38) Narita, M.; Tomotake, Y.; Isokawa, S.; Matsuzawa, T.; Miyauchi, T.
Macromolecules 1984, 17, 1903-1906.
(39) Peptides 51 and 52 were prepared starting from glycine-preloaded 2-chloro-
trityl polystyrene resin 21 with a loading of 0.78 mmol/g.
(40) Hyde, C.; Johnson, T.; Sheppard, R. C. J. Chem. Soc., Chem. Commun.
1992, 1573-1575.
(41) (a) Toniolo, C.; Bonora, G. M.; Mutter, M.; Pillai, V. N. R. Makromol.
Chem. 1981, 182, 2007-2014. (b) Rijkers, D. T. S.; Ho¨ppener, J. W. M.;
Posthuma, G.; Lips, C. J. M.; Liskamp, R. M. J. Chem. Eur. J. 2002, 8,
4285-4291. (c) Wu, C. W.; Kirshenbaum, K.; Sanborn, T. J.; Patch, J. A.;
Huang, K.; Dill, K. A.; Zuckermann, R. N.; Barron, A. E. J. Am. Chem.
Soc. 2003, 125, 13525-13530.
(32) In these reactions, the ratio of products was determined by NMR analysis
of the crude product mixture. In instances when the analysis of crude NMR
data did not allow an accurate estimate of this ratio, products were analyzed
by HPLC-ESI.
(33) When the resin-bound AcValAzySer(tBu)AlaGly pentapeptide was swelled
in DMF containing 20% DMSO (v/v) and then reacted with EtSH and
DBU, the same product distribution was observed.
(34) A similar distribution of products was observed in the reaction of these
two coupling partners in 10% DMSO in DMF (v/v) and in the presence of
2 M ethylenecarbonate in DMF.
9
J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005 7365

A R T I C L E S
Scheme 5 a
Galonic et al.
v/v). The ligation was complete in less than 3 h, and the product
was purified by RP-HPLC to afford glycosylated decapeptide
56 in 83% yield. The successful preparation of 56, commencing
with the conjugation of unprotected carbohydrate thiol 37 and
aziridine-containing peptide 32, followed by the extension of
the peptide backbone with the cysteine-containing peptide,
demonstrates the utility of this sequential ligation approach in
the convergent assembly of complex peptide thioconjugates.
Conclusion
The development of a novel method for site- and stereo-
selective peptide modification based on incorporation of the
aziridine-2-carboxylic acid moiety into peptides has been
described. To allow for the rapid construction of diverse Azy-
containing peptides, FmocAzyOH has been prepared and
incorporated into peptides via a modified Fmoc-based solid-
phase synthesis protocol. The unique electrophilic nature of this
nonproteinogenic residue allows for site-selective conjugation
with thiol nucleophiles in a highly selective aziridine ring-
opening process. A wide range of thiol nucleophiles have been
coupled with Azy-containing peptides, both in solution and on
solid support. This method is applicable to the site-specific
labeling of peptides with valuable biochemical probes and is
amenable to the preparation of posttranslationally modified
peptides, such as farnesylated peptides and thioglycoconjugates
derived from either protected or unprotected carbohydrate
nucleophiles. This methodology, combined with native chemical
ligation, also allows for the generation of complex thioglyco-
conjugates in which the stereochemical integrity of the carbo-
hydrate donor and the R-carbon stereocenter of the amino acid
is preserved. Present studies are focused on delineation of the
factors governing the regioselectivity of the process and
application of this methodology for the generation of stereo-
defined â-amino acids. This facile route to Azy-containing
peptides and the efficiency with which these substrates undergo
ligation also shows promise for the convergent synthesis of
multivalent glycopeptide displays and application in functional
proteomics studies.
a Reagents and conditions: (a) DBU, DMF; (b) (CF3)2CHOH/CH2Cl2
(20% v/v); (c) BnSH, EDC‚HCl, HOBt, DMF (44% over 10 steps from
21; 92%/step); (d) TFA/CH2Cl2/iPr3SiH/HS(CH2)2SH, 10:10:1:1 (88%); (e)
HCysAlaSerPheAspOH, Gn‚HCl, PhSH, sodium phosphate buffer, pH 7.5
(83%).
terminal to the aziridine moiety (Table 4), direct application of
the thiol-aziridine ligation strategy to construct internal ho-
mogeneous cysteine conjugates of extended peptide substrates
may be hampered by the formation of constitutional isomers.
This potential complication notwithstanding, it is clear that the
ligation of thiols with aziridine-containing peptides possessing
Azy as the second amino acid from the resin proceeds with
exceptionally high efficiency and regio- and stereoselectivity
for the cysteine conjugates (Table 3, entries 1-13). In fact, the
convergency of this strategy is by no means compromised when
it is applied to the preparation of more complex peptide
conjugates given the prospect of further extension at the
C-terminus via subsequent peptide ligation. Native chemical
ligation42 proceeds under mild conditions and is compatible with
the presence of glycosylated amino acids,6h,43 although ligation
efficiency in the presence of a bulky glycosylated residue in
proximity to the C-terminal coupling fragment has heretofore
not been assessed. Thus, unprotected â-thioglucopyranose 37
was reacted with aziridine-containing pentapeptide 32 (Scheme
5), prepared according to the standard iterative procedure (vide
supra). Following the carbohydrate-peptide ligation, the resin
was removed by treatment with hexafluoro-2-propanol in
dichloromethane (20% v/v), and the glycopeptide conjugate was
then condensed at the C-terminus with benzylthiol,44 providing
the desired glycopeptide thioester 55 in characteristically high
overall yield (44% over 10 steps from 21, ∼92% per step).
Deprotection of the side-chain protecting groups in thioester
55 was accomplished with TFA in the presence of ethanedithiol
and tri-iso-propyl silane. This generated the corresponding fully
deprotected glycopeptide thioester, which was subsequently
purified by RP-HPLC. The ligation of AcGluGlyLysCys(â-
Glucose)Gly-SBn (55) and HCysAlaSerPheAspOH, prepared
and purified by the standard solid-phase synthesis protocol, was
performed in sodium phosphate buffer (100 mM, pH 7.5),
containing 6 M guanidine hydrochloride and thiophenol (4%
Experimental Section
General. All reactions were performed in dry modified Schlenk
(Kjeldahl shape) flasks fitted with a glass stopper under positive pressure
of argon, unless otherwise noted. Air- and moisture-sensitive liquids
were transferred via syringe. Organic solutions were concentrated by
rotary evaporation below 30 °C at ca. 25 Torr. Flash column
chromatography was performed on 230-400 mesh silica gel. Thin-
layer chromatography was performed on glass plates precoated to a
depth of 0.25 mm with 230-400 mesh silica gel impregnated with a
fluorescent indicator (254 nm). When necessary, solvents were degassed
by the freeze-pump thaw method (>3 cycles).
Dichloromethane, toluene, acetonitrile, 1,2-dimethoxyethane, and
tetrahydrofuran were purified by passage through two packed columns
of neutral alumina under an argon atmosphere. Chloroform, pyridine,
diisopropylethylamine, and triethylamine were distilled from calcium
hydride at 760 Torr. Methanol was distilled from magnesium oxide at
760 Torr. DMF was dried over activated 4 Å molecular sieves. Millipore
water was used for reactions in an aqueous solvent or cosolvent.
Infrared (IR) spectra were obtained on a Perkin-Elmer Spectrum BX
spectrophotometer referenced to a polystyrene standard. Data are
presented as frequency of absorption (reciprocal centimeters). Proton
and carbon-13 nuclear magnetic resonance (1H NMR and 13C NMR)
(42) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994,
266, 776-779.
(43) 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.
(44) Matsumura, S.; Takahashi, T.; Ueno, A.; Mihara, H. Chem. Eur. J. 2003,
9, 4829-4837.
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7366 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005

Aziridine-2-carboxylic Acid-Containing Peptides
A R T I C L E S
spectra were recorded on a Varian 400, a Varian 500, or a Varian Inova
500 NMR spectrometer; chemical shifts are expressed in parts per
million (δ scale) downfield from tetramethylsilane and are referenced
to residual protium in the NMR solvent [CHCl3, δ 7.27; HDO, 4.80;
C6D4HN, 7.22; CHD2OD, 3.31; DCON(CD3)CHD2, 2.75]. Data are
presented as follows: chemical shift, multiplicity (s ) singlet, d )
doublet, t ) triplet, q ) quartet, m ) multiplet and/or multiple
resonances), coupling constants in Hertz (Hz), and integration.
RP-HPLC purification and analyses were carried out on a Rainin
HPLC system (model SD 200), equipped with UV absorbance detector
(model UV 1), at a wavelength of 220 nm. The products were analyzed
and purified on Vydac C18 columns (analytical, 250 × 4.6 mm;
preparative, 250 × 22 mm). Products were eluted in 0.1% TFA in
acetonitrile/water in a linear gradient optimized for every compound
at flow rates of 1 mL min-1 for analytical and 8 mL min-1 on the
preparative column.
General SPPS Procedure: AcGlyGlyPheAzyAla-2-Chlorotrityl
Resin (27). Alanine-preloaded 2-chlorotrityl resin 26 (228 mg, 0.15
mmol, 1 equiv; loading 0.64 mmol/g) was swollen in CH2Cl2/DMF
(30% v/v) in a solid-phase peptide synthesis reaction vessel (see Ace
Glass) for 1 h. After swelling, the resin was rinsed with DMF (10 ×
2 mL). FmocAzyOH (190 mg, 0.6 mmol, 4 equiv), HBTU (227 mg,
0.6 mmol, 4 equiv), and 0.4 M NMM in DMF (6 mL, 2.4 mmol, 16
equiv) were premixed in a vial and added to the resin. Nitrogen was
bubbled through the reaction vessel for 2.5 h, after which the resin
was rinsed with DMF (10 × 2 mL). To ensure complete reaction, the
coupling was repeated (FmocAzyOH, 93 mg, 0.3 mmol, 2 equiv;
HBTU, 114 mg, 0.3 mmol, 2 equiv; 0.4 M NMM/DMF, 3 mL, 1.2
mmol, 8 equiv; reaction time 1 h). The resin was subsequently rinsed
with DMF (10 × 2 mL), and Fmoc was removed with 1% DBU/DMF
(6 mL, 0.4 mmol, 2.7 equiv) for 1 min and then rinsed with DMF (10
× 2 mL). Fmoc deprotection was repeated twice in the same manner.
FmocPheOH (236 mg, 0.6 mmol, 4 equiv), HBTU (228 mg, 0.6 mmol,
4 equiv), and 0.4 M NMM/DMF (6 mL, 2.4 mmol, 16 equiv) were
premixed in a vial and added to the reaction vessel containing resin.
Nitrogen was bubbled through for 2.5 h, the resin was rinsed with DMF
(10 × 2 mL), and the coupling was repeated to ensure complete
acylation of the secondary amine within the Azy residue (FmocPheOH,
234 mg, 0.6 mmol, 4 equiv; HBTU, 227 mg, 0.6 mmol, 4 equiv; 0.4
M NMM/DMF, 6 mL, 2.4 mmol, 16 equiv; reaction time 1 h). The
resin was subsequently rinsed with DMF (10 × 2 mL), and Fmoc was
removed with 1% DBU/DMF (6 mL, 0.4 mmol, 2.7 equiv) for 1 min
and then rinsed with DMF (10 × 2 mL). Fmoc deprotection was
repeated twice in the same manner. FmocGlyGlyOH (219 mg, 0.6
mmol, 4 equiv), HBTU (230 mg, 0.6 mmol, 4 equiv), and 0.4 M NMM/
DMF (6 mL, 2.4 mmol, 16 equiv) were premixed in a vial and added
to the reaction vessel containing resin. Nitrogen was bubbled through
for 2 h, and the resin was rinsed with DMF (10 × 2 mL). Fmoc was
removed with 1% DBU/DMF (6 mL, 0.4 mmol, 2.7 equiv) for 1 min,
and the resin was rinsed with DMF (10 × 2 mL). Fmoc deprotection
was repeated twice in the same manner. A solution of Ac2O in pyridine
(1:3 v/v; 15.5 mL, 37.5 mmol, 250 equiv) was then added to the SPPS
reaction vessel with the resin, through which nitrogen was bubbled for
15 min. Following acetylation, the resin was washed with DMF and
CH2Cl2 and stored at -10 °C under Ar until it was used for ligations.
A portion of the product was cleaved from the resin with 20% (v/v)
hexafluoro-2-propanol (HFIPA) in dichloromethane. 1H NMR analysis
of a crude product showed ca. 85-90% purity for the desired peptide.
1H NMR (500 MHz, DMF-d7) δ ) 8.64 (d, J ) 6.9 Hz, 1H), 8.49 (br
s, 1H), 8.33 (t, J ) 5.5 Hz, 1H), 8.21 (t, J ) 6.0 Hz, 1H), 8.18 (d, J
) 7.2 Hz, 1H), 7.33-7.21 (m, 5H), 4.68 (m, 1H), 4.39 (pentet, J )
7.1 Hz, 1H), 3.89-3.76 (m, 4H), 3.30 (dd, J ) 5.6 and 3.2 Hz, 1H),
3.25 (dd, J ) 14.3 and 4.5 Hz, 1H), 3.01 (dd, J ) 14.0 and 9.3 Hz,
1H), 2.65 (dd, J ) 5.6 and 2.0 Hz, 1H), 2.45 (dd, J ) 3.2 and 1.9 Hz,
1H), 1.96 (s, 3H), 1.41 (d, J ) 7.2 Hz, 3H). HRMS (ESI)+ m/z calcd
for C21H28N5O7 [M + H]+, 462.1989; found, 462.1992.
General Procedure for Conjugation with Azy-containing Peptides
in Solution: Reaction of BocAlaAzyAlaOBn with EtSH. DBU (1.0
µL, 0.006 mmol, 0.1 equiv) was added to a solution of BocAlaAzy-
AlaOBn 7 (27 mg, 0.06 mmol, 1 equiv), and ethanethiol (6 µL, 0.08
mmol, 1.20 equiv) in acetonitrile (315 µL) at 23 °C. Formation of a
white precipitate was observed after 12 min. The resulting solution
was stirred at this temperature for 3.5 h and then concentrated in vacuo.
The residue was purified by silica gel flash chromatography (25% ethyl
acetate in dichloromethane) to afford the C3 adduct (27 mg, 85%), as
1
a white solid and C2 adduct (0.4 mg, 1%), as a white solid (crude H
NMR ratio of C3:C2 adduct ) 20:1).
BocAlaCys(Et)AlaOBn. Mp ) 53 °C; Rf ) 0.30 (33% ethyl acetate
in dichloromethane); 1H NMR (500 MHz, CDCl3) δ ) 7.39-7.33 (m,
5 H), 7.26 (br s, 1H), 7.06 (d, J ) 6.4 Hz, 1H), 5.18 (ABq, JAB ) 12.3
Hz, 2H), 4.96 (br s, 1H), 4.59 (pentet, J ) 7.2 Hz, 1H), 4.54 (m, 1H),
4.13 (m, 1H), 3.08 (dd, J ) 14.8 and 2.5 Hz, 1H), 2.77 (dd, J ) 14.0
and 7.1 Hz, 1H), 1.46 (s, 9H), 1.44 (d, J ) 7.1 Hz, 3H), 1.39 (d, J )
7.3 Hz, 3H), 1.26 (t, J ) 7.5 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ
) 172.9, 172.4, 170.1, 156.0, 135.5, 128.8, 128.6, 128.4, 80.7, 67.4,
52.4, 50.9, 48.7, 33.5, 28.5, 26.5, 18.2, 18.0, 14.9; FTIR (neat film)
3317, 1737, 1680, 1645, 1532, 1166, 850 cm-1. HRMS (FAB)+ m/z
calcd for C23H36N3O6S [M + H]+, 482.2325; found, 482.2318.
BocAlaâ2EtS-HGlyAlaOBn. Rf ) 0.17 (33% ethyl acetate in
dichloromethane); 1H NMR (500 MHz, CDCl3) δ ) 7.50 (d, J ) 6.98
Hz, 1H), 7.40-7.34 (m, 5H), 7.18 (br s, 1H), 5.22 (ABq, JAB ) 12.2
Hz, 2H), 5.04 (d, J ) 4.3 Hz, 1H), 4.66 (pentet, J ) 7.7 Hz, 1H), 4.03
(pentet, J ) 7.7 Hz, 1H), 3.93 (m, 1H), 3.55 (m, 2H), 2.56 (q, J ) 7.4
Hz, 2H), 1.52 (d, J ) 7.2 Hz, 3H), 1.44 (s, 3H), 1.32 (d, J ) 7.0 Hz,
3H), 1.23 (t, J ) 7.5 Hz, 3H); FTIR (neat film) 3291, 1651, 1546,
1037 cm-1. HRMS (ESI)+ m/z calcd for C23H36N3O6S [M + H]+,
482.2325; found, 482.2325.
General Procedure for Conjugation with 3-MeAzy-containing
Peptides in Solution: BocGly-3-MeCys(Et)NHBn. DBU (13.0 µL,
0.087 mmol, 1.0 equiv) was added to a solution of BocGly-3-
MeAzyNHBn (29.2 mg, 0.084 mmol, 1.0 equiv) and ethanethiol (52.2
µL, 0.840 mmol, 10.0 equiv) in chloroform (420 µL) at 23 °C. The
resulting clear solution was stirred at this temperature for 2 h and then
concentrated in vacuo. The residue was purified by silica gel flash
chromatography (40% ethyl acetate in dichloromethane) to afford the
C3 conjugation adduct (13 mg, 36%) as a colorless oil. Rf ) 0.37 (50%
General Procedure for Conjugation with Aziridine-containing
Peptides on Solid Support. Method A: AcGlyGlyPheCys(Et)AlaOH
(38). DBU (2.4 µL, 0.016 mmol, 1 equiv) was added to a suspension
of AcGlyGlyPheAzyAla-2-chlorotrityl resin 27 (32 mg, 0.016 mmol,
1 equiv) and ethanethiol (12 µL, 0.16 mmol, 10 equiv) in DMF (650
µL) at 23 °C. The resulting suspension was stirred at this temperature
for 16 h, then the resin was washed with CH2Cl2 (25 × 1.5 mL), and
the crude product mixture was cleaved from the resin with 20% (v/v)
HFIPA/CH2Cl2 (9 mL, 15 min, 23 °C). Concentration in vacuo and
purification of the residue by preparative RP-HPLC (10% CH3CN in
H2O to 70% CH3CN in H2O + 0.1% TFA, in 50 min) provided
conjugate 38 (4 mg, 52% from HAla-preloaded resin 26; 9 steps, 93%/
step), as a white solid. Rt (10% CH3CN in H2O to 70% CH3CN in
1
ethyl acetate in dichloromethane); H NMR (500 MHz, CDCl3) δ )
7.42 (m, 1H), 7.36-7.24 (m, 5H), 7.19 (d, J ) 7.5 Hz, 1H), 5.22 (m,
1H), 4.60 (dd, J ) 7.7 and 2.6 Hz, 1H), 4.54 (dd, J ) 14.7 and 6.2 Hz,
1H), 4.39 (dd, J ) 14.7 and 5.3 Hz, 1H), 3.80 (m, 2H), 3.60 (m, 1H),
2.62 (m, 1H), 2.49 (m, 1H), 1.41 (s, 9H), 1.20 (m, 6H); 13C NMR (125
MHz, CDCl3) δ ) 169.5, 169.3, 156.7, 138.0, 128.8, 128.2, 127.6,
81.0, 56.9, 45.0, 43.9, 41.3, 28.4, 26.0, 18.4, 15.0; FTIR (neat film)
3305, 2976, 2930, 1650, 1518, 1455, 1367, 1250, 1169 cm-1. HRMS
(ESI)+ m/z calcd for C20H32N3O4S [M + H]+, 410.2114; found,
410.2132.
1
H2O + 0.1% TFA, in 50 min, analytical column) ) 14.60 min. H
NMR (500 MHz, CD3OD) δ ) 8.35 (t, J ) 5.4 Hz, 1H), 8.20 (d, J )
7.3 Hz, 1H), 8.16 (d, J ) 8.0 Hz, 1H), 8.13 (d, J ) 7.6 Hz, 1H), 7.27-
9
J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005 7367

A R T I C L E S
Galonic et al.
7.19 (m, 5 H), 4.63 (ddd, J ) 9.6, 7.9, and 5.1 Hz, 1H), 4.52 (m, 1H),
4.36 (pentet, J ) 7.5 Hz, 1H), 3.80 (m, 4H), 3.21 (dd, J ) 14.0 and
5.1 Hz, 1H), 3.04 (dd, J ) 13.9 and 5.2 Hz, 1H), 2.97 (dd, J ) 14.0
and 9.6 Hz, 1H), 2.80 (dd, J ) 14.0 and 9.0 Hz, 1H), 2.59 (qd, J ) 7.5
and 1.7 Hz, 2H), 2.02 (s, 3H), 1.41 (d, J ) 7.4 Hz, 3H), 1.24 (t, J )
7.3 Hz, 3H). HRMS (ESI)+ m/z calcd for C23H34N5O7S [M + H]+,
524.2179; found, 524.2186.
was concentrated in vacuo to provide 16 mg of the crude product. 1-(3-
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC‚HCl,
42 mg, 0.22 mmol, 12 equiv) was added to a suspension of the obtained
crude AcGlu(tBu)GlyLys(Boc)Cys(â-Glc)GlyOH (14 mg), 1-hydroxy-
benzotriazole hydrate (HOBt, 29 mg, 0.21 mmol, 12 equiv), and
benzylthiol (62 µL, 0.62 mmol, 30 equiv) in DMF (1.25 mL) at 0 °C.
The resulting solution was stirred at this temperature for 24 h, and
then concentrated in vacuo. Purification of the residue by preparative
RP-HPLC (10% CH3CN in H2O to 70% CH3CN in H2O + 0.1% TFA,
in 30 min) provided thioester 55 (8 mg, 44% from HGly-preloaded
resin 21; 10 steps, 92%/step), as a colorless oil. Rt (10% CH3CN in
H2O to 70% CH3CN in H2O + 0.1% TFA, in 30 min, analytical column)
Method B: AcGlyGlyPheCys(Et)AspOH (39). DBU (4.4 µL, 0.03
mmol, 1 equiv) was added to a suspension of AcGlyGlyPheAzyAsp-
(tBu)-2-chlorotrityl resin 29 (52 mg, 0.03 mmol, 1 equiv) and ethanethiol
(22 µL, 0.29 mmol, 10 equiv) in DMF (1.2 mL) at 23 °C. The resulting
suspension was stirred at this temperature for 22 h, and then the resin
was filtered and washed with dichloromethane (25 × 1.5 mL). The
ligation product was cleaved from the resin by treatment with TFA/
CH2Cl2/iPr3SiH/HS(CH2)2SH (10:10:1:1, 10 mL) for 1.5 h at 23 °C.
The resin was filtered off, and the filtrate was coevaporated sequentially
with toluene (2 × 4 mL), followed by cold Et2O (5 mL). The resulting
white precipitate was washed with hexanes (3 × 2 mL), and cold Et2O
(3 × 1 mL). Purification of the residue by preparative RP-HPLC (10%
CH3CN in H2O to 70% CH3CN in H2O + 0.1% TFA, in 50 min)
provided conjugate 39 (6 mg, 35% from HAsp(tBu)-preloaded resin
28; 9 steps, 89%/step). Rt (10% CH3CN in H2O to 70% CH3CN in
1
) 22.8 min. H NMR (500 MHz, DMF-d7) δ ) 8.44 (t, J ) 6.1 Hz,
1H), 8.42 (t, J ) 5.5 Hz, 1H), 8.28 (m, 2H), 8.16 (d, J ) 8.3 Hz, 1H),
7.36-7.27 (m, 5 H), 6.69 (t, J ) 5.1 Hz, 1H), 4.71 (m, 1H), 4.44 (d,
J ) 9.5 Hz, 1H), 4.33 (m, 2H), 4.15 (m, 4H), 3.89 (m, 3H), 3.62 (m,
1H), 3.38 (dd, J ) 8.6 and 3.1 Hz, 1H), 3.32-3.23 (m, 3H), 3.02 (m,
3H), 2.92 (dd, J ) 13.7 and 8.8 Hz, 1H), 2.37 (m, 2H), 2.07 (m, 1H),
1.97 (s, 3H), 1.89 (m, 1H), 1.82 (m, 1H), 1.71 (m, 1H), 1.45 (m, 4H),
1.42 (s, 9H), 1.40 (s, 9H). HRMS (ESI)+ m/z calcd for C42H67N6O15S2
[M + H]+, 959.4106; found, 959.4070.
AcGluGlyLysCys(Glcâ1)GlySBn. A mixture of TFA/CH2Cl2/
iPr3SiH/HS(CH2)2SH ) (10:10:1:1, 11 mL) was added to a flask
containing AcGlu(tBu)GlyLys(Boc)Cys(â-Glc)GlySBn (55, 8 mg, 0.008
mmol, 1 equiv) at 23 °C. The resulting solution was stirred at this
temperature for 1.5 h and then concentrated in vacuo. The residue was
coevaporated with cold diethyl ether (5 mL). The resulting white
precipitate was washed with cold diethyl ether (4 × 1 mL). The residue
was purified by preparative RP-HPLC (5% CH3CN in H2O to 32%
CH3CN in H2O + 0.1% TFA, in 35 min), providing fully deprotected
thioester (6 mg, 88%) as a white solid. Rt (5% CH3CN in H2O to 32%
CH3CN in H2O + 0.1% TFA, in 30 min, analytical column) ) 20.0
1
H2O + 0.1% TFA, in 50 min, analytical column) ) 13.80 min. H
NMR (500 MHz, CD3OD) δ ) 8.22 (m, 2 H), 8.11 (d, J ) 7.6 Hz,
1H), 8.22 (m, 2 H), 7.26 (m, 5H), 4.72 (m, 1H), 4.64 (m, 1H), 4.54
(m, 1H), 3.82 (m, 4H), 3.21 (dd, J ) 14.0 and 4.7 Hz, 1H), 3.03 (dd,
J ) 14.0 and 5.4 Hz, 1H), 2.97 (dd, J ) 14.0 and 9.6 Hz, 1H), 2.85
(m, 3H), 2.58 (qd, J ) 7.4 and 1.0 Hz, 2H), 2.03 (s, 3H), 1.24 (t, J )
7.6 Hz, 3H). HRMS (ESI)+ m/z calcd for C24H34N5O9S [M + H]+,
568.2077; found, 568.2073.
Method C: AcGlyGlyPheCys(GalNAcr1)AlaOH (45). DBU (7.2
µL, 0.05 mmol, 1 equiv) was added to a suspension of AcGlyGlyPhe-
AzyAla-2-chlorotrityl resin 27 (99 mg, 0.05 mmol, 1 equiv) and thio
2-N-acetylamino-3,4,6-tri-O-acetyl-1,2-dideoxy-R-D-galactopyranose 16
(91 mg, 0.24 mmol, 5 equiv) in DMF (3.4 mL) at 60 °C. The resulting
suspension was stirred at this temperature for 22 h, and then the resin
was filtered and washed with dichloromethane (25 × 1.5 mL). The
ligation product was cleaved from the resin with 20% (v/v) HFIPA/
CH2Cl2 (15 mL, 15 min, 23 °C). The residue was concentration in vacuo
and dissolved in methanol (30 mL). A solution of NaOMe in MeOH
(0.1 M) was added until pH 8.5, and the resulting solution was stirred
for 80 min at 23 °C. Subsequently, a 1 M solution of AcOH in MeOH
was added until pH 6. Concentration in vacuo and purification of the
residue by preparative RP-HPLC (7% CH3CN in H2O to 26% CH3CN
in H2O + 0.1% TFA, in 40 min) provided conjugate 45 (7 mg, 21%
from HAla-preloaded resin 26; 10 steps, 86%/step). Rt (7% CH3CN in
H2O to 30% CH3CN in H2O + 0.1% TFA, in 40 min, analytical column)
) 15.01 min. 1H NMR (500 MHz, CD3OD) δ ) 8.36 (d, J ) 8.6 Hz,
1H), 8.27 (t, J ) 6.2 Hz, 1H), 8.22 (d, J ) 6.8 Hz, 1H), 8.14 (d, J )
7.6 Hz, 1H), 8.05 (d, J ) 7.6 Hz, 1H), 7.29-7.19 (m, 5 H), 5.59 (d,
J ) 5.5 Hz, 1H), 4.67 (m, 1H), 4.61 (m, 1H), 4.43 (m, 1H), 4.38 (pentet,
J ) 7.2 Hz, 1H), 4.17 (dd, J ) 7.8 and 3.9 Hz, 1H), 3.87 (m, 6H),
3.72 (dd, J ) 11.4 and 4.3 Hz, 1H), 3.65 (dd, J ) 11.4 and 3.2 Hz,
1H), 3.23 (dd, J ) 13.8 and 4.8 Hz, 1H), 3.11 (dd, J ) 13.8 and 4.4
Hz, 1H), 3.00 (dd, J ) 14.6 and 4.4 Hz, 1H), 2.95 (dd, J ) 14.0 and
9.6 Hz, 1H), 2.02 (s, 3H), 1.98 (s, 3H), 1.41 (d, J ) 7.4 Hz, 3H).
HRMS (ESI)+ m/z calcd for C29H43N6O12S [M + H]+, 699.2660; found,
699.2656.
1
min. H NMR (500 MHz, D2O) δ ) 7.23-7.16 (m, 5 H), 4.52 (dd, J
) 8.0 and 6.5 Hz, 1H), 4.41 (d, J ) 9.9 Hz, 1H), 4.18 (dd, J ) 8.8 and
5.6 Hz, 1H), 4.14 (dd, J ) 8.8 and 5.6 Hz, 1H), 4.04-3.95 (m, 4H),
3.75 (m, 3H), 3.52 (dd, J ) 12.4 and 6.1 Hz, 1H), 3.33 (m, 2H), 3.23
(q, J ) 9.4 Hz, 1H), 3.16 (t, J ) 9.5 Hz, 1H), 3.12 (dd, J ) 14.3 and
6.4 Hz, 1H), 2.86 (dd, J ) 14.3 and 8.4 Hz, 1H), 2.78 (m, 2H), 2.31
(m, 2H), 1.95 (m, 1H), 1.87 (s, 3H), 1.81 (m, 1H), 1.65 (m, 1H), 1.56
(m, 1H), 1.48 (m, 2H), 1.23 (m, 2H). HRMS (ESI)+ m/z calcd for
C33H51N6O13S2 [M + H]+, 803.2956; found, 803.2974.
AcGluGlyLysCys(Glcâ1)GlyCysAlaSerPheAspOH (56). Sodium
phosphate buffer (100 mM, pH 7.5, 4.3 mL,), containing 6 M guanidine
hydrochloride and PhSH (4% v/v) was added to a mixture of
AcGluGlyLysCys(â-Glc)GlySBn (3.2 mg, 0.004 mmol, 1 equiv) and
HCysAlaSerPheAspOH (4.5 mg, 0.008 mmol, 2.1 equiv) at 23 °C. The
resulting solution was stirred for 28 h, and the reaction was monitored
by analytical C18 RP-HPLC. Following reaction, the product was
purified by preparative RP-HPLC (7% CH3CN in H2O to 16% CH3CN
in H2O + 0.1% TFA, in 40 min) by direct injections of crude reaction
mixture, and fractions containing product were lyophilized. Repurifi-
cation of the product by the same method provided glycoconjugate
56 (4.1 mg, 83%), as a white solid. Rt (7% CH3CN in H2O to 16%
CH3CN in H2O + 0.1% TFA, in 35 min, analytical column) ) 23.0
1
min. H NMR (500 MHz, D2O) δ ) 7.21-7.09 (m, 5H), 4.52 (dd, J
) 7.8 and 6.3 Hz, 1H), 4.48 (m, 1H), 4.43 (m, 2H), 4.37 (t, J ) 6.3
Hz, 1H), 4.24-4.16 (m, 4H), 3.80 (m, 3H), 3.61 (m, 2H), 3.53 (dd, J
) 12.4 and 6.7 Hz, 1H), 3.34 (m, 2H), 3.26-3.17 (m, 2H), 3.06 (dd,
J ) 14.1 and 7.3 Hz, 1H), 3.02 (dd, J ) 13.8 and 6.0 Hz, 1H), 2.90
(m, 2H), 2.82 (t, J ) 7.6 Hz, 2H), 2.77-2.65 (m, 4H), 2.33 (t, J ) 7.8
Hz, 2H), 1.95 (m, 1H), 1.88 (s, 3H), 1.83 (m, 1H), 1.68 (m, 1H), 1. 60
(m, 1H), 1.51 (m, 2H), 1.26 (m, 2H), 1.20 (d, J ) 7.3 Hz, 3H). HRMS
(ESI)+ m/z calcd for C48H74N11O22S2 [M + H]+, 1220.4451; found,
1220.4435.
AcGlu(tBu)GlyLys(Boc)Cys(Glcâ1)GlySBn (55). DBU (3.2 µL,
0.021 mmol, 1 equiv) was added to a suspension of AcGlu(tBu)GlyLys-
(Boc)AzyGly-2-chlorotrityl resin 32 (51 mg, 0.021 mmol, 1 equiv) and
1-thio-â-D-glucopyranose 37 (21 mg, 0.11 mmol, 5 equiv) in DMF
(1.4 mL) at 23 °C. The resulting suspension was stirred at this
temperature for 3.5 days, then the resin was washed with CH2Cl2 (25
× 1.5 mL), and the crude product mixture was cleaved from the resin
with 20% (v/v) HFIPA/CH2Cl2 (8 mL, 15 min, 23 °C). The residue
Acknowledgment. This research was supported by the NIH
(GM58833 and GM58822). Procter and Gamble fellowships to
9
7368 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005

Aziridine-2-carboxylic Acid-Containing Peptides
A R T I C L E S
D.P.G. and N.D.I. are acknowledged. We thank Filip Petron-
ijevic´ for the preparation of viable quantities of FmocAzyOH.
preparation and analytical characterization of prepared com-
pounds. This information is available free of charge via the
Internet at http://pubs.acs.org.
Supporting Information Available: List of abbreviations,
complete citation for ref 4b, and experimental details for the
JA050304R
9
J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005 7369