A novel neoglycopeptide linkage compatible with native chemical ligation

Article abstract of DOI:10.1039/b607200c

The straightforward synthesis of a novel class of neoglycopeptide and its fusion with a larger peptide thioester using sequential chemoselective ligations is described. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b607200c

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A novel neoglycopeptide linkage compatible with native chemical ligation†
Derek Macmillan* and Javier Blanc
Received 22nd May 2006, Accepted 25th May 2006
First published as an Advance Article on the web 3rd July 2006
DOI: 10.1039/b607200c
The straightforward synthesis of a novel class of neogly-
copeptide and its fusion with a larger peptide thioester using
sequential chemoselective ligations is described.
The synthesis of glycosylated proteins is a formidable challenge
and it is fair to say that synthetic glycoproteins have had modest
impact, thus far, on the study of glycobiology when compared
to genetic methods. This is largely due to the well documented
difficulties associated with synthesis of oligosaccharides and
glycopeptides such as the requirement for extensive protection and
deprotection strategies and the stereoselective formation of glyco-
sidic linkages. We, and others, have pursued alternative method-
ologies for the assembly of modified peptides and proteins.1
Using the click “ligation”2 for bioconjugation was of particular
interest due to its reliability and compatibility with the aqueous
milieu.3 We had previously shown that glycosyl iodoacetamides
can react with the sulfhydryl groups of cysteine on solid-phase
to afford glycopeptide mimetics and that this methodology was
compatible with useful protein assembly methods such as native
chemical ligation (NCL).4 We were keen to investigate whether
the coupling of glycosyl azides with peptides displaying acetylene
dipolarophiles may also be useful for this purpose since the
glycosyl azides are more stable and can be prepared in fewer steps
than the corresponding glycosyl iodoacetamides (Scheme 1).
Initially we prepared the peracetylated glycopyranosylazides of
N-acetylglucosamine (1) N-acetyllactosamine (2) and chitobiose
(3) which are all constituents of the N-linked class of glyco-
proteins. We investigated conditions for their union with the
heterobifunctional adaptor 2-bromoacetyl propargylamide (4)5
Scheme 1 Construction of novel neoglycopeptides.
Table 1 Synthesis of neoglycopeptide precursor bromoacetamides
and the reaction of these saccharides with 4 proceeded smoothly
under conditions reported in the recent literature,5,6 the 1,4-
addition product being favoured by the presence of a Cu(I) catalyst
(Table 1). The crude products did not require purification by
column chromatography.
Pleased with the facility with which such glycoconjugates could
be prepared we next aimed to expose the bromoacetamides to
conditions typically encountered in peptide chemistry and native
chemical ligation. We were particularly interested in the potential
to modify the thiol groups of cysteine residues. Additionally, to
demonstrate the versatility of the approach, we explored the pos-
sibility of reacting thiol groups directly with the bromoacetamide
products 5–7 on solid phase and the reaction of cysteine thiols
with 4 such that click chemistry could subsequently be investigated
in solution or on solid-phase with peptides displaying acetylenes
Saccharide
Catalyst
Product Yield (%)c
1, R = Ac
Cu(I)I (5 eq.)a
5
5
6
7
100
97
91
1, R = Ac
Cu(II)SO4 (0.1 eq.)b
Cu(II)SO4 (0.1 eq.)
2, R = (OAc)4-b-Gal-
3, R = (OAc)3 b-GlcNAc- Cu(II)SO4 (0.1 eq.)
87
a Methanol as solvent. b Active copper species generated in the presence of
1.1 equivalent sodium ascorbate in 9 : 1 : 1 CHCl3–EtOH–H2O as solvent.
c Isolated yield.
University College London, Department of Chemistry, Christopher In-
gold Laboratories, 20 Gordon Street, London, UK WC1H 0AJ. E-mail:
d.macmillan@ucl.ac.uk; Tel: +44 (0)20 7679 4684
(Scheme 2). As anticipated, 4 and 5 reacted cleanly with benzyl
mercaptan forming model thioethers 8 and 9 in 84% and 75%
yield respectively. 8 also reacted cleanly with peracetylated 2-
acetamido-2-deoxy-D-glucopyranosyl azide, affording 9 in 91%
† Electronic supplementary information (ESI) available: experimental
procedures and analytical data for all products (compounds 4–14). See
DOI: 10.1039/b607200c
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yield. To establish whether the products might be stable to the
usual acidic peptide cleavage conditions 9 was subjected to 95%
aqueous TFA for 3 h. NMR analysis of the crude material after
evaporation showed no decomposition had taken place. Finally
the acetyl esters were cleanly removed upon exposure to 2% v/v
hydrazine hydrate in EtOH for 72 h and the fully deprotected
compound 10 was obtained.7 Encouraged by the preliminary
results we assembled a peptide fragment (11), similar in sequence
to human erythropoietin (residues 21–32), plus an N-terminal
cysteine residue, and furnished with two disulfide bond protected
cysteine residues at pre-determined positions (Scheme 3). The
peptide was assembled using standard protocols for Fmoc solid-
phase peptide synthesis and in an automated fashion. The cysteine
residues were deprotected on solid-phase by exposure to 10% w/v
dithiothreitol (DTT) containing 2.5% v/v DIPEA to expose the
thiol functional groups.4 N-Acetylglucosamine and the disaccha-
ride chitobiose were then incorporated by exposure of the resin to
bromoacetamides 5 or 7, employing three equivalents 5 or 7 per
thiol in each reaction. After 16 h reaction at room temperature,
cleavage of a small resin sample indicated that the reaction was
complete as the starting material was not observed. After cleavage
from the solid support by treatment of the resin with 95% TFA,
2.5% ethanedithiol and 2 5% H2O for four hours the crude
products were purified by semi-preparative HPLC, lyophilized,
and treated with 2% v/v aqueous hydrazine hydrate containing
5% w/v DTT to obtain the fully deprotected products 12 and 13
in quantitative yield (determined by HPLC). Bromoacetamide 6
and acetylenic bromoacetamide 4 could also be incorporated into
synthetic peptides in an identical fashion.
Scheme 2 Model reactions with benzylmercaptan demonstrate that the
order in which the reactions take place appears unimportant. Reagents and
conditions: i) BnSH, Et3N, DMF, 16 h, 84% and 75% for 8 and 9 respectively
ii) sodium ascorbate (1.1 eq.), Cu(II)SO4·5H2O (0.1 eq.), CHCl3, EtOH,
H2O (9 : 1 : 1), 37 ◦C, 16 h, 91%, iii) 2% v/v hydrazine monohydrate EtOH,
72 h, 66%.7
Scheme 3 Reagents and conditions: i) 10% w/v DTT, 2.5% DIPEA, DMF, 16 h, ii) 5 or 7 (3 eq. per thiol), 2.5% v/v Et3N, DMF, 16 h, iii) 95% TFA,
2.5% ethanedithiol, 2.5% H2O, 4 h, iv) 2% v/v aqueous hydrazine monohydrate, 1 h.
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Scheme 4 The neoglycopeptides are compatible with native chemical ligation reactions.7 Reagents and conditions: i) 6 M guanidine HCl, 1% w/v
MESNA, 300 mM Na phosphate buffer (pH 8.0), 10 mM TCEP, ii) 2% aqueous H2N-NH2.
Fragment 13 was then coupled to a peptide thioester in a
native chemical ligation reaction.8 The construction of the peptide
thioester, corresponding to human erythropoietin residues 1–19,
and its release from the solid support were monitored using the
dual-linker approach recently described by Unverzagt and co-
workers.9 In the ligation reaction equimolar quantities of each
peptide were combined in 0.25 ml of 6 M guanidine hydrochloride
containing 300 mM sodium phosphate buffer; pH 8.0, 1%
w/v mercaptoethanesulfonic acid (MESNA) and 10 mM tris-
carboxyethylphosphine (TCEP) for 36 h with shaking at room
temperature (Scheme 4).7 After this time the reaction mixture
was purified by directly loading it onto a semi-preparative HPLC
column. The ligated product (14) was the only species observed by
HPLC.7
new modes of presentation of carbohydrates (or other appendages)
should be explored concomitantly.1 Such neoglycoconjugates may
improve the pharmacokinetic profile of therapeutic glycopro-
teins. The fact that the neoglycopeptides described are simple
to prepare, homogeneous, and are also compatible with native
chemical ligation, may render them attractive building blocks for
neoglycoprotein assembly.
Acknowledgements
The authors would like to acknowledge The Royal Society, The
BBSRC, The University of Edinburgh and UCL for financial
support.
In summary we have developed a novel class of neoglycopeptide
that is compatible with modification of cysteine mutant proteins,
with synthetic peptides, and native chemical ligation. Furthermore
the fusion of glycosyl azides with peptides displaying acetylenes
may be of particular interest since NCL has been shown to fail
when large oligosaccharide appendages are located proximal to
the ligation site.10 Therefore, if acetylenes can be installed so
as to “encode” for glycosylation then bulky saccharide motifs
may be installed after ligation using click chemistry in solution.
We have already observed that the reaction between peptides
displaying acetylenes derived from 4 and glycosylazides proceeds
on solid-support.7 Although the linkage between the carbohydrate
and peptide moiety is unnatural, difficulties associated with the
synthesis of native glycopeptides and glycoproteins dictates that
Notes and references
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555; (c) D. Crich, V. Krishnamurthy and T. K. Hutton, J. Am. Chem.
Soc., 2006, 128(8), 2544–2545.
2 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, Angew.
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3 (a) A. J. Link, M. K. S. Vink and D. A. Tirrell, J. Am. Chem. Soc., 2004,
126, 10598–10602; (b) H. Lin and C. T. Walsh, J. Am. Chem. Soc., 2004,
126, 13998–14003; (c) J. A. Prescher and C. R. Bertozzi, Nat. Chem.
Biol., 2005, 1(1), 13–21.
4 D. Macmillan, A. M. Daines, M. Bayrhuber and S. L. Flitsch, Org.
Lett., 2002, 4(9), 1467–1470.
5 S. Chittaboina, F. Xie and Q. Wang, Tetrahedron Lett., 2005, 46, 2331–
2336.
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6 M. C. Bryan, F. Fazio, H.-K. Lee, C.-Y. Huang, A. Chang, M. D.
Best, D. A. Calarese, O. Blixt, J. C. Paulson, D. Burton, I. A.
Wilson and C.-H. Wong, J. Am. Chem. Soc., 2004, 126(28), 8640–
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7 See supporting information for HPLC and mass spectrometry data for
products 12 and 13 and NCL product 14†.
8 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. Kent, Science, 1994,
266(5186), 776–779.
9 S. Mezzato, M. Schaffrath and C. Unverzagt, Angew. Chem., Int. Ed.,
2005, 44, 1650–1654.
10 M. Mandal, V. Y. Dudkin, X. Geng and S. J. Danishefsky, Angew.
Chem., Int. Ed., 2004, 43, 2557–2561.
2850 | Org. Biomol. Chem., 2006, 4, 2847–2850
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