A One-Pot approach to neoglycopeptides using orthogonal native chemical ligation and click chemistry

Article abstract of DOI:10.1021/ol902131n

The powerful combination of native chemical ligation and click chemistry has been used to affect a one-pot synthesis of neogiycopeptiaes from propargyl-containing peptides using GaINAc-N₃ as the glycan component. A versatile chemical toolkit fo

Full text of DOI:10.1021/ol902131n

ORGANIC
LETTERS
2009
Vol. 11, No. 22
5270-5273
A One-Pot Approach to
Neoglycopeptides using Orthogonal
Native Chemical Ligation and Click
Chemistry
Dong Jun Lee,† Kalyaneswar Mandal,‡ Paul W. R. Harris,† Margaret A. Brimble,*,†
and Stephen B. H. Kent*,‡
Department of Chemistry, The UniVersity of Auckland, 23 Symonds Street, Auckland,
New Zealand, and Department of Chemistry, Department of Biochemistry & Molecular
Biology, Institute for Biophysical Dynamics, The UniVersity of Chicago,
929 East 57th Street, Chicago, Illinois 60637
skent@uchicago.edu; m.brimble@auckland.ac.nz
Received September 24, 2009
ABSTRACT
The powerful combination of native chemical ligation and click chemistry has been used to affect a one-pot synthesis of neoglycopeptides
from propargyl-containing peptides using GalNAc-N3 as the glycan component. A versatile chemical toolkit for the fully convergent synthesis
of neoglycoproteins using click chemistry, native chemical ligation, and kinetically controlled ligation is thus demonstrated.
Glycosylation is the most common and complex post-
translational modification of proteins.1-3 Glycoproteins are
essential in many biological processes including immune
defense, cell growth, and inflammation.1 For these reasons,
there is considerable interest in the structure-function
relationships of glycoproteins, the investigation of which
requires the systematic, controlled variation of glycoprotein
structure.2-5 Recombinant techniques for expression of
glycoproteins are problematic because glycosylation is
organism-specific and artificial cultivation conditions may
result in incorrect or inconsistent glycosylation patterns.2-5
Glycoproteins of defined chemical structure can be prepared
by total or semisynthesis based on modern chemical ligation
methods,6 and chemistry can then be used to control both
the site and structure of glycosylation.5,7,8
† The University of Auckland.
‡ The University of Chicago.
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10.1021/ol902131n CCC: $40.75
Published on Web 10/20/2009
 2009 American Chemical Society

To date, glycoconjugate mimetics, or neoglycoconjugates,
containing a variety of unnatural linkages between the
carbohydrate and aglycone moieties have also been ex-
plored.9,10 This mimicry allows rapid and convenient access
to a wide variety of neoglycopeptides and neoglycoproteins
that may also maintain, or even enhance, the biological
activity of the natural glycoproteins. One of the most studied
of these unnatural linkages is the triazole ring formed via
1,3-dipolar cycloaddition (click chemistry) of an organic
azide to a terminal alkyne.11-14 There are a number of
extensive reviews on this topic.15-18 Previous work by Rutjes
et al.19 made use of protected building blocks to synthesize
a series of triazole-linked glycosyl amino acids and dipep-
tides. Danishefsky’s group20 and Walsh et al.21 have
independently reported the union of unprotected carbohy-
drates and peptides containing a limited range of side chains
using click chemistry.22 Macmillan’s group reported that the
triazole linkage itself was compatible with conditions used
in native chemical ligation (6 M guanidine HCl, 0.3 M
Na2PO4 buffer pH 8.0, 10 mM tris(2-carboxyethyl)phosphine,
1% w/v sodium 2-mercaptoethanesulfonate (MESNA)).23
Davis et al.24,25 have utilized click chemistry to demonstrate
the diversity of post-translational chemical protein modifica-
tion. Semisynthetic lipoproteins have also been synthesized
using click chemistry by Moroder et al.26 Although the
ligation of peptides using click chemistry has been reported,27
somewhat surprisingly, a detailed investigation of reaction
conditions for click reactions, at useful scale with isolation
of pure products and full product characterization, between
sugar azides and alkyne-containing unprotected peptides that
contain all 20 amino acids found in proteins has not been
carried out. Furthermore, a one-pot approach that combines
native chemical ligation and click chemistry to yield neogly-
copeptides has not been explored. Combining these two
chemistries would provide a powerful tool to synthesize
neoglycopeptides as it avoids low yielding intervening HPLC
purifications steps.
Herein we report our studies on the use of click chemistry
to attach an azido sugar to propargyl-containing unprotected
peptides. The compatibility of the key click reaction with
the thiazolidine (Thz-), Cys-, -thioester, and acetamidomethyl
(Acm) moieties used in total protein synthesis by thioester-
mediated amide-forming chemical ligation methods and most
importantly, for the first time, the use of orthogonal native
chemical ligation and copper(I)-mediated alkyne-azide
cycloaddition reactions in a one-pot approach demonstrate
a convenient method to access highly sophisticated as-
semblies of neoglycopeptides efficiently (Figure 1).
Figure 1
chemistry.
. Proposed one-pot native chemical ligation and click
(7) Pratt, M. R.; Bertozzi, C. R. Chem. Soc. ReV. 2005, 34, 58–68
(8) Davis, B. G. Angew. Chem., Int. Ed. 2009, 48, 4674–4678
(9) Specker, D.; Wittmann, V. Top. Curr. Chem. 2007, 267, 65–107
(10) Nicotra, F.; Cipolla, L.; Peri, F.; La Ferla, B.; Redaelli, C. AdV.
Carbohydr. Chem. Biochem. 2007, 61, 353–398
(11) Toenøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057–3064
(12) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599
(13) Huisgen, R. Chem. Ber. 1967, 100, 2494–2507
(14) Huisgen, R. Pure Appl. Chem. 1989, 61, 613–628
(15) Meldal, M.; Tornoe, C. W. Chem. ReV. 2008, 108, 2952–3015
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To illustrate this strategy, we designed and synthesized
three propargyl-containing model peptides using in situ Boc
chemistry solid phase peptide synthesis (SPPS) (Figure 2).28
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(16) Kolb, H. C.; Sharpless, K. B. Drug DiscoVery Today 2003, 8, 1128–
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P. J. L. M.; Keereweer, A. R.; Groothuys, S.; Kuijpers, B. H. M. Org. Lett.
2004, 6, 3123–3126.
(20) Danishefsky, S. J.; Chen, G.; Chen, J.; Wan, Q. J. Org. Chem.
2006, 71, 8244–8249.
(21) Walsh, C. T.; Lin, H. J. Am. Chem. Soc. 2004, 126, 13998–14003.
(22) For reviews dedicated to click neoglycoconjugates, see: (a) Dondoni,
A. Chem. Asian J. 2007, 2, 700–708. (b) Field, R. A.; Nepogodiev, S. A.;
Dedola, S. Org. Biomol. Chem. 2007, 5, 1006–1017.
Figure 2. Synthesized peptides 1, 2, and 3.
(23) Macmillan, D.; Blanc, J. Org. Biomol. Chem. 2006, 4, 2847–2850.
(24) Davis, B. G.; van Kasteren, S. I.; Kramer, H. B.; Gamblin, D. P.
These three peptides contained all 20 genetically encoded
amino acids found in proteins, a thioester moiety (peptide
1), a Thz- moeity (peptide 2), and a Cys(Acm) (peptide 3).
Nat. Protocols 2007, 2, 3185–3194
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(25) Davis, B. G.; van Kasteren, S. I.; Kramer, H. B.; Jensen, H. H.;
Campbell, S. J.; Kirkpatrick, J.; Oldham, N. J.; Anthony, D. C. Nature 2007,
446, 1105–1109
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(26) Moroder, L.; Musiol, H.; Dong, S.; Kaiser, M.; Bausinger, R.;
Zumbusch, A.; Bertsch, U. ChemBioChem 2005, 6, 625–628.
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(28) Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H.
Int. J. Pept. Res. Ther. 2007, 13, 31–44.
Org. Lett., Vol. 11, No. 22, 2009
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Table 1. Synthesis of Neoglycopeptides via Click Reaction of Peptides 1 and 3 with GalNAc-N3
entry
peptidea
catalyst
solvent
temperature
time
product yield (%)b
1
2
3
4
5
3
3
3
1
1
20 mM CuI/30 mM DIEA
DMF
MeCN/H2O 1:1
6 M GnHCl/0.2 M Na2HPO4, pH 7
MeCN/H2O 1:1
6 M GnHCl/0.2 M Na2HPO4, pH 7
25 °C
25 °C
25 °C
35 °Cc
35 °Cc
>12 h
4 h
3 h
7 h
5 h
<40
>85
>95
>85
>95
20 mM CuSO4/30 mM NaAsc
20 mM CuSO4/30 mM TCEP
20 mM CuSO4/30 mM NaAsc
20 mM CuSO4/30 mM TCEP
a Reactions were performed on 3 mM concentration of peptide using GalNAc-N3 (5 mM). b Based on integration of HPLC traces, not isolated. c No
reaction was observed after 1 h at 25 °C, at which point the temperature was increased to 35 °C.
N-Boc-L-propargylglycine (Boc-L-Pra-OH) was synthesized
using established methods and was readily incorporated into
the synthetic peptides. The propargyl group was entirely
compatible with in situ Boc SPPS conditions, commonly used
for the synthesis of peptide thioesters as well as the final
HF treatment for deprotection and cleavage of the peptide
from the resin support. N-Acetylgalactosamine azide (Gal-
NAc-N3) was chosen as the azide-containing sugar compo-
nent. GalNAc is a common and well-studied motif for
O-glycans found in higher eukaryotes,29 thus it was deemed
a useful neoglycan for our model study.
With the propargyl-containing peptides in hand, we set
out to investigate the use of click chemistry on peptides 1
and 3 containing unprotected side chains (Table 1). After
extensive investigation, it was found that a ratio of 20 mM
of Cu(I) “catalyst” to 3 mM of unprotected peptide was
optimal for the click reaction to occur within an acceptable
time period (hours). It was also found that in situ reduction
of CuSO4 by NaAsc or TCEP proved to be a much better
catalyst than the use of the CuI/DIEA system. Data for the
click product from these two peptides are in Supporting
Information, Figures S2 and S3. The successful addition of
GalNAc-N3 to peptides 1 and 3 demonstrated that click
reactions were compatible with all the unprotected side chain
functionalities, as well as with an N-terminal Cys residue, a
Cys(Acm), and a peptide-thioester moiety, all of which are
important functionalities for the synthesis of proteins using
native chemical ligation.
after 1 h at room temperature (see Supporting Information).
Thus, the click reaction cannot be used to directly make a
Thz-neoglycopeptide from a Thz-(propargyl)peptide. How-
ever, if necessary, the resulting Cys-neoglycopeptide can
subsequently be converted to the Thz-neoglycopeptide with
exquisite specificity simply by addition of formaldehyde.30
The results reported above show that click reactions with
GalNAc-N3 can be successfully carried out on unprotected
propargyl-containing peptides that contain any of the 20
amino acids found in proteins, in the presence of high
concentrations of CuSO4 and TCEP, in 6 M GnHCl/0.2 M
Na2HPO4 buffer at pH 7. These are the same solvent
conditions used for native chemical ligation. It has become
standard practice to use TCEP as an additive in native
chemical ligation reactions, to avoid the formation of mixed
disulfides between the (4-carboxymethyl)thiophenol catalyst
(mercaptophenylacetic acid, MPAA) and cysteine-containing
reactant/product peptides.33 We therefore postulated that it
should be possible to devise conditions under which con-
secutive native chemical ligation and click reactions could
be carried out in a one-pot fashion, without intervening
isolations.
Peptide-thioester 1 (3 mM) and Cys-peptide 3 (3 mM)
were dissolved in 6 M GnHCl/0.2 M Na2HPO4 (200 µL)
with TCEP (40 mM) and degassed with helium for 30 min.
MPAA (20 mM) was added, and the pH was adjusted to
7.0. Ligated product was detected within 5 min (Figure 3A),
and after overnight reaction the ligation was complete (Figure
3B). CuSO4 (20 mM) was added to the reaction mixture,
followed by GalNAc-N3 (10 mM). Within 2 h at room
temperature more than 50% of the starting ligated peptide
was converted into the desired double triazole product, and
two different single triazole products (from reaction at Pra9
and Pra19) were also observed (Figure 3C). After 6 h, reaction
was complete, and the desired double triazole ligated
neoglycopeptide product was readily purified by reverse-
phase HPLC (Figure 3D and 3E). The desired product, a 23
The Thz- group is also important for the synthesis of large
proteins by native chemical ligation.30-32 To test whether
Thz- to Cys- conversion was occurring under optimized click
conditions, the Thz-peptide 2 was reacted with GalNAc-N3
in the presence of CuSO4 and TCEP. Both the Thz- to Cys-
conversion and the click reaction were essentially complete
(29) Haase, C.; Seitz, O. Top. Curr. Chem. 2007, 267, 1–36.
(30) Rose, K.; Vizzavona, J.; Villain, M. Chem. Biol. 2001, 8, 673–
679
.
(31) Bang, D.; Kent, S. B. H. Angew. Chem., Int. Ed. 2004, 43, 2534–
2538
.
(32) Durek, T.; Torbeev, V. Y.; Kent, S. B. H. Proc. Natl. Acad. Sci.
(33) Johnson, E. C.; Kent, S. B. H. J. Am. Chem. Soc. 2006, 128, 6640–
6646.
2007, 104, 4846–4851
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Org. Lett., Vol. 11, No. 22, 2009

Figure 3. One-pot consecutive native chemical ligation plus click reactions to give a double triazole neoglycopeptide ligated product.
Analytical HPLC profiles (λ ) 214 nm) together with ESI MS data (inset). (A) t ) 5 min. (B) t ) overnight reaction. At this stage, CuSO4
(20 mM) and GalNAc-N3 (10 mM) were added. (C) t ) 2 h after beginning of click reaction. Peak 1 is the desired double triazole product;
peaks 2 and 3 are single triazole products at different reaction sites; and peak 4 is the starting ligated peptide. (D) Total crude products at
t ) 6 h; formation of the double trizaole neoglycopeptide product was completed. (The broad, early eluting peaks in (C) and (D) are
chromatography artifacts.) (E) Purified 23 residue double trizaole neoglycopeptide product from one-pot native chemical ligation plus click
chemistry reactions.
residue neoglycopeptide, had an observed mass of 3252.0
( 0.4 Da (theoretical mass 3252.6 Da (average isotope
composition)).
ligation, and kinetically controlled ligation.34 Syntheses
involving the separate functionalization of each peptide
building block with distinct sugars or using more complex
carbohydrate moieties are underway in our laboratories, using
erythopoietin as an initial target.
In conclusion, we have described a novel combination of
click chemistry and native chemical ligation for the fully
convergent synthesis of neoglycopeptides. The two ligation
chemistries are fully compatible, and can be carried out in
any order. The optimized click conditions reported here are
compatible with all 20 genetically encoded amino acids,
N-terminal Cys, the peptide-thioester moiety, and a Cys(Acm)
residue, all of which are used for chemical protein synthesis
by native chemical ligation. Native chemical ligation and
click chemistry have been successfully combined to affect
an efficient one-pot synthesis of a neoglycopeptide containing
two sugars, starting from three unprotected building blocks
(two peptides and a sugar azide). These findings provide a
versatile chemical toolkit for the fully convergent synthesis
of neoglycoproteins using click chemistry, native chemical
Acknowledgment. We thank the Maurice Wilkins Centre
for Molecular Discovery for financial assistance.
Supporting Information Available: Experimental data
for syntheses of building blocks, model peptides 1, 2, and
3, and the click chemistry and ligation studies (including
HPLC traces). This material is available free of charge via
the Internet at http://pubs.acs.org.
OL902131N
(34) Bang, D.; Pentelute, B. L.; Kent, S. B. H. Angew. Chem., Int. Ed.
2006, 45, 3985–3988.
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