Synthesis of MUC1 Neoglycopeptides using efficient microwave-enhanced chaotrope-assisted click chemistry

Article abstract of DOI:10.1039/c0ob01043j

The first synthesis of click neoglycopeptide analogues of the biologically relevant MUC1 sequence is reported. In the process, microwave-enhanced chaotrope-assisted click reaction conditions that may be used on a routine basis for the synthesis of click p

Full text of DOI:10.1039/c0ob01043j

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 1621
www.rsc.org/obc
PAPER
Synthesis of MUC1 Neoglycopeptides using efficient microwave-enhanced
chaotrope-assisted click chemistry†
Dong Jun Lee, Paul W. R. Harris and Margaret A. Brimble*
Received 18th November 2010, Accepted 29th November 2010
DOI: 10.1039/c0ob01043j
The first synthesis of click neoglycopeptide analogues of the biologically relevant MUC1 sequence is
reported. In the process, microwave-enhanced chaotrope-assisted click reaction conditions that
may be used on a routine basis for the synthesis of click peptide conjugates have been developed. The
convergent route for the synthesis of neoglycopeptides using these reaction conditions enables the facile,
rapid, and highly efficient preparation of focused neoglycopeptide libraries of defined chemical
structure for biological evaluation.
of an organic azide with a terminal alkyne.7 Rutjes et al.8 made
use of protected building blocks to synthesize a series of triazole-
Introduction
linked glycosyl amino acids and dipeptides. Danishefsky’s group9
and Walsh et al.10 have independently reported the union of
unprotected carbohydrates and peptides containing a limited
range of side chains using click chemistry. Macmillan’s group
reported the synthesis of a glycosyl amino acid linked via a
triazole linkage which was incorporated into solid-phase peptide
synthesis.11 Moroder et al.12 have utilized click chemistry to
synthesize semi-synthetic lipoproteins. Davis et al.13 have also
utilized click chemistry to demonstrate the diversity of post-
translational chemical protein modification. Besides these ex-
amples, click chemistry is also widely used in other areas such
as analytical and material sciences.14,15 An excellent example
of this was demonstrated by Ravoo et al. who used click
chemistry to prepare carbohydrate microarrays by microcontact
printing of carbohydrate alkyne conjugates onto azide self-
assembled monolayers.15 Recently we reported the synthesis of
neoglycopeptides using one-pot native chemical ligation and
click chemistry using unprotected sugar-azides and propargylated
peptides.16 Click analogues of anti-freeze glycopeptides have also
been reported by our group with azido-alanine incorporated in
the peptide chain; however, one limitation is that the azide moiety
is susceptible to elimination during Fmoc deprotection cycles in
SPPS.17
We therefore envisaged that the complementary click chemistry
using unprotected sugar-azides and peptides containing one or
more propargyl groups would facilitate rapid synthesis of a library
of MUC1 neoglycopeptides with a variable number of GalNAca1-
O-Thr/Ser mimics for biological evaluation (Fig. 1). Surprisingly,
click mimics of the biologically relevant MUC1 sequence have
not been reported to date. Importantly, the chemistry reported
herein is amenable to the synthesis of a focused library of
neoglycopeptides of defined structures, thereby overcoming the
problems of working with native glycoproteins that often exist as
a variety of glycoforms.18
The membrane-bound tumor-associated MUC1 glycoproteins
occur ubiquitously in almost all epithelial tissues of most or-
gans, and are also excessively expressed on tumor tissue.1 They
are also involved in cell signalling events, and their aberrant
expression has been found to aid the metastatic spread of
tumor cells.2 The extracellular domain of MUC1 contains a
variable number of tandem repeat domains of 20 amino acids
(HGVTSAPDTRPAPGSTAPPA), possessing five potential O-
glycosylation sites on Thr and Ser.3 The altered glycan patterns of
MUC1 on tumour cells are caused by down-regulation of b6-N-
acetylglucosaminyltransferase (b6GnT) and the concomitant up-
regulation of a3-sialyltransferase (a3ST) resulting in shortened
sialylated oligosaccharides (sialyl-TN and TN antigens) instead
of the longer chains found to be expressed on normal cells.4
Over-expression of these antigens on tumor cells renders them
attractive candidates for selective cancer vaccines, and these have
been studied by several research groups.5
Difficulties experienced in making the native GalNAca1-O-
Thr/Ser building blocks in the large quantities needed for Fmoc
SPPS prompted us to explore more convergent methods to obtain
MUC1-like glycopeptides. Previously, glycoconjugates containing
a variety of unnatural linkages between the carbohydrate and
aglycone moieties have been explored.6 When carried out in a
convergent manner this mimicry allows rapid and efficient access
to a wide variety of neoglycopeptides that may retain 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)
The University of Auckland, 23 Symonds Street, Auckland, New Zealand.
E-mail: m.brimble@auckland.ac.nz
† Electronic supplementary information (ESI) available: Details of syn-
thesis of propargylated peptides, click reaction procedures, HPLC and
ESI-MS profiles of click neoglycopeptides are provided. See DOI:
10.1039/c0ob01043j
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1621–1626 | 1621
©

View Article Online
Table 1 Summary of click reactions. All click reactions were carried out
in 6 M GnHCl/0.2 M Na2HPO4 buffer at pH 7 with microwave irradiation
(25 W)
Peptide
(3mM) CuSO4 conc./mM TCEP conc./M Time takena/h Yield (%)b
8
20
40
40
60
80
100
20
40
40
60
80
100
1.0
2.5
4.0
4.5
3.5
4.0
95 (43)
95 (36)
95 (34)
95 (28)
95 (34)
90 (30)
9
Fig. 1 Synthesis of click neoglycopeptides of MUC1 sequence.
10
11
12
13
Synthesis of sugar-azide and propargylated peptides
The key GalNAca1-O-Thr/Ser neoglycosyl amino acid mimic 6
was designed to incorporate an N-acetyl group at C-2, and an
a-O-glycosidic link at the C-1 anomeric position (Scheme 1).
Azido-acetate 1 was formed from galactosamine hydrochloride
via diazo-transfer then acetylation. Glycosylation of alcohol 2
with 1 afforded tosylate 3 as a 2 : 1 mixture of a : b anomers,
which were separated to give the a-anomer in 41% yield. The
a-anomer was subjected to reductive acetylation in 87% yield,
then tosylate 4 underwent subsequent azide displacement and
deacetylation to give the desired 3¢-azido-1¢-propyl-2-acetamido-
2-deoxy-a-D-galactopyranoside 6 ready for reaction with the
synthetic propargylated peptides.
a Time taken to completion observed by HPLC. b % Conversion from
starting peptide to product observed by HPLC. Isolated yield after reverse
phase HPLC chromatography and lyophilisation, depicted in parentheses.
quantities of CuSO4/NaAsc are undesirable in bioconjugation
due to copper-mediated generation of reactive oxygen species.19
Dehydroascorbate and other ascorbate byproducts can also react
with lysine amino and arginine guanidino groups, resulting in
covalent modification and aggregation of proteins.20 Given the
drawbacks, we therefore focused on a much milder and biologically
friendly reductant, namely tris(carboxyethyl)phosphine (TCEP).
TCEP is often used in bioconjugation chemistry and native
chemical ligation reactions,21 and its oxidised byproduct, TCEP
oxide, is relatively inert. When aqueous CuSO4 and TCEP
were premixed before addition to the peptide solution, blue
aggregates were observed suggesting incomplete reduction of
CuSO4 by TCEP. After exploring several different solvent systems,
it was established that use of 6 M guanidine hydrochloride
(GnHCl)/0.2 M Na2HPO4 solution helped to solubilise the blue
aggregates facilitating complete reduction of the CuSO4 almost
immediately upon addition. In addition to its use as a common
solvent for the native chemical ligation of peptides,22 GnHCl is an
important chaotrope used to minimize aggregation in proteins and
to facilitate chemical modification.23 In our study, use of GnHCl
was critical to the success of the click chemistry. It appeared to
accelerate the reduction of CuSO4 by TCEP, and importantly the
Cu(I) species formed seemed to be less susceptible to air oxidation.
Furthermore, it is noteworthy that a broad peak was observed
on the HPLC chromatograms (see Scheme 3A and the ESI†)
where CuSO4 and TCEP coexisted with GnHCl that was not
present in aqueous buffer solutions lacking GnHCl. When the
reaction mixture was blue (i.e. suggesting that the Cu(II) species
was present) this broad peak was not observed by HPLC (see the
ESI†) and the click reaction did not progress to completion.
Click reactions on all peptides 8–13 using 20 mM of both CuSO4
and TCEP per 3 mM of propargyl moiety proceeded cleanly to
afford the desired click peptides at both room temperature and
50 ◦C. In addition, microwave irradiation reduced the reaction
times, often by 2–4 fold compared to the use of conventional heat-
ing, with slightly improved purities. Using microwave conditions,
the key click reactions were successfully executed on all mono-,
di-, tri-, tetra-, and penta-propargylated peptides in a matter of
hours (Table 1). Scheme 3 highlights the success of our multiple,
contiguous click reaction on a penta-propargylated peptide 13.
The reaction progression was monitored by the presence of
multiple peaks in the HPLC chromatogram at t = 30 min that
corresponded to mono-, di-, tri-, and tetra-clicked intermediates
Scheme
1
(a) i. Imidazole-1-sulfonyl azide hydrochloride, K2CO3,
CuSO4·5H2O, MeOH, 3 h, rt.; ii. pyridine/Ac2O, DMAP, 12 h, rt., 83%;
(b) BF3·OEt2, DCM, 48 h, 40 ◦C, 41% (a purified); (c) THF/Ac2O/AcOH
(3/2/1 (v/v/v)), powdered Zn, aq. CuSO4 (sat.), 1 h, rt., 87%; (d) NaN3,
nBu4N+HSO4-, H2O/DCM, 16 h, rt., 80%; (e) 1 M NaOMe, 3 h, rt., 98%.
A series of propargylated peptide derivatives of the MUC1
sequence were synthesized on amino-functionalized polystyrene
resin containing the acid labile hydroxymethylphenoxypropionic
acid (HMPP) linker using microwave enhanced Fmoc SPPS
(Scheme 2). Fmoc-L-propargylglycine16 7 was incorporated into
the peptide chain using the conditions shown in Scheme 2,
replacing Thr/Ser at selected positions in the MUC1 sequence.
These replacements were chosen to aid the determination of which
specific sites were important for incorporation of the GalNAc
mimic.
Click neoglycopeptides
Initial attempts to carry out click reactions using catalytic amounts
of CuSO4 and sodium ascorbate (NaAsc) in aqueous phosphate
buffer were unsuccessful, even at elevated temperatures using
microwave irradiation. Our previous studies16 of click reactions
involving unprotected peptides and sugars highlighted the need
for more than equimolar amounts of Cu(I) species per propargyl
group, possibly due to chelation. The desired click product
was only observed when a large excess of CuSO4/NaAsc was
used; however, significant formation of side products compli-
cated subsequent purification (see the ESI†). Furthermore, large
1622 | Org. Biomol. Chem., 2011, 9, 1621–1626
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Scheme 2 Synthesis of propargylated peptides. (a) 20% (v/v) piperidine in DMF, MW 25 W; (b) Fmoc-Aaa-OH (incl. 7), HBTU, DIPEA in DMF, MW
25 W; (c) TFA/TIS/H2O (38/1/1), 2.5 h.
with only a minor amount of starting peptide remaining (Scheme
3B). At t = 4 h, the reaction was essentially complete affording the
desired penta-clicked neoglycopeptide 14 (Scheme 3C), which was
isolated by solid phase extraction and then lyophilized. The crude
product was formed in 90% yield, and this was further purified
by reverse phase HPLC to give the neoglycopeptide in high purity
and isolated yield (Table 1).
reaction conditions developed in this study enable the facile,
rapid, and highly efficient preparation of focused neoglycopeptide
libraries of defined chemical structure for biological evaluation.
Access to neoglycopeptides of defined structures overcomes the
inherent problems of working with native glycoproteins that are
often complicated by their existence in a variety of glycoforms.
Biological evaluation of the synthetic click neoglycopeptides
prepared herein is in progress.
Conclusions
Experimental Section
In conclusion, we have successfully synthesized the first click
neoglycopeptide analogues of the biologically relevant MUC1
sequence. In the process, a new combination of click reaction
conditions that may be used on a routine basis for the synthesis
of click neopeptides using unprotected sugar-azides and peptides
has been developed. This convergent route for the synthesis of
neoglycopeptide analogues using click chemistry and the improved
General Information
All reagents were purchased as reagent grade and used without
further purification. Analytical thin layer chromatography was
performed using 0.2 mm plates of Kieselgel F254 (Merck) and
compounds were visualised by ultra-violet fluorescence or by
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1621–1626 | 1623
©

View Article Online
Scheme 3 Synthesis of penta-clicked product 14 with reaction profile HPLC traces. A) t = 5 min. * = Broad peak observed when CuSO4/TCEP was
added to 6 M GnHCl/0.2 M Na2HPO4 solution. ** = GalNAca1-propylazide 6 peak. B) t = 30 min. Multiple peaks eluting before the starting peptide
peak correspond to mono-, di-, tri-, and tetra-clicked peptide intermediates. C) t = 240 min. Penta-clicked neoglycopeptide peak observed with essentially
no starting/intermediate peptide peaks remaining. D) Purified penta-clicked neoglycopeptide by RP-HPLC.
staining with 4% sulfuric acid in ethanol or ethanolic ninhydrin
solution (0.3% ninhydrin in ethanol + 1% v/v acetic acid), followed
by heating the plate for a few minutes. Flash chromatography was
performed using Kieselgel F254 S 0.063–0.1 mm (Riedel de Hahn)
silica gel with indicated solvents. Infrared spectra were obtained
using a Perkin Elmer Spectrum One Fourier Transform infrared
spectrometer as a thin film between sodium chloride plates.
Absorption maxima are expressed in wavenumbers (cm-1) with
the following abbreviations: s = strong, m = medium, w = weak,
br = broad and v = varying. Optical rotations were determined
at the sodium D line (589 nm) at 20 ◦C with a Perkin Elmer
341 polarimeter and are given in units of 10-1 deg cm2 g-1.
Nuclear magnetic resonance (NMR) spectra were recorded as
indicated on a Bruker AVANCE DRX300 (1H, 300 MHz, 13C,
75 MHz). Chemical shifts are reported in parts per million
(ppm) relative to the tetramethylsilane signal recorded at dH
0.00 ppm in CDCl3/SiMe4 solvent or were referenced to the
residual water signal at dH 4.79 ppm in D2O solvent. The 13C
values were referenced to the residual chloroform signal at dC
77.0 ppm in CDCl3/SiMe4 solvent. 1H NMR shift values are
reported as chemical shift (dH), relative integral, multiplicity (s,
singlet; d, doublet; t, triplet; m, multiplet; br s, broad singlet;
dd, doublet of doublets, ddd, doublet of doublets of doublets),
coupling constant (J in Hz) and assignments. 13C values are
1624 | Org. Biomol. Chem., 2011, 9, 1621–1626
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
reported as chemical shift (dC), degree of hybridisation and
assignment.
in a mixture of THF/Ac2O/AcOH (3/2/1 (v/v/v), 5 mL), then
powdered zinc (100 mg, 1.53 mmol) was added. After addition of
sat. CuSO4 (200 mL) the reaction mixture was left to stir for 1 h at
room temperature. The reaction mixture was then filtered through
Synthesis of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-a,b-D-
galactopyranosyl acetate 1
R
Celiteꢀ, diluted with CH2Cl2 and co-evaporated with PhMe (2 ¥
20 mL). The crude product was purified by flash chromatography
to afford the title compound 4 as a yellow oil (71.0 mg, 0.13 mmol,
87%): (Found: MH+, 560.1790, C24H34NO12S requires 560.1796);
[a]D20 +76.5 (c 1.0 in CHCl3); vmax (film)/cm-1 2964 (s, C–H alkane),
1742 (s, C O ester), 1661 (s, C O amide), 1537 (m, C O amide),
Imidazole-1-sulfonyl azide hydrochloride24 (2.52 g, 12.0 mmol)
was added to D-galactosamine hydrochloride (2.15 g, 10.0 mmol),
K2CO3 (3.73 g, 27.0 mmol), and CuSO4·5H2O (25.0 mg,
0.010 mmol) in MeOH (50 mL) and the mixture stirred at room
temperature for 2 h. The mixture was concentrated and co-
evaporated with PhMe (2 ¥ 50 mL). The resulting residue was
suspended in pyridine (40 mL) at 0 ◦C, then acetic anhydride
(30 mL) was added and the reaction mixture stirred at room
temperature for 16 h. The reaction mixture was diluted with
EtOAc (200 mL), washed with 10% HCl(aq) (2 ¥ 50 mL), sat.
NaHCO3 (50 mL), dried with MgSO4 then concentrated. The
crude product was purified by flash chromatography to afford
the title compound 1 as a pale-yellow oil (3.08 g, 8.25 mmol, 83%
(both a and b anomers (2 : 1 a : b) were obtained). 1H NMR data
was in agreement with that reported in the literature.25
1436 (m, ArC C), 1368, 1175 (s, O
S O), 1229, 1132 (s, C–O),
1041 (s, C–N); dH (300 MHz, CDCl3) 1.88–1.90 (2H, m, -CH2-
CH2-OTs), 1.93, 1.95, 1.99, 2.11 (4 ¥ 3H, s, COCH3), 2.41 (3H,
s, PhCH3), 3.47–3.54 (1H, m, -O–CH2-CH2-CH2-OTs), 3.72-3.79
(1H, m, -O–CH2-CH2-CH2-OTs), 3.99–4.07 (4H, m, -CH2-OTs,
H-6A, B), 4.20–4.27 (1H, m, H-5), 4.51–4.59 (1H, m, H-2), 4.81
(1H, d, J 3.3, H-1), 5.07 (1H, dd, J 11.4, 3.3, H-3), 5.30 (1H, d, J
3.0, H-4), 5.99 (1H, d, J 9.6, NH), 7.32 (2H, d, J 8.1, ArH), 7.73
(2H, d, J 8.1, ArH); dC (75 MHz, CDCl3) 20.5, 20.6, 20.6, 21.5
(COCH3), 23.0 (PhCH3), 28.5 (-CH2-CH2-OTs), 47.4 (C-2), 61.8
(O-CH2-CH2-CH2-OTs), 63.4 (-CH2-OTs), 66.7 (C-5), 66.8 (C-6),
67.2 (C-4), 68.3 (C-3), 97.6 (C-1), 127.6 (Ar-CH), 129.9 (Ar-CH),
132.7 (quat., Ar–C), 145.0 (quat., Ar–C), 170.2, 170.3, 170.4, 170.7
(quat., COCH3).
Synthesis of 3¢-p-toluenesulfonyl-1¢-propyl-3,4,6-tri-O-acetyl-2-
azido-2-deoxy-a-D-galactopyranoside 3
3,4,6-Tri-O-acetyl-2-azido-2-deoxy-a,b-D-galactopyranosyl ace-
tate
1 (0.80 g, 2.14 mmol) and 3¢-hydroxy-1¢-propyl p-
Synthesis of 3¢-azido-1¢-propyl-3,4,6-tri-O-acetyl-2-acetamido-2-
deoxy-a-D-galactopyranoside 5
toluenesulfonate 2 (0.74 g, 3.21 mmol) were dried together in the
presence of P2O5 under high vacuum for 16 h prior to the reaction.
The mixture was dissolved in dry CH2Cl2 (25 mL) and stirred
under argon for 30 min at 0 ◦C. BF3·Et2O (2.63 mL, 21.4 mmol)
Tetrabutylammonium hydrogen sulfate (137 mg, 0.40 mmol) was
added to a solution of 3¢-p-toluenesulfonyl-1¢-propyl-3,4,6-tri-
O-acetyl-2-acetamido-2-deoxy-a-D-galactopyranoside 4 (150 mg,
0.27 mmol) in CH2Cl2 (10 mL). NaN3 (170 mg, 2.62 mmol) in
H2O (10 mL) was added and the reaction mixture was stirred
vigorously for 16 h. The aqueous layer was extracted with CH2Cl2
(2 ¥ 50 mL), and the combined organic layer was dried with
MgSO4 and concentrated. The crude product was purified by flash
chromatography to afford the title compound 5 as a yellow oil
(92.2 mg, 0.21 mmol, 80%): (Found: MH+, 431.1757, C17H27N4O9
requires 431.1773); [a]2D0 +80.1 (c 1.0 in CHCl3); vmax (film)/cm-1
2935 (s, C–H alkane), 2097 (s, N3), 1743 (s, C O ester), 1660 (s,
◦
was added slowly and the reaction mixture was stirred at 40 C
for 48 h. The solution was cooled to room temperature then
neutralized with sat. NaHCO3. The product was extracted with
CH2Cl2 (2 ¥ 75 mL), washed with sat. NaHCO3 (50 mL), brine
(25 mL), dried with MgSO4 then concentrated. The crude product
was purified by flash chromatography to afford the title compound
3 as a colorless solid (0.48 g, 0.88 mmol, 41%): (Found: [M+Na]+,
566.1422, C22H29N3NaO11S requires 566.1415); mp 105–107 ◦C;
[a]D20 +94.9 (c 1.0 in CHCl3); vmax (film)/cm-1 2964 (s, C H alkane),
2110 (s, N3), 1750, 1734 (s, C O ester), 1465, 1404 (m, ArC C),
C
O amide), 1535 (m, C O amide), 1435 (m, ArC C), 1219,
1364, 1175 (s, O
S O), 1229, 1128 (s, C–O), 1035 (s, C–N); dH
1131 (s, C–O), 1042 (s, C–N); dH (300 MHz, CDCl3) 1.93 (2H, p,
J 6.3, -CH2-CH2–N3), 1.97, 1.99, 2.05, 2.16 (4 ¥ 3H, s, COCH3),
3.38–3.48 (2H, m, -CH2-N3), 3.51–3.58 (1H, m, -O–CH2-CH2-
CH2–N3), 3.78–3.86 (1H, m, -O–CH2-CH2-CH2–N3), 4.06–4.19
(3H, m, H-5, H-6A, B), 4.52–4.60 (1H, m, H-2), 4.91 (1H, d, J 3.6,
H-1), 5.14 (1H, dd, J 11.4, 3.3, H-3), 5.38 (1H, d, J 3.0, H-4), 5.97
(1H, d, J 9.6, NH); dC (75 MHz, CDCl3) 20.4, 20.4, 20.4, 22.9
(COCH3), 28.3 (-CH2-CH2–N3), 47.5 (C-2), 48.2 (-CH2–N3), 61.8
(C-6), 65.1 (O-CH2-CH2-CH2–N3), 66.6 (C-5), 67.1 (C-4), 68.1
(C-3), 97.5 (C-1), 169.9, 170.0, 170.2, 170.6 (quat., COCH3).
(300 MHz, CDCl3) 2.01–2.02 (2H, m, -CH2-CH2-OTs), 2.04, 2.05,
2.13 (3 ¥ 3H, s, COCH3), 2.45 (3H, s, PhCH3), 3.51–3.56 (1H, m, -
O–CH2-CH2-CH2-OTs), 3.60 (1H, dd, J 11.0, 3.6, H-2), 3.78–3.85
(1H, m, -O–CH2-CH2-CH2-OTs), 4.04–4.12 (4H, m, -CH2-OTs,
H-6A, B), 4.17 (1H, t, J 6.0, H-5), 4.93 (1H, d, J 3.3, H-1), 5.25
(1H, dd, J 11.1, 3.3, H-3), 5.38 (1H, d, J 3.0, H-4), 7.37 (2H, d,
J 7.8, ArH), 7.80 (2H, d, J 8.1, ArH); dC (75 MHz, CDCl3) 20.4,
20.4, 20.5 (COCH3), 21.4 (PhCH3), 28.9 (-CH2-CH2-OTs), 57.2
(C-2), 61.6 (-CH2-OTs), 64.0 (O-CH2-CH2-CH2-OTs), 66.6 (C-5),
66.9 (C-6), 67.4 (C-4), 67.9 (C-3), 98.0 (C-1), 127.7 (Ar-CH), 129.8
(Ar-CH), 132.7 (quat., Ar–C), 144.8 (quat., Ar–C), 169.6, 169.9,
170.3 (quat., COCH3).
Synthesis of 3¢-azido-1¢-propyl-2-acetamido-2-deoxy-a-D-
galactopyranoside 6
Synthesis of 3¢-p-toluenesulfonyl-1¢-propyl-3,4,6-tri-O-acetyl-2-
acetamido-2-deoxy-a-D-galactopyranoside 4
3¢-Azido-1¢-propyl-3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-a-D-
galactopyranoside 5 (220 mg, 0.51 mmol) was stirred in 1 M
NaOMe (5 mL, pH 11.5) for 3 h at room temperature. The
reaction mixture was acidified with H+ Dowex resin then filtered.
3¢-p-Toluenesulfonyl-1¢-propyl-3,4,6-tri-O-acetyl-2-azido-2-deo-
xy-a-D-galactopyranoside 3 (80.0 mg, 0.15 mmol) was dissolved
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1621–1626 | 1625
©

View Article Online
The filtrate was concentrated, diluted with Milli Q water (15 mL)
and lyophilized to afford the pure title compound 6 as a colorless
solid (152 mg, 0.50 mmol, 98%): (Found: [M+◦Na]+, 327.1273,
C11H20N4NaO6 requires 327.1275); mp 142–144 C; [a]2D0 +160.7
(c 1.0 in H2O); vmax (film)/cm-1 3297 (s, O–H), 2924 (s, C–H alkane),
2097 (s, N3), 1616 (s, C O amide), 1553 (m, C O amide), 1448
(m, ArC C), 1115, 1065, 1017 (s, alcohol C–O), 1037 (s, C–N);
dH (300 MHz, D2O) 1.96 (2H, p, -CH2-CH2–N3), 2.11 (3H, s,
COCH3), 3.46–3.64 (2H, m, -CH2-N3, -O–CH2-CH2-CH2–N3),
3.79–3.91 (3H, m, -O–CH2-CH2-CH2–N3, H-6A, B), 3.96–4.06 (3H,
m, H-3, H-4, H-5), 4.22 (1H, dd, J 12.0, 3.0, H-2), 4.97 (1H, d, J
3.0, H-1); dC (75 MHz, D2O) 22.0 (COCH3), 28.0 (-CH2-CH2–N3),
48.2 (-CH2–N3), 50.0 (C-2), 61.3 (C-6), 65.0 (O-CH2-CH2-CH2–
N3), 67.7 (C-5), 68.5 (C-4), 71.0 (C-3), 97.0 (C-1).
49, 3688–3692; (c) C. K. Y. Chun and R. J. Payne, Aust. J. Chem., 2009,
62, 1339–1343; (d) B. L. Wilkinson, C. K. Y. Chun and R. J. Payne, J.
Pept. Sci., 2010, DOI: 10.1002/bip.21471; (e) B. L. Wilkinson, L. R.
Malins, C. K. Y. Chun and R. J. Payne, Chem. Commun., 2010, 46,
6249–6251.
6 (a) D. Specker and V. Wittmann, Top. Curr. Chem., 2007, 267, 65–107;
(b) F. Nicotra, L. Cipolla, F. Peri, B. La, Ferla and C. Redaelli, Adv.
Carbohydr. Chem. Biochem., 2007, 61, 353–398.
7 (a) C. W. Tornoe, C. Christensen and M. Meldal, J. Org. Chem., 2002,
67, 3057–3064; (b) V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B.
Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596–2599; (c) R. Huisgen,
Chem. Ber., 1967, 100; (d) R. Huisgen, Pure Appl. Chem., 1989, 61;
(e) M. Meldal and C. W. Tornøe, Chem. Rev., 2008, 108, 2952–3015;
(f) H. C. Kolb and K. B. Sharpless, Drug Discovery Today, 2003, 8,
1128–1137; (g) V. D. Bock, H. Hiemstra and J. H. van Maarseveen,
Eur. J. Org. Chem., 2006, 2006, 51–68; (h) J. Lutz, Angew. Chem., Int.
Ed., 2007, 46, 1018–1025; (i) G. o. Pint, I. Bereczki, G. Batta, R. a. v, F.
Sztaricskai, E. e. Roth, E. Ostorhi, F. Rozgonyi, L. Naesens, M. Szarvas,
Z. Boda and P. Herczegh, Bioorg. Med. Chem. Lett., 20, 2713–2717;
(j) A. Dondoni, Chem.–Asian J., 2007, 2, 700–708; (k) S. Dedola, S. A.
Nepogodiev and R. A. Field, Org. Biomol. Chem., 2007, 5, 1006–1017.
8 F. P. J. T. Rutjes, F. L. van Delft, R. H. Blaauw, P. J. L. M. Quaedflieg,
A. R. Keereweer, S. Groothuys and B. H. M. Kuijpers, Org. Lett., 2004,
6, 3123–3126.
General click chemistry procedure
Peptide (3 mM) and CuSO4/TCEP (20 mM per 3 mM of propargyl
group) were mixed in a degassed solution of 6 M GnHCl/0.2 M
Na2HPO4 buffer. After 30 min, 3¢-azido-1¢-propyl-2-acetamido-2-
deoxy-a-D-galactopyranoside 6 (1.5 equiv. per propargyl group)
was added and the reaction was carried out under argon with mi-
crowave irradiation (25 W). The reaction progress was monitored
by analytical RP-HPLC, and upon completion of the reaction
the crude product was isolated by solid phase extraction then
lyophilized. The crude product was purified by semi-prep RP-
HPLC to afford the desired clicked neoglycopeptide products.
9 Q. Wan, J. Chen, G. Chen and S. J. Danishefsky, J. Org. Chem., 2006,
71, 8244–8249.
10 H. Lin and C. T. Walsh, J. Am. Chem. Soc., 2004, 126, 13998–14003.
11 J. Blanc and D. Macmillan, Org. Biomol. Chem., 2006, 4, 2847–2850.
12 L. Moroder, H. Musiol, S. Dong, M. Kaiser, R. Bausinger, A.
Zumbusch and U. Bertsch, ChemBioChem, 2005, 6, 625–628.
13 (a) D. P. Gamblin, H. B. Kramer, S. I. van Kasteren and B. G. Davis,
Nat. Protoc., 2007, 2, 3185–3194; (b) D. C. Anthony, H. B. Kramer,
H. H. Jensen, S. J. Campbell, J. Kirkpatrick, N. J. Oldham, S. I. van
Kasteren and B. G. Davis, Nature, 2007, 446, 1105–1109.
14 Y. Zhang, S. Luo, Y. Tang, L. Yu, K.-Y. Hou, J.-P. Cheng, X. Zeng and
P. G. Wang, Anal. Chem., 2006, 78, 2001–2008.
15 O. Michel and B. J. Ravoo, Langmuir, 2008, 24, 12116–12118.
16 D. J. Lee, K. Mandal, P. W. R. Harris, M. A. Brimble and S. B. H. Kent,
Org. Lett., 2009, 11, 5270–5273.
17 G. M. Williams and M. A. Brimble, Org. Lett., 2010, 12, 1375–1376.
18 N. Miller, G. M. Williams and M. A. Brimble, Int. J. Pept. Res. Ther.,
2010, 16, 125–132.
Acknowledgements
We thank the Maurice Wilkins Centre for Molecular Discovery
for financial assistance.
Notes and references
19 (a) H. Vu, P. Stanislav, I. M. Celia and M. G. Finn, Angew. Chem.,
Int. Ed., 2009, 48, 9879–9883; (b) L. Pei-Yan, J. Ning, Z. Ji, W. Xi, L.
Hong-Hui and Y. Xiao-Qi, Chem. Biodiversity, 2006, 3, 958–966.
20 (a) R. L. Matthew, C. K. Christopher, C. Champak and A. v. d. D.
Wilfred, ChemBioChem, 2009, 10, 911–919; (b) R. H. Nagaraj, D. R.
Sell, M. Prabhakaram, B. J. Ortwerth and V. M. Monnier, Proc. Natl.
Acad. Sci. U. S. A., 1991, 88, 10257–10261.
21 E. C. Johnson and S. B. H. Kent, J. Am. Chem. Soc., 2006, 128, 6640.
22 (a) P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. Kent, Science,
1994, 266, 776–779; (b) S. B. H. Kent, Chem. Soc. Rev., 2009, 38, 338–
351.
1 F.-G. Hanisch and S. Muller, Glycobiology, 2000, 10, 439–449.
2 (a) J. Hilkens, M. n. J. L. Ligtenberg, H. L. Vos and S. V. Litvinov,
Trends Biochem. Sci., 1992, 17, 359–363; (b) A. P. Sherblom and C. E.
Moody, Cancer Research, 1986, 46, 4543–4546.
3 S. J. Gendler, C. A. Lancaster, J. Taylor-Papadimitriou, T. Duhig, N.
Peat, J. Burchell, L. Pemberton, E. N. Lalani and D. Wilson, J. Biol.
Chem., 1990, 265, 15286–15293.
4 B. Inka, Y. Ji-Mao, B. Joy, W. Caroline and T.-P. Joyce, Eur. J. Biochem.,
1995, 233, 607–617.
5 (a) A. Kaiser, N. Gaidzik, U. Westerlind, D. Kowalczyk, A. Hobel, E.
Schmitt and H. Kunz, Angew. Chem., Int. Ed., 2009, 48, 7551–7555;
(b) A. Kaiser, N. Gaidzik, T. Becker, C. Menge, K. Groh, H. Cai, Y.-M.
Li, B. Gerlitzki, E. Schmitt and H. Kunz, Angew. Chem. Int. Ed., 2010,
23 R. F. Greene and C. N. Pace, J. Biol. Chem., 1974, 249, 5388–5393.
24 E. D. Goddard-Borger and R. V. Stick, Org. Lett., 2007, 9, 3797–3800.
25 B. Ralph, M. M. Laura, W. J. Robert and W. Thomas, Eur. J. Org.
Chem., 2001, 2001, 2697–2705.
1626 | Org. Biomol. Chem., 2011, 9, 1621–1626
This journal is
The Royal Society of Chemistry 2011
©