Synthesis of multivalent neoglyconjugates of MUC1 by the conjugation of carbohydrate-centered, triazole-linked glycoclusters to MUC1 peptides using click chemistry

Article abstract of DOI:10.1021/jo3013435

The efficient synthesis of multivalent neoglycoconjugates of MUC1 is reported, which utilizes Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuACC) of azide-functionalized GlcNAc-centered neoglycotetrasaccharide clusters to the MUC1 peptide sequence that was equipped with a propargylglycine residue for click chemistry ?. In turn the azido-GlcNAc-centered neoglycoclusters were assembled by reaction of a GlcNAc core containing peripheral propargyl functionalities with an appropriate azido-functionalized monosaccharide. The resulting suitably substituted tetrasaccharyl triazole cluster can be easily appended to a range of acetylene-functionalized peptides to produce neoglycoconjugates of biologically important glycopeptides. As proof of principle, the click neoglycoclusters prepared herein were ligated to the MUC1 peptide sequence.

Full text of DOI:10.1021/jo3013435

Article
pubs.acs.org/joc
Synthesis of Multivalent Neoglyconjugates of MUC1 by the
Conjugation of Carbohydrate-Centered, Triazole-Linked
Glycoclusters to MUC1 Peptides Using Click Chemistry
Dong Jun Lee, Sung-Hyun Yang, Geoffrey M. Williams, and Margaret A. Brimble*
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland, New Zealand
S
* Supporting Information
ABSTRACT: The efficient synthesis of multivalent neoglycoconjugates of MUC1 is reported, which utilizes Cu(I)-catalyzed
azide−alkyne 1,3-dipolar cycloaddition (CuACC) of azide-functionalized GlcNAc-centered neoglycotetrasaccharide clusters to
the MUC1 peptide sequence that was equipped with a propargylglycine residue for “click chemistry”. In turn the azido-GlcNAc-
centered neoglycoclusters were assembled by reaction of a GlcNAc core containing peripheral propargyl functionalities with an
appropriate azido-functionalized monosaccharide. The resulting suitably substituted tetrasaccharyl triazole cluster can be easily
appended to a range of acetylene-functionalized peptides to produce neoglycoconjugates of biologically important glycopeptides.
As proof of principle, the click neoglycoclusters prepared herein were ligated to the MUC1 peptide sequence.
to different scaffolds to probe the specific interactions involved
in these carbohydrate−protein bindings. A variety of multi-
valent architectures7 have been explored such as polymers,8
peptide dendrimers,9 poly(amidoamine) (PAMAM) den-
drimers,10 cyclopeptides,11 oligonucleotides,12 cyclodextrins,13
calixarenes,14 silsesquioxanes,15 fullerenes,16 nanoparticles,17
liposomes and beads.6 These glycomimics offer exciting
opportunities for biotechnology, pharmaceutical and medical
applications6 including the prevention of early adhesion of
neutrophils to endothelial surfaces,18 inhibitors of bacterial
adhesion,19 neutralization of viruses and toxins,20 immunomo-
dulators and angiogenesis inhibitors,21 and carbohydrate-based
anticancer therapy.22
INTRODUCTION
■
Cell-surface oligosaccharides play a vital role in biological
processes such as cell adhesion, signal transduction, inflamma-
tion, cancer metathesis as well as bacterial and viral
infection.1−3 Carbohydrates mediate specific multivalent
interactions with soluble or membrane proteins called lectins.4
However, our understanding of the carbohydrate interactions
with their lectin receptors is still limited, mainly due to the
complexity and difficulty in obtaining homogeneous samples in
sufficient quantities for biological testing and analysis. Total
chemical synthesis of complex glycoconjugates also remains a
major challenge, mainly hindered by the necessity to carry out
extensive protection/deprotection of hydroxyl groups, and
glycosylation reactions are also usually inconsistent and poor
yielding. To further complicate matters, monovalent inter-
actions between the carbohydrates and their receptors are
usually weak (the Kd value being ca. 10−3−10−6 M) and
unspecific. In nature this weak monovalent binding is
compensated by multiple interactions between “clusters” of
glycans and receptors resulting in high selectivity and strong
binding, a phenomenon now often referred to as the “glycoside
cluster effect”.5 For these reasons, synthetic chemists have
strived to prepare artificial multivalent carbohydrate models or
glycomimics6 by conjugation of the carbohydrate ligand moiety
Our research group23 is interested in the synthesis of
glycopeptide mimetics using Cu(I)-catalyzed azide−alkyne
cycloaddition24 (“click chemistry”). We recently reported the
synthesis of a series of click analogues of the MUC1
glycopeptide.23c The membrane-bound tumor-associated
MUC1 glycoproteins occur ubiquitously in epithelial tissues
of most organs and are also excessively expressed on tumor
tissue.25 They are also involved in cell signaling events, and
Received: June 28, 2012
Published: August 9, 2012
© 2012 American Chemical Society
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Figure 1. Structures of MUC1 neoglycopeptides prepared by appendage of different neoglycotetrasaccharides to the MUC1 peptide sequence.
their aberrant expression facilitates the metastatic spread of
tumor cells. A number of excellent developments using MUC1
glycopeptides as immunotherapeutics have been made, notably
by the Kunz and Payne groups.26
addressing problems associated with the inherent difficulties
in synthesizing highly glycosylated molecules such as chemical
incompatibility, poor regio- and stereoselectivity, incomplete
reactions, and purification issues. One approach to achieve even
stronger binding of glycoclusters to their native lectins is to
increase the density of the monosaccharide units on the
carbohydrate binding motif that is appended to a peptide
scaffold thereby improving the chances of initiating the binding
event. We therefore herein report our investigations into the
synthesis of MUC1 neoglycopeptide analogues decorated with
more complex “click neoglycotetrasaccharides” (Figure 1). In
essence click chemistry is used to prepare both the
carbohydrate ligand binding motifs and to append these motifs
to a peptide scaffold thus demonstrating a powerful synthetic
armory for the generation of structurally diverse complex
neoglycopeptide clusters.
As part of our continued endeavor23 to develop methods to
obtain highly glycosylated MUC1 peptides more easily, our
synthetic program resulted in the successful synthesis of a
“penta-clicked” MUC1 neoglycopeptide assembled by addition
of a pentapropargylglycine-substituted peptide to a monomeric
sugar-azide mimic of the natural threonine or serine O-linked
N-acetylgalactosamine (TN antigen). Importantly, the click
chemistry approach affords a focused library of neoglycopep-
tides of defined structures, thereby overcoming the purification
problems of working with native glycoproteins that often exist
as a variety of glycoforms. Furthermore, it is a chemoselective
reaction, thus avoiding the necessary iterative deprotection/
protection steps required for the synthesis of native
glycopeptides.
Several groups have used click chemistry for the synthesis of
a diverse range of oligosaccharide glycoclusters. Examples
include the facile synthesis and molecular recognition of
Optimization of the binding affinity and/or potency of
glycoclusters depends on the nature and number of the
carbohydrate moieties. Accordingly, a wide variety of scaffolds
of varying topology, valency and density for attaching
appropriate carbohydrate motifs have been investigated.7−16
However, tuning the carbohydrate structure still requires
carbohydrate-centered multivalent glycoclusters by a plant
27
lectin RCA120
.
Santoyo-Gonzalez et al.28 reported an
extensive study on the synthesis of click multivalent neo-
glycoconjugates as activators in cell adhesion and stimulation of
monocyte/macrophage cell lines. Linhardt and Bera29 reported
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a
Scheme 1
a
Reagents and conditions: (a) NaH, DMF, rt, 2 h and then 0 °C, propargyl bromide, 10 min, 58% (α:β = 1:3); (b) p-thiocresol, CH2Cl2, reflux, 16 h,
71%; (c) 3-hydroxyprop-1-yl-p-toluenesulfonate, AgOTf, NIS, 4 Å MS, CH2Cl2, 0 °C → rt, 58%; (d) (i) sugar azide 11, 12 or 13, CuSO4·5H2O,
sodium-L-ascorbate, CH2Cl2:H2O:tBuOH (1:1:1, v/v/v), μW (100 W), 15 min; (ii) NaN3, MeCN:H2O (4:1, v/v), reflux, 5 h, 8 (89% over 2 steps),
9 (89% over 2 steps), 10 (85% over 2 steps); (e) 1 M NaOMe, MeOH, rt, 1 h and then Dowex resin, 0.5 h, 1 (95%), 2 (95%), 3 (95%).
the synthesis of unnatural heparosan and chondroitin building
blocks using both linear and convergent click chemistry.
Importantly, none of these glycoclusters were appended to a
peptide scaffold using click chemistry.
linkage. We also report an efficient method for the preparation
of tetrasaccharyl neoglycoclusters that have different types of
monosaccharides in the periphery, which is usually where
receptor-recognition takes place.
The synthetic route to our carbohydrate-centered neo-
glycoclusters is depicted in Scheme 1. N-Acetyl-D-glucosamine
(GlcNAc) armed with peripheral propargyl groups was used as
the core molecule as most oligosaccharides present in
glycopeptides extend from this core, which in turn is N-linked
to the peptide backbone. Thus, three azido-functionalized
unprotected neoglycoclusters 1, 2, and 3 each bearing three
triazole-linked β-D-galactosyl, α-1-O-propyl-D-N-acetylgalacto-
syl, and β-D-glucosyl moieties respectively, were synthesized
ready for direct click conjugation onto a propargylated MUC1
peptide sequence.
RESULTS AND DISCUSSION
■
As part of our continuous search for improved methods to
prepare glycopeptide mimetics that exhibit similar or better
biological activity than the native glycopeptides/glycoproteins,
we designed an efficient method to not only synthesize
complex tetrasaccharyl neoglycan moieties but also to append
the tetrasaccharyl neoglycans to a peptide backbone. To
showcase our concept, we herein describe the synthesis of
MUC1 neoglycopeptides containing various carbohydrate-
centered glycoclusters conjugated to the peptides via a triazole
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a
Scheme 2
a
Reagents and conditions: (a) 20% (v/v) piperidine in DMF, μW (25 W); (b) Fmoc-Aaa−OH (incl. 15), HBTU, DIPEA in DMF, μW (25 W); (c)
TFA:TIS:H2O (38:1:1, v/v/v), 2.5 h; (d) CuSO4·5H2O (20 mM), TCEP (20 mM), 6 M Gn·HCl/0.2 M Na2HPO4 (pH 7.2), μW (25 W), 1, 2, or 3.
The synthesis of neoglycoclusters 1, 2 and 3 started from
readily available D-GlcNAc (4) (Scheme 1) that was reacted
with sodium hydride and propargyl bromide in DMF at room
temperature for 2 h to afford tetra-O-propargyl derivative 5 as a
mixture of α:β anomers (1:3). Treatment of this mixture with
p-thiocresol in dichloromethane under reflux for 16 h resulted
in formation of β-thioglycoside (6) in 71% yield. Glycosylation
of 6 with 3-hydroxyprop-1-yl-p-toluenesulfonate, activated by
silver(I) triflate and N-iodosuccinimide in dichloromethane in
the presence of 4 Å molecular sieves at 0 °C afforded tosylate
(7) in 58% yield. The three propargyl groups in 7 then
underwent smooth Cu(I)-catalyzed 1,3-dipolar cycloaddition
with monosaccharyl azides 11, 12, or 13, respectively, using
CuSO4·5H2O, sodium-L-ascorbate, CH2Cl2:H2O:tBuOH
(1:1:1, v/v/v) using microwave irradiation (100 W) for 15
min followed by displacement of the tosylate using sodium
azide in acetonitrile:H2O (4:1) under reflux for 5 h to afford
azides 8, 9 and 10 in 85−89% yield over two steps. The
previously reported monosaccharyl azides 11,30,31 1223c or 1332
in turn were readily prepared from the corresponding per-O-
acylated sugars using known procedures. Finally global
deprotection of the acetate groups on all of the three
monosaccharides in neoglycoclusters 8, 9 and 10 afforded the
corresponding unprotected neoglycoside clusters 1, 2 and 3 in
95% yield.
With neoglycoclusters 1, 2 and 3 in hand, attention turned to
their subsequent ligation with 14, a propargylated peptide
derivative of the MUC1 sequence (Scheme 2). The peptide 14
was synthesized on amino-functionalized polystyrene resin
containing the acid labile hydroxymethylphenoxypropionic acid
(HMPP) linker using microwave enhanced Fmoc SPPS.23c
Using the conditions shown (Scheme 2), Fmoc-L-propargyl-
glycine 1523d was incorporated into the peptide chain as a
replacement for the threonine residue, a site of glycosylation in
the native MUC1 sequence.
Initial attempts to carry out click reactions between the
azide-functionalized neoglycotetrasaccharides 1, 2 and 3 and
propargyl-containing MUC1 peptide 14 using catalytic
amounts of CuSO4 and sodium ascorbate in aqueous phosphate
buffer were unsuccessful, even at elevated temperatures using
microwave irradiation. Therefore these click reactions were
carried out using protocols previously developed within our
research group.23c CuSO4·5H2O (20 mM) and TCEP (20
mM) were premixed with heating before addition to the
peptide 14 (3 mM) in 6 M guanidine hydrochloride (GnHCl)/
0.2 M Na2HPO4 solution at pH 7.2. Click reactions on peptide
14 with neoglycosyl azides 1, 2 and 3 proceeded cleanly under
microwave conditions (25 W) at 50 °C for 5 h to afford the
desired click neoglycopeptides 16, 17 and 18 in excellent yields
(Scheme 2 and Figure 2).
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Importantly, the neoglycoconjugates thus prepared have a well-
defined architecture, and the nature of the saccharide epitope
can also be varied. The flexibility and efficiency of the “click
chemistry” used to prepare these well-defined and custom-
made click multivalent neoglycoconjugates provide a powerful
tool for the development of neoglycoconjugates with
therapeutic and materials science applications.
EXPERIMENTAL SECTION
■
General Methods. Unless stated, all solvents and reagents were
used as supplied from commercial sources. Aminomethyl polystyrene
resin was synthesized using literature methods.33 Analytical thin layer
chromatography (TLC) was performed using Kieselgel F254 0.2 mm
silica plates with visualization by ultraviolet irradiation (254 nm)
followed by staining with potassium permanganate or vanillin. Flash
chromatography was performed using Kieselgel S 63−100 μm silica
gel. NMR spectra were recorded at room temperature in deuterated
1
solvents operating at 300 MHz for H nuclei and 75 MHz for 13C
nuclei. Unless otherwise noted the chemical shifts were referenced to δ
7.26 and 77.0 ppm from chloroform for 1H and 13C, respectively. The
multiplicities of 1H signals are designated by the following
abbreviations: s = singlet; d = doublet; t = triplet; q = quartet; m =
multiplet; br = broad. All coupling constants J are reported in Hertz.
All 13C NMR spectra were acquired using broadband decoupled mode,
and assignments were determined using heteronuclear correlation
spectra. Infrared spectra were obtained as neat samples, and absorption
maxima are expressed in wavenumbers (cm−1) and recorded using a
range of 450 to 4000 cm−1. Melting points were recorded on an
electrothermal melting point apparatus and are uncorrected. Optical
rotations were measured at 20 °C at λ = 598 nm and are given in units
of deg cm3 g−1 dm−1 with the concentration of the solution measured
in grams per 100 mL. High resolution mass spectra (HRMS) were
recorded on a mass spectrometer at a nominal accelerating voltage of
70 eV. High performance liquid chromatography (HPLC) was
performed using an analytical 50 × 2.00 mm Phenomenex column
with a guard column attached using the conditions outlined in the
relevant experimental procedure. Semipreparative RH-HPLC was
performed using 250 × 10.0 mm Phenomenex column.
General Procedure for SPPS of Peptides Following the Fmoc
Strategy. Solid phase peptide synthesis was performed using a Liberty
Microwave Peptide Synthesizer (CEM Corporation, Mathews, NC)
using the Fmoc/t-Bu strategy. The Fmoc group was deprotected with
20% v/v piperidine in DMF for 30 s followed by a second
deprotection for 3 min using a microwave power of 60 W for both
deprotections. The maximum temperature for both deprotections was
set at 75 °C. The coupling step was performed with 5 equiv of Fmoc
protected amino acid in DMF (0.2 M), 4.5 equiv of HBTU in DMF
(0.45 M) and 10 equiv of DIPEA in NMP (2 M). The building block
Fmoc-Pra−OH 15 (0.11 g, 1.5 equiv) in DMF (2 mL) was manually
added, and the following coupling conditions were used: 1.45 equiv of
HATU, 1.5 equiv of HOAt, 5.0 equiv of 2,4,6-collidine and catalytic
amounts of DMAP. All couplings were performed for 5 min at 25 W at
a maximum temperature of 75 °C except for the following amino
acids: Fmoc-Arg(Pbf)−OH was double coupled using a 25 min room
temperature coupling followed by a 5 min period at 25 W; Fmoc-Pra−
OH 15 couplings were performed for 20 min at 25 W, at a maximum
temperature of 75 °C. Following completion of the sequence, peptides
were released from the resin with concomitant removal of protecting
groups by treatment with TFA/TIPS/H2O (38/1/1, v/v/v) at room
temperature for 2.0 h. The crude peptide was triturated with cold
diethyl ether, isolated by centrifugation, washed with cold diethyl
ether, dissolved in 1:1 (v/v) acetonitrile:H2O containing 0.1% TFA,
and then lyophilized.
Figure 2. HPLC traces for click reactions between neoglycote-
trasaccharides 1, 2 and 3 and propargyl-containing MUC1 peptide 14.
(A) Click reaction between 1 and 14: (i) t = 1 min; (ii) t = 180 min;
(iii) purified (m/z (2+): 1457.9). (B) Click reaction between 2 and 14:
(i) t = 1 min; (ii) t = 300 min; (iii) purified (m/z (2+): 1606.7). (C)
Click reaction between 3 and 14: (i) t = 1 min; (ii) t = 180 min; (iii)
purified (m/z (2+): 1457.9). Column: Gemini C18 (5 μ; 2.0 × 50
mm) column (Phenomenex). Gradient: Ai−ii, Bi−ii and Ci−ii were
run at 0.2 mL/min with a gradient of 5−65% B over 25 min, and Aiii,
Biii and Ciii were run at 0.2 mL/min with a shallow gradient of 5−45%
B over 40 min. The solvent system used was A (0.1% TFA in H2O)
and B (0.1% TFA in MeOH).
CONCLUSIONS
■
In conclusion, a full account of our synthetic efforts culminating
in the successful completion of MUC1 neoglycopeptides 16, 17
and 18 has been reported herein. The versatility and efficiency
of this methodology using multiple applications of the Cu(I)-
catalyzed 1,3-dipolar cycloaddition of alkynes and azides
provides a useful synthetic strategy for convenient assembly
of neoglycan containing peptides using a modular approach.
1,3,4,6-Tetra-O-propargyl-2-acetamido-2-deoxy-α,β-D-glucopyr-
anoside 5. To a suspension of D-GlcNAc 4 (1.0 g, 4.52 mmol) in
DMF (20 mL) at room temperature was added sodium hydride (60%
in mineral oil, 1.1 g, 27.1 mmol), and the mixture was left to stir for 2
h. The reaction mixture was cooled down to 0 °C followed by addition
of propargyl bromide (80% in toluene, 4.7 mL, 31.6 mmol), and then
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stirring continued for a further 10 min. The reaction mixture was
diluted with ethyl acetate (50 mL) and washed with H2O (3 × 20
mL). The organic extracts were dried over anhydrous MgSO4 and
concentrated in vacuo. Purification by silica gel flash column
chromatography using dichloromethane/methanol (98:2) as eluent
afforded an inseparable anomeric mixture of compound 5 as a brown
solid (0.98 g, 58%, α:β anomer 1:3). This compound was used in the
next step without further purification of anomers.
product was taken up in acetonitrile:H2O (4:1, 30 mL), and sodium
azide was added, which was then heated under reflux for 5 h. The
reaction mixture was diluted with ethyl acetate (30 mL), washed with
H2O (3 × 20 mL), and the organic extracts were dried over anhydrous
MgSO4 and concentrated in vacuo.
1-Azidopropyloxy-3,4,6,-tri-O-[(2′,3′,4′,6′-tetra-O-acetyl-β-D-gal-
actopyranosyl)-1″H-1″,2″,3″-triazol-1″-ylmethyl]-2-acetamido-2-
deoxy-β-D-glucopyranoside 8. Compound 8 was prepared according
to the General procedure A using tosylate 7 and azide 11 (0.24 g, 0.64
mmol). Purification by silica gel flash column chromatography using
dichloromethane/methanol (95:5) as eluent afforded compound 8 as
an off-white solid (0.25 g, 89% over 2 steps): mp 217.7−218.5 °C;
[α]D20 = −22.5 (c 0.622, MeOH); νmax (neat)/cm−1 2916, 2100, 1745,
p-Tolyl 3,4,6-tri-O-propargyl-2-acetamido-2-deoxy-β-D-1-thio-
glucopyranoside 6. To a solution of tetra-propargylated sugar 5
(2.5 g, 6.69 mmol) in dichloromethane (50 mL) at room temperature
was added p-thiocresol (4.16 g, 33.4 mmol) in dichloromethane (25
mL), and the reaction mixture was heated under reflux for overnight.
The reaction mixture was quenched by the slow addition of saturated
aq NaHCO3 (30 mL), and the aqueous phase was extracted with
dichloromethane (3 × 30 mL). The combined organic extracts were
dried over anhydrous MgSO4 and concentrated in vacuo. Purification
by silica gel flash column chromatography using dichloromethane/
methanol (99:1) as eluent afforded compound 6 as an off-white solid
1
1674, 1368, 1211, 1041; H NMR (300 MHz, CDCl3) δ = 1.78−1.85
(m, 11H), 1.94 (s, 3H), 1.97−2.03 (m, 18H), 2.17−2.23 (m, 9H),
3.28−3.38 (m, 2H), 3.40−3.66 (m, 4H), 3.71−3.84 (m, 3H), 3.84−
3.93 (m, 1H), 4.08−4.28 (m, 9H), 4.54 (d, J = 8.2 Hz, 1H), 4.66−4.78
(m, 3H), 4.85−4.94 (m, 3H), 5.20−5.30 (m, 3H), 5.45−5.66 (m, 6H),
5.78−5.91 (m, 3H), 6.19 (d, J = 8.2 Hz, 1H), 7.88 (s, 1H), 7.89 (s,
1H), 7.97 (s, 1H); 13C NMR (75 MHz, CDCl3) δ = 20.1 (CH3 × 2),
20.3 (CH3), 20.41 (CH3), 20.43 (CH3 × 2), 20.54 (CH3), 20.57 (CH3
× 3), 20.60 (CH3), 20.61 (CH3), 23.3 (CH3), 28.9 (CH2), 48.0
(CH2), 55.8 (CH), 61.0 (CH2 × 3), 64.6 (CH2), 64.7 (CH2), 65.1
(CH2), 65.7 (CH2), 66.7 (CH), 66.8 (CH × 2), 67.7 (CH), 67.8
(CH), 68.1 (CH), 69.1 (CH2), 70.5 (CH), 70.7 (CH), 70.8 (CH),
73.81 (CH), 73.85 (CH), 73.9 (CH), 74.6 (CH), 78.2 (CH), 79.3
(CH), 86.06 (CH), 86.09 (CH), 86.1 (CH), 100.6 (CH), 121.4 (CH),
121.7 (CH), 121.9 (CH), 145.1 (quat.), 145.4 (quat.), 145.7 (quat.),
168.8 (quat.), 168.9 (quat.), 169.3 (quat.), 169.72 (quat.), 169.77
(quat.), 169.79 (quat.), 169.8 (quat.), 170.00 (quat.), 170.03 (quat.),
170.23 (quat.), 170.26 (quat.), 170.28 (quat.), 170.3 (quat.); HRMS
(ESI+) C62H83N13O33Na, [M + Na+] calcd 1560.5108, found
1560.5086.
20
(2.1 g, 71%): mp 154.6−156.1 °C; [α]D = +5.1 (c 0.53, MeOH);
νmax (neat)/cm−1 3289, 3262, 3274, 3088, 2933, 2863, 2116, 1652,
1557, 1492; 1H NMR (300 MHz, DMSO-d6) δ 1.92 (s, 3 H), 2.33 (s,
3H), 3.29−3.37 (m, 1H), 3.44−3.52 (m, 4H), 3.57−3.74 (m, 3H),
3.76−3.84 (m, 1H), 4.17−4.25 (m, 2H), 4.35−4.46 (m, 4H), 4.82 (d, J
= 9.90 Hz, 1H), 7.15−7.23 (m, 2H), 7.35−7.41 (m, 2H), 8.08 (d, J =
8.80 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 20.4 (CH3),
22.9 (CH3), 53.2 (CH), 57.6 (CH2), 59.0 (CH2), 59.1 (CH2), 68.2
(CH2), 76.7 (CH × 3), 77.1 (CH), 77.2 (CH), 79.9 (quat.), 80.0
(quat.), 80.2 (quat.), 82.8 (CH), 85.7 (CH), 129.4 (CH), 130.1
(quat.), 130.6 (CH), 136.4 (quat.), 168.9 (quat); HRMS (ESI+)
C24H28NO5S, [M + H+] calcd 442.1683, found 442.1673.
3-[(3′,4′,6′-Tri-O-propargyl-2′-acetamido-2′-deoxy-β-D-
glucopyranosyl)oxy]-p-toluenesulfonate 7. To a solution of
compound 6 (0.5 g, 1.13 mmol), 3-hydroxyprop-1-yl-p-toluenesulfo-
nate (1.0 g, 4.53 mmol) and preactivated 4 Å molecular sieve (0.5 g) in
dichloromethane (10 mL) at 0 °C were added AgOTf (0.08 g, 0.34
mmol) and N-iodosuccinimide (0.3 g, 1.35 mmol). The reaction
mixture was left to stir for 5 min at this temperature before being
warmed to room temperature and stirred for further 2 h. The reaction
mixture was quenched by addition of saturated aq NaHCO3:saturated
aq Na2S2O3 (1:1, 10 mL), and the aqueous phase was extracted with
dichloromethane (3 × 10 mL). The combined organic extracts were
dried over anhydrous MgSO4 and concentrated in vacuo. Purification
by silica gel flash column chromatography using ethyl acetate/hexane
(1:1) afforded compound 7 as an off-white solid (0.36 g, 58%): mp
1-Azidopropyloxy-3,4,6,-tri-O-{[(3′,4′,6′-tri-O-acetyl-2′-acetami-
do-2′-deoxy-α-D-galacto pyranosyl)oxy]propyl-1″H-1″,2″,3″-tria-
zol-1″-ylmethyl}-2-acetamido-2-deoxy-β-D-glucopyranoside 9.
Compound 9 was prepared according to the general procedure A
using tosylate 7 and azide 12 (0.28 g, 0.65 mmol). Purification by silica
gel flash column chromatography using dichloromethane/methanol
(93:7) as eluent afforded compound 9 as an off-white solid (0.27 g,
20
85% over 2 steps): mp 204.5−205.9 °C; [α]D = +79.4 (c 0.545,
MeOH); νmax (neat)/cm−1 2914, 2098, 1746, 1662, 1542, 1372, 1234,
1132, 1048; 1H NMR (300 MHz, CDCl3) δ 1.67−1.82 (m, 2H),
1.84−2.01 (m, 30H), 2.08 (s, 9H), 2.10−2.23 (m, 6H), 3.25−3.41 (m,
5H), 3.41−3.62 (m, 3H), 3.62−3.94 (m, 6H), 3.94−4.17 (m, 9H),
4.32−4.89 (m, 21H), 4.95−5.10 (m, 3H), 5.30 (br s, 3H), 6.40−6.49
(m, 1H, NH), 6.54−6.73 (m, 3H, NH), 7.68 (s, 1H), 7.74 (s, 2H); 13C
NMR (75 MHz, CDCl3) δ 20.52 (CH3 × 6), 20.57 (CH3 × 3), 22.9
(CH3 × 3), 23.1 (CH3), 28.7 (CH2), 29.6 (CH2), 29.7 (CH2 × 2),
46.8 (CH2), 47.0 (CH2 × 2), 47.3 (CH), 47.4 (CH × 2), 47.9 (CH2),
56.0 (CH), 61.7 (CH2 × 2), 61.8 (CH2), 64.4 (CH2), 64.5 (CH2),
64.61 (CH2), 64.68 (CH2), 65.1 (CH2), 65.3 (CH2), 65.9 (CH2), 66.5
(CH × 3), 67.0 (CH × 3), 68.1 (CH × 3), 68.9 (CH2), 74.4 (CH),
77.3 (CH), 80.9 (CH), 97.71 (CH), 97.75 (CH), 97.8 (CH), 100.6
(CH), 122.8 (CH), 123.4 (CH × 2), 144.6 (quat.), 145.0 (quat.),
145.2 (quat.), 170.20 (quat. × 2), 170.22 (quat.), 170.32 (quat.),
170.35 (quat. × 2), 170.51 (quat. × 2), 170.56 (quat.), 170.6 (quat. ×
3), 170.7 (quat.); HRMS (ESI+) C71H104N16O33Na2, [M + 2Na2+]
calcd 877.3368, found 877.3362.
20
99.8−100.5 °C; [α]D = +3.7 (c 0.577, MeOH); νmax (neat)/cm−1
3265, 3246, 3092, 2872, 2116, 1653, 1552, 1354, 1169; 1H NMR (300
MHz, CDCl3) δ 1.81−1.88 (m, 2H), 1.92 (s, 3H), 2.40 (s, 3H), 2.42−
2.46 (m, 3H), 3.33−3.57 (m, 5H), 3.69−3.80 (m, 3H), 3.81−3.90 (m,
1H), 3.92−4.02 (m, 1H), 4.11−4.23 (m, 3H), 4.32−4.39 (m, 4H),
4.54 (d, J = 8.2 Hz, 1H), 6.16 (d, J = 8.2 Hz, 1H), 7.27−7.36 (m, 2H),
7.68−7.75 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 21.4 (CH3), 23.4
(CH3), 28.8 (CH2), 55.6 (CH), 58.4 (CH2), 59.62 (CH2) 59.66
(CH2), 64.7 (CH2), 67.2 (CH2), 68.1 (CH2), 74.0 (CH), 74.41 (CH),
74.46 (CH), 74.7 (CH), 77.7 (CH), 79.3 (quat.), 79.6 (quat.), 80.2
(quat.), 80.7 (CH), 100.5 (CH), 127.6 (CH), 129.8 (CH), 132.7
(quat.), 144.7 (quat.), 170.5 (quat.); HRMS (ESI+) C27H33NO9SNa,
[M + Na+] calcd 570.1768, found 570.1771.
General Procedure A for the Synthesis of Neoglycoclusters
8−10. A 10 mL microwave reaction vessel was charged with tosylate 7
(0.1 g, 0.18 mmol, 1.0 equiv) and sugar azides 11−13 (0.63 mmol, 3.5
equiv) in a mixture of dichloromethane:H2O:tBuOH (1.05 mL, 1:1:1
v/v/v) followed by the addition of a premixed solution of
CuSO4·5H2O (37 μL, 1.0 M sol., 0.2 equiv) and sodium-L-ascorbate
(73 μL, 1.0 M sol., 0.4 equiv). The reaction vessel was sealed with a
cap containing a silicon septum and heated in the microwave reactor
(Discover CEM) at 70 °C (100 W) for 15 min. After cooling, the
reaction mixture was diluted with EtOAc (10 mL) and washed with
saturated aq NH4Cl (3 × 5 mL). The organic extracts were dried over
anhydrous MgSO4 and concentrated in vacuo. The resultant crude
1-Azidopropyloxy-3,4,6,-tri-O-[(2′,3′,4′,6′-tetra-O-acetyl-β-D-glu-
copyranosyl)-1″H-1″,2″,3″-triazol-1″-ylmethyl]-2-acetamido-2-
deoxy-β-D-glucopyranoside 10. Compound 10 was prepared
according to the General procedure A using tosylate 7 and azide 13
(0.24 g, 0.64 mmol). Purification by silica gel flash column
chromatography using dichloromethane/methanol (95:5) as eluent
afforded compound 10 as an off-white solid (0.25 g, 89% over 2 steps):
20
mp 179.5−181.3 °C; [α]D = −29.6 (c 0.54, MeOH); νmax (neat)/
cm−1 2918, 2097, 1751, 1654, 1367, 1219, 1040; 1H NMR (300 MHz,
CDCl3) δ 1.65 (s, 3H), 1.80 (s, 8H), 1.89−2.10 (m, 30H), 3.31−3.39
(m, 2H), 3.39−3.48 (m, 2H), 3.49−3.58 (m, 2H), 3.65−3.73 (m, 1H),
7569
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Article
3.85−3.97 (m, 3H), 3.98−4.07 (m, 2H), 4.08−4.19 (m, 4H), 4.23−
4.35 (m, 3H), 4.59−4.71 (m, 2H), 4.75−4.94 (m, 4H), 5.19−5.30 (m,
1H), 5.37−5.46 (m, 4H), 5.46−5.56 (m, 2H), 5.56−5.68 (m, 2H),
5.98 (d, J = 9.3 Hz, 1H), 5.99 (d, J = 9.3 Hz, 1H), 6.07 (d, J = 7.8 Hz,
NH, 1H), 6.15 (d, J = 9.5 Hz, 1H), 8.00 (s, 1H), 8.31 (s, 1H), 8.37 (s,
1H); 13C NMR (75 MHz, CDCl3) δ = 20.03 (CH3), 20.09 (CH3),
20.1 (CH3), 20.4 (CH3 × 4), 20.5 (CH3 × 2), 20.6 (CH3 × 3), 23.5
(CH3), 28.9 (CH2), 48.1 (CH2), 56.9 (CH), 61.4 (CH2), 61.5 (CH2),
61.6 (CH2), 64.2 (CH2), 64.5 (CH2), 65.3 (CH2), 65.9 (CH2), 67.6
(CH), 67.7 (CH × 2), 69.4 (CH2), 70.0 (CH × 2), 70.2 (CH), 72.7
(CH), 72.9 (CH), 73.1 (CH), 74.2 (CH), 74.7 (CH), 74.9 (CH × 2),
76.9 (CH), 78.2 (CH), 85.3 (CH), 85.4 (CH), 85.7 (CH), 100.1
(CH), 121.3 (CH), 122.6 (CH), 122.9 (CH), 144.7 (quat.), 145.1
(quat.), 145.6 (quat.), 168.7 (quat.), 168.9 (quat.), 169.0 (quat.),
169.2 (quat.), 169.4 (quat.), 169.6 (quat.), 169.94 (quat.), 169.98
(quat.), 170.0 (quat.), 170.4 (quat.), 170.51 (quat.), 170.57 (quat.),
170.7 (quat.); HRMS (ESI+) C62H83N13O33Na2, [M + 2Na2+] calcd
791.7500, found 791.7518.
General Procedure B for the Synthesis of Unprotected
Neoglycoclusters 1−3. To a solution of acetylated neoglycoclusters
8−10 (0.16 mmol) in methanol (1.0 mL) at room temperature was
added freshly prepared sodium methoxide (1.0 M, 3.0 mL), and the
mixture was left to stir until analytical RP-HPLC analysis showed
complete conversion to the corresponding product (approximately 1
h). The reaction mixture was diluted with methanol (30 mL) and
quenched by stirring with acidified Dowex resins (0.5 g) for 30 min.
The resin was then filtered and washed with methanol, and then
combined organic extracts were concentrated in vacuo. The crude
product was redissolved in H2O (10 mL), lyophilized and used
without further purification.
174.44 (quat.); HRMS (ESI+) C53H86N16O24Na2, [M + 2Na2+] calcd
688.2893, found 688.2896.
1-Azidopropyloxy-3,4,6,-tri-O-[(β-D-glucopyranosyl)-1″H-
1″,2″,3″-triazol-1″-ylmethyl]-2-acetamido-2-deoxy-β-D-glucopyra-
noside 3. Compound 3 was prepared according to the General
procedure B using acetylated neoglycocluster 10 (0.25 g, 0.16 mmol).
Lyophilization of the reaction mixture afforded compound 3 as a white
20
solid (0.16 g, 95%): mp 179.1−181.5 °C; [α]D = −6.6 (c 0.507,
H2O); νmax (neat)/cm−1 3274, 2882, 2101, 1645, 1557, 1374, 1094,
1045; 1H NMR (400 MHz, D2O) δ 1.77−1.85 (m, 2H), 1.91 (s, 3H),
3.31−3.37 (m, 2H), 3.54−3.70 (m, 8H), 3.70−3.83 (m, 11H), 3.85−
3.94 (m, 4H), 3.96−4.06 (m, 3H), 4.49 (d, J = 7.95 Hz, 1H), 4.68−
4.87 (m, 6H), 5.73−5.79 (m, 3H), 8.22 (s, 1H), 8.27 (s, 1H), 8.30 (s,
1H); 13C NMR (100 MHz, D2O) δ = 22.0 (CH3), 28.0 (CH2), 47.7
(CH2), 54.7 (CH), 60.33 (CH2), 60.37 (CH2 × 2), 63.1 (CH2), 64.4
(CH2), 64.8 (CH2), 67.2 (CH2), 67.9 (CH2), 68.8 (CH × 3), 72.2
(CH × 3), 73.2 (CH), 75.8 (CH × 2), 75.9 (CH), 77.4 (CH), 78.7
(CH), 78.8 (CH × 2), 81.5 (CH), 87.4 (CH × 3), 100.7 (CH), 124.1
(CH), 124.4 (CH × 2), 143.7 (quat.), 144.0 (quat.), 144.3 (quat.),
174.0 (quat.); HRMS (ESI+) C38H59N13O21Na, [M + Na+] calcd
1056.3841, found 1056.3807.
General Procedure C for the CuAAC between Peptide 14
and Unprotected Neoglyclusters 1−3. To a degassed solution of
6.0 M GnHCl/0.2 M Na2HPO4 buffer at room temperature was added
CuSO4·5H2O (20 mM):TCEP (20 mM), and pH was adjusted to
approximately 7.1−7.2 by the addition of 1.0 M NaOH. The resultant
solution was then incubated at 80 °C for 30 min and centrifuged prior
to use. To this solution (177 μL) was then added peptide 14 (1.0 mg,
3 mM, 1.0 equiv) and unprotected neoglycoclusters 1−3 (4.5 mM, 1.5
equiv), and the reaction was carried out under nitrogen atmosphere
using microwave reactor (Discover CEM) at 50 °C (25 W). The
reaction progress was monitored by analytical RP-HPLC, and after 3−
5 h the reaction was complete, and the crude product was isolated by
solid phase extraction using a RP-C18 cartridge and then lyophilized.
Click Neoglycopeptide 16. Neoglycopeptide 16 was prepared
according to the general procedure C using unprotected neo-
glycocluster 1 (0.82 mg) and peptide 14 (1.0 mg). Purification by
semipreparative RP-HPLC afforded compound 16 as a white solid
(0.54 mg, 32%).
Click Neoglycopeptide 17. Neoglycopeptide 17 was prepared
according to the general procedure C using unprotected neo-
glycocluster 2 (1.06 mg) and peptide 14 (1.0 mg). Purification by
semipreparative RP-HPLC afforded compound 17 as a white solid
(0.72 mg, 42%).
Click Neoglycopeptide 18. Neoglycopeptide 18 was prepared
according to the general procedure C using unprotected neo-
glycocluster 3 (0.82 mg) and peptide 14 (1.0 mg). Purification by
semipreparative RP-HPLC afforded compound 18 as a white solid
(0.43 mg, 28%).
1-Azidopropyloxy-3,4,6,-tri-O-[(β-D-galactopyranosyl)-1″H-
1″,2″,3″-triazol-1″-ylmethyl]-2-acetamido-2-deoxy-β-D-glucopyra-
noside 1. Compound 1 was prepared according to the General
procedure B using acetylated neoglycocluster 8 (0.25 g, 0.16 mmol).
Lyophilization of the reaction mixture afforded compound 1 as a white
20
solid (0.16 g, 95%): mp 155.6−156.2 °C; [α]D = −12.9 (c 0.400,
H2O); νmax (neat)/cm−1 3281, 2885, 2101, 1649, 1555, 1373, 1090,
1050; 1H NMR (400 MHz, D2O) δ 1.78−1.86 (m, 2H), 1.92 (s, 3H),
3.32−3.40 (m, 2H), 3.56−3.74 (m, 5H), 3.74−3.84 (m, 8H), 3.87−
3.95 (m, 4H), 3.99−4.06 (m, 3H), 4.09−4.14 (m, 3H), 4.21−4.30 (m,
3H), 4.51 (d, J = 8.32 Hz, 1H), 4.71−4.90 (m, 6H), 5.69−5.77 (m,
3H), 8.27 (s, 1H), 8.33 (s, 1H), 8.35 (s, 1H); 13C NMR (100 MHz,
D2O) δ 22.0 (CH3), 28.0 (CH2), 47.7 (CH2), 54.7 (CH), 60.7 (CH2
× 3), 63.1 (CH2), 64.5 (CH2), 64.8 (CH2), 67.2 (CH2), 67.9 (CH2),
68.51 (CH × 2), 68.54 (CH), 69.6 (CH × 3), 72.91 (CH × 2), 72.94
(CH), 73.3 (CH), 77.5 (CH), 78.2 (CH × 3), 81.4 (CH), 87.9 (CH ×
3), 100.7 (CH), 124.0 (CH), 124.2 (CH × 2), 143.8 (quat.), 144.0
(quat.), 144.3 (quat.), 174.0 (quat.); HRMS (ESI+) C38H59N13O21Na,
[M + Na+] calcd 1056.3841, found 1056.3860.
1-Azidopropyloxy-3,4,6,-tri-O-{[(2′-acetamido-2′-deoxy-α-D-
galactopyranosyl)oxy]propyl-1″H-1″,2″,3″-triazol-1″-ylmethyl}-2-
acetamido-2-deoxy-β-D-glucopyranoside 2. Compound 2 was
prepared according to the general procedure B using acetylated
neoglycocluster 9 (0.27 g, 0.16 mmol). Lyophilization of the reaction
mixture afforded compound 2 as a white solid (0.2 g, 95%): mp
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Copies of H and 13C NMR spectra for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
20
126.4−127.8 °C; [α]D = +73.2 (c 0.492, H2O); νmax (neat)/cm−1
3285, 2884, 2099, 1647, 1548, 1374, 1119, 1043; 1H NMR (400 MHz,
D2O) δ 1.78−1.85 (m, 2H), 1.94 (s, 3H), 2.04−2.08 (m, 9H), 2.16−
2.25 (m, 6H), 3.32−3.42 (m, 5H), 3.57−3.78 (m, 17H), 3.81−3.89
(m, 6H), 3.89−3.98 (m, 4H), 4.11−4.18 (m, 3H), 4.50−4.61 (m, 7H),
4.63−4.85 (m, 8H), 8.00 (s, 1H), 8.02 (s, 1H), 8.10 (s, 1H); 13C NMR
(100 MHz, D2O) δ 21.9 (CH3 × 3), 22.0 (CH3), 28.0 (CH2), 29.0
(CH2), 29.13 (CH2), 29.16 (CH2), 47.5 (CH2), 47.62 (CH2), 47.64
(CH2), 47.7 (CH2), 49.8 (CH × 3), 54.9 (CH), 61.1 (CH2 × 3), 63.0
(CH2), 64.3 (CH2), 64.43 (CH2), 64.46 (CH2), 64.5 (CH2), 64.9
(CH2), 67.1 (CH2), 67.6 (CH × 3), 67.9 (CH2), 68.4 (CH × 3), 70.9
(CH × 3), 73.3 (CH), 77.2 (CH), 81.7 (CH), 97.1 (CH × 3), 100.7
(CH), 124.83 (CH), 124.85 (CH), 125.2 (CH), 143.5 (quat.), 143.6
(quat.), 143.9 (quat.), 173.9 (quat.), 174.42 (quat.), 174.43 (quat.),
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: m.brimble@auckland.ac.nz.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial assistance from the Maurice Wilkins Centre for
Molecular Biodiscovery is gratefully acknowledged.
7570
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Article
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