Synthesis of 1,2,3-triazolo-linked octyl (1 → 6)-α-d- oligomannosides and their evaluation in mycobacterial mannosyltransferase assay

Article abstract of DOI:10.1021/bc100421g

The synthesis of conjugates consisting of two or three mannose units interconnected by a 1,2,3-triazole linker installed by the "click" reaction is reported. These conjugates were evaluated in mycobacterial mannosyltransferase (ManT) assay. Detailed analysis of the reaction products showed that these compounds with triazole linker between sugar moieties were tolerated by the enzyme, which elongated them by one or two sugar units with α-(1→6) linkage. The effectiveness of this transfer was reduced in comparison to that observed for the acceptor analogues containing a glycosidic linkage, but still, this is the first report on such unnatural compounds serving as substrates for mycobacterial ManT. The ability of the studied compounds to function as acceptors for the ManT suggests that the relative distance and spatial orientation of acceptor octyl hydrophobic aglycone (optimal length for the ManT) and free primary C-6 hydroxy group of the nonreducing terminal mannose unit (to which glycosyl residue is transferred by the mycobacterial ManT) are important for ManT activity, but at the same time, their variations are tolerated by the enzyme in a relatively wide range.

Full text of DOI:10.1021/bc100421g

ARTICLE
pubs.acs.org/bc
Synthesis of 1,2,3-Triazolo-Linked Octyl (1f6)-R-D-Oligomannosides
and Their Evaluation in Mycobacterial Mannosyltransferase Assay
Monika Polꢀakovꢀa,*,† Martina Belꢀaꢁnovꢀa,‡ Katarína Mikuꢁsovꢀa,‡ Erika Lattovꢀa,§ and Hꢀelꢂene Perreault§
†Institute of Chemistry, Center for Glycomics, GLYCOMED, Slovak Academy of Sciences, Dꢀubravskꢀa cesta 9, SK-845 38 Bratislava,
Slovakia
‡Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynskꢀa dolina, CH1, SK-842 15 Bratislava, Slovakia
§Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba R3T 2N2, Canada
S Supporting Information
b
ABSTRACT: The synthesis of conjugates consisting of two
or three mannose units interconnected by a 1,2,3-triazole
linker installed by the “click” reaction is reported. These
conjugates were evaluated in mycobacterial mannosyltrans-
ferase (ManT) assay. Detailed analysis of the reaction
products showed that these compounds with triazole linker
between sugar moieties were tolerated by the enzyme, which
elongated them by one or two sugar units with R-(1f6)
linkage. The effectiveness of this transfer was reduced in
comparison to that observed for the acceptor analogues containing a glycosidic linkage, but still, this is the first report on such
unnatural compounds serving as substrates for mycobacterial ManT. The ability of the studied compounds to function as acceptors
for the ManT suggests that the relative distance and spatial orientation of acceptor octyl hydrophobic aglycone (optimal length for
the ManT) and free primary C-6 hydroxy group of the nonreducing terminal mannose unit (to which glycosyl residue is transferred
by the mycobacterial ManT) are important for ManT activity, but at the same time, their variations are tolerated by the enzyme in a
relatively wide range.
’ INTRODUCTION
The 1,3-dipolar cycloaddition reaction (“click” reaction) is a
powerful means for linking two units, one with azide and the
other consisting of an alkyne function. The reaction is also widely
used for linking a carbohydrate with another carbohydrate or
noncarbohydrate unit. Recently, Sharpless et al.1 and Meldal
et al.2 found out that the use of Cu(I) as catalyst led to an
Figure 1. Example of Cu(I)-catalyzed Husigen azide-alkyne 1,3-
dipolar cycloaddition reaction.
exclusive formation of 1,4-disubstituted 1,2,3-triazole (Figure 1).
potential antitubercular agents.28 A series of arabino glycosyl
This finding has raised interest in the use of this kind of
chemistry, which is furthermore advantageous in terms of com-
triazoles with varying length of the hydrophobic group has been
patibility with various functionalities and of its broad applicability
in different solvent systems.
prepared in order to evaluate their inhibitory activity against
Mycobacterium bovis. The screening has revealed their low to
moderate antimycobacterial activity, which was strongly depen-
dent on the nature of the hydrophobic group.29 Small molecules
for screening a glycosyltransferase activity have also been pre-
pared by the use of the “click” reaction. Thus, a diverse set of
organic azides or alkynes was coupled with an appropriate
mannose-based alkyne (propargyl function at C-1) or azide (at
C-6) derivatives, respectively, in order to construct a library of
modified compounds, which were utilized to study the substrate
In the field of carbohydrate chemistry, click chemistry has
been used for the synthesis of a large number of compounds
ranging from small molecules, such as simple glycosides, e.g.,
N-glycosyltriazoles,3-5 through oligosaccharide mimetics6-15 to
homogeneous and heterogeneous neoglycoconjugates. The latter
group of neoglycoconjugates includes glycomacrocycles,16-18
glycoclusters,19,20 glycodendrimers,21-24 glycocyclodextrins,25,26
and glycopolymers.27
Click chemistry has also found applications in the field of
mycobacterial investigation. Carbohydrate-derived triazoles with
arabino and ribo-configurations in which the triazole unit was
built up at position 5 have been recently synthesized and tested as
Received: September 23, 2010
Revised:
December 17, 2010
Published: January 20, 2011
r
2011 American Chemical Society
289
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Figure 2. Reaction catalyzed by the polyprenolphosphomannose-dependent R-(1f6)-mannosyltransferase (ManT).
specificity of the leishmanian β-1,2-mannosyltransferases.30 Very
recently, Dondoni’s group31 synthesized a set of C-oligomanno-
sides having the triazole ring as the interglycosidic linker and
being deoxygenated at the 6-position of the nonreducing terminal
mannose residue, and reported their inhibitory activity against
mycobacterial ManT.
241 polarimeter at 20 °C. High-resolution mass spectra (HRMS)
and CID spectra were recorded on a MALDI-QqTOF mass
spectrometer operating in positive ion mode. Typically, the samples
were spotted onto 2,5-dihydroxybenzoic acid matrix predeposited
on the surface of a MALDI target. MS/MS spectra were interpreted
manually. For the assignment of fragment ions, the general nomen-
clature established by Domon and Costello was followed.34 ESI-
MS experiments were carried out on a Varian 500 LC-MS Ion
Trap equipped with an ESI source. The samples were introduced
with carrying solvent acetonitrile-water (1:1). The nebulizing
gas pressure was 50 psi and drying gas pressure was 39 psi at
350 °C. Mass spectra were recorded in the positive mode with a
scan rate of 3 s/scan. Online LC-MS analysis was performed using
an HPLC system (Varian, ProStar). Eluent A was 5% acetonitrile
in water and eluent B was 0.01 M formic acid in 90% acetonitrile in
water. The proportion of 5% B was maintained for 5 min and then
linearly increasedto40%over 55min. The sample was separatedat
a flow rate of 0.4 mL/min on Vydac 218-TP54 C18 analytical
column (Grace, Hesperia, USA). The column effluent was intro-
duced directly into the mass spectrometer equipped with an
electrospray ionization source operated in positive ion mode.
Octyl 2,3,4-tri-O-acetyl-6-O-tosyl-R-D-mannopyranoside (2). To
a stirred solution containing (1)35,36 (0.52 g, 1.78 mmol) in
pyridine (7 mL) cooled down in an ice bath, tosyl chloride (0.44
g, 2.31 mmol) in CH2Cl2 (5 mL) was added dropwise. The
resulting mixture was brought to rt and the stirring was continued
for 16 h. The reaction mixture was diluted with CH2Cl2 (30 mL);
washed with 1 M HCl (5 ꢀ 10 mL), satd NaHCO3 (3 ꢀ 10 mL),
and water (20 mL); dried with anhydrous Na2SO4; filtered; and
concentrated. The crude product was dissolved in pyridine
(4 mL) and cooled down in an ice bath. Acetic anhydride
(7 mL) was added dropwise. The resulting mixture was brought
to rt and the stirring was continued for 16 h. The reaction mixture
was diluted with water (10 mL) and CH2Cl2 (30 mL). The
organic phase was separated, washed with 1 M HCl (3 ꢀ 15 mL)
and satd NaHCO3 (3 ꢀ 10 mL), dried with anhydrous Na2SO4,
filtered, and concentrated. The crude product was purified by
column chromatography (hexanes/EtOAc 5:1f2:1) to give (2)
(0.52 g, 51% over 2 steps) as an oil. [R]D þ42 (c 1, CHCl3). 1H
NMR (400 MHz, CDCl3): δ 7.80 (d, 2H, J 8.2 Hz, Ts), 7.35 (d,
The aim of this study was to use the Cu(I)-catalyzed azide-
alkyne cycloaddition (CuAAC) reaction for the synthesis of 1,2,
3-triazolo-linked octyl (1f6)-R-D-oligomannosides and to investi-
gate whether they are processed by the mycobacterial mannosyl-
transferase (ManT), whose action is schematically shown in
Figure 2. These conjugates consist of two or three mannose
moieties, wherein each glycosidic linkage between mannose units
is replaced by 1,2,3-triazole spacer, while a glycosidic linkage
between the aglycone and the first sugar unit remained intact.
Thus, the obtained novel conjugates possess structural features
characteristic for the already confirmed synthetic substrates for
the ManT: hydrophobic aglycone attached by a glycosidic bond
and free primary hydroxyl group at C-6 atom of the nonreducing
terminal mannose residue. In these terms, they are similar to
octyl (1f6)-R-D-mannooligosaccharides, efficient synthetic
acceptor analogues, which have been screened in ManT assay
previously.32,33
’ EXPERIMENTAL PROCEDURES
General Methods. TLC was performed on aluminum sheets
precoated with silica gel 60 F254 (Merck). Flash column chro-
matography was carried out on silica gel 60 (0.040-0.060 mm,
Merck) with distilled solvents (hexanes, ethyl acetate dichloro-
methane, chloroform, methanol). Reaction solvents were dried
and distilled before use. All reactions containing sensitive reagents
were carried out under argon atmosphere. 1H NMR and 13C NMR
spectra were recorded at 25 °C with a VNMRS 400 MHz Varian
spectrometer; chemical shifts are referenced to either TMS (δ 0.00,
CDCl3 for 1H) or HOD (δ 4.87, CD3OD for 1H) and to internal
CDCl3 (δ 77.23) or CD3OD (δ 49.15) for 13C. The assignment
of resonances in the 1H and 13C NMR spectra was made by two-
dimensional homonuclear and heteronuclear shift correlation
experiments. Optical rotations were measured on a Perkin-Elmer
290
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Bioconjugate Chemistry
ARTICLE
2H, J 8.1 Hz, Ts), 5.32 (dd, 1H, J3,4 10.0 Hz, H-3), 5.19 (dd, 1H,
J2,3 3.4 Hz, H-2), 5.14 (t, 1H, J4,5 10.0 Hz, H-4), 4.72 (d, 1H, J1,2
1.4 Hz, H-1), 4.13-4.11 (m, 2H, H-6a, H-6b), 4.02 (m, 1H,
H-5), 3.62 (dt, 1H, J 6.7, 9.6 Hz, OCH2C7H15), 3.37 (dt, 1H,
J 6.6, 9.6 Hz, OCH2C7H15), 2.45 (s, 3H, CH3 (Ts)), 2.12, 1.99,
1.98 (each s, each 3H, 3 ꢀ CH3CO), 1.58-1.53 (m, 2H, OCH2-
CH2C6H13), 1.33-1.27 (m, 10H, OCH2CH2(CH2)5CH3), 0.89
(t, 3H, J 6.8 Hz, O(CH2)7CH3). 13C NMR (100 Mz, CDCl3): δ
170.3, 170.1, 170.0 (3 ꢀ CH3CO), 145.1, 133.0, 130.0, 128.3 (Ar),
97.6 (C-1), 69.8 (C-2), 69.2 (C-3), 68.8 (OCH2C7H15), 68.5
(2ꢀ) (C-5, C-6), 66.7 (C-4), 32.0, 29.5, 29.4 (2ꢀ), 26.2, 22.9
(OCH2(CH2)6CH3), 21.9 (CH3(Ts)), 21.1, 20.8 (2ꢀ) (3 ꢀ
CH3CO), 14.3 (O(CH2)7CH3). HRMS (MALDI): m/z calcd
for [C27H40O11S]Naþ: 595.2189. Found: 595.2199.
Octyl 2,3,4-tri-O-acetyl-6-azido-6-deoxy-R-D-mannopyranoside
(3). Compound (2) (0.50 g, 0.87 mmol) and NaN3 (0.28 g, 4.36
mmol) in DMF (5 mL) were stirred at 80 °C for 40 h. The
reaction mixture was cooled to rt, diluted with CH2Cl2 (40 mL),
and extracted with water (3 ꢀ 10 mL) and brine (3 ꢀ 10 mL).
The organic phase was dried with anhydrous Na2SO4 and
concentrated. Purification by column chromatography (hexanes/
EtOAc 5:1f3:1) afforded (3) (0.31 g, 80%) as an oil. [R]D þ56
(c 1, CHCl3). 1H NMR (400 MHz, CDCl3): δ 5.35 (dd, 1H, J3,4
10.0 Hz, H-3), 5.23 (dd, 1H, J2,3 3.5 Hz, H-2), 5.21 (t, 1H, J4,5
10.1 Hz, H-4), 4.80 (d, 1H, J1,2 1.2 Hz, H-1), 3.95 (ddd, 1H, J5,6a
2.4 Hz, H-5), 3.72 (dt, 1H, J 6.8, 9.5 Hz, OCH2C7H15), 3.47
(dt, 1H, J 6.6, 9.6 Hz, OCH2C7H15), 3.37 (dd, 1H, J5,6b 6.9 Hz,
J6a,6b -13.3 Hz, H-6b), 3.27 (dd, 1H, H-6a), 2.16, 2.05, 2.00
(each s, each 3H, 3 ꢀ CH3CO), 1.63-1.60 (m, 2H, OCH2CH2-
C6H13), 1.37-1.25 (m, 10H, OCH2CH2(CH2)5CH3), 0.89 (t,
3H, J 6.9 Hz, O(CH2)7CH3). 13C NMR (100 Mz, CDCl3): δ
170.3, 170.1 (2 ꢀ ) (3ꢀ CH3CO), 97.6 (C-1), 70.1 (C-5), 69.9
(C-2), 69.2 (C-3), 68.9 (OCH2C7H15), 67.6 (C-4), 51.4 (C-6),
32.0, 29.5 (2ꢀ), 29.4, 26.3, 22.9 (OCH2(CH2)6CH3), 21.1,
21.0, 20.9 (3 ꢀ CH3CO), 14.3 (O(CH2)7CH3). HRMS (MA-
LDI): m/z calcd for [C20H33N3O8]Naþ: 466.2166. Found:
466.2183.
CH), 20.8, 20.7, 20.6 (2ꢀ) (4 ꢀ CH3CO). HRMS (MALDI):
m/z calcd for [C17H22O10]Naþ 409.1110. Found 409.12.
2-Propynyl R-D-mannopyranoside (6). Compound 5 was
dissolved in MeOH/CH2Cl2 (7.5 mL/2 mL) and 1 M MeONa
(0.5 mL) was added. The reaction mixture was stirred for 24 h at
rt, neutralized with DOWEX 50 Hþ-form, filtered, and concen-
trated. The crude product was purified by column chromatogra-
phy (CH2Cl2/MeOH 10:1f5:1) to give (6) as white crystals
(0.75 g, 89%). mp = 117-118 °C; lit. 122-123 °C. [R]D þ123
(c 1, methanol). 1H NMR (400 MHz, CD3OD): δ 5.00 (d, 1H,
J1,2 1.3 Hz, H-1), 4.31 (d, 2H, J 2.4 Hz, OCH2CtCH), 3.86 (dd,
1H, J5,6a 2.2 Hz, J6a,6b -11.8 Hz, H-6a), 3.82 (dd, 1H, J2,3 3.0 Hz,
H-2), 3.75 (dd, 1H, J5,6b 5.9 Hz, H-6b), 3.72 (dd, 1H, J3,4 9.3 Hz,
H-3), 3.65 (t, 1H, J4,5 9.3 Hz, H-4), 3.54 (m, 1H, H-5), 2.88 (t,
1H, J 2.4 Hz, OCH2CtCH). 13C NMR (100 Mz, CD3OD): δ
99.9 (C-1), 80.2 (OCH2CtCH), 76.1 (OCH2CtCH), 75.2 (C-
5), 72.6 (C-3), 72.2 (C-2), 68.6 (C-4), 63.0 (C-6), 54.9 (OCH2-
CtCH). HRMS (MALDI): m/z calcd for [C9H14O6]Naþ
241.0688. Found 241.0675.
2-Propynyl 2,3,4-tri-O-acetyl-6-O-tosyl-R-D-mannopyrano-
side (7). To a stirred solution containing (6) (0.12 g, 0.55 mmol)
in pyridine (4 mL) cooled down on an ice bath, tosyl chloride
(0.31 g, 1.65 mmol) in CH2Cl2 (2 mL) was added dropwise. The
resulting mixture was brought to rt and the stirring was continued
for 16 h. The reaction mixture was diluted with CH2Cl2 (20 mL);
washed with 1 M HCl (5 ꢀ 10 mL), satd NaHCO3 (3 ꢀ 15 mL),
and water (20 mL); dried with anhydrous Na2SO4; filtered; and
concentrated. The crude product was dissolved in pyridine (3
mL) and cooled down on an ice bath. Acetic anhydride (4 mL)
was added dropwise. The resulting mixture was brought to rt and
stirring was continued for 16 h. The reaction mixture was diluted
with water (10 mL) and CH2Cl2 (30 mL). The organic phase was
separated, washed with 1 M HCl (3 ꢀ 15 mL) and satd NaHCO3
(3 ꢀ 10 mL), dried with anhydrous Na2SO4, filtered, and
concentrated. The crude product was purified by column chro-
matography (hexanes/EtOAc 2:1f1:1) to give (7) (0.13 g, 47%
over two steps) as an oil. [R]D þ54 (c 1, CHCl3). 1H NMR (400
MHz, CDCl3): δ 7.79 (d, 2H, J 8.2 Hz, Ts), 7.35 (d, 2H, J 8.1 Hz,
Ts), 5.30 (dd, 1H, J3,4 10.0 Hz, H-3), 5.23 (dd, 1H, J2,3 3.4 Hz,
H-2), 5.16 (t, 1H, J4,5 10.0 Hz, H-4), 4.94 (d, 1H, J1,2 1.5 Hz,
H-1), 4.21 (d, 2H, J 2.1 Hz, OCH2CtCH), 4.13-4.11 (m, 2H,
H-6a, H-6b), 4.03 (m, 1H, H-5), 2.47-2.44 (m, 4H, CH3(Ts),
OCH2CtCH), 2.13, 1.99, 1.98 (each s, each 3H, 3 ꢀ CH3CO).
13C NMR (100 Mz, CDCl3): δ 170.1, 170.0, 169.9 (3 ꢀ
CH3CO), 145.2, 132.9, 130.1, 128.3 (Ar), 96.2 (C-1), 78.1
(OCH2CtCH), 75.9 (OCH2CtCH), 69.4 (C-2), 69.1
(C-5), 68.9 (C-3), 68.4 (C-6), 66.4 (C-4), 55.2 (OCH2Ct
CH), 21.9 (CH3(Ts)), 21.0, 20.8 (2ꢀ) (3 ꢀ CH3CO). HRMS
(MALDI): m/z calcd for [C22H26O11S]Naþ 521.1093. Found
521.1096.
2-Propynyl 2,3,4,6-tetra-O-acetyl-R-D-mannopyranoside (5). To
a stirred solution containing 1,2,3,4,6-penta-O-acetyl-D-manno-
pyranose (4) (1.50 g, 3.84 mmol) in CH2Cl2 (20 mL) propargyl
alcohol (1.08 g, 1.12 mL, 19.21 mmol) was added. The reaction
mixture was stirred for 20 min, cooled down on an ice bath, and
BF3 OEt2 (5.46 g, 4.86 mL, 38.43 mmol) was added dropwise.
3
The resulting mixture was stirred for 15 min, brought to rt, and
the stirring was continued for 24 h. The reaction mixture was
diluted with CH2Cl2 (100 mL) and poured into ice-cold water
(150 mL) under stirring. The organic phase was separated,
washed with satd NaHCO3 (3 ꢀ 100 mL) and water (100 mL),
dried with anhydrous Na2SO4, filtered, and concentrated. The
crude product was purified by column chromatography (hexanes/
EtOAc 4:1f2.5:1) to give (5) as white crystals (1.34 g, 90%).
mp = 103-104 °C; lit.30 104-106 °C; [R]D þ68 (c 1,
4-(2,3,4,6-Tetra-O-acetyl-R-D-mannopyranosyloxymethyl)-
1-(octyl-2,3,4-tri-O-acetyl-6-deoxy-R-D-mannopyranoside-6-
yl)-1H-1,2,3-triazole (8). Compound (3) (0.12 g, 0.26 mmol)
and compound (5) (0.10 g, 0.26 mmol) were dissolved in DMF/
H2O (3 mL/1 mL). Copper sulfate (17.2 mg, 0.11 mmol) and
sodium ascorbate (42.8 mg, 0.22 mmol) were added, and the
resulting mixture was stirred at rt for 4 h. The mixture was poured
into satd. NH4Cl/H2O solution (10 mL, 1:1) and extracted with
EtOAc (4 ꢀ 10 mL). The organic phase was dried with
anhydrous Na2SO4 and concentrated. Purification by column
chromatography (hexanes/EtOAc 5:1f1:5) afforded (8) as a
colorless oil (0.19 g, 89%). [R]D þ28 (c 0.5, CHCl3).
1
chloroform); lit.37 þ66.5 (c 0.77, chloroform). H NMR (400
MHz, CDCl3): δ 5.35 (dd, 1H, J3,4 10.0 Hz, H-3), 5.26 (t, 1H, J3,4
10.0 Hz, H-4), 5.23 (dd, 1H, J2,3 3.3 Hz, H-2), 5.01 (d, 1H, J1,2 1.6
Hz, H-1), 4.32-4.26 (m, 3H, H-6b, CH2CtCH), 4.11 (dd, 1H,
J5,6a 2.4 Hz, J6a,6b -12.1 Hz, H-6a), 4.00 (ddd, 1H, J5,6b 5.2 Hz,
H-5), 2.48 (t, 1H, J 2.4 Hz, CH2CtCH), 2.16, 2.11, 2.05, 2.00
(each s, each 3H, 4 ꢀ CH3CO). 13C NMR (100 Mz, CD3OD): δ
170.6, 169.9, 169.8, 169.7 (4 ꢀ CH3CO), 96.2 (C-1, 1JC-1,H-1 175.0
Hz), 77.9 (OCH2CtCH), 75.6 (OCH2CtCH), 69.3 (C-2),
69.0 (C-3), 68.9 (C-5), 66.0 (C-4), 62.3 (C-6), 54.9 (OCH2Ct
291
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ARTICLE
1H NMR (400 MHz, CDCl3): δ 7.74 (s, 1H, CHN), 5.36 (dd,
1H, J2,3 3.4 Hz, J3,4 10.0 Hz, H-3), 5.32-5.29 (m, 2H, H-30,
H-40), 5.24-5.22 (m, 2H, H-2, H-20), 5.14 (t, J4,5 10.0 Hz, H-4),
J1 ,2 1.3 Hz, H-1 ), 4.80 (d, 1H, J -12.3 Hz, OCH2CdCH), 4.76
(d, 1H, J1,2 1.5 Hz, H-1), 4.67 (d, 1H, J -12.3 Hz,
OCH2CdCH), 4.62 (dd, J5,6a 2.4 Hz, J6a,6b -14.2 Hz, H-6a),
4.43 (dd, J5,6b 8.9 Hz, H-6b), 4.17 (m, 1H, H-5), 4.11-4.04 (m,
3H, H-50, H-60a, H-60b), 3.22 (m, 2H, OCH2C7H15), 2.46 (s, 3H,
CH3 (Ts)), 2.15, 2.11 (2ꢀ), 2.00, 1.98, 1.96 (each s, each 3H, 6
ꢀ CH3CO), 1.45-1.39 (m, 2H, OCH2CH2C6H13), 1.30-1.16
(m, 10H, OCH2CH2(CH2)5CH3), 0.88 (t, 3H, J 6.9 Hz, O-
(CH2)7CH3). 13C NMR (100 Mz, CDCl3): δ 170.4, 170.2,
170.0, 169.9 (2ꢀ), 169.8 (6 ꢀ CH3CO), 162.8 (NCdCH),
145.3, 132.8, 130.1, 128.3 (Ar), 125.0 (NCdCH), 97.5 (C-1),
97.1 (C-10), 69.8, 69.5, 69.2, 69.1, 69.0 (2ꢀ), 68.7, 66.5 (C-2,
C-20, C-3, C-30, C-4, C-40, C-5, C-50), 68.5 (OCH2C7H15), 67.8
(C-60), 61.3 (OCH2CdCH), 51.3 (C-6), 32.0, 29.5, 29.4, 29.3,
26.2, 22.8 (OCH2(CH2)6CH3), 21.9 (CH3(Ts)), 21.1, 21.0 (2ꢀ),
20.9, 20.8 (2ꢀ) (6 ꢀ CH3CO), 14.3 (O(CH2)7CH3). HRMS
(MALDI): m/z calcd for [C42H59N3O19S]Hþ 942.3536. Found
942.3526.
0
0
0
4.95 (d, 1H, J1 ,2 1.5 Hz, H-10), 4.87 (d, 1H, J -12.3 Hz,
OCH2CdCH), 4.75 (d, 1H, J1,2 1.6 Hz, H-1), 4.67 (d, 1H,
J -12.3 Hz, OCH2CdCH), 4.62 (dd, J5,6a 2.4 Hz, J6a,6b -14.3
0
0
0
0
Hz, H-6a), 4.40 (dd, J5,6b 8.9 Hz, H-6b), 4.30 (dd, J5 ,6 b 5.1 Hz,
H-60b), 4.18 (dd, H-5), 4.13 (dd, J5 ,6 a 2.4 Hz, J6 a,6 b -11.9 Hz,
H-60a), 4.05 (m, 1H, H-50), 3.22 (m, 2H, OCH2C7H15), 2.16,
2.15, 2.12 (2ꢀ), 2.04, 2.00, 1.98 (each s, each 3H, 7 ꢀ CH3CO),
1.46-1.40 (m, 2H, OCH2CH2C6H13), 1.31-1.17 (m, 10H,
OCH2CH2(CH2)5CH3), 0.88 (t, 3H, J 6.9 Hz, O(CH2)7CH3).
13C NMR (100 Mz, CDCl3): δ 170.9, 170.4, 170.2, 170.1, 170.0
(2ꢀ), 169.9 (7 ꢀ CH3CO), 143.7 (NCdCH), 124.7 (NCd
CH), 97.6 (C-1), 97.2 (C-10), 69.8, 69.6, 69.3, 69.2, 69.0 (2ꢀ),
67.9, 66.2 (C-2, C-20, C-3, C-30, C-4, C-40, C-5, C-50), 68.7
(OCH2C7H15), 62.6 (C-60), 61.4 (OCH2CdCH), 51.3 (C-6),
32.0, 29.5, 29.4, 29.3, 26.2, 22.8 (OCH2(CH2)6CH3), 21.1, 21.0,
20.9 (2ꢀ), 20.8, 20.7 (2ꢀ) (7 ꢀ CH3CO), 14.3 (O(CH2)7-
CH3). HRMS (MALDI): m/z calcd for [C37H55N3O18]Hþ
830.3558. Found 830.3550.
0
0
0
0
4-(2,3,4-Tri-O-acetyl-6-azido-6-deoxy-R-D-mannopyrano-
syloxymethyl)-1-(octyl 2,3,4-tri-O-acetyl-6-deoxy-R-D-manno-
pyranoside-6-yl)-1H-1,2,3-triazole (11). To a solution of com-
pound (10) (0.14 g, 0.15 mmol) in DMF (4 mL), NaN3 (52 mg,
0.80 mmol) and 15-crown-5 (0.18 g, 0.16 mL, 0.8 mmol) were
added. The reaction mixture was stirred at 60 °C for 16 h. The
reaction mixture was cooled to rt, diluted with CH2Cl2 (30 mL),
and extracted with water (3 ꢀ 10 mL) and brine (1 ꢀ 10 mL).
The organic phase was dried with anhydrous Na2SO4 and con-
centrated. Purification by column chromatography (hexanes/
EtOAc 5:1f1:1) gave (11) (0.11 g, 92%) as an oil. [R]D þ36
4-(R-D-Mannopyranosyloxymethyl)-1-(octyl 6-deoxy-R-D-
mannopyranoside-6-yl)-1H-1,2,3-triazole (9). Compound (8)
(67 mg, 0.08 mmol) was dissolved in MeOH (5 mL), and 1 M
MeONa (0.2 mL) was added. The reaction mixture was stirred at
rt for 16 h, neutralized with DOWEX 50 Hþ-form, filtered, and
concentrated. The crude product was purified by column chro-
matography (CHCl3/MeOH 20:1f3:1). Evaporation and lyo-
philization gave the title compound (9) (40 mg, 93%). [R]D þ55
(c 0.2, methanol). 1H NMR (400 MHz, CD3OD): δ 8.05 (s, 1H,
CHN), 4.89-4.85 (m, 2H, H-10, H-6a), 4.80 (d, 1H, J -12.3 Hz,
OCH2CdCH), 4.68 (d, 1H, J1,2 1.4 Hz, H-1), 4.64 (d, 1H,
J -12.3 Hz, OCH2CdCH), 4.50 (dd, 1H, J5,6b 8.7 Hz,
1
(c 0.5, CHCl3). H NMR (400 MHz, CDCl3): δ 7.75 (s, 1H,
CHN), 5.36 (dd, 1H, J2,3 3.4 Hz, J3,4 9.9 Hz, H-3), 5.32-5.26 (m,
2H, H-30, H-40), 5.25-5.21 (m, H-2, H-20), 5.14 (t, 1H, J4,5 10.0
Hz, H-4), 4.95 (d, 1H, J1 ,2 1.3 Hz, H-10), 4.90 (d, 1H, J -12.3
Hz, OCH2CdCH), 4.74 (d, 1H, J1,2 1.1 Hz, H-1), 4.69 (d, 1H,
J -12.3 Hz, OCH2CdCH), 4.62 (dd, J5,6a 2.4 Hz, J6a,6b -14.2
Hz, H-6a), 4.40 (dd, J5,6b 8.9 Hz, H-6b), 4.17 (m, 1H, H-5), 4.02
(m, 1H, H-50), 3.38-3.34 (m, 2H, H-60a, H-60b), 3.22 (m, 2H,
OCH2C7H15), 2.15 (2ꢀ), 2.12, 2.04, 2.00, 1.98 (each s, each 3H,
6 ꢀ CH3CO), 1.46-1.39 (m, 2H, OCH2CH2C6H13), 1.32-
1.17 (m, 10H, OCH2CH2(CH2)5CH3), 0.88 (t, 3H, J 6.9 Hz,
O(CH2)7CH3). 13C NMR (100 Mz, CDCl3): δ 170.4, 170.2,
170.1, 170.0, 169.9 (2ꢀ) (6 ꢀ CH3CO), 143.5 (NCdCH),
124.8 (NCdCH), 97.6 (C-1), 96.9 (C-10), 70.5, 69.8, 69.6, 69.3,
69.0, 68.9, 67.8, 67.2 (C-2, C-20, C-3, C-30, C-4, C-40, C-5, C-50),
68.7 (OCH2C7H15), 61.3 (OCH2CdCH), 51.3, 51.2 (C-6,
C-60), 32.0, 29.5, 29.4, 29.3, 26.2, 22.8 (OCH2(CH2)6CH3),
21.1-20.9 (6 ꢀ CH3CO), 14.3 (O(CH2)7CH3). HRMS
(MALDI): m/z calcd for [C35H52N6O16]Hþ 813.3512. Found
813.3538.
0
0
0
0
0
0
J6a,6b -14.1 Hz, H-6b), 3.87 (dd, J5 ,6 a 1.9 Hz, J6 a,6 b -11.8
Hz, H-60a), 3.82 (dd, J5,6a 2.2 Hz, H-5), 3.79-3.76 (m, 2H, H-2,
H-20), 3.72 (dd, 1H, J5 ,6 b 6.0 Hz, H-60b), 3.69-3.56 (m, 4H,
H-3, H-30, H-40, H-50), 3.51 (t, 1H, J3,4 9.4 Hz, J4,5 9.8 Hz, H-4),
3.22 (m, 2H, OCH2C7H15), 1.45-1.20 (m, 12H, OCH2-
(CH2)6CH3), 0.90 (t, 3H, J 6.9 Hz, O(CH2)7CH3). 13C NMR
(100 MHz, CD3OD): δ 145.1 (NCdCH), 127.1 (NCdCH),
101.6 (C-1), 100.6 (C-10), 75.1, 73.1, 72.6, 72.5, 72.2, 72.1, 69.8,
68.8 (C-2, C-20, C-3, C-30, C-4, C-40, C-5, C-50), 68.7
(OCH2C7H15), 63.1 (C-60), 60.6 (OCH2CdCH), 52.6 (C-6),
33.1, 30.6 (2ꢀ), 30.5, 27.5, 23.9 (OCH2(CH2)6CH3), 14.6
(O(CH2)7CH3). HRMS (MALDI): m/z calcd for [C23H41-
N3O11]Hþ 536.2814. Found 536.2811.
0
0
4-(2,3,4-Tri-O-acetyl-6-O-tosyl-R-D-mannopyranosyloxymethyl)-
1-(octyl 2,3,4-tri-O-acetyl-6-deoxy-R-D-mannopyranoside-6-
yl)-1H-1,2,3-triazole (10). Compound (3) (0.09 g, 0.20 mmol)
and compound (7) (0.1 g, 0.20 mmol) were dissolved in DMF/
H2O (3 mL/1 mL). Copper sulfate (12.8 mg, 0.08 mmol) and
sodium ascorbate (31.8 mg, 0.16 mmol) were added, and the
resulting mixture was stirred at rt for 4 h. The mixture was poured
into satd. NH4Cl/H2O solution (10 mL, 1:1) and extracted with
EtOAc (5 ꢀ 10 mL). The organic phase was dried with anhydrous
Na2SO4 and concentrated. The residue was purified by column
chromatography (hexanes/EtOAc 6:1f1:5) to give (10) as a
colorless oil (0.16 g, 85%). [R]D þ38 (c 0.5, CHCl3). 1H NMR
(400 MHz, CDCl3): δ 8.02 (s, 1H, CHN), 7.80 (d, 2H, J 8.2 Hz,
4-{2,3,4-Tri-O-acetyl-6-deoxy-6-[4-(2,3,4,6-tetra-O-acetyl-R-
D-mannopyranosyloxymethyl)-1H-1,2,3-triazol-1-yl]-R-D-man-
nopyranosyloxymethyl}-1-(octyl 2,3,4-tri-O-acetyl-6-deoxy-R-
D-mannopyranoside-6-yl)-1H-1,2,3-triazole (12). Compound
(5) (44 mg, 0.11 mmol) and compound (11) (93 mg, 0.11 mmol)
were dissolved in DMF/H2O (2.1 mL/0.7 mL). Copper sulfate
(3.6 mg, 0.023 mmol) and sodium ascorbate (9.0 mg, 0.046
mmol) were added, and the resulting mixture was stirred at rt for
4 h. The mixture was poured into satd. NH4Cl/H2O solution (10
mL, 1:1) and extracted with EtOAc (5 ꢀ 10 mL). The organic
phase was dried with anhydrous Na2SO4 and concentrated. The
residue was purified by column chromatography (hexanes/
EtOAc 4:1f1:9) to give (12) as a colorless oil (0.11 g, 80%).
Ts), 7.37 (d, 2H, J 8.1 Hz, Ts), 5.36 (dd, 1H, J3,4 9.9 Hz, H-3),
0
0
0
0
0
5.27 (dd, 1H, J2 ,3 3.4 Hz, J3 ,4 10.0 Hz, H-3 ), 5.22 (dd, 1H, J2,3
3.4 Hz, H-2), 5.19-5.11 (m, 3H, H-20, H-4, H-40), 4.88 (d, 1H,
292
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Bioconjugate Chemistry
ARTICLE
Scheme 1a
a Reagents and conditions: (a) 1. TsCl, CH2Cl2, py, 16 h, rt; 2. Ac2O, py, 16 h, rt, 51% over 2 steps for 2; 47% for 7; (b) NaN3, DMF, 80 °C; 40 h, 80%;
(c) BF3 OEt2, propargyl alcohol, CH2Cl2, 24 h, rt, 90%; (d) MeONa, MeOH/CH2Cl2, 24 h, rt, 89%.
3
[R]D þ40 (c 0.5, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.86,
7.67 (each s, each 1H, 2 ꢀ CHN), 5.35 (dd, 1H, J3,4 10.0 Hz, J2,3
3.6 Hz, H-3), 5.31-5.27 (m, 3H, H-30, H-300, H-400), 5.22 (dd,
27.5, 23.9 (OCH2(CH2)6CH3), 14.6 (O(CH2)7CH3). HRMS
(MALDI): m/z calcd for [C32H54N6O16]Hþ 779.3669; found
779.3673.
1H, H-2), 5.20-5.18 (m, H-20, H-200), 5.16 (t, 1H, J3 ,4 9.2 Hz,
0
0
In Vitro Mannosyltransferase Assay and Preparation of the
Enzyme Reaction Products for Structural Characterization.
ManT reactions were performed with mycobacterial membranes
and cell wall fractions as the source of the enzyme, as described.36
The reaction mixtures contained 1.5 mg membrane protein, 1 mg
cell wall protein, 0.05 μCi GDP-[14C]mannose (Amersham, 275
mCi/mmol), 60 μM ATP, DMSO in final concn of 0.8% (v/v), 4
mM synthetic acceptors, and buffer A (50 mM MOPS pH 7.9, 10
mM MgCl2, 5 mM β-mercaptoethanol) in the final volume of
160 μL. The reactions were incubated for 1 h at 37 °C and then
stopped by an addition of 1 mL of 96% ethanol. The reaction
products were obtained by n-butanol/water partitioning, as
described before.36 The butanol extracts were dried under N2
and resuspended in n-butanol. 10% of these extracts were
analyzed by TLC as described below for evaluation of possible
inhibitory effect of the tested compounds on the production of
the natural mannolipids: PIMs and polyprenylphosphoryl man-
noses in the in vitro assay. The rest of the sample was subjected to
mild acid and mild alkali hydrolyses36 to reveal the products
formed from the synthetic substrates. TLC analysis of the
reaction products was performed on aluminum-coated silica 60
F254 plate (Merck) developed in CHCl3/MeOH/conc NH4OH/
H2O (65:25:0.5:4; by vol.). The TLC plates were exposed to
X-ray film (Kodak Bio-Max MR) and incorporation of radio-
active label to the individual products was quantified by scraping
the silica from the corresponding bands to scintillation vials and
measuring the radioactivity, as described.36
Milligram-scale reactions were carried out with 4 mM com-
pounds (9) and (13) using the same conditions as described
above in the volume of 320 μL, using 1.5 mg membrane protein,
1 mg cell wall protein, and 200 μM cold GDP-Man instead of the
radioactive substrate. Dried ethanol extracts collected from 3
reactions, in the case of 13, and 5 reactions, in the case of 9, were
redissolved in 2 mL of water and loaded on a C18 Sep-Pak
cartridge (Waters) preequilibrated with methanol and water.
Then, the cartridge was first thoroughly washed with water and
finally eluted with methanol. Methanol fractions containing
carbohydrate compounds were pooled, dried under nitrogen,
and analyzed by mass spectrometry.
J4 ,5 9.6 Hz, H-400)0 , 5.12 (t, 1H, J4,5 9.8 Hz, H-4), 4.95 (d, 1H,
0
0
0
00 00
0
0
J1 ,2 1.6 Hz, H-1 ), 4.91 (d, 1H, J1 ,2 1.2 Hz, H-1 ), 4.87 (d, 1H,
J -12.2 Hz, OCH2CdCH), 4.75 (d, 1H, J1,2 1.6 Hz, H-1), 4.68
(d, 1H, J -12.3 Hz, OCH2CdCH), 4.62 (dd, J5,6a 2.6 Hz,
0
0
0
0
J6a,6b -14.1 Hz, H-6a), 4.58 (dd, 1H, J5 ,6 a 2.7 Hz, J6 a,6 b -13.9
Hz, H-60a), 4.47 (dd, 1H, J5 ,6 b 8.6 Hz, H-60b), 4.43 (d, 2H,
J -11.8 Hz, OCH2CdCH), 4.40 (dd, 1H, J5,6b 8.6 Hz, H-6b),
4.32-4.27 (m, 2H, H-50, H-600b), 4.16 (m, 1H, H-5), 4.12 (dd,
0
0
1H, J5 ,6 a 2.2 Hz, J6 a,6 b -12.3 Hz, H-600a), 4.06 (m, 1H,
H-500), 3.22 (m, 2H, OCH2C7H15), 2.15, 2.14, 2.13, 2.12, 2.11
(2ꢀ), 2.03, 2.00, 1.98, 1.97 (each s, each 3H, 10 ꢀ CH3CO),
1.47-1.40 (m, 2H, OCH2CH2C6H13), 1.32-1.19 (m, 10H,
OCH2CH2(CH2)5CH3), 0.88 (t, 3H, J 6.9 Hz, O(CH2)7CH3).
13C NMR (100 Mz, CDCl3): δ 170.9, 170.3-169.9 (9ꢀ) (10 ꢀ
CH3CO), 143.8, 143.2 (2 ꢀ NCdCH), 125.0, 124.9 (2 ꢀ
NCdCH), 97.5 (C-1), 97.1 (C-100), 96.8 (C-10), 69.8, 69.6,
69.4 (2ꢀ), 69.2, 69.1, 69.0, 68.9, 68.8, 67.8, 67.6, 66.2 (C-2, C-20,
C-200, C-3, C-30, C-300, C-4, C-40, C-400, C-5, C-50, C-500), 68.7
(OCH2C7H15), 62.6 (C-600), 61.2, 60.9 (2 ꢀ OCH2CdCH),
51.2 (2ꢀ) (C-6, C-60), 32.0, 29.5, 29.3 (2ꢀ), 26.2, 22.8 (6 ꢀ
OCH2(CH2)6CH3), 21.1-20.8 (10 ꢀ CH3CO), 14.3 (O(CH2)7-
CH3). HRMS (MALDI): m/z calcd for [C52H74N6O26]Hþ
1199.4725. Found 1199.4728.
00 00
00
00
4-{6-Deoxy-6-[4-(R-D-mannopyranosyloxymethyl)-1H-1,2,
3-triazol-1-yl]-R-D-mannopyranosyloxymethyl}-1-(octyl 6-deoxy-
R-D-mannopyranoside-6-yl)-1H-1,2,3-triazole (13). Compound
(12) (80 mg, 0.067 mmol) was dissolved in MeOH/CH2Cl2 (2.2mL:
0.25 mL) and 1 M MeONa (0.12 mL) was added. The reaction
mixture was stirred for 16 h, then neutralized with DOWEX 50 Hþ-
form, filtered, concentrated, and lyophilized to give the title com-
pound (13) (42 mg, 81%). [R]D þ50 (c 0.2, methanol). 1H NMR
(400 MHz, CD3OD): δ 8.10, 7.83 (each s, each 1H, 2 ꢀ CHN),
4.85-4.73 (m, 3H), 4.72 (d, 1H, J -12.3 Hz, OCH2CdCH), 4.71
(d, 1H, J 1.0 Hz), 4.61 (d, 1H, J 0.9 Hz), 4.57 (d, 1H, J -12.3 Hz,
OCH2CdCH), 4.48-4.40 (m, 2H), 4.28 (q, 2H, J -12.3 Hz,
OCH2CdCH), 3.81-3.37 (m, 14H), 3.14 (m, 2H, OCH2C7H15),
1.35-1.11 (m, 12H, OCH2(CH2)6CH3), 0.80 (t, 3H, J 6.9 Hz,
O(CH2)7CH3). 13C NMR (100 MHz, CD3OD): δ 145.3, 144.7
(2 ꢀ NCdCH), 127.1, 126.9 (2 ꢀ NCdCH), 101.8, 100.9, 100.8
(C-1, C-10, C-100), 75.2, 73.3, 73.0, 72.6 (2ꢀ), 72.5, 72.3, 72.1, 72.0,
69.8 (2ꢀ), 68.8 (C-2, C-20, C-200, C-3, C-30, C-300, C-4, C-40, C-400,
C-5, C-50, C-500), 68.8 (OCH2C7H15), 63.2 (C-600), 60.7 (2ꢀ)
(2 ꢀ OCH2CdCH), 52.7 (2ꢀ) (C-6, C-60), 33.1, 30.6 (2ꢀ), 30.5,
’ RESULTS AND DISCUSSION
Synthesis of the Conjugates. The synthetic route to azido
and alkyne building blocks used in the CuAAC reaction is
depicted in Scheme 1.
293
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Bioconjugate Chemistry
ARTICLE
Scheme 2a
δ = 128-145 and for methyl group of the tosyl function at δ =
21.9 were present. In the next step, the tosyl group of 10 was
smoothly replaced by reaction with sodium azide under a
promotion of 15-crown-5 in DMF at 60 °C providing conjugate
(11) in almost quantitative yield (92%). In this compound, the
chemical shift of C-60 (δ ≈ 51) was markedly upfielded which
clearly indicated the introduction of azide. Repetition of the
CuAAC reaction utilizing azido (11) and alkyne (5) building
blocks led to a formation of conjugate (12) consisting of three
mannose units interconnected with 1,2,3-triazole linkers. The
presence of two 1,2,3-triazole linkers was assigned from two
singlets (δ = 7.86 and δ = 7.67) of the olefinic protons. Final
Zemplen deprotection of 12 proceeded smoothly giving the
target conjugate (13).
a Reagents and conditions. (a) CuSO4, sodium ascorbate, DMF/H2O
3:1, 4 h, rt, 89%; (b) MeONa, MeOH, 16 h, rt, 93%.
The alkyne building block, 2-propynyl 2,3,4,6-tetra-O-acetyl-
R-D-mannopyranoside (5)30,37 was accessible with a yield of
∼90% in one step by glycosylation of propargyl alcohol with
The CuAAC reaction provided simple and regioselective access
to the conjugates in which the 1,2,3-triazole ring acts as a bridge
between positions 1 and 6 of neighboring mannose units.
Keeping structural similarity with natural acceptors, these con-
jugates are promising compounds for evaluation in ManT assay.
In our recent study,36 octyl R-D-mannopyranoside (14) along
with octyl R-D-mannopyranosyl-(1f6)-R-D-mannopyranoside
(15) were recognized as the acceptor substrates in ManT assay,
and both compounds (Figure 3) were chosen as the reference
synthetic substrates.
Biological Evaluation of the Synthesized Compounds.
The synthetic conjugates (9) and (13) and the reference synthetic
substrates (14) and (15) were tested in the in vitro assay monitoring
ManT activity of the mycobacterial membranes. First, we eval-
uated the possible inhibitory effect of these compounds on
ManT activity by TLC analysis of the full range of radioactive
lipids having incorporated [14C]-Man from GDP-[14C]-Man.
Similarly to our previous observation for 14 and 15,36 the
presence of 9 or 13 in the reaction mixtures did not affect
production of labeled phosphatidylinositol mannosides or poly-
prenylphosphoryl mannoses (data not shown). In a recent study
of Lo Conte et al.,31 the inhibitory effect of triazolo-linked C-
oligomannosides (dimer to hexadecamer) were, on the contrary,
examined in the ManT assay using synthetic octyl (1f6)-R-D-
mannodisaccharide as the acceptor substrate and M. smegmatis
membranes as the enzyme source. Hexamer and octamer were
the most potent conjugates showing about 95% inhibition at
1 mM concentration. While elongation of these compounds could
not be expected due to their deoxygenation at position 6 of the
nonreducing terminal mannose residue, the novel triazolo-linked
oligomannosides (9) and (13) presented in our study possess
free hydroxyl group at position 6, which is necessary for their
elongation by the mycobacterial R-(1f6)-ManT. Therefore, we
aimed to investigate this possibility.
mannose pentaacetate (4) in the presence of BF3 OEt2 at rt. The
3
synthesis of azide (3) bearing hydrophobic octyl aglycone was
accomplished by a three-step protocol. Octyl R-D-mannopyrano-
side (1) was converted to acetylated 6-O-tosyl derivative (2) by a
similar procedure as we described recently36 followed by a
treatment of the latter with an excess of NaN3 in DMF at
80 °C providing the azido building block (3). On the basis of
previous reports describing a successful improvement of the
CuAAC reaction protocol, the coupling of 3 and 5 was carried
out at rt for 4 h in DMF/H2O (3:1) solvent mixture using
copper(II) sulfate and sodium ascorbate.38 Under these condi-
tions, the conjugate (8) was obtained in good yield (89%) as a
single product (Scheme 2). Its structure was identified based on
1
the characteristic signals in the H NMR spectrum: olefinic
proton associated with the 1,2,3-triazole moiety at δ = 7.74 as
a singlet, and a pair of proton doublets at δ = 4.87 and δ = 4.67
assigned to methylene group linking O-10 atom to the triazole
moiety. In the 13C NMR spectrum, the following signals were
observed: at δ = 97.6 and δ = 97.2 for two anomeric carbons,
and at δ = 124.7 and δ = 143.7 assigned to olefinic carbons.
Characteristic signals for two hydrogens (OCH2C7H15) of octyl
chain were slightly shifted and detected as a multiplet at δ = 3.22,
while in the starting azide (3), they gave a characteristic pattern
of dt at δ = 3.72 and δ = 3.47 (each 1H). Removal of acetyl
groups from 8 with MeONa in MeOH yielded the unprotected
conjugate (9), wherein the olefinic proton showed a slight
downfield shift (singlet at δ = 8.05).
The construction of conjugate (12) consisting of three
mannose units interconnected through the 1,2,3-triazole linkers
was achieved by a strategy outlined in Scheme 3, starting from a
tosylated intermediate conjugate (10), since tosyl function
remains intact in the CuAAC reaction and can be subsequently
subjected to a nucleophilic substitution. The tosylated alkynyl
building block (7) was prepared by a protocol used for the
synthesis of 2, i.e., conversion of the primary hydroxyl group at
C-6 of 6 to the tosylate followed by conventional acetylation of
secondary hydroxyl groups (Ac2O/py). The CuAAC reaction
between azide (3) and alkyne (7) was accomplished under the
same experimental conditions as mentioned above giving con-
jugate (10) in high yield (85%). Its structure was clearly
Interestingly, both conjugates (9) and (13) served as acceptor
substrates for the ManT, although their acceptor ability was
significantly different. In fact, incorporation of the radioactive
label into compound (13) was hardly detectable by TLC (Figure 4).
In two separate experiments, the conjugate (9) having two mannose
units was about a 20ꢀ less effective acceptor than the disacchar-
ide analogue (15), but about a 2ꢀ better acceptor than the
monosaccharide (14). In addition, TLC analysis of the reaction
mixture containing conjugate (9) showed a ladder-like profile
suggesting stepwise mannose addition (Figure 4).
1
established as follows: In the H NMR spectrum, the olefinic
proton was identified as a singlet at δ = 8.02. The signals of tosyl
function were seen as two doublets (2H each) at δ = 7.80 and
δ = 7.37 and as a singlet at δ = 2.46 corresponding to a methyl
group. In the 13C NMR spectrum, signals for aromatic carbons at
Since TLC analysis indicated that both conjugates (9) and (13)
were transformed by the ManT, the enzymatic reactions of 9 and
13 with unlabeled GDP-Man were conducted on a larger scale to
obtain the reaction products in an amount sufficient for detailed
294
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Bioconjugate Chemistry
ARTICLE
Scheme 3a
a Reagents and conditions. (a) CuSO4, sodium ascorbate, DMF/H2O 3:1, 4 h, rt, 85%; (b) NaN3, 15-crown-5, DMF, 60 °C; 40 h, 92%; (c) (3), CuSO4,
sodium ascorbate, DMF/H2O 3:1, 4 h, rt, 80%; (d) MeONa, MeOH/CH2Cl2, 16 h, rt, 81%.
Figure 3. Structures of the reference synthetic substrates 14 and 15.
structural characterization. HPLC analysis of the products iso-
lated from the reaction mixture containing 9 showed three peaks
(Figure 5, a). The mass spectrum obtained from a dominant
chromatographic peak (marked as peak 3) with retention time of
24.5 min gave molecular [MþH]þ ion for conjugate (9) at m/z
536.5 (Figure 5, a,b3). The chromatographic peak with elution
time of 23.5 min (marked as peak 2) produced a mass spectrum
with molecular [MþH]þ ion at m/z 698.5, consistent with an
expected monomannosylated product (Figure 5, a,b2). Moreover,
the chromatographic peak eluting prior to the peaks mentioned
above (marked as peak 1, retention time of 22 min) produced an
Figure 4. TLC analysis of the reaction products formed from synthetic
acceptors 9, 13, 14, and 15 by mycobacterial ManT. The profile is a
representation of two replicates of the experiment carried out at 4 mM
[MþH]þ ion at m/z 860.5 corresponding to a transfer of two
mannosyl residues to the acceptor (9) (Figure 5, a,b1). The
detection of the latter doubly mannosylated product identified by
LC/MS is in agreement with the ladder-like TLC profile of the
reaction products obtained with this acceptor. Similarly, LC-MS
analysis carried out for the reaction mixture of 13 gave three
chromatographic peaks with retention times of 23.5, 22.6, and
22.1 min. The molecular [MþH]þ ions obtained from these
peaks at m/z values 779.6, 941.8, and 1103.8 (Figure S1, see
Supporting Information) clearly indicated the presence of 13 and
its monomannosylated and dimannosylated products. The dou-
bly mannosylated 13 detected by LC-MS but not apparent from
TLC thus gives additional information about the process activity
of the mycobacterial ManT.
concentration of the tested compounds. “-“ stands for control experi-
ment without any acceptor.
To determine the structure of the monomannosylated
product detected in the reaction mixture of 9, its molecular
ion was subjected to fragmentation. The product under
MALDI-MS conditions gave [MþNH4-H2O]þ molecular
ion at m/z 697.33 (calculated monoisotopic mass is 697.32),
which yielded the MS/MS fragmentation pattern shown in
Figure 6. Y- and Z-fragment ions at m/z 535.23 (Y4), 373.14
(Y3), and m/z 357.15 (Z3) resulted from a characteristic loss
of monosaccharide residue(s). The most abundant fragments
originated from the mannose cross-ring cleavages (X and
A-type fragment ions) and provided additional information
on the structure. The majority of the X4 cross ring ions is
isobaric and cannot be used to easily distinguish the linkage. In
this regard, the most important ion is that observed at m/z
505.25 (5,6X4), which in association with the Z4 fragment ion
observed at m/z 519.24 could be unambiguously assigned to a
In our recent study,36 in which the same enzyme preparations
exhibiting ManT activity were used, we observed that 6-deox-
ygenated derivatives were not elongated. Therefore, the ManT
reaction can be hypothesized to occur at position 6 of the non-
reducing terminal mannose unit of conjugates (9) and (13). This
assumption is also supported by previously published reports,32,33
wherein R-(1f6)-ManT activity of the enzyme was demonstrated.
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Figure 5. LC-MS analysis of the enzymatic reaction mixture of 9. Panel a: HPLC profile. Panel b: ESI mass spectra obtained from peaks 1, 2, and 3. For
assignment of the fragment ions, see upper part of Figure 6.
new (1f6) linkage in the monomannosylated product of 9
formed by the ManT reaction.
triazole linker is much bulkier than a single glycosidic oxygen
atom. In addition, the difference in the number of atoms connecting
the sugar residues may also result in an alteration of their spatial
orientation, which might affect interaction with the enzyme.
It seems that the natural glycosidic linkage is important for
keeping the ability of the compound to function as an efficient
mannosyl acceptor for the ManT. On the other hand, the
transformation of both conjugates performed by the ManT
demonstrates that the enzyme has a certain freedom to catalyze
the transfer of glycosyl residue(s). The enzyme has at least two
structural requirements for the acceptors: hydrophobic aglycone
and free primary hydroxyl group at the nonreducing terminal
mannose residue. Both these requirements are met not only with
the best synthetic acceptors, octyl (1f6)-R-D-Man2 (15) and
octyl (1f6)-R-D-Man3, but also with conjugates (9) and (13).
The differences in the ability of the four evaluated compounds to
function as acceptors for the ManT suggest that the relative
The size of the carbohydrate unit of the acceptor undoubtedly
plays an important role in ManT catalysis. As has been previously
shown by Lowary et al.,32 a disaccharide appears to be preferred
by the ManT, and octyl (1f6)-R-D-mannodisaccharide (15) has
been found to serve as the best synthetic acceptor. The enlarge-
ment of the acceptor to trisaccharide did not lead to a significant
increase in activity, since it was recognized as the acceptor with
similar efficiency as the disaccharide (15).32 In the case of
triazolo-linked oligomannosides, 13 bearing three mannose units
is a much poorer acceptor than 9, which comprises two man-
noses. The latter is elongated by the ManT slightly better than
monosaccharide (14) but markedly worse than disaccharide
(15). Hence, the acceptor ability increases in the order (13) ,
(14) < (9) < (15) = octyl (1f6)-R-D-mannotrisaccharide. All
these data can be explained by the size of the acceptors, since the
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Figure 6. MALDI-MS/MS spectrum and the proposed fragmentation scheme for the monomannosylated product (MW 697.33) generated from 9 by
the ManT.
distance and spatial orientation of these structural requirements
are important for ManT activity, but at the same time, their
variations are tolerated by the enzyme in a relatively wide range.
’ REFERENCES
(1) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B.
(2002) A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed
regioselective “ligation” of azides and terminal alkynes. Angew. Chem.,
Int. Ed. 41, 2596–2599.
’ ASSOCIATED CONTENT
(2) Tørnoe, C. W., Christensen, C., and Meldal, M. (2002) Pepti-
dotriazoles on solid phase: [1,2,3]-Triazoles by regiospecific copper(I)-
catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org.
Chem. 67, 3057–3064.
(3) Rossi, L. L., and Basu, A. (2005) Glycosidase inhibition by
1-glycosyl-4-phenyl triazoles. Bioorg. Med. Chem. Lett. 15, 3596–
3599.
(4) Wilkinson, B. L., Bornaghi, L. F., and Houston, T. A. (2006)
Synthetic utility of glycosyltriazoles in carbohydrate chemistry. Tetra-
hedron 62, 8115–8125.
(5) Kuipers, B. H. M., Dijkmans, G. C. T., Groothuys, S., Quaedflieg,
P. J. L. M., Blaauw, R. H., van Delft, F. L., and Rutjes, F. P. J. T. (2005)
Copper (I)-mediated synthesis of trisubstituted 1,2,3-triazoles. Synlett
3059–3062.
(6) Nepogodiev, S. A., Dedola, S., Marmuse, L., de Oliveira, M. T.,
and Field, R. A. (2007) Synthesis of triazole-linked pseudo-starch
fragments. Carbohydr. Res. 342, 529–540.
(7) Marmuse, L., Nepogodiev, S. A., and Field, R. A. (2005) ”Click
chemistry” en route to pseudo-starch. Org. Biomol. Chem. 3, 2225–
2227.
(8) Muthana, S., Yu, H., Cao, H., Cheng, J., and Chen, X. (2009)
Chemoenzymatic synthesis of a new class of macrocyclic oligosacchar-
ides. J. Org. Chem. 74, 2928–2936.
(9) Bodine, K. D., Gin, D. Y., and Gin, M. S. (2005) Highly
convergent synthesis of C3- or C2- symmetric carbohydrate macrocycles.
Org. Lett. 7, 4479–4482.
Supporting Information. 1H NMR and 13C NMR spec-
S
b
tra of compounds 2, 3, 7, 8, 9, 10, 11, 12, and 13. Figure S1. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Corresponding author. Tel: þ421 259410272. Fax: þ421
259410222. E-mail address: Monika.Polakova@savba.sk.
’ ACKNOWLEDGMENT
This work was supported by the APVV-51-046505 and
VEGA-2/0199/09 grants, the Slovak State Programme Pro-
ject No. 2003SP200280203 and by the European Commission
under contract LSHP-CT-2005-018923“NM4TB”. Drs. Vladimír
Puchart and Jana Kordulꢀakovꢀa are appreciated for helpful
discussion. Filip Ekholm (Åbo Akademi University) is thanked
for the computational analysis of the 1H NMR spectra of
conjugates by the PERCH NMR software. Dr. Tom Ward
(University of Manitoba) is thanked for help with LC-MS
experiments.
297
dx.doi.org/10.1021/bc100421g |Bioconjugate Chem. 2011, 22, 289–298

Bioconjugate Chemistry
ARTICLE
(10) Bodine, K. D., Gin, D. Y., and Gin, M. S. (2004) Synthesis of
readily modifiable cyclodextrin analogues via cyclodimerization of an
alkynyl-azido trisaccharide. J. Am. Chem. Soc. 126, 1638–1639.
(11) Hohta, S., and Kashyap, S. (2006) “Click chemistry” inspired
synthesis of pseudo-oligosaccharides and amino acid glycoconjugates.
J. Org. Chem. 71, 364–367.
(12) Pꢀerion, R., Ferriꢂeres, V., García-Moreno, M., Mellet, I., Duval,
C. O., R., Fernꢀandez, J. M. G., and Plusquellec, D. (2005) 1,2,3-Triazoles
and related glycoconjugates as new glycosidase inhibitors. Tetrahedron
61, 9118–9128.
(13) Hasegawa, T., Umeda, M., Numata, M., Li, C., Bae, A.-H.,
Fujisawa, T., Haraguchi, S., Sakurai, K., and Shinkai, S. (2006) `Click
chemistry’ on polysaccharides: a convenient, general, and monitorable
approach to develop (1f3)-β-D-glucans with various functional ap-
pendages. Carbohydr. Res. 341, 35–40.
(29) Wilkinson, B. L., Long, H., Sim, E., and Fairbanks, A. J. (2008)
Synthesis of Arabino glycosyl triazoles as potential inhibitors of myco-
bacterial cell wall biosynthesis. Bioorg. Med. Chem. Lett. 18, 6265–6267.
(30) van der Peet, P., Gannon, C. T., Walker, I., Dinev, Z., Angelin,
M., Tam, S., Ralton, J. E., McConville, M. J., and Williams, S. J. (2006)
Use of click chemistry to define the substrate specificity of Leishmania
β-1,2-mannosyltransferases. ChemBioChem 7, 1384–1391.
(31) Lo Conte, M., Marra, A., Chambery, A., Gurcha, S. S., Besra,
G. S., and Dondoni, A. (2010) Modular approach to triazole-linked 1,
6-R-D-oligomannosides to the discovery of inhibitors of Mycobacterium
tuberculosis cell wall synthetase. J. Org. Chem. 75, 6326–6336.
(32) Tam, P.-H., Besra, G. S., and Lowary, T. L. (2008) Exploring
the substrate specificity of a mycobacterial polyprenol monophospho-
mannose-dependent R-(1f6)-mannosyltransferase. ChemBioChem 9,
267–278.
(33) Brown, J. R., Field, R. A., Barker, A., Guy, M., Grewal, R., Khoo,
K.-H., Brennan, P. J., Besra, G. S., and Chatterjee, D. (2001) Synthetic
mannosides act as acceptors for mycobacterial R1-6 mannosyltransfer-
ase. Bioorg. Med. Chem. 9, 815–824.
(34) Domon, B., and Costello, C. E. (1988) A systematic nomen-
clature for carbohydrate fragmentations in FAB MS-MS spectra of
glycoconjugates. Glycoconjugate J. 5, 397–409.
(14) Temelkoff, D. P., Zeller, M., and Norris, P. (2006) N-Glycoside
neoglycotrimers from 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide.
Carbohydr. Res. 341, 1081–1090.
(15) Cheshev, P., Marra, A., and Dondoni, A. (2006) First synthesis
of 1,2,3-triazolo-linked (1,6)-R-D-oligomannoses (triazolomannoses)
by iterative Cu(I)-catalyzed alkyne-azide cycloaddition. Org. Biomol.
Chem. 4, 3225–3227.
(35) Watt, J. A., and Williams, S. J. (2005) Rapid, iterative assembly
of octyl R-1,6-oligomannosides and their 6-deoxy equivalents. Org.
Biomol. Chem. 3, 1982–1992.
(16) Agren, J. K. M., Billing, J. F., Grundberg, H. E., and Nilsson, U. J.
(2006) Synthesis of a chiral and fluorescent sugar-based macrocycle by
1,3-dipolar cycloaddition. Synthesis 3141–3145.
(36) Polꢀakovꢀa, M., Belꢀaꢁnovꢀa, M., Petruꢁs, L., and Mikuꢁsovꢀa, K.
(2010) Synthesis of alkyl and cycloalkyl R-D-mannopyranosides and
derivatives thereof and their evaluation in the mycobacterial mannosyl-
transferase assay. Carbohydr. Res. 345, 1339–1347.
(37) Wardrop, D. J., Zhang, W., and Fritz, J. (2002) Stereospecific
entry to [4.5]spiroketal glycosides using alkylidenecarbene C-H inser-
tion. Org. Lett. 4, 489–492.
(38) Gouin, S. G., Bultel, L., Falentin, C., and Kovensky, J. (2007) A
simple procedure for connecting two carbohydrate moieties by click
chemistry techniques. Eur. J. Org. Chem. 1160–1167.
(17) Jarosz, S., Lewandowski, B., and Listkowski, A. (2008) Towards
sucrose macrocycles with higher symmetry via a ’Click Chemistry’ route.
Synthesis 913–916.
(18) Ray, A., Manoj, K., Bhadbhade, M. M., Mukhopadhyay, R., and
Bhattachrajya, A. (2006) Cu(I)-Catalyzed cycloaddition of constrained
azido-alkynes: access to 12- to 17-membered monomeric triazolophanes
incorporating furanoside rings. Tetrahedron Lett. 47, 2775–2778.
(19) Dondoni, A., and Marra, A. (2006) C-Glycoside clustering on
calix[4]arene, adamantane, and benzene scaffolds through 1,2,3-triazole
linkers. J. Org. Chem. 71, 7546–7557.
(20) Katajisto, J., Karskela, T., Heinonen, P., and Lonnberg, H.
(2002) An orthogonally protected R,R-bis(aminomethyl)-β-alanine
building block for the construction of glycoconjugates on a solid
support. J. Org. Chem. 67, 7995–8001.
(21) Fernandez-Megia, E., Correa, J., and Riguera, R. (2006) “Click-
able” PEG-dendritic block copolymers. Biomacromolecules 7, 3104–
3111.
ꢁ
(22) Niederhafner, P., Sebestík, J., and Jeꢁzek, J. (2008) Glycopeptide
dendrimers. Part I. J. Pept. Sci. 14, 2–43.
(23) Deguise, I., Lagnoux, D., and Roy, R. (2007) Synthesis of
glycodendrimers containing both fucoside and galactoside residues and
their binding properties to Pa-IL and PA-IIL lectins from Pseudomonas
aeruginosa. New J. Chem. 31, 1321–1331.
(24) Wolfenden, M. L., and Cloninger, M. J. (2005) Mannose/
glucose-functionalized dendrimers to investigate the predictable tun-
ability of multivalent interactions. J. Am. Chem. Soc. 127, 12168–12169.
(25) Vargas-Berenguel, A., Ortega-Caballero, F., and Casas-Solvas,
J. M. (2007) Supramolecular chemistry of carbohydrate clusters with
cores having guest binding abilities. Mini-Rev. Org. Chem. 4, 1–14.
(26) Gꢀomez-García, M., Benito, J. M., Rodríguez-Lucena, D., Yu,
J. X., Chmurski, K., Mellet, C. O., Gutiꢀerrez-Gallego, R., Maestre, A.,
Defaye, J., and García Fernꢀandez, J. M. (2005) Probing secondary
carbohydrate-protein interactions with highly dense cyclodextrin-
centered heteroglycoclusters: The heterocluster effect. J. Am. Chem.
Soc. 127, 7970–7971.
(27) Ladmiral, V., Mantovani, G., Clarkson, G. J., Cauet, S., Irwin,
J. L., and Haddleton, D. M. (2006) Synthesis of neoglycopolymers by a
combination of “click chemistry” and living radical polymerization.
J. Am. Chem. Soc. 128, 4823–4830.
(28) Singh, B. S., Yadav, A. K., Kumar, B., Gaikwad, A., Sinha, S. K.,
Chaturvedi, V., and Tripathi, R. P. (2008) Preparation and reactions of
sugar azides with alkynes: synthesis of sugar triazoles as antitubercular
agents. Carbohydr. Res. 343, 1153–1162.
298
dx.doi.org/10.1021/bc100421g |Bioconjugate Chem. 2011, 22, 289–298