Modular approach to triazole-linked 1,6-α-d-oligomannosides to the discovery of inhibitors of mycobacterium tuberculosis cell wall synthetase

Article abstract of DOI:10.1021/jo100928g

Aiming at developing inhibitors of mannosyltransferases, the enzymes that participate in the biosynthesis of the cell envelope of Mycobacterium tuberculosis, the synthesis of a range of designed triazole-linked 1,6-oligomannosides up to a hexadecamer has been accomplished by a modular approach centered on the Cu(I)-catalyzed azide-alkyne cycloaddition as key process. The efficiency and fidelity of the cycloaddition are substantiated by high yields (76-96%) and exclusive formation of the expected 1,4-disubstituted triazole ring in all oligomer assembling reactions. Key features of oligomers thus prepared are the anomeric carbon-carbon bond of all mannoside residues and the 6-deoxymannoside capping residue. Suitable bioassays with dimer, tetramer, hexamer, octamer, decamer, and hexadecamer showed variable inhibitor activity against mycobacterial α-(1,6)-mannosyltransferases, the highest activity (IC₅₀ = 0.14-0.22 mM) being registered with the hexamannoside and octamannoside.

Full text of DOI:10.1021/jo100928g

pubs.acs.org/joc
Modular Approach to Triazole-Linked 1,6-r-D-Oligomannosides
to the Discovery of Inhibitors of Mycobacterium tuberculosis
Cell Wall Synthetase
Mauro Lo Conte,† Alberto Marra,† Angela Chambery,‡ Sudagar S. Gurcha,§
Gurdyal S. Besra,§ and Alessandro Dondoni*,†
†
ꢀ
Dipartimento di Chimica, Laboratorio di Chimica Organica, Universita di Ferrara, Via L. Borsari 46,
I-44100 Ferrara, Italy, Dipartimento di Scienze della Vita, II Universita di Napoli, Via Vivaldi 43,
‡
ꢀ
§
I-81100 Caserta, Italy, and School of Biosciences, The University of Birmingham, Edgbaston,
Birmingham B15 2TT, U.K.
adn@unife.it
Received May 20, 2010
Aiming at developing inhibitors of mannosyltransferases, the enzymes that participate in the
biosynthesis of the cell envelope of Mycobacterium tuberculosis, the synthesis of a range of designed
triazole-linked 1,6-oligomannosides up to a hexadecamer has been accomplished by a modular
approach centered on the Cu(I)-catalyzed azide-alkyne cycloaddition as key process. The efficiency
and fidelity of the cycloaddition are substantiated by high yields (76-96%) and exclusive formation
of the expected 1,4-disubstituted triazole ring in all oligomer assembling reactions. Key features of
oligomers thus prepared are the anomeric carbon-carbon bond of all mannoside residues and the
6-deoxymannoside capping residue. Suitable bioassays with dimer, tetramer, hexamer, octamer,
decamer, and hexadecamer showed variable inhibitor activity against mycobacterial R-(1,6)-
mannosyltransferases, the highest activity (IC50 = 0.14-0.22 mM) being registered with the hexa-
mannoside and octamannoside.
Introduction
incompatibility. Various drugs are currently available with
different modes of action to treat TB.3 Nevertheless, drug
resistance continues to emerge, and therefore, there is an
increasing demand for rapid development of new antituber-
cular drugs4 targeting essential functions of its etiological
agent, Mycobacterium tuberculosis (Mtb). As this bacterium
invades and colonizes macrophage cells, its ability to survive
within this inhospitable environment has been attributed to
its robust cell wall.5 Therefore, an ideal TB drug target is the
biosynthesis of the mycobacterial cell envelope. Indeed, two
Among infectious diseases that were thought to be de-
feated in the last century, tuberculosis (TB) has reappeared
overbearingly1 and is presently a leading cause of mortality
in many parts of the world, including Western countries.
Tuberculosis is especially diffused in sub-Saharan Africa,
where it is epidemic because of an increased susceptibility
conferred by HIV infection.2 The convergence of TB and
HIV poses difficult problems because antiviral and antitu-
bercular drugs given together may present high levels of
(3) Lenaerts, A. J.; De Groote, M. A.; Orme, I. M. Trends Microbiol.
2008, 16, 48–54.
(1) (a) The Return of the White Plague: Global Poverty and the New
Tuberculosis; Gaudy, M., Zumla, A., Eds.; Verso: London, 2003. (b) Ryan, F.
The Forgotten Plague: How the Battle Against Tuberculosis Was Won - and Lost;
Back Bay Books: New York, 1994.
(2) (a) Corbett, E. L.; Marston, B.; Churchyard, G. J.; De Cock, K. M.
Lancet 2006, 367, 926–937. (b) Maartens, G.; Wilkinson, R. J. Lancet 2007,
370, 2030–2043.
(4) (a) Zhao, Y.; Bacher, A.; Illarionov, B.; Fischer, M.; Georg, G.; Ye,
Q.-Z.; Fanwick, P. E.; Franzblau, S. G.; Wan, B.; Cushman, M. J. Org.
Chem. 2009, 74, 5297–5303. (b) Hung, A. W.; Silvestre, H. L.; Wen, S.; Ciulli,
A.; Blundell, T. L.; Abell, C. Angew. Chem., Int. Ed. 2009, 48, 8452–8456.
(c) Tan, L. P.; Wu, H.; Yang, P.-Y.; Kalesh, K. A.; Zhang, X.; Hu, M.;
Srinivasan, R.; Yao, S. Q. Org. Lett. 2009, 11, 5102–5105.
6326 J. Org. Chem. 2010, 75, 6326–6336
Published on Web 09/07/2010
DOI: 10.1021/jo100928g
r
2010 American Chemical Society

Lo Conte et al.
JOCFeatured Article
various instances.10 Moreover, in a series of papers, Lowary
and co-workers have described the preparation and bio-
chemical evaluation of fluoro, methoxy, amino, and deoxy
analogues11 of octyl 1,6-R-D-dimannoside12 (R-D-Manp-(1,6)-
R-D-Manp-O(CH2)7CH3) through selective substitution of
the C6 hydroxyl group. Compounds have been also prepared
in which two of the hydroxyl groups on the nonreducing
residue of the above disaccharide were replaced.13 These
compounds were all considered potential substrates and
inhibitors for R-1,6-mannosyltransferase (R-(1,6)-ManT)
activity required for the biosynthesis of LM and LAM
mannan cores. Along the same vein, Watt and Williams
prepared a series of hydrophobic octyl 6-deoxy R-1,6-linked
oligomannosides up to a tetramer via iterative glycosylation.14
The rationale behind these synthetic efforts was that deoxy-
genation of the 6-position of the oligomannosyl chain should
prevent these compounds acting as substrates for the R-(1,6)-
ManTs. Oligomannosides prepared by Watt and Williams
featured the natural O-glycosidic anomeric linkage and,
therefore, were prone to undergo a facile cleavage by hydro-
lases. Moreover, the chain lengths of these compounds might
be too short for an efficient mimicry of the oligomannoside
chains of LMs and LAMs, which are in fact constituted of
10-20 mannose units. Hence, we have developed a new
approach to more stable and higher oligomers 1, all pre-
served from hydrolytic degradation by virtue of anomeric
carbon-carbon bonds and triazole rings as interglycosidic
linkers (Figure 1). In fact, we envisaged the Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC), first chronicled by
Sharpless and Meldal groups,15 as a suitable tool for the
assembly of a number of mannose residues via linear and/or
convergent strategies. The usefulness of this valuable reac-
tion for oligomannoside assembly was demonstrated in
recent work from our laboratory.16 Moreover, we thought
that in the oligomers thus obtained the triazole ring could act
not only as a robust linker impervious to chemical and
enzymatic degradation but could also participate in hydrogen-
bonding and dipole interactions, thereby favoring molecular
recognition processes.17
FIGURE 1. Structures of lipomannans LMs and lipoarabinoman-
nans (LAMs) of Mtb cell walls (R stands for various fatty acid acyl
groups) and designed triazole-tethered oligomannosides 1 featuring
a capping 6-deoxymannose fragment.
of the available antibiotics used to treat TB, ethambutol and
isoniazid, act by preventing the formation of a complete cell
wall.6 This is composed of various glycophospholipids such
as mycolyl-arabinogalactan (mAG)7 and phosphatidylino-
sitol mannosides (PIMs).8 The latter, in addition to being
important in their own right, may also be hyperglycosylated
to form other wall components such lipomannans (LMs) and
lipoarabinomannans (LAMs).9 These glycolipids all contain
a common R-1,6-linked mannoside core as shown, for example,
in LMs and LAMs (Figure 1). Synthetic approaches to oligo-
mannan fragments of the Mtb cell wall were described in
Results and Discussion
We set out to prepare triazole-linked oligomannosides
1 (TOM) by a modular approach involving CuAAC reac-
tions as shown in Scheme 1. While this scheme exemplifies
only the synthesis of a high oligomer featuring 16 mannose
(5) (a) Brennan, P. J.; Crick, D. C. Curr. Top. Med. Chem. 2007, 7, 475–
488. (b) Berg, S.; Kaur, D.; Jackson, M.; Brennan, P. J. Glycobiology 2007,
17, 35R–56R. (c) Tam, P.-H.; Lowary, T. L. Curr. Opin. Chem. Biol. 2009, 13,
618–625.
(6) Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479–2513.
(7) Crick, D. C.; Mahapatra, S.; Brennan, P. J. Glycobiology 2001, 11,
107R–118R.
(8) Morita, Y. S.; Patterson, J. H.; Billman-Jacobe, H.; McConville, M. J.
Biochem. J. 2004, 378, 589–597.
(11) (a) Subramaniam, V.; Gurcha, S. S.; Besra, G. S.; Lowary, T. L.
Bioorg. Med. Chem. 2005, 13, 1083–1094. (b) Tam, P.-H.; Lowary, T. L.
Carbohydr. Res. 2007, 342, 1741–1772. (c) Tam, P.-H.; Lowary, T. L. Org.
Biomol. Chem. 2010, 8, 181–192.
(9) (a) Chatterjee, D.; Hunter, S. W.; McNeil, M.; Brennan, P. J. J. Biol.
Chem. 1992, 267, 6228–6233. (b) Khoo, K. H.; Dell, A.; Morris, H. R.;
Brennan, P. J.; Chatterjee, D. Glycobiology 1995, 5, 117–127. (c) Alderwick,
L. J.; Birch, H. L.; Mishra, A. K.; Eggeling, L.; Besra, G. S. Biochem. Soc.
Trans. 2007, 35, 1325–1328.
(12) This disaccharide is a known substrate for R-(1,6)-ManT, the enzyme
promoting the biosynthesis of the mannan core in LM and LAM of Mtb. See:
Brown, J. R.; Field, R. A.; Barker, A.; Guy, M.; Grewal, R.; Khoo, K.-H.;
Brennan, P. J.; Besra, G. S.; Chatterjee, D. Bioorg. Med. Chem. 2001, 9, 815–
824.
(10) (a) Jayaprakash, K. N.; Lu, J.; Fraser-Reid, B. Angew. Chem., Int.
Ed. 2005, 44, 5894–5898. (b) Fraser-Reid, B.; Lu, J.; Jayaprakash, K. N.;
(13) Subramaniam, V.; Gurcha, S. S.; Besra, G. S.; Lowary, T. L.
Tetrahedron: Asymmetry 2005, 16, 553–567.
ꢁ
ꢁ
Cristobal Lopez, J. Tetrahedron: Asymmetry 2006, 17, 2449–2463. (c) Liu, X.;
Stocker, B. L.; Seeberger, P. H. J. Am. Chem. Soc. 2006, 128, 3638–3648.
(d) Hoelemann, A.; Stocker, B. L.; Seeberger, P. H. J. Org. Chem. 2006, 71,
8071–8088. (e) Jayaprakash, K. N.; Chaudhuri, S. R.; Murty, C. V. S. R.;
Fraser-Reid, B. J. Org. Chem. 2007, 72, 5534–5545. (f) Fraser-Reid, B.;
Chaudhuri, S. R.; Jayaprakash, K. N.; Lu, J.; Ramamurty, C. V. S. J. Org.
Chem. 2008, 73, 9732–9743. (g) Boonyarattanakalin, S.; Liu, X.; Michieletti,
M.; Lepenies, B.; Seeberger, P. H. J. Am. Chem. Soc. 2008, 130, 16791–16799.
(h) Patil, P. S.; Hung, S.-C. Chem.;Eur. J. 2009, 15, 1091–1094.
(14) Watt, J. A.; Williams, S. J. Org. Biomol. Chem. 2005, 3, 1982–1992.
(15) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599. (b) Tornøe, C. W.; Christensen,
C.; Meldal, M. J. Org. Chem. 2002, 67, 3057–3062.
(16) (a) Cheshev, P.; Marra, A.; Dondoni, A. Org. Biomol. Chem. 2006, 4,
3225–3227. (b) Lo Conte, M.; Chambery, A.; Marra, A.; Dondoni, A. Synlett
2009, 2679–2681.
(17) Horne, W. S.; Yadav, M. K.; Stout, C. D.; Ghadiri, M. R. J. Am.
Chem. Soc. 2004, 126, 15366–15367.
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SCHEME 1. Convergent Synthesis of Triazole-Linked Hexadecamannoside (R = Protective Group)
SCHEME 2
residues, it can be realized that the lower oligomers can be
equally prepared from suitable precursors showed therein.
Details on each reaction sequence constituted of the CuAAC
and functional group transformation as well as the synthesis
of a range of oligomers are presented below.
Preparation of Starting Monosaccharidic Building Blocks.
Although previously reported in a succinct manner,16 the
synthetic routes to these building blocks are presented briefly
in Scheme 2. Compounds 5 and 9 were finalized to act as a
capping residue and a platform, respectively, in the prepared
oligosaccharides, while 4 and 7 were the first pair of elongat-
ing units. The methyl group in 5 constituted a key structural
motif to prevent the enzyme-promoted oligomerization, while
the protected hydroxyethyl group at the anomeric carbon of 9
formed a stable terminal C-glycoside unit. Upon removal of
the methoxymethyl (MOM) group by acid hydrolysis, the
free hydroxyethyl group could serve as a point of the
attachment of fatty acids via esterification. The 3-hydroxy-
3-methylbutynyl (HMB) group in 7 acted as a masked ethynyl
group. In fact, HMB is a formal adduct of acetone to the
ethynyl group from which the former can be easily removed
(deacetonation) simply by basic treatment. This protection/
deprotection strategy avoided homopolymer formation via
intermolecular Cu(I)-catalyzed cycloaddition.18 The use of
HMB represented two optimized conditions, i.e., stability
under the CuAAC conditions and ease of deacetonation.
(18) It is well known that the CuAAC takes place with terminal alkynes.
Exceptionally, iodoalkynes do react as well. See: Hein, J. E.; Tripp, J. C.;
Krasnova, L.; Sharpless, B. K. B.; Fokin, V. V. Angew. Chem., Int. Ed. 2009,
48, 8018–8021.
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SCHEME 3
SCHEME 4
On the other hand, the ethynyl protection as cobalt hexa-
carbonyl complex or trimethylsilyl derivative16b appeared
unsatisfactory as these groups were partially removed under
the CuAAC conditions.
As shown in Scheme 2, the preparation of all starting
building blocks relied on the use of tetra-O-benzylmanno-
pyranosyl acetate 2 as the common precursor. Succinctly, the
3-hydroxy-3-methylbutynyl group was introduced by glycosy-
lation of 2 with 1-tributylstannyl-3-methyl-3-trimethylsilyloxy-
1-butyne followed by sequential desilylation-acetylation of
the hydroxy group. Compound 6 thus obtained was selec-
tively debenzylated, then deacetylated, and finally trans-
formed into the target azide 7 by selective activation of the
primary hydroxy group as tosylate and reaction with sodium
azide.16b Coupling of 2 with 1-tributylstannyl-2-trimethylsilyl-
ethyne, followed by desilylation, yielded the ethynyl glyco-
side 3 that was selectively debenzylated to afford 4.16a This
compound was transformed into the target 6-deoxy deriva-
tive 5 by free hydroxy group iodination and hydride reduc-
tion.16b The preparation of the azide 9 involved first the
protection of the free hydroxy group of 4 as silyl derivative
and then hydroboration-oxidation of the alkyne function to
give the hydroxyethyl C-mannoside 8. The latter was con-
verted into the methoxymethyl (MOM) derivative that in
turn was transformed into the target azide 9 by desilylation
and treatment with diphenyl phosphoryl azide (DPPA) in the
presence of 1,8-diazabicyclo[5.4.0.]undec-7-ene (DBU).16a
Synthesis of Triazole-Tethered 1,6-r-D-Oligomannoside
Building Blocks and Assembly of the Hexadecamer. To demon-
strate the feasibility of the synthetic strategy outlined in
Scheme 1, the synthesis of the hexadecasaccharide 1f was first
performed. This involved execution of four consecutive cycles
each one being constituted of CuAAC and azidation or/and
deacetonation reactions. All cycloadditions were performed
under the conditions optimized in our recent work,16b that is,
by microwave heating at 80 °C (external vessel wall temper-
ature) for 15 min of equimolar azide and alkyne mixture in
DMF in the presence of catalytic CuI (0.2 equiv) and N,N-
diisopropylethylamine (2 equiv) as a ligand. The deacetona-
tion of the 3-hydroxy-3-methylbutynyl group to give the free
ethynyl moiety was efficiently carried out under mild basic
conditions (K2CO3, crown ether) in refluxing toluene. Finally,
the transformation of the primary hydroxy group into azide
was carried out by either direct azidation with DPPA or by
one-pot tosylation and substitution with sodium azide.
First Cycle. This involved (a) coupling of alkyne 5 with
azide 7 followed by deacetonation to give the dimannoside 10
(Scheme 3, eq a); (b) coupling of alkyne 4 with azide 9
followed by azidation to give 11 (Scheme 3, eq b); and (c)
coupling of alkyne 4 with azide 7 to give 12 and transformation
of the latter into 13 and 14 by deacetonation and azidation,
respectively (Scheme 4).
Second Cycle. This involved (a) coupling of alkyne 10 with
azide 14 followed by deacetonation to give the tetramanno-
side 15 (Scheme 5, eq a); (b) coupling of alkyne 13 with azide
11 followed by azidation to give 16 (Scheme 5, eq b);
(c) coupling of alkyne 13 with azide 14 to give 17 and trans-
formation of the latter into 19 and 18 by azidation and
deacetonation, respectively (Scheme 6).
Third Cycle. This involved (a) coupling of alkyne 15 with
azide 19 followed by deacetonation to give the octamanno-
side 20 (Scheme 7, eq a); (b) coupling of alkyne 18 with azide
16 followed by azidation to give 21 (Scheme 7, eq b).
Fourth Cycle. This involved only the final Cu(I)-catalyzed
coupling of octamannosides 20 and 21 to give the protected
hexadecamannoside 22 (Scheme 8). MALDI-TOF mass
spectrometry of 22 revealed m/z = 7761.40 for the corres-
ponding hydrogen adduct, as compared to the calculated
m/z = 7761.15. This compound was subjected to the protect-
ing group removal (demethoxymethylation by CF3CO2H
and debenzylation by BCl3) and acetylation by Ac2O to give
the peracetylated derivative 23, which was purified and char-
acterized by NMR and MS analyses. Finally the removal of
all acetyl protecting groups from 23 under mild basic condi-
tions using NH3 in methanol and water at room temperature
afforded the target TOM 1f (calcd m/z = 3413.10 [M þ Na]þ,
found m/z = 3412.99) in 9.4% overall yield from the mono-
saccharidic building blocks.
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SCHEME 5
SCHEME 7
SCHEME 6
SCHEME 8
that all intermolecular CuAAC reactions took place with great
efficiency regardless of the complexity of the substrates to give
exclusively a single cycloadduct. The structure of the 1,4-
disubstituted 1,2,3-triazole moieties of the oligosaccharides
was confirmed by 13C NMR spectroscopy.19 It can be also
noticed that the two functional group transformations, i.e.,
deacetonation and azidation, were simple and efficient processes
that were performed without any damage of the substrates.
Synthesis of a Set of TOMs (1). From an inspection of
Scheme 1 it can be easily realized that the Cu(I)-catalyzed
(19) The 1,4-disubstitution pattern of the triazole ring in all prepared
oligomannosides was supported by the large and positive Δ(δC4 - δC5) value
(17-21 ppm) in their 13C NMR spectra as observed in other compounds
obtained by click CuAAC in our laboratory. See ref 16a and: (a) Dondoni, A.;
Marra, A. J. Org. Chem. 2006, 71, 7546–7557. (b) Dondoni, A.; Giovannini,
P. P.; Massi, A. Org. Lett. 2004, 6, 2929–2932. (c) Marra, A.; Vecchi, A.;
Chiappe, C.; Melai, B.; Dondoni, A. J. Org. Chem. 2008, 73, 2458–2461.
Before closing this section, a few aspects regarding
Schemes 3-8 are worth considering. First, it can be noticed
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cycloadditions of the alkynes on the left side with the azides on
the right side can give rise to the formation of all oligomers of
type 1 starting from the dimannoside up to the hexadeca-
mannoside. Even higher oligomers should be accessible by
the extension of Scheme 1. Nevertheless, considering that the
aim of the present work consisted of the discovery of Mtb cell
envelope synthetase inhibitors, we decided to prepare only a
set of compounds that could give some inside on the effect of
the chain length on their biological activity. Suitable reaction
partners required for the CuAAC-based synthesis of the
target oligomers were synthesized as shown in Schemes 2, 3,
and 5. Thus, the CuAAC of alkyne 5 with azide 9 afforded
the fully protected dimannoside 24 (Scheme 9, eq a). The
removal of MOM and benzyl protective groups and acetyla-
tion transformed this compound into the peracetylated
derivative 25, which was purified and characterized by
NMR and MS. Finally, the removal of all acetyl groups from
25 by NH3 in methanol afforded the target TOM 1a (71.5%
yield from 5), suitable for the planned biological tests. The
same procedure was followed for the preparation of the
tetramannoside 1b starting from the cycloaddition of alkyne
10 with azide 11 (Scheme 9, eq b) and octamannoside 1d
starting from the cycloaddition of alkyne 15 with azide 16
(Scheme9, eq c). The oligomannosides1b and 1d were obtained
in 43.5% and 32.8% overall yield, respectively, from the mono-
saccharidic building blocks.
SCHEME 9
In vitro r-(1,6)-Mannosyltransferases Inhibition by TOMs.
The inhibition of the mycobacterial R-(1,6)-mannosyltrans-
ferases by triazole-linked oligomannosides TOM-2 (1a),
TOM-4 (1b), TOM-8 (1d), and TOM-16 (1f), as well as
hexamannoside TOM-6 (1c) and decamannoside TOM-10
(1e), these being synthesized as reported,16b was evaluated in
vitro using a well-established assay11-13 based on membrane
extracts from Mycobacterium smegmatis. These membrane
extracts contain polyprenolphosphomannose (PPM)-depen-
dent R-(1,6)-mannosyltransferases that are involved in the
synthesis of the R-1,6-linked mannoside core present in the
mycobacterial cell wall LAMs.20 It has been demonstrated
that an octyl dimannoside [R-Manp(1,6)-R-Manp-O(CH2)7-
CH3] is the minimum acceptor substrate12 for the above
R-(1,6)-mannosyltransferases, whereas their donor substrate,
i.e., phosphodecaprenyl β-D-mannopyranoside, is formed
from guanosine disphosphate (GDP)-mannose.21
Each triazole-linked oligomannoside 1a-f (at 1.0 mM)
was incubated at 37 °C for 30 min with 14C1-labeled phospho-
decaprenyl β-D-mannopyranoside (generated in situ from
GDP-[14C]mannose), M. smegmatis membrane fractions,
and R-Manp(1,6)-R-Manp-O(CH2)7CH3 disaccharidic ac-
ceptor (at 0.2 mM). After these experiments, we were pleased
to observe that all the oligomannosides led to a significant
inhibition of [14C]Manp incorporation from decaprenyl
P-[14C]mannose onto the disaccharidic acceptor (101 cpm, no
enzyme control; 13,627 cpm, no inhibitor; 8,174 cpm, TOM-
2 (1a); 7,498 cpm, TOM-4 (1b); 401 cpm, TOM-6 (1c); 656
cpm, TOM-8 (1d); 6,680 cpm, TOM-10 (1e); 4,739 cpm,
TOM-16 (1f). It has to be noted, however, that the hexam-
annoside TOM-6 (1c) and the octamannoside TOM-8 (1d),
consistently over several independent experiments (Figure 2),
showed the highest activity (ca. 95% inhibition). Subsequent
experiments where TOM concentrations were varied pro-
vided inhibitory IC50 values for TOM-2 (1.24 mM), TOM-4
(1.15 mM), TOM-6 (0.14 mM), TOM-8 (0.22 mM), TOM-10
(0.95 mM), and TOM-16 (0.84 mM). These values demon-
strated that TOM-6 and TOM-8 were stronger R-(1,6)-
ManTs inhibitors than the octyl 60-amino-O-disaccharides
prepared by Tam and Lowary (IC50 = 0.8-1.2 mM).11c Very
recent attempts to prepare other mycobacterial mannosyl-
transferase inhibitors were met with failure.22 In particular, a
(20) Besra, G. S.; Morehouse, C. B.; Rittner, C. M.; Waechter, C. J.;
Brennan, P. J. J. Biol. Chem. 1997, 272, 18460–18466.
(21) Gurcha, S. S.; Baulard, A. R.; Kremer, L.; Locht, C.; Moody, D. B.;
Muhlecker, W.; Costello, C. E.; Crick, D. C.; Brennan, P. J.; Besra, G. S.
Biochem. J. 2002, 365, 441–450.
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acceptor up to 4 mM but also a weak inhibitor of the R-(1,6)-
ManTs (IC50 = 0.96 mM). Thus, while the deoxygenated
6-position at the reducing end ensures the total inertness of
TOMs toward mannosyltransferases, this structural feature
appears to be not crucial for inhibition.
Conclusion
In summary, a modular strategy has been established for
the preparation of a set of C-oligomannosides featuring the
triazole ring as the interglycosidic linker. In fact, the key
reaction for the assembly of these oligomers consists of the
very efficient click azide-alkyne cycloaddition (CuAAC).
The efficiency and fidelity of this reaction were maintained
regardless of the structural complexity of building blocks to
be assembled. The biological experiments indicated that
TOM-6 (1c) and TOM-8 (1d) are endowed with the optimal
chain length for the interaction with PPM-dependent
R-(1,6)-mannosyltransferases. Although the core-R-(1,6)-
LM is around 20 Man units in length, a TOM with a minor
number of Man units binds efficiently to the R-(1,6)-man-
nosyltransferase, suggesting that the binding groove of the
TOM-6 oligosaccharide fits optimally into the protein. Very
likely the 1,4-disubstituted triazole ring spacers contribute
substantially to the overall length of these non-natural oligo-
mannosides. In addition, it is worth noting that the presence
of a non-natural C-glycosidic linkage and triazole linker in
oligomannosides 1a-f does not perturb their molecular
recognition properties toward these mycobacterial R-(1,6)-
mannosyltransferases.
FIGURE 2. In vitro inhibition of mycobacterial R-(1,6)-mannosyl-
transferase enzyme activity by TOM-2-TOM-16 (1a-f). The assay
utilizes M. smegmatis membranes, R-Manp(1,6)-R-Manp-O(CH2)7-
CH3 disaccharidic acceptor (0.2 mM), 0.25 μCi of [14C]GDP-Man,
and 1a-f (1.0 mM). The results are expressed as percent inhibition
of the incorporation of [14C]mannose into the acceptor and are
representative of three independent experiments with each assay
performed in triplicate.
SCHEME 10
Experimental Section
General Experimental Section. All moisture-sensitive reac-
tions were performed under a nitrogen atmosphere using oven-
dried glassware. Anhydrous solvents were dried over standard
drying agents23 and freshly distilled prior to use. Reactions were
monitored by TLC on silica gel 60 F254 with detection by
charring with sulfuric acid. Flash column chromatography24
was performed on silica gel 60 (40-63 μm). Optical rotations
were measured at 20 ( 2 °C in the stated solvent; [R]D values are
given in deg mL g-1 dm-1. 1H NMR (300 and 400 MHz) and
3
3
3
13C NMR spectra (75 MHz) were recorded from CDCl3 solu-
tions at room temperature unless otherwise specified. Peak
assignments were aided by 1H-1H COSY and gradient-HMQC
experiments. In the 1H NMR spectra reported below, the n and
m values quoted in geminal or vicinal proton-proton coupling
constants Jn,m refer to the number of the corresponding sugar
protons. The closed vessel MW experiments were performed
using a single-mode cavity Biotage Initiator microwave reactor;
the temperature was measured externally on the outside vessel
wall by an IR sensor; the reaction time was counted when the
reaction mixture reached the preset temperature. Monosacchar-
ides 4,16a 5,16b 6,16b 7,16b 8,16a and 916a and disaccharides 10,16b
11,16a and 3016a were prepared as described in our previous
papers.
MS Analysis. For accurate mass measurements, the com-
pounds were analyzed in positive-ion mode by electrospray
hybrid quadrupole orthogonal acceleration time-of-flight mass
spectrometer (Q-TOF) fitted with a Z-spray electrospray ion
source. The capillary source voltage and the cone voltage were
set at 3500 and 35 V, respectively; the source temperature was
series of 15 monosaccharidic alkyl and cycloalkyl R-D-man-
nopyranoside derivatives, including 6-deoxygenated analo-
gues, were prepared and evaluated as inhibitors. However,
all these compounds were totally inactive toward the R-(1,6)-
ManTs.22
Following these results, it was important to establish
whether the presence of a deoxygenated capping residue
was a key structural feature for the mycobacterial R-(1,6)-
mannosyltransferase inhibition. To this end, the triazole-
linked 1,6-dimannoside 31 was prepared from the known16a
protected derivative 30 (Scheme 10) and tested by the
standard assay described for TOM-2. Quite surprisingly,
this compound turned out to be not only a poor mannosyl
(23) Armarego, W. L. F.;Chai, C. L. L. Purification of Laboratory Chemicals,
5th ed., Butterworth-Heinemann: Amsterdam, 2003.
(24) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923–2925.
ꢁ
ꢁ
ꢁ
ꢁ
(22) Polakova, M.; Belanova, M.; Petrus, L.; Mikusova, K. Carbohydr.
ꢁ
Res. 2010, 345, 1339–1347.
6332 J. Org. Chem. Vol. 75, No. 19, 2010

Lo Conte et al.
JOCFeatured Article
kept at 80 °C; nitrogen was used as a drying gas at a flow rate of
ca. 50 L/h. The time-of-flight analyzer was externally calibrated
with NaI from m/z 300 to 2000 to yield an accuracy near to
5 ppm. When necessary an internal lock mass was used to further
increase the mass accuracy. Accurate mass data were collected
by directly infusing samples (10 pmol/μL in 1:1 CH3CN-H2O
containing 10 mM ammonium formate) into the system at a flow
rate of 5 μL/min. Compounds 1b, 1d, 1f, 20-23, 28, and 29 were
analyzed by MALDI TOF mass spectrometry using a pulsed
nitrogen laser (λ = 337 nm). Each compound was dissolved in
H2O (1b, 1d, 1f) or 1:1 CHCl3-acetonitrile (20-23, 28, 29), and
prior to the acquisition of spectra, 1 μL of this solution was
diluted with 1 μL of saturated R-cyano-4-hydroxycinnamic acid
matrix solution (10 mg/mL in 1:1 EtOH-H2O, containing 0.1%
of CF3CO2H). Ca. 1 μL of the resulting mixture was placed onto
the mass spectrometer’s sample target and dried at room
temperature. Once the liquid was completely evaporated, the
sample was loaded into the mass spectrometer and analyzed.
The instrument was operated in positive ion reflectron mode
with the source voltage set to 12 kV. The pulse voltage was
optimized at 1999 V, and the detector and reflectron voltages
were set to 5200 and 2350 V, respectively. Measurements were
performed in the mass range m/z 800-5000 with a suppression
mass gate set to m/z 500 to prevent detector saturation from
matrix cluster peaks and an extraction delay of 600 ns. The
instrument was externally calibrated using a polyethylene glycol
mix as standard. A mass accuracy near to the nominal (50 ppm)
was achieved for each standard. The protonated monoisotopic
mass of ACTH peptide (m/z 2465.199) was used as internal lock
mass to further improve the peptide mass accuracy near to
10-20 ppm. The monoisotopic masses were calculated accord-
ing to the reported25 atomic weights of the elements.
3,7-Anhydro-4,5,6-tri-O-benzyl-1,2,8-trideoxy-8-[4-(20,30,40-tri-
O-benzyl-r-D-mannopyranosyl)-1H-1,2,3-triazol-1-yl]-D-glycero-
D-talo-oct-1-ynitol (13). A mixture of 12 (355 mg, 0.36 mmol),
anhydrous K2CO3 (20 mg, 0.15 mmol), 18-crown-6 ether (19 mg,
0.07 mmol), and toluene (3 mL) was stirred at 110 °C for 4 h and
then concentrated. The residue was eluted from a column of
silica gel with 2:1 cyclohexane-AcOEt to give 13 (308 mg, 92%)
as an amorphous solid: [R]D = þ28.9 (c 1.1, CHCl3); 1H NMR
(400 MHz) selected data δ 7.68 (s, 1H, H-5 tr), 7.43-7.21 (m,
30H, Ar), 5.25 (d, 1H, J1 ,2 = 2.0 Hz, H-10), 4.54 (dd, 1H,
J7,8a = 2.4, J8a,8b = 14.3 Hz, H-8a), 4.07 (ddd, 1H, J6,7 = 9.7,
J7,8b = 5.2 Hz, H-7), 4.04 (dd, 1H, J4,5 = 3.0, J5,6 = 9.3 Hz,
0
0
H-5), 4.00 (dd, 1H, J3 ,4 = 9.4, J4 ,5 = 9.0 Hz, H-40), 3.91 (dd,
0
0
0
0
1H, J2 ,3 = 2.9 Hz, H-30), 3.51 (dd, 1H, H-5), 3.48 (ddd, 1H,
0
0
J5 ,6 a = 3.2, J5 ,6 b = 5.2 Hz, H-50), 2.49 (d, 1H, J1,3 = 2.3, H-1),
2.07 (bs, 1H, OH); 13C NMR δ 144.6 (C-4 tr), 138.4 (C), 138.0
(C), 137.8 (C), 137.5 (C), 128.4 (CH), 128.3 (CH), 127.9 (CH),
127.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 123.9 (C-5 tr),
80.2 (CH), 80.0 (CH), 78.2 (CH), 77.6 (C), 76.1 (CH), 75.3
(CH2), 75.2 (CH), 75.0 (CH2), 74.8 (CH), 74.2 (CH), 74.1 (CH),
73.1 (CH2), 72.7 (CH2), 72.1 (CH2), 72.0 (CH2), 71.8 (CH2), 71.4
(CH), 66.1 (CH), 62.7 (CH2), 50.6 (CH2); HRMS (ESI/Q-TOF)
m/z calcd for C58H60N3O9 (M þ H)þ 942.4330, found 942.4366.
3,7-Anhydro-8-[4-(60-azido-20,30,40-tri-O-benzyl-60-deoxy-r-D-
mannopyranosyl)-1H-1,2,3-triazol-1-yl]-4,5,6-tri-O-benzyl-1,2,8-
trideoxy-1-C-[2-(2-hydroxy)propyl]-D-glycero-D-talo-oct-1-ynitol
(14). A mixture of alcohol 12 (440 mg, 0.44 mmol), freshly dis-
tilled Et3N (0.19 mL, 1.32 mmol), and p-toluensulfonyl chloride
(126 mg, 0.66 mmol) in anhydrous CH2Cl2 (4 mL) was stirred at
room temperature for 3 h and then diluted with CH2Cl2 (40 mL),
washed with 1 M phosphate buffer at pH 7 (2 ꢀ 15 mL), dried
(Na2SO4), and concentrated. The residue was filtered through a
short column of silica gel with 2:1 cyclohexane-AcOEt to give
the 80-O-tosyl derivative as a white solid (545 mg). A mixture of
this compound, sodium azide (143 mg, 2.20 mmol), and anhy-
drous DMF (3 mL) was stirred at 55 °C for 19 h and then cooled
to room temperature, diluted with AcOEt (40 mL), washed with
H2O (2 ꢀ 15 mL), dried (Na2SO4), and concentrated. The resi-
due was eluted from a column of silica gel with 3:1 cyclohexane-
AcOEt to give 14 (352 mg, 78%) as a syrup: [R]D = þ32.1 (c 0.8,
CHCl3); 1H NMR (400 MHz) selected data δ 7.81 (s, 1H, H-5
0
0
0
0
3,7-Anhydro-4,5,6-tri-O-benzyl-1,2,8-trideoxy-1-C-[2-(2-hydroxy)-
propyl]-8-[4-(20,30,40-tri-O-benzyl-r-D-mannopyranosyl)-1H-1,2,3-
triazol-1-yl]-D-glycero-D-talo-oct-1-ynitol (12). A mixture of alkyne 4
(420 mg, 0.92 mmol), azide 7 (497 mg, 0.92 mmol), N,N-diisopro-
pylethylamine (320 μL, 1.84 mmol), CuI (34 mg, 0.18 mmol), and
DMF (4 mL) in a vial sealed with a Teflon septum and aluminum
crimp was subjected to microwave irradiation for 15 min at
80 °C and then cooled to room temperature, diluted with AcOEt
(150 mL), washed with an aqueous solution of EDTA disodium salt
(0.05M,3ꢀ 30 mL), dried (Na2SO4), and concentrated. The residue
was eluted from a column of silica gel with 3:2 cyclohexane-
tr), 7.44-7.20 (m, 30H, Ar), 5.24 (d, 1H, J1 ,2 = 1.7 Hz, H-10),
4.96 and 4.72 (2 d, 2H, J = 10.7 Hz, PhCH2), 4.89 and 4.57 (2 d,
0
0
2H, J = 11.0 Hz, PhCH2), 4.81 (dd, 1H, J2 ,3 = 3.0 Hz, H-20),
4.77 (d, 1H, J3,4 = 2.0 Hz, H-3), 3.72 (dd, 1H, J4,5 = 3.5 Hz, H-4),
AcOEt to give 12 (818 mg, 89%) as an amorphous solid: [R]D
=
0
0
-13.6 (c 0.9, CHCl3); 1H NMR (400 MHz) δ 7.72 (s, 1H, H-5 tr),
0
3.53 (ddd, 1H, J4 ,5 = 9.5, J5 ,6a = 7.5, J5 ,6b = 2.3 Hz, H-50),
0
0
7.46-7.26 (m, 30H, Ar), 5.23 (d, 1H, J1 ,2 = 1.8 Hz, H-1 ), 4.96
and 4.71 (2 d, 2H, J = 11.0 Hz, PhCH2), 4.89 and 4.59 (2 d, 2H,
J = 11.0 Hz, PhCH2), 4.82 and 4.77 (2 d, 2H, J = 12.5 Hz,
0
0
0
0
0
0
3.38 (dd, 1H, J6a ,6b = 13.0 Hz, H-60a), 3.32 (dd, 1H, H-60b),
2.40 (s, 1H, OH), 1.32 and 1.31 (2 s, 6H, 2 CH3); 13C NMR δ
144.3 (C-4 tr), 138.2 (C), 137.9 (C), 137.7 (C), 137.6 (C), 137.5
(C), 128.3 (CH), 128.2 (CH), 127.9 (CH), 127.7 (CH), 127.6
(CH), 127.5 (CH), 127.4 (CH), 124.3 (C-5 tr), 94.9 (C), 80.3
(CH), 79.3 (CH), 76.4 (CH), 75.2 (CH), 75.2 (CH2), 74.9 (CH2),
74.39 (CH), 74.35 (CH), 73.9 (CH), 73.1 (CH), 72.5 (CH2), 71.9
(CH2), 71.69 (CH2), 71.65 (CH2), 71.1 (CH), 66.2 (CH), 64.4 (C),
51.5 (CH2), 50.6 (CH2), 31.1 (CH3), 30.8 (CH3); HRMS (ESI/
Q-TOF) m/z calcd for C61H65N6O9 (M þ H)þ 1025.4813, found
1025.4814.
0
0
0
0
PhCH2), 4.77 (dd, 1H, J2 ,3 = 3.0 Hz, H-2 ), 4.74 (d, 1H, J3,4
0
=
2.0 Hz, H-3), 4.72 and 4.61 (2 d, 2H, J = 11.5 Hz, PhCH2), 4.68
(dd, 1H, J7,8a = 2.2, J8a,8b = 14.3 Hz, H-8a), 4.66 (s, 2H,
PhCH2), 4.62 and 4.59 (2 d, 2H, J = 12.0 Hz, PhCH2), 4.52 (dd,
1H, J7,8b = 7.0 Hz, H-8b), 4.00-3.90 (m, 4H, H-30, H-5, H-6,
H-7), 3.80 (dd, 1H, J5 ,6 a = 3.0, J6 a,6 b = 12.0 Hz, H-60a), 3.72
0
0
0
0
0
0
(dd, 1H, J4,5 = 3.0 Hz, H-4), 3.68 (dd, 1H, J5 ,6 b = 6.2 Hz,
0
H-60b), 3.62 (dd, 1H, J3 ,4 = J4 ,5 = 9.0 Hz, H-4 ), 3.43(ddd, 1H,
H-50), 2.85 (s, 1H, OH), 2.20 (bs, 1H, OH), 1.31 (s, 6H, 2 CH3);
13C NMR δ 144.5 (C-4 tr), 138.4 (C), 138.0 (C), 137.9 (C), 137.70
(C), 137.67 (C), 128.5 (CH), 128.4 (CH), 128.3 (CH), 127.9
(CH), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.4 (CH), 123.7
(C-5 tr), 95.1 (C), 80.4 (CH), 79.4 (CH), 76.4 (CH), 75.3 (CH2),
75.1 (CH2), 75.0 (CH2), 74.8 (CH), 74.7 (CH), 74.3 (CH), 73.2
(CH), 72.8 (CH2), 72.1 (CH2), 71.9 (CH2), 71.5 (CH), 66.4 (CH),
64.4 (C), 62.9 (CH2), 50.8 (CH2), 31.2 (CH3), 30.9 (CH3); HRMS
(ESI/Q-TOF) m/z calcd for C61H66N3O10 (M þ H)þ 1000.4748,
found 1000.4714.
0
0
0
0
Tetramannoside Alkyne (15). A mixture of alkyne 10 (110 mg,
0.12 mmol), azide 14 (122 mg, 0.12 mmol), N,N-diisopropy-
lethylamine (42 μL, 0.24 mmol), CuI (5 mg, 24 μmol), and DMF
(0.6 mL) in a vial sealed with a Teflon septum and aluminum
crimp was subjected to microwave irradiation for 15 min at
80 °C and then cooled to room temperature, diluted with AcOEt
(50 mL), washed with 0.05 M aqueous solution of EDTA (3 ꢀ
10 mL), dried (Na2SO4), and concentrated. A mixture of this
residue, anhydrous K2CO3 (7 mg, 50 μmol), 18-crown-6 ether
(6 mg, 24 μmol), and toluene (1 mL) was stirred at 110 °C for 4 h
and then concentrated. The residue was eluted from a column of
(25) Pure Appl. Chem. 1991, 63, 975-990.
J. Org. Chem. Vol. 75, No. 19, 2010 6333

Lo Conte et al.
JOCFeatured Article
silica gel with 2:1 cyclohexane-AcOEt to give 15 (176 mg, 78%),
identical to the product described by us in a previous paper.16b
Tetramannoside azide (16). A mixture of alkyne 13 (68 mg,
65 μmol), azide 11 (62 mg, 65 μmol), N,N-diisopropylethylamine
(23 μL, 0.13 mmol), CuI (2.5 mg, 13 μmol), and DMF (1 mL) in a
vial sealed with a Teflon septum and aluminum crimp was
subjected to microwave irradiation for 15 min at 80 °C and then
cooled to room temperature, diluted with AcOEt (50 mL), washed
with 0.05 M aqueous solution of EDTA (2 ꢀ 15 mL), dried
(Na2SO4), and concentrated. A mixture of this residue, diphenyl
phosphoryl azide (43 μL, 0.20 mmol), 1,8-diazabicyclo[5.4.0.]
undec-7-ene (19 μL, 0.13 mmol), sodium azide (22 mg, 0.33), and
anhydrous DMF (1 mL) in a vial sealed with a Teflon septum
and aluminum crimp was subjected to microwave irradiation for
2 h at 130 °C and then cooled to room temperature, diluted with
AcOEt (50 mL), washed with saturated aqueous Na2CO3 (20 mL),
dried (Na2SO4), and concentrated. The residue was eluted from
a column of silica gel with 3:1 cyclohexane-AcOEt to give 16
(81 mg, 61%), identical to the product described by us in a
previous paper.16a
(CH), 64.2 (C), 61.8 (CH2), 51.8 (CH2), 51.3 (CH2), 51.0 (CH2),
50.5 (CH2), 31.2 (CH3), 30.9 (CH3); HRMS (ESI/Q-TOF) m/z
calcd for (C119H124N12O17)/2 (M þ 2H)2þ 996.4604, found
996.4620.
Octamannoside Alkyne (20). The alkyne 15 (97 mg, 0.05 mmol)
was allowed to react with azide 19 (102 mg, 0.05 mmol) as
described for the preparation of 15 to give, after trituration
with CH3OH, 20 (101 mg, 52%) as an amorphous solid: [R]D
=
-172.3 (c 0.7, CHCl3); 1H NMR (400 MHz) selected data
δ 7.84 (s, 1H, H-5 tr), 6.02 (bs, 2H, 2 anom H), 5.96, 5.88, 5.80,
5.47, and 5.41 (5 bs, 5H, 5 anom H), 1.40 (d, 3H, J = 6.0 Hz,
CH3); 13C NMR selected data δ 145.0 (C-4 tr), 143.5 (C-4 tr),
143.3 (C-4 tr), 143.0 (C-4 tr), 139.6-137.5 (C), 128.5-126.7
(CH), 124.9-123.9 (C-5 tr), 18.5 (CH3); MALDI-TOF MS m/z
calcd for C232H234N21O32 (M þ H)þ 3828.53, found 3828.41.
Octamannoside Azide (21). The alkyne 18 (55 mg, 0.03 mmol)
was allowed to react with azide 16 (58 mg, 0.03 mmol) as
described for the preparation of 16 to give, after trituration
with CH3OH, 21 (71 mg, 63%) as an amorphous solid: [R]D
=
-138.6 (c 1.1, CHCl3); 1H NMR (400 MHz) selected data δ 7.92
(s, 1H, H-5 tr), 5.98 (bs, 2H, 2 anom. H), 5.94, 5.86, 5.71, 5.50,
and 5.44 (5 bs, 5H, 5 anom. H), 3.10 (s, 3H, CH3); 13C NMR
selected data: δ 144.5-143.1 (C-4 tr), 139.5-137.6 (C), 128.3-
126.7 (CH), 125.5-123.9 (C-5 tr); MALDI-TOF MS m/z calcd
for C234H241N24O34 (M þ H)þ 3933.63, found 3933.40.
Hexadecamannoside (22). The alkyne 20 (50.6 mg, 13 μmol)
was allowed to react with azide 21 (52 mg, 13 μmol) as described
for the preparation of 12 to give, after trituration with CH3CN,
22 (93 mg, 92%) as an amorphous solid: [R]D = -3.2 (c 0.5,
CHCl3); 1H NMR (400 MHz) selected data δ 7.88 (s, 1H, H-5
tr), 3.17 (s, 3H, CH3); 13C NMR selected data δ 143.4 (C-4 tr),
139.8-137.5 (C), 128.2-126.7 (CH), 124.8 (C-5 tr); MALDI-
TOF MS m/z calcd for C466H474N45O66 (M þ H)þ 7761.15,
found 7761.40.
Protected Tetramannoside Alkyne (17). The coupling of azide
14 (178 mg, 0.17 mmol) with alkyne 13 (165 mg, 0.14 mmol) was
performed as described for the synthesis of 12 to give, after column
chromatography on silica gel (from 2:1 to 1:1 cyclohexane-
AcOEt), 17 (259 mg, 76%) as an amorphous solid: [R]D
=
þ6.8 (c 0.8, CHCl3); 1H NMR (400 MHz) selected data δ 7.79 (s,
1H, H-5 tr), 7.39-7.12 (m, 62H, Ar, 2 H-5 tr), 5.36, 5.25, and
0
0
00 00
000,2000
5.23 (3 d, 3H, J1 ,2 = 1.5 Hz, J1 ,2 = 1.5 Hz, J1
= 1.5 Hz,
H-10, H-100, H-1000), 4.96 (d, 1H, J3,4 = 1.5 Hz, H-3), 3.03 (s, 1H,
000
000
OH), 2.40 (dd, 1H, J6 a,OH = 5.0, J6 b,OH = 7.2 Hz, OH), 1.24
and 1.22 (2 s, 6H, 2 CH3); 13C NMR δ 144.5 (C-4 tr), 143.4 (C-4
tr), 138.5 (C), 138.3 (C), 138.1 (C), 138.0 (C), 137.9 (C), 137.6
(C), 128.7-127.5 (CH), 124.3 (C-5 tr), 124.0 (C-5 tr), 95.0 (C),
80.0 (CH), 79.5 (CH), 76.6 (CH), 75.3 (CH), 75.1 (CH2), 74.9
(CH2), 74.6 (CH), 73.1 (CH), 72.6 (CH2), 72.4 (CH2), 72.3 (CH),
71.8 (CH2), 71.6 (CH2), 71.4 (CH2), 71.1 (CH), 66.1 (CH), 64.2
(C), 62.7 (CH2), 51.1 (CH2), 50.5 (CH2), 31.1 (CH3), 30.9 (CH3);
HRMS (ESI/Q-TOF) m/z calcd for (C119H125N9O18)/2 (M þ 2H)2þ
983.9571, found 983.9586.
Hexadecamannoside (23). To a solution of 22 (75 mg, 9.7 μmol)
in anhydrous CH2Cl2 (5 mL) was added trifluoroacetic acid
(200 μL). The solution was kept at room temperature for 3 h
and then concentrated. To a stirred, cooled (-60 °C) solution
of the residue in anhydrous CH2Cl2 (2 mL) was added a 1 M
solution of BCl3 in CH2Cl2 (930 μL, 0.93 mmol). The solution
was stirred at -60 °C for 1 h and, after an additional 1 h stirring
at 0 °C, diluted with MeOH (0.5 mL), stirred at 0 °C for 30 min,
diluted with Et3N (0.5 mL), concentrated, and dried under high
vacuum to give a white solid. A suspension of the residue in
pyridine (2 mL) and acetic anhydride (1 mL) was stirred at
room temperature for 24 h, diluted with MeOH (1 mL), and
concentrated. The residue was eluted from a column of silica
gel with AcOEt to give 23 (18 mg, 40%) as an amorphous solid:
[R]D = þ32.9 (c 0.4, CHCl3); 1H NMR (300 MHz) selected data
δ 7.99 (s, 1H, H-5 tr), 7.73-7.62 (14 s, 14H, 14 H-5 tr); MALDI-
TOF MS: m/z calcd for C226H279N45NaO114 (M þ Na)þ 5472.93,
found 5472.88.
Tetramannoside Alkyne (18). The deprotection of tetraman-
noside 17 (75 mg, 0.04 mmol) was performed as described for the
synthesis of 13 to give, after column chromatography on silica
gel (2:1 cyclohexane-AcOEt), 18 (65 mg, 89%) as an amor-
phous solid: [R]D = þ2.8 (c 1.1, CHCl3); 1H NMR (400 MHz)
selected data δ 7.79 (s, 1H, H-5 tr), 7.47 (s, 1H, H-5 tr),
7.42-7.17 (m, 61H, Ar, 1 H-5 tr), 5.35, 5.26, and 5.23 (3 d,
3H, J1 ,2 = 1.7 Hz, J1 ,2 = 1.7 Hz, J1
= 1.7 Hz, H-10, H-100,
0
0
00 00
000,2000
H-1000), 2.33 (bs, 1H, OH), 2.32 (d, 1H, J1,3 = 2.2 Hz, H-1); 13
C
NMR selected data δ 144.6 (C-4 tr), 143.8 (C-4 tr), 143.6 (C-4 tr),
138.5-137.5 (C), 128.9-127.6 (CH), 124.4 (C-5 tr), 124.2 (C-5
tr), 124.0 (C-5 tr), 67.8 (CH), 62.8 (CH2), 51.1 (CH2), 50.9
(CH2), 50.5 (CH2); HRMS (ESI/Q-TOF) m/z calcd for
(C116H119N9O17)/2 (M þ 2H)2þ 954.9362, found 954.9412.
Tetramannoside Azide (19). The alcohol 17 (150 mg, 0.08 mmol)
was azidated as described for the preparation of 14 to give, after
column chromatography on silica gel (2:1 cyclohexane-AcOEt),
Hexadecamannoside (1f). To a solution of 23 (18 mg, 3.8 μmol)
in MeOH(2 mL) was added a 2 M solution ofammonia inMeOH
(2 mL). Thesolutionwas keptat roomtemperature overnight and
then concentrated to give 1f (10 mg, 91%) as an amorphous solid:
[R]D = þ4.7 (c 0.3, DMSO); 1H NMR (300 MHz, D2O) selected
data: δ 7.80-7.36 (15 s, 15H, 15 H-5 tr) MALDI-TOF MS m/z
calcd for C128H181N45NaO65 (M þ Na)þ 3413.10, found 3412.99.
3,7-Anhydro-4,5,6-tri-O-benzyl-2,8-dideoxy-1-methoxymethyl-
8-[4-(20,30,40-tri-O-benzyl-60-deoxy-r-D-mannopyranosyl)-1H-
1,2,3-triazol-1-yl]-D-glycero-D-talo-octitol (24). The coupling of
azide 9 (12 mg, 0.023 mmol) with alkyne 5 (10 mg, 0.023 mmol)
was performed as described for the synthesis of 12 to give, after
column chromatography on silica gel (3:1 cyclohexane-AcOEt),
24 (21 mg, 96%) as an amorphous solid; [R]D = þ21.7 (c 1.1,
CHCl3); 1H NMR (400 MHz) selected data δ 7.65 (s, 1H, H-5
1
19 (118 mg, 79%) as a syrup: [R]D = þ4.3 (c 1.1, CHCl3); H
NMR (400 MHz) selected data δ 7.91 (1s, 1H, H-5 tr), 741-7.06
0
0
(m, 62H, Ar, 2 H-5tr), 5.40, 5.27, and 5.26 (3 d, 3H, J1 ,2 =1.5Hz,
= 1.5 Hz, H-10, H-100, H-1000), 4.99 (d, 1H,
00 00
J1 ,2 = 1.5 Hz, J1
000,2000
J
3,4 = 2.0 Hz, H-3), 2.80 (s, 1H, OH), 1.23 and 1.19 (2 s, 6H, 2
CH3); 13C NMR: δ 144.5 (C-4 tr), 143.5 (C-4 tr), 138.5 (C), 138.4
(C), 138.3 (C), 138.1 (C), 138.0 (C), 137.6 (C), 128.7-127.5 (CH),
124.7 (C-5 tr), 124.4 (C-5 tr), 124.1 (C-5 tr), 95.0 (C), 80.3 (CH),
80.2(CH), 80.0(CH), 79.6(CH), 77.2 (CH), 76.7 (CH), 75.5(CH),
75.2 (CH), 75.0 (CH2), 74.7 (CH), 74.5 (CH), 73.9 (CH), 73.2
(CH), 72.6 (CH), 72.5 (CH2), 72.3 (CH), 71.8 (CH2), 71.6 (CH2),
71.4 (CH2), 71.1 (CH), 70.6 (CH2), 70.3 (CH2), 70.0 (CH2), 66.1
tr), 7.43-7.22 (m, 30H, Ar), 5.18 (d, 1H, J1 ,2 = 2.0 Hz, H-10),
0
0
6334 J. Org. Chem. Vol. 75, No. 19, 2010

Lo Conte et al.
JOCFeatured Article
4.92 and 4.60 (2 d, 2H, J = 10.8 Hz, PhCH2), 4.74 (dd, 1H,
J7,8a = 6.5, J8a,8b = 14.3 Hz, H-8a), 4.47 (dd, 1H, J7,8b = 3.0 Hz,
H-8b), 4.18 (ddd, 2H, J = 4.3, 5.0, 9.0 Hz, 2 H-1), 3.80 (dd, 1H,
J3,4 = 3.0, J4,5 = 7.5 Hz, H-4), 3.62 (dd, 1H, H-3), 3.54 (dd, 1H,
125.0-124.0 (C-5 tr), 96.0 (CH2), 64.2 (CH2), 54.8 (CH), 18.5
(CH3);MALDI-TOFMSm/zcalcd for C234H242N21O34 (Mþ H)þ
3892.62, found 3892.57.
Octamannoside (29). The deprotection of 28 (34 mg, 8.7 μmol)
was performed as described for the synthesis of 23 to give, after
column chromatography on silica gel (AcOEt), 29 (19 mg, 78%)
as an amorphous solid: [R]D = -52.1 (c 0.5, CHCl3). 1H NMR
(300 MHz) selected data δ 7.98 (s, 1H, H-5 tr), 7.72 (s, 2H, 2 H-5
tr), 7.70 (s, 2H, 2 H-5 tr), 7.68 and 7.63 (2 s, 2H, 2 H-5 tr), 1.20 (d,
3H, J = 6.0 Hz, CH3); MALDI-TOF MS m/z calcd for
C114H143N21NaO58 (M þ Na)þ 2758.49, found 2758.48.
Octamannoside (1d). The deprotection of 29 (9 mg, 3.4 μmol)
was performed as described for the synthesis of 1f to give 1d
(6 mg, quantitative) as an amorphous solid: HRMS (MALDI-
TOF) m/z calcd for C64H94N21O33 (M þ H)þ 1684.6323, found
1684.5648.
J5,6 = 6.8 Hz, H-5), 3.28 (s, 3H, CH3), 1.80-1.70 (m, 2H, 2 H-2),
1.33 (d, 3H, J = 6.0 Hz, 3 H-60); 13C NMR δ 145.2 (C-4 tr), 138.7
(C), 138.6 (C), 138.0 (C), 137.8 (C), 128.5 (CH), 128.4 (CH),
128.2 (CH), 128.1 (CH), 127.9 (CH), 127.8 (CH), 127.7 (CH),
127.5 (CH), 127.4 (CH), 123.8 (C-5 tr), 96.4 (CH2), 80.5 (CH),
80.2 (CH), 77.2 (CH), 75.9 (CH), 75.1 (CH2), 74.9 (CH), 74.0
(CH2), 72.6 (CH), 72.5 (CH2), 72.4 (CH2), 71.8 (CH2), 71.0
(CH), 70.4 (CH), 70.2 (CH), 63.6 (CH2), 55.2 (CH), 50.5 (CH2),
29.8 (CH2), 26.9 (CH2), 18.2 (CH3); HRMS (ESI/Q-TOF) m/z
calcd for C60H67N3NaO10 (Mþ Na)þ 1012.4724, found 1012.4786.
1,4,5,6-Tetra-O-acetyl-3,7-anhydro-2,8-dideoxy-8-[4-(20,30,40-
tri-O-acetyl-60-deoxy-r-D-mannopyranosyl)-1H-1,2,3-triazol-1-yl]-
D-glycero-D-talo-octitol (25). The deprotection of 24 (18 mg,
18 μmol) was performed as described for the synthesis of 23 to
give, after column chromatography on silica gel (AcOEt), 25
(10 mg, 81%) as an amorphous solid: [R]D = þ118.7 (c 0.6,
CHCl3); 1H NMR (300 MHz) selected data δ 7.83 (s, 1H, H-5
tr); 13C NMR selected data δ 170.1 (CdO), 141.8 (C-4 Tr), 124.3
(C-5 Tr), 50.5 (CH2), 29.8 (CH2), 27.9 (CH2), 17.6 (CH3);
HRMS (ESI/Q-TOF) m/z calcd for C30H42N3O16 (M þ H)þ
700.2565, found 700.2619.
3,7-Anhydro-2,8-dideoxy-8-[4-(r-D-mannopyranosyl)-1H-1,2,3-
triazol-1-yl]-D-glycero-D-talo-octitol (31). To a solution of 30
(22 mg, 0.02 mmol) in anhydrous CH2Cl2 (5 mL) was added
trifluoroacetic acid (400 μL). The solution was kept at room
temperature for 3 h and then concentrated. To a solution of the
residue in 4:2:1 CH3OH-THF-H2O (4 mL) were added am-
monium formate (110 mg, 1.75 mmol) and 10% palladium on
carbon (212 mg, 0.20 mmol). The mixture was stirred at 50 °C
for 18 h in a sealed vial and then cooled to room temperature,
filtered through a pad of Celite, and concentrated. The residue
was eluted from a column of Sephadex LH20 (1 ꢀ 20 cm, d ꢀ h)
with 3:1 H2O-CH3OH to give 31 (4 mg, 45%) as an amorphous
3,7-Anhydro-2,8-dideoxy-8-[4-(60-deoxy-r-D-mannopyranosyl)-
1H-1,2,3-triazol-1-yl]-D-glycero-D-talo-octitol (1a). The depro-
tection of 25 (8 mg, 11 μmol) was performed as described for the
synthesis of 1f to give, after a column chromatography on
Sephadex LH20 (1:1 CH2Cl2-MeOH), 1a as an amorphous
1
solid: [R]D = -6.1 (c 0.4, H2O); H NMR (400 MHz, D2O)
0
0
selected data δ 7.97 (s, 1H, H-5 tr), 5.10 (d, 1H, J1 ,2 = 2.2 Hz,
1
H-10), 4.46 (dd, 1H, J7,8b = 9.0, J8a,8b = 14.2 Hz, H-8b), 4.41 (dd,
solid (4.2 mg, 92%): H NMR (300 MHz, D2O) selected data
1H, J2 ,3 = 3.2 Hz, H-2 ); 13C NMR (D2O) δ 146.5 (C-4 tr), 128.3
(C-5 tr), 78.2 (CH), 77.7 (CH), 75.7 (CH), 75.2 (CH), 74.1 (CH),
73.7 (CH), 73.4 (CH), 72.7 (CH), 71.4 (CH), 69.9 (CH), 63.7
(CH2), 60.3 (CH2), 54.2 (CH2), 32.3 (CH2); HRMS (ESI/Q-TOF)
m/z calcd for C16H28N3O10 (M þ H)þ 422.1775, found 422.1767.
Evaluation of Inhibitor Activity of 1a-f in the PPM1/R(1,6)-
Mannosyltransferase Assay. The R-Manp(1,6)-R-Manp-O(CH2)7-
CH3 disaccharide acceptor12 (0.8 μL, stored as a 20 mM ethanol
stock) was dried under a stream of argon in a microcentrifuge
tube (1.5 mL), placed in a vacuum desiccator for 15 min to remove
any residual solvent, and then resuspended in 8 μL of a 1%
aqueous solution of Igepal. Assays were performed by incubating
each TOM compound (1a-f) at a final concentration of 1.0 mM
with 2.4 μM GDP-[U-14C]mannose (321 mCi/mmol, 0.25 μCi),
62.5 μM ATP, 10 μM MgCl2, 62.5 μM DTT, 12.5 μM NaF,
0.25 mM decaprenolphosphate (in 1% CHAPS) and M. smeg-
matis membrane fractions corresponding to 500 μg. The final
volume of the assays was adjusted to 80 μL with 50 mM MOPS
(pH 7.9), with a 0.2 mM final concentration of R-Manp(1,6)-R-
Manp-O(CH2)7CH3 acceptor. The reaction mixtures were then
incubated at 37 °C for 30 min. A 1:1 CHCl3/CH3OH (533 μL)
solution was added to the incubation tubes, and the entire
contents were centrifuged at 18000g. The supernatant was recovered
and dried under a stream of argon, resuspended in 1:1 EtOH/
H2O (1 mL), and loaded onto a pre-equilibrated (1:1 EtOH/H2O)
1 mL Whatmann strong anion-exchange (SAX) cartridge, which
was washed with 3 mL of EtOH. The eluate was dried, and the
resulting products were partitioned between the two phases
arising from a mixture of n-butanol (3 mL) and H2O (3 mL).
The resulting organic phase was recovered following centrifuga-
tion at 3500g, the aqueous phase was again extracted twice with
3 mL of water-saturated n-butanol, and the pooled extracts were
back-washed twice with water-saturated n-butanol (3 mL). The
water-saturated n-butanol fraction was dried and resuspended in
200 μL of n-butanol. The total counts per minute of radiolabeled
material extractable into the n-butanol phase was measured by
scintillation counting using 10% of the labeled material and
0
δ 7.96 (s, 1H, H-5 tr), 5.10 (bs, 1H, H-10); HRMS (ESI/Q-TOF)
m/z calcd for C16H28N3O9 (M þ H)þ 406.1826, found 406.1815.
Tetramannoside (26). The coupling of azide 11 (15 mg, 0.015
mmol) with alkyne 10 (13.8 mg, 0.015 mmol) was performed as
described for the synthesis of 12 to give, after column chromato-
graphy on silica gel (2:1 cyclohexane-AcOEt), 26 (26 mg, 88%)
as an amorphous solid: [R]D = -34.5 (c 0.7, CHCl3); 1H NMR
(400 MHz) selected data δ 7.80, 7.69, and 7.43 (3 s, 3H, 3 H-5 tr),
0
0
0
0
7.42-7.14 (m, 60H, Ar) 5.29, 5.23, and 5.20 (3 d, 3H, J1 ,2 = 1.8
00 00
000,2000
Hz, J1 ,2 = 1.8 Hz, J1
= 1.8 Hz, H-10, H-100, H-1000), 3.22 (s,
3H, CH3), 1.34 (d, 3H, J = 6.0 Hz, CH3); 13C NMR selected
data δ 145.2 (C-4 tr), 143.9 (C-4 tr), 143.8 (C-4 tr), 138.7-137.9
(C), 128.5-127.3 (CH), 124.4 (C-5 tr), 124.3 (C-5 tr), 123.9 (C-5
tr), 96.4 (CH2), 63.8 (CH2), 55.2 (CH), 50.8 (CH2), 50.7 (CH2),
29.7 (CH2), 18.4 (CH3).
Tetramannoside (27). The deprotection of 26 (20 mg, 10 μmol)
was performed as described for the synthesis of 23 to give, after
column chromatography on silica gel (AcOEt), 27 (10 mg, 73%)
as an amorphous solid: [R]D = þ3.9 (c 0.7, CHCl3); 1H NMR
(400 MHz) selected data δ 8.15, 7.77, and 7.68 (3 s, 3H, 3 H-5 tr);
13C NMR selected data δ 170.5-170.0 (CdO), 29.7 (CH2),
20.8-20.7 (CH3), 17.6 (CH3); HRMS (ESI/Q-TOF) m/z calcd
for C58H76N9O30 (M þ H)þ 1378.4698, found 1378.4749.
Tetramannoside (1b). The deprotection of 27 (9 mg, 6.5 μmol)
was performed as described for the synthesis of 1f to give 1b as
an amorphous solid (5 mg, quantitative): HRMS (MALDI-
TOF) m/z calcd for C32H49N9NaO17 (M þ Na)þ 854.3144,
found 854.3214.
Octamannoside (28). The coupling of azide 16 (21 mg, 10.6 μmol)
with alkyne 15 (20 mg, 10.6 μmol) was performed as described
for the synthesis of 12 to give, after trituration with CH3OH, 28
(35 mg, 85%) as an amorphous solid: [R]D = -150.4 (c 1.0,
1
CHCl3); H NMR (400 MHz) selected data δ 7.88, 6.50, 6.38,
6.36, and 6.24 (5 s, 5H, 5 H-5 tr), 6.00 (bs, 2H, 2 anom H), 5.94,
5.87, 5.71, 5.46, and 5.41 (5 bs, 5H, 5 anom. H), 3.09 (s, 3H,
CH3), 1.39 (d, 3H, J = 6.0 Hz, CH3); 13C NMR selected data
δ 145.0-143.1 (C-4 tr), 139.5-137.5 (C), 128.4-126.7 (CH),
J. Org. Chem. Vol. 75, No. 19, 2010 6335

Lo Conte et al.
JOCFeatured Article
10 mL of EcoScintA. The incorporation of [14C]Man was deter-
mined by subtracting counts present in control assays (incubation
of the reaction components in the absence of R-Manp(1,6)-R-
Manp-O(CH2)7CH3).
Royal Society Wolfson Research Merit Award, as a former
Lister Institute-Jenner Research Fellow, the Medical Research
Council, and The Wellcome Trust (081569/Z/06/Z).
Supporting Information Available: Copies of the NMR
spectra of the new products. This material is available free of
charge via the Internet at http://pubs.acs.org.
Acknowledgment. G.S.B. acknowledges support in the
form of a Personal Research Chair from Mr. James Bardrick,
6336 J. Org. Chem. Vol. 75, No. 19, 2010