Synthetic 3-O-methylmannose-containing polysaccharides (sMMPs): Design and synthesis

Article abstract of DOI:10.1021/jo061991n

With the hope of mimicking the chemical and biological properties of natural 3-O-methylmannose-containing polysaccharides (MMPs), synthetic 3-O-methylmannose-containing polysaccharides (sMMPs) were designed and synthesized in a convergent manner. With lit

Full text of DOI:10.1021/jo061991n

Synthetic 3-O-Methylmannose-Containing Polysaccharides
(sMMPs): Design and Synthesis
Margaret C. Hsu, Jinhwa Lee, and Yoshito Kishi*
Department of Chemistry and Chemical Biology, HarVard UniVersity, 12 Oxford Street,
Cambridge, Massachusetts 02138
kishi@chemistry.harVard.edu
ReceiVed September 27, 2006
With the hope of mimicking the chemical and biological properties of natural 3-O-methylmannose-
containing polysaccharides (MMPs), synthetic 3-O-methylmannose-containing polysaccharides (sMMPs)
were designed and synthesized in a convergent manner. With little modification of the Mukaiyama
glycosidation, high R-selectivity (>50:1∼>20:1) and yields (79∼74%) were achieved for the key
glycosidation steps. The exceptionally high R-selectivity observed was shown to be consequent to the
selective anomerization of â- to R-anomer under the glycosidation conditions. This glycosidation is well
suited for a highly convergent oligosaccharide synthesis, particularly because of excellent chemical yields
even when using approximately equal-sized donors and acceptors in an approximately 1:1 molar ratio.
An iterative reaction sequence allowed the growing oligosaccharide to double in size after each cycle
and led to an efficient synthesis of sMMP 8-, 12-, and 16-mers 18-20.
Introduction
they differ in the number of lipophilic side chains attached to
each molecule.3 The side chains include acetate, propionate,
isobutyrate, octanoate, and succinates, but the precise distribution
of the acyl groups remains unknown. Saponification of each
MGLP is known to yield the same 6-O-methyl-D-glucose-
containing polysaccharide (MGP) that contains 11 internal,
contiguous 6-O-methyl-D-glucose residues and a 3-O-methyl-
D-glucose residue at the nonreducing terminus.4
Extracts of Mycobacterium smegmatis cells provide two series
of polysaccharides: the 3-O-methyl-D-mannose-containing
polysaccharides (MMPs) and the 6-O-methyl-D-glucose-contain-
ing lipopolysaccharides (MGLPs) (Figure 1).1 MMPs consist
of 11-14 R-(1f4)-linked mannosides with most rings bearing
a 3-O-methyl ether functionality.2 The reducing end of the
oligosaccharides is capped by a methyl aglycon. The four
members of the MGLP family share the polysaccharide back-
bone whose reducing end is capped with a glyceric acid, but
(3) For isolation and structural characterization of MGLP/MGP, see: (a)
Lee, Y. C.; Ballou, C. E. J. Biol. Chem. 1964, 239, PC3602. (b) Saier, M.
H., Jr.; Ballou, C. E. J. Biol. Chem. 1968, 243, 4332. (c) Smith, W. L.;
Ballou, C. E. J. Biol. Chem. 1973, 248, 7118. (d) Forsberg, L. S.; Dell, A.;
Walton, D. J.; Ballou, C. E. J. Biol. Chem. 1982, 257, 3555. Regarding the
structural heterogeneity of MGP, Ballou commented that at least two forms
of MGP containing 21 hexoses exist: Kamisango, K.; Dell, A.; Ballou, C.
E. J. Biol. Chem. 1987, 262, 4580. Also see: Tuffal, G.; Albigot, R.;
Monsarrat, B.; Ponthus, C.; Picard, C.; Rivie`re, M.; Puzo, G. J. Carbohydr.
Chem. 1995, 14, 631.
(4) The structure of MG(L)P shown in Figure 1 is the revised structure
suggested by Rivie`re on the basis of the structure of polysaccharide isolated
from Mycobacterium boVis BCG. See: Tuffal, G.; Albigot, R.; Rivie`re,
M; Puzo, G. Glycobiology 1998, 8, 675-684.
(1) For reviews on MMP and MGLP/MGP, see: (a) Bloch, K. AdV.
Enzymol. 1977, 45, 1. (b) Ballou, C. E. Acc. Chem. Res. 1968, 1, 366. (c)
Ballou, C. E. Pure Appl. Chem. 1981, 53, 107.
(2) For isolation and structural characterization of MMP, see: (a) Gray,
G. R.; Ballou, C. E. J. Biol. Chem. 1971, 246, 6835. (b) Maitra, S. K.;
Ballou, C. E. J. Biol. Chem. 1977, 252, 2459. (c) Weisman, L. S.; Ballou,
C. E. J. Biol. Chem. 1984, 259, 3457. (d) Weisman, L. S.; Ballou, C. E. J.
Biol. Chem. 1984, 259, 3464. (e) Ilton, M.; Jevans, A. W.; McCarthy, E.
D.; Vance, D.; White, H. B., III; Bloch, K. Proc. Natl. Acad. Sci. U.S.A.
1971, 68, 87.
10.1021/jo061991n CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/03/2007
J. Org. Chem. 2007, 72, 1931-1940
1931

Hsu et al.
FIGURE 2. Structures of synthetic analogues of mycobacterial
polysaccharides, sMMPs (synthetic polysaccharides composed of 3-O-
methyl-D-mannose), and sMGPs (synthetic polysaccharides composed
of 6-O-methyl-D-glucose).
Because sMMP and sMGP are R-(1f4)-linked oligomers of
monomethylated mannose and glucose, respectively, we envi-
sioned that their oligosaccharide backbones could be built by
iterative glycosidations. For the overall efficiency of synthesis,
we desired to incorporate the highest degree of convergence in
the synthetic design and planned to synthesize sMMP and sMGP
from an appropriate combination of a glycosyl donor and a
glycosyl acceptor. To execute this plan with high overall
efficiency, it is critically important to identify a glycosylation
reaction that is not only highly stereoselective but also achiev-
able with an approximately 1:1 mixture of a glycosyl donor
and a glycosyl acceptor of approximately the same size. In this
paper, using sMMP (R ) n-Pr) with n ) 8, 12, and 16 as
examples, we demonstrate the feasibility of this synthetic plan.
We should note that this project was initiated in 1992,8 when
the chemical synthesis of oligosaccharides was still in the
relatively underdeveloped stage. However, for the past one and
a half decades, we have witnessed a remarkable development
in this general area. A number of glycosyl donors, including
halo sugars, pentenyl glycosides, thioglycosides, isopropenyl
glycosides, orthoesters, 1-O-acyl sugars, 1-O-pentenoyl sugars,
trichloroacetimidates, glycosyl sulfoxides, glycosyl sulfones,
glycosyl thiocyanates, glycosyl dialkylphosphites, glycosyl
phosphorodithioates, glycosyl tetramethylphosphorodiamidates,
glycals and 1,2-anhydrosugars, seleno glycosides, and glycosyl
diazirines, are now known to effect the glycosidation in a
stereoselective manner in solution- and/or solid-phase synthe-
ses.9,10 Many of these synthetic methods have successfully been
FIGURE 1. Structures of mycobacterial polysaccharides 3-O-methyl-
D-mannose-containing polysaccharides (MMPs) and 6-O-methyl-D-
glucose-containing lipopolysaccharides (MGLPs)/6-O-methyl-D-glucose-
containing polysaccharides (MGPs).
Both MMP and MGLP/MGP are known to affect profoundly
the fatty acid biosynthesis in Mycobacterium smegmatis.1a They
increase the overall rate of fatty acid biosynthesis, change the
product distribution, and prevent product inhibition of the fatty
acid synthetase.2e,5,6 They form stoichiometric complexes with
C16- or longer acyl-CoA.7 Bloch proposed a structural model
for complex formation between palmitoyl-CoA and MMP.1a,7
In this model, the conformationally flexible glycosidic bonds
of MMP rotate to achieve a helical conformation with appropri-
ate dimensions for accommodating the aliphatic side chain of
acyl-CoA. In this conformation, the 3-O-methyl ether groups
are directed toward the core of the helical carbohydrate, thereby
increasing the hydrophobic nature of the host and stabilizing
the proposed complex via hydrophobic interactions between
MMP and the aliphatic side chain of acyl-CoA. Coincidentally,
the remaining hydroxyl groups on the 2- and 6-positions of the
sugar rings are located on the outside of the helix and can
interact with either water molecules or the polar functionalities
of acyl-CoA via hydrogen bonding.
We were interested in gaining mechanistic insights for the
intriguing biological role(s) of MMP and MGLP/MGP. How-
ever, we felt that naturally occurring MMP and MGLP/MGP
are not necessarily ideal substrates for our study, as they were
isolated as complex mixtures of closely related polysaccha-
rides.2,3 For this reason, we chose to use synthetic polysaccha-
rides structurally related to natural MMP and MGLP/MGP for
two major reasons: (1) synthetic polysaccharides should be
available as chemically well-defined and homogeneous materials
and (2) synthetic polysaccharides should be structurally tunable
for the needs of our investigation. Obviously, the most unique
structural feature of natural MMP and MGLP/MGP is the
polymeric form of O-methylated mannose and glucose. Thus,
we decided to incorporate this structural feature in the synthetic
polysaccharides and selected the polymers composed of 3-O-
methyl-D-mannose and 6-O-methyl-D-glucose ( Figure 2).
(8) M.C.H. started the sMMP synthesis as her thesis work in 1992. This
paper is largely based on her Dissertation: Hsu, M. C. Synthetic 3-O-
Methylmannose Polysaccharides (sMMP): Complex Formation and Mo-
lecular Recognition with Fatty Acids. Dissertation, Harvard University, 1997.
(9) A number of comprehensive monographs on “Chemical Synthesis
of Glycosides” are available. For example, see: (a) Carbohydrates in
Chemistry and Biology; Ernst, B., Hart, G. W., Sinay¨, P., Eds.; Wiley-
VCH: New York, 2000; Vol. 1. The book contains 18 relevant reviews by
the leading practitioners. (b) Carbohydrate-based Drug DiscoVery; Wong,
C.-H., Ed.; Wiley-VCH: Weinheim, Germany, 2003; Vol. 1. This contains
three relevant reviews. (c) Glycoscience: Chemistry and Chemical Biology;
Fraser-Reid, B. O., Tatsuta, K., Thiem, J., Eds.; Springer-Verlag: Berlin,
Germany; Vols. 1 and 2. These contain five relevant reviews. In addition,
the December issue of Chemical Reviews (2000, 100) was dedicated to
Carbohydrate Chemistry, which contains four relevant reviews.
(10) For recent reviews on “Chemical Synthesis of Glycosides and
Oligosaccharides” by the experts in the field, see: (a) Schmidt, R. R. (i)
page 5 of the monograph quoted in ref 9a; (ii) Chem. ReV. 2000, 100, 4423;
(iii) Chem. ReV. 2006, 106, 160. (b) Danishefsky, S. J. (i) page 61 of the
monograph quoted in ref 9a; (ii) Acc. Chem. Res. 1998, 31, 685; (iii) Angew.
Chem., Int. Ed. Engl. 1996, 35, 1380. (c) Fraser-Reid, B. (i) page 135 of
the monograph quoted in ref 9a. (d) Wong, C.-H. (i) page 117 of the
monograph quoted in ref 9a; (ii) Chem. ReV. 2000, 100, 4465; (iii) Science,
2001, 291, 2344. (e) Seeberger, P. H. (i) page 103 of the monograph quoted
in ref 9b; (ii) Chem. ReV. 2000, 100, 4349; (iii) Eur. J. Org. Chem. 2002,
595. (f) Hanessian, S. Chem. ReV. 2000, 100, 4443. (g) Ley, S. V. (i) page
427 of the monograph quoted in ref 9a; (ii) Chem. ReV. 2001, 101, 53.
(5) Flick, P. K.; Bloch, K. J. Biol. Chem. 1974, 249, 1031.
(6) Knoche, H.; Esders, T. W.; Koths, K.; Bloch, K. J. Biol. Chem. 1973,
248, 2317.
(7) (a) Machida, Y.; Bloch, K. Proc. Natl. Acad. Sci. U.S.A. 1973, 70,
1146. (b) Bergeron, M.; Machida, Y.; Bloch, K. J. Biol. Chem. 1975, 250,
1223.
1932 J. Org. Chem., Vol. 72, No. 6, 2007

Synthetic 3-O-Methylmannose-Containing Polysaccharides
SCHEME 1a
applied for the synthesis of a broad range of complex oligosac-
charides, most notably mannose-containing oligosaccharides.11
Results and Discussion
The synthetic analysis briefly given above calls for ap-
propriately substituted monosaccharides as the starting material.
To execute effectively the designed synthesis, we needed to
select a minimum of three different protecting groups to
differentiate the hydroxyl groups present in A. Taking into
a Reagents and conditions: (a) (1) Sc(OTf)3, allyl-OH, 80%; (2)
PhCH(OMe)2, HBF4, 83%; (b) (1) n-Bu2SnO, MeOH, 83%; (2) MeI, 74%;
(c) (1) NaH, BnBr, imidazole, 66%; (2) TiCl4, NaBH3CN, 73%.
Treatment of D-mannose with Sc(OTf)3 (3 mol %) in allyl
alcohol at 100 °C furnished the desired allyl glycoside in 80%
isolated yield, with ca. 10:1 stereoselectivity (Scheme 1).16
Addition of fluoroboric acid (catalytic) by a syringe pump to a
solution of allyl mannoside and benzaldehyde dimethyl acetal
in DMF gave the known acetal 2.17 The competing side reaction
was the formation of the five-membered acetal between the C2
and C3 hydroxyl groups. A selective methylation of the C3
hydroxyl group of 2 was achieved via a cyclic stannyl ether,18
to give 3 in 74% isolated yield. After protecting the C2 hydroxyl
as a benzyl ether, the minor amount of the â-anomer was
removed at this stage. The cyclic acetal was regioselectively
opened by NaBH3CN/TiCl4,19 to provide a chromatographically
inseparable ca. 4:1 mixture of the desired product 4 and its C6
regioisomer. However, selective silylation of the primary alcohol
present in the undesired regioisomer with t-butyldiphenylchlo-
rosilane (TBDPSCl) facilitated separation/isolation, to furnish
allyl R-mannoside 4 in 73% isolated yield.
The properly protected mannoside 4 gave an easy access to
both the glycosyl acceptor 5 and donor 6. To identify the optimal
C4 protecting group, the glycosyl donors 6a-d, with four types
of protecting groups, triisopropylsilyl (TIPS), 4-methoxyphen-
ylmethyl (MPM), phenylcarbonate, or benzoyl, were prepared
(Scheme 2). Their behaviors under the Mukaiyama conditions
(5 + 6, SnCl4-AgClO4 (10 mol %), 0 °C, 36 h, MeCN or Et2O)
were tested, thereby demonstrating the following: (1) either the
TIPS group or the MPM group was not compatible with the
glycosidation conditions; (2) a good R/â-selectivity (10:1) but
a poor chemical yield (28%) was observed for 6c; and (3)
excellent R/â-selectivity and chemical yield were observed for
6d (R/â-selectivity ) 16:1 or 17:1, isolated yield ) 79% or
76% in MeCN or Et2O, 20 h, 0 °C).20 From these preliminary
account the anticipated reactions for execution of the plan, we
were decisive in choosing an allyl group as P1 and a benzyl
group as P2. However, we postponed the decision on the
protecting group P3 until the first glycosidation was studied.
Using glucose-derived glycosyl donors and acceptors, we then
conducted preliminary studies to develop the key glycosidation
reaction.12 However, as discussed in a separate paper,13 we later
realized that the profile of the glycosidation in the mannose
series was pronouncedly different from that in the glucose series,
and therefore some of the conclusions derived from the
preliminary experiments on the glucose series should not be
extrapolated to the mannose series. Nonetheless, on the basis
of these preliminary studies, it became evident that, among
several methods evaluated, the Mukaiyama glycosidation14 best
met our needs. Donors with various aliphatic and aromatic esters
at C1 were tested for the Mukaiyama glycosidation, and the
monomethyl phthalate ester (cf. 6 in Scheme 2) was found to
give the most satisfactory results (chemical yield and R/â-
selectivity).15 With these preliminary results as our guide, we
began the synthesis of suitably protected mannoses.
(11) For recent examples, see the following papers and references cited
therein: (a) Merritt, J. R.; Naisang, E.; Fraser-Reid, B. J. Org. Chem. 1994,
59, 4443. (b) Grice, P.; Ley, S. V.; Pietruszka, J.; Osborn, H. M. I.; Priepke,
H. W. M.; Warriner, S. L. Chem.-Eur. J. 1997, 3, 431. (c) Lee, H.-K.;
Scanlan, C. N.; Huang, C.-Y.; Chang, A. Y.; Calarese, D. A.; Dwek, R.
A.; Rudd, P. M.; Burton, D. R.; Wilson, I. A.; Wong, C.-H. Angew. Chem.,
Int. Ed. 2004, 43, 1000. (d) Danishefsky and co-workers synthesized several
biologically important oligosaccharides containing mannosides. For the most
recent work from the Danishefsky laboratory, see: Geng, X.; Dudkin, V.
Y.; Mandal, M.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2004, 43, 2562.
(e) Jiang, L.; Chan, T. H. Can. J. Chem. 2005, 83, 693. (f) Watt, J. A.;
Williams, S. J. Org. Biomol. Chem. 2005, 3, 1982. (g) Seeberger and co-
workers synthesized several biologically important oligosaccharides contain-
ing mannosides. For the most recent work from the Seeberger laboratory,
see: Liu, X.; Stocker, B. L.; Seeberger, P. H. J. Am. Chem. Soc. 2006,
128, 3638.
(12) Among the representative glycosidation methods known in 1992,
the Schmidt glycosidation gave excellent results in terms of chemical yield
and stereoselectivity, but its potential drawback for the current work was
that, probably due to the instability of trichloroacetimidates, we had to use
an excess of the donor to ensure a high yield for the glycosidation.
(13) Wang, Y.; Cheon, H.-S.; Lee, J.; Kishi, Y., in preparation.
(14) (a) Mukaiyama, T.; Takashima, T.; Katsurada, M.; Aizawa, H. Chem.
Lett. 1991, 533. (b) Mukaiyama, T.; Katsurada, M.; Takashima, T. Chem.
Lett. 1991, 985.
(15) This screening had been conducted before the second generation
of the Mukaiyama ester, 2-(2-methoxyethoxy)acetate, was reported: Mat-
subara, K.; Sasaki, T.; Mukaiyama, T. Chem. Lett. 1993, 1373. In the
mannose series, the monomethyl phthalate and the second generation of
the Mukaiyama ester gave comparable results (chemical yield and stereo-
selectivity), except for the rate of glycosidation, as the second generation
of the Mukaiyama ester gave a much faster rate than with the monomethyl
phthalate.
(16) The similar stereoselectivity (∼10:1 R/â) was observed for formation
of allyl mannoside with Dowex-H+ resin: Lee, R. T.; Lee, Y. C. Carbohydr.
Res. 1974, 37, 193. However, the Sc(OTf)3 conditions furnished allyl
mannoside in a higher yield with less side products and shorter reaction
times.
(17) (a) Albert, R.; Dax, K.; Pleschko, R.; Stutz, A. E. Carbohydr. Res.
1985, 137, 282. (b) Vasella, A.; Witzig, C.; Waldraff, C.; Uhlmann, P.;
Briner, K.; Bernet, B.; Panza, L.; Husi, R. HelV. Chim. Acta 1993, 76, 2847.
(c) Winnik, F. M.; Carver, J. P.; Krepinski, J. J. J. Org. Chem. 1982, 47,
2701.
(18) Nashed, M. A. Carbohydr. Res. 1978, 60, 200.
(19) Adam, G.; Seebach, D. Synthesis 1988, 373.
1
(20) The R- and â-anomers were distinguishable by H NMR; i.e., the
anomeric proton of R- and â-anomers resonated at 5.35 and 4.73 ppm,
respectively. The stereoselectivity of glycosidation was estimated from the
relative intensity of these signals.
J. Org. Chem, Vol. 72, No. 6, 2007 1933

Hsu et al.
SCHEME 4. Reaction Pathways of the Mannosyl Donor
SCHEME 2 a
(6d) and Acceptor (5) under the Mukaiyama Glycosidation
Conditions
a Reagents and conditions: (a) TMSOTf, Et3N, 98%; (b) (1) protection
with R; (2) (i) (Ph3P)3RhCl, DABCO; (ii) HCl; (3) monomethyl phthalate,
EDCI, DMAP; (c) SnCl4-AgClO4 (10 mol %), 0 °C.
SCHEME 3. Products Formed from â-7d under the
Mukaiyama Glycosidation Conditions
anomerization was not observed in the gluco series, and more
extensive mechanistic studies will be reported in a separate
paper.13
The selective anomerization suggests a possibility that only
the desired R-disaccharide R-7d would be obtained, if a
sufficiently long reaction time was given for the glycosidation.
However, this was not a practical option because a significant
amount of “scrambled” products were formed when a longer
reaction time was employed. To gain insight into this scram-
bling, the stability of 5, 6d, and R-7d under the glycosidation
conditions was then tested, thereby showing: (1) the acceptor
5 yielded the homocoupling product along with the desilylated
starting material; (2) the disaccharide R-7d gave a small amount
of the cleaved monosaccharides; and (3) the donor 6d was
susceptible to decomposition. Subsequently, a 1:1 mixture of
R-7d and 5 was found to yield a mixture of scrambled products
composed of trisaccharide (17%), tetrasaccharide (2%), and the
homocoupling product (7%) under the standard glycosidation
conditions. These results are summarized in Scheme 4, in which
the reaction pathways on the left side show productive paths to
the desired disaccharide R-7d, whereas the reaction pathways
on the right side indicate paths to the scrambled products.
Overall, as the glycosidation of 5 with 6d is significantly faster
than the glycosidation of 5 with 7d or 5 itself, the byproduct
formation via the latter couplings is negligible in a short reaction
time but becomes considerable in a prolonged reaction time.
Thus, a fine tuning of the reaction time was required. In passing,
we noticed that the acceptor reactivity between the alcohol 4
and trimethylsilyl ether 5 showed no appreciable difference in
glycosidation, whereas there was considerable difference in
reactivity between R-6d and â-6d (kâ . kR).
experiments, it became evident that the benzoate-protected donor
(6d as ca. a 2:1 mixture of R- and â-anomers)21 was the best
choice.
Having identified the best combination of glycosyl donor and
acceptor, we conducted a time course study to gain insight into
the glycosidation. This study showed that the R/â-selectivity
was only 3 in 1 h but improved to 5, 9, and 20 in 8, 22, and
30 h, respectively, thereby suggesting that the â-disaccharide
â-7d anomerized into the R-disaccharide R-7d under the
glycosidation conditions. Indeed, under the condition of 10 mol
% of SnCl4-AgClO4 for 36 h, the â-disaccharide â-7d was
converted to a mixture of R-glycoside R-7d (78%) and two
monosaccharides (Scheme 3). Interestingly, however, the cor-
responding R-disaccharide R-7d was found not to anomerize
to the â-disaccharide â-7d under the same conditions. Thus,
the observed high stereoselectivity is attributed to the selective
anomerization from the â- to R-anomer.22 Interestingly, this
(21) Although a considerable difference was observed in their reactivity,
both anomers â-6d and R-6d were found equally effective for the
glycosidation.
(22) It is generally recognized that â-mannosides are prone to facile
anomerization. However, it is worth noting that the Mukaiyama catalyst
(AgClO4-SnCl4) was found to promote the anomerization only from the
â-mannosides to the corresponding R-mannosides. To the best of our
knowledge, the anomerization with the observed level of selectivity is rare.
Perhaps, the example reported by Lee and co-workers (Lee, C. K.; Kim, E.
J.; Lee, I.-S. H. Carbohydr. Res. 1993, 240, 197) is most relevant to this
case.
With information on the profile of glycosidation, the optimal
conditions were identified to obtain the desired R-7d: coupling
of 5 (1.0 equiv) with 6d (1.0 equiv) in the presence of SnCl4-
AgClO4 (10 mol %) in diethyl ether at 0 °C for 36 h gave the
1934 J. Org. Chem., Vol. 72, No. 6, 2007

Synthetic 3-O-Methylmannose-Containing Polysaccharides
SCHEME 5 a
SCHEME 7 a
a Reagents and conditions: (a) SnCl4-AgClO4, Et2O, 0 °C, 76%.
a Reagents and conditions: (a) (1) (i) (Ph3P)3RhCl, DABCO; (ii) HCl,
81%; (2) monomethyl phthalate, EDCI, DMAP, 100%; (b) (1) NaOH,
MeOH, 92%; (2) TMSOTf, Et3N, 85%; (c) SnCl4-AgClO4, Et2O,
-20 °Cf0 °C, 79%.
SCHEME 6 a
a Reagents and conditions: (a) (1) (i) (Ph3P)3RhCl, DABCO; (ii) HCl,
81%; (2) monomethyl phthalate, EDCI, DMAP, 98%; (b) (1) NaOH, MeOH,
92%; (2) TMSOTf, Et3N, 99%; (c) SnCl4-AgClO4, Et2O, -20 °Cf0 °C,
76%.
FIGURE 3. 1H NMR spectra of the MeO signals of 13 (400 MHz,
CDCl3). Reaction A was quenched after 21.5 h. Reaction B was
quenched after 9.5 h.
disaccharide as a 20:1 mixture of R/â-anomers, from which the
desired R-disaccharide R-7d was isolated in 76% yield in a pure
form (Scheme 5).
For the synthesis of the tetrasaccharide R-10, R-7d became
the common source of the disaccharide donor and acceptor
(Scheme 6). Rh-assisted deallylation23 of R-7d, followed by
esterification with monomethyl phthalate, provided the disac-
charide donor 8. The glycosyl acceptor 9 was prepared via
saponification of the benzoate and TMS ether formation. The
glycosidation proceeded in the same manner as the previous
case. An 8:1 mixture of R/â-tetrasaccharides was formed in 3 h
at 0 °C, whereas a >50:1 mixture was obtained in 26 h at 0 °C.
After the minor â-tetrasaccharide was removed from the crude
reaction mixture by HPLC (µ-porasil), purification by JAI (Japan
Analytical Industry Co., Ltd., vide infra) gave the R-tetrasac-
charide R-10 in 76% yield with g99% purity.
Tetrasaccharide R-10 was converted to both tetrasaccharide
donor 11 and tetrasaccharide acceptor 12 under conditions
similar to those described for the smaller fragments (Scheme
7).
Glycosidation of 11 with 12 met with complications when
the reaction was left unquenched for over 10 h. The product
obtained under such conditions exhibited a clean 1H NMR
spectrum, except the MeO signals (rxn A in Figure 3). Mass
spectroscopy analysis (FAB-MS) showed the product contained
a broad range of oligomers. Assuming that the oligomers have
similar ionization potentials, the major components in the
mixture included the hexamer (ca. 35%), heptamer (ca. 22%),
octamer (desired; ca. 24%), nanomer (ca. 9%), and decamer
(ca. 6%). Knowing the profile of glycosidation and considering
the total number of the glycosidic bonds present in R-13, this
result was not totally surprising. Quenching the reaction within
10 h provides high chemical conversion with significantly less
contamination of scrambled products (FAB-MS: relative con-
1
tribution of the octamer was .70%). The H NMR spectrum
showed six sharp signals corresponding to eight methyoxyls.
Subsequent purification by JAI recycling HPLC provided the
octasaccharide R-13 in at least 90% purity (rxn B in Figure 3).
For preparative purposes, the glycosidation of 11 and 12 was
1
quenched after 9.5 h. The H NMR spectrum of the crude
product showed that the R/â-stereoselectivity was >50:1 and
also that a small amount of unreacted donor 11 and desilylated
acceptor remained. Although the three components had similar
Rf values by TLC, R-13 could be isolated in 79% yield by silica
gel chromatography. The more preparatively acceptable method
of separation involved two chemical derivatization steps: (1)
treatment of the crude product with TMSOTf/NEt3 regenerated
12 from the unreacted acceptor, which was readily recovered
by chromatography, and (2) base hydrolysis facilitated column-
chromatographic separation of R-13 from the remaining depro-
tected form of 11.
(23) (a) Corey, E. J.; Suggs, W. J. J. Org. Chem. 1973, 38, 3224. (b)
Gent, P. A.; Gigg, R. J. Chem. Soc., Chem. Commun. 1974, 277.
J. Org. Chem, Vol. 72, No. 6, 2007 1935

Hsu et al.
SCHEME 8 a
SCHEME 10 a
a Reagents and conditions: (a) (1) NaOH, MeOH; (2) Pd(OH)2 on C,
H2.
SCHEME 11 a
a Reagents and conditions: (a) (1) NaOH, MeOH, 83%; (2) TMSOTf,
Et3N, 100%; (b) SnCl4-AgClO4, Et2O, 0 °C, 75%.
SCHEME 9 a
a Reagents and conditions: (a) (1) (i) (Ph3P)3RhCl, DABCO; (ii) HCl,
77%; (2) monomethyl phthalate, EDCI, DMAP, 94%; (b) (1) NaOH, MeOH,
83%; (2) TMSOTf, Et3N, 100%; (c) SnCl4-AgClO4, Et2O, 0 °C, 74%.
a Reagents and conditions: (a) SnCl4-AgClO4, Et2O. Note: 12-mer was
synthesized via a combination of donor 4-mer and acceptor 8-mer.
Octasaccharide R-13 was converted to the octasaccharide
acceptor 14 in two steps (Scheme 8), and coupling of 14 with
tetrasaccharide donor 11 was accomplished under established
glycosidation conditions. Minimizing formation of scrambled
products, this reaction was quenched after 8.5 h. Crude NMR
again showed >50:1 (R/â) selectivity. The protected dodecasac-
charide R-15 was isolated in 75% yield.
Following procedures similar to those mentioned for the
preparation of other donors, octasaccharide R-13 was converted
to the octasaccharide donor 16 (Scheme 9). Reaction of donor
16 with acceptor 14 allowed the formation of R-hexadecasac-
charide R-17 (>50:1 R/â-selectivity) in 9.5 h under the standard
conditions (74% yield).
(hexadecasaccharide) with the purity of >98%, >98%, >90%,
>85%, and >75%, respectively.24
Complete deprotection of the oligosaccharides was ac-
complished in two steps to furnish sMMP 8-, 12-, and 16-mers
18-20, respectively (Scheme 10). Hydrolysis of the benzoate
proceeded smoothly with methanolic NaOH. The final step was
the hydrogenolysis of all benzyl ethers accompanied by the
reduction of the allyl aglycon using Pearlman’s catalyst and
hydrogen. The products were isolated by reverse-phase chro-
matography on C18 silica gel. As discussed, the oligosaccharides
R-13, R-15, and R-17 were contaminated with scrambled
products even after JAI chromatographic purification. However,
the purity of sMMP 8-, 12-, and 16-mers 18-20 was at least
95% by FAB-MS and 1H NMR analyses, implying that
contaminated scrambled products were removed during the
deprotection and purification. This may be one of the reasons
the overall yield of the deprotection and purification declined
from the sMMP 8-mer to the sMMP 16-mer.25
The purification method of oligo- and polysaccharides by JAI
(Japan Analytical Industry Co., Ltd.) HPLC deserves a com-
ment. The glucosidation products were purified by a JAI LC-
908 preparative liquid chromatographic unit using two poly-
styrene-based gel permeation columns in tandem. The polystyrene
column utilizes the principle of size exclusion, and this
chromatographic technique was indispensable for preparative
separation/purification of the polysaccharides. As one might
speculate from the principle used for this separation technique,
the capacity of separating a polysaccharide from the next lower
or higher polysaccharide decreases with an increase of its
molecular size. Nonetheless, this chromatographic technique
allowed us to isolate R-7d (disaccharide), R-10 (tetrasaccharide),
R-13 (octasaccharide), R-15 (dodecasaccharide), and R-17
Conclusion
A highly convergent synthesis of sMMP has been achieved
as summarized in Scheme 11. Beginning with D-mannose, the
monosaccharide with the appropriate protecting groups installed
(B with n ) 1) was secured in six steps. B served as the source
of glycosyl donor C and glycosyl acceptor D, each obtainable
1936 J. Org. Chem., Vol. 72, No. 6, 2007

Synthetic 3-O-Methylmannose-Containing Polysaccharides
and heated overnight at 50 °C. The crude product was flushed
through a silica gel column under reduced pressure (9:1 hexanes/
EtOAc, 4:1 hexanes/EtOAc, 1:1 hexanes/EtOAc), to yield 3 (30.6 g,
in two steps. Glycosidation under the modified Mukaiyama
protocol gave E, which is in fact identical to B except for n.
The key steps of this synthesis are the five glycosidations, which
proceeded with high R-selectivities and yields. This glycosida-
tion was well suited for a highly convergent oligosaccharide
synthesis, particularly because of excellent chemical yields even
when using approximately equal-sized donors and acceptors in
a molar ratio of approximately 1:1. An iterative reaction
sequence allowed the growing oligosaccharide to double in size
after each cycle and led to an efficient synthesis of sMMP 8-,
12-, and 16-mers 18-20.
sMMP 8-, 12-, and 16-mers 18-20 thus obtained provided
us, for the first time, with an opportunity to study the chemical
and biological properties of synthetic MMP. To our delight, the
preliminary experiments demonstrated that synthetic and natural
MMPs exhibit identical, or at least very similar, properties.26
With this encouraging result, we felt it to be critically important
to address the scalability of sMMP synthesis to ensure the supply
of the materials. Recognizing a potential problem with (1) the
scalability of glycosidation and (2) the isolation of a glycosi-
dation product free from scrambled products, we initiated a study
on the second generation of sMMP synthesis, resulting in an
improved, scalable synthesis of this class of polysaccharides.27
1
74%). H NMR of the major anomer (CDCl3, 400 MHz): 7.53-
7.47 ppm (m, 2 H), 7.40-7.31 (m, 3 H), 5.92 (m, 1 H), 5.60 (s, 1
H), 5.32 (m, 1 H), 5.24 (m, 1 H), 4.95 (d, J ) 1.0 Hz, 1 H), 4.27
(dd, J ) 3.5, 8.8 Hz, 1 H), 4.21 (m, 1 H), 4.14 (m, 1 H), 4.05-
3.98 (m, 2 H), 3.91-3.81 (m, 2 H), 3.72 (dd, J ) 3.5, 9.6 Hz, 1
H), 3.56 (s, 3 H), 2.58 (d, J ) 1.2 Hz, 1 H). 13C NMR (CDCl3,
100 MHz): 137.5 ppm, 133.5, 129.0, 128.2, 126.1, 117.9, 101.8,
99.2, 78.8, 69.3, 68.9, 68.3, 63.3, 58.7. HRMS (FAB) calcd for
(C17H22O6Na)+ 345.1314, found 345.1304. Select IR peaks (neat):
3463 cm-1, 2912, 1456, 1379, 1218, 1172, 1125, 1096, 1049, 1036,
1009. [R]25 +77.1 (c 0.72, CH2Cl2).
D
Secondary Alcohol 4. To a solution of 3 (30.6 g, 95.0 mmol)
in THF-DMF (4:1, 500 mL) were slowly added NaH (60%
dispersion in mineral oil, 7.6 g, 0.19 mol), imidazole (0.65 g, 9.5
mmol), and benzyl bromide (BnBr, 23 mL, 0.19 mol). After 1 h,
the reaction was quenched by addition of excess MeOH and water,
followed by removal of solvents in vacuo. The crude mixture was
dissolved in a CH2Cl2-water mixture, and the two layers were
separated. The aqueous layer was extracted with CH2Cl2 (3×), and
the organic extracts were dried over MgSO4 and concentrated to
dryness. The R-anomer (25.8 g, 66%) was isolated by silica gel
chromatography under reduced pressure (9:1 hexanes/Et2O).28
To a solution of benzyl ether (25.8 g, 62.5 mmol) and NaBH3-
CN (4.3 g, 69 mmol) in CH3CN (160 mL) at 0 °C was added TiCl4
(7.6 mL, 69 mmol) dropwise. The reaction mixture was stirred at
0 °C for an additional 3 h, then concentrated in vacuo. A solution
of NaOH (2 N, ca. 200 mL) was added to the crude mixture,
followed by a saturated NaHCO3 solution. The aqueous layer was
extracted with CH2Cl2 (3×), and the organic extracts were dried
over MgSO4 and concentrated to dryness.
Experimental Section
Allyl 4,6-O-Benzylidene-R-D-mannopyranoside (2). To a solu-
tion of D-mannose (50.1 g, 278 mmol) in allyl alcohol (185 mL)
was added Sc(OTf)3 (412 mg, 3 mol %). The reaction mixture was
heated to reflux for 6 h. The reaction mixture was concentrated to
dryness and flushed through a silica gel column under reduced
pressure (9:1 CH2Cl2-MeOH), to yield allyl mannopyranoside
(49.2 g, 80%, >10:1 R/â mix).28
The crude product was dissolved in DMF (125 mL) and stirred
overnight with t-butyldiphenylchlorosilane (TBDPSCl, 16 mL, 63
mmol) and imidazole (2.1 g, 31 mmol). After removal of solvent,
4 (18.9 g, 73%) was isolated by silica gel chromatography (4:1
A solution of allyl mannopyranoside (46.3 g, 210 mmol) and
benzaldehyde dimethyl acetal (38 mL, 0.25 mol) in DMF (1.0 L)
was stirred vigorously. Fluoroboric acid (HBF4, 54% in Et2O,
19 mL, 0.11 mmol) was added over 120 min via a syringe pump.
After stirring for an additional hour, Et3N (88 mL, 0.63 mol) was
added and the reaction mixture was concentrated to dryness. Silica
gel chromatography (1:1 EtOAc/hexanes, EtOAc) gave the product
2 (53.5 g, 83%), a known compound in the literature.17 1H NMR
of the major anomer (CDCl3, 400 MHz): 7.54-7.45 ppm (m, 2
H), 7.43-7.34 (m, 3 H), 5.91 (m, 1 H), 5.58 (s, 1 H), 5.32 (m, 1
H), 5.23 (m, 1 H), 4.93 (d, J ) 1.0 Hz, 1 H), 4.28 (dd, J ) 3.4, 9.0
Hz, 1 H), 4.22 (m, 1 H), 4.13 (dd, J ) 3.5, 9.2 Hz, 1 H), 4.09 (dd,
J ) 1.4, 3.5 Hz, 1 H), 4.02 (m, 1 H), 3.94 (dd, J ) 9.1, 9.2 Hz, 1
H), 3.84 (m, 2 H), 2.60 (bs, 2 H). 13C NMR (CDCl3, 100 MHz):
137.2 ppm, 133.4, 129.3, 128.4, 126.2, 117.8, 102.3, 99.3, 77.9,
71.0, 68.8, 68.3, 63.1. HRMS (FAB) calcd for (C16H20O6Na)+
331.1158, found 331.1158. IR (neat): 3341 cm-1, 3230, 2923, 2903,
2866, 1454, 1397, 1378, 1285, 1219, 1133, 1112, 1098, 1069, 1029.
1
hexanes/Et2O, 13:7 hexanes/Et2O). H NMR (CDCl3, 400 MHz):
7.41-7.24 ppm (m, 10 H), 5.88 (m, 1 H), 5.25 (m, 1 H), 5.18 (m,
1 H), 4.95 (d, J ) 1.8 Hz, 1 H), 4.69 (dd, J ) 12.3, 16.9 Hz, 2 H),
4.61 (dd, J ) 12.1, 19.1 Hz, 2 H), 4.19 (m, 1 H), 4.03-3.93 (m,
2 H), 3.84-3.72 (m, 4 H), 3.49 (dd, J ) 3.2, 9.5 Hz, 1 H), 3.35 (s,
3 H), 2.58 (br s, 1 H). 13C NMR (CDCl3, 100 MHz): 138.4 ppm,
138.3, 133.9, 128.3, 127.9, 127.7, 127.6, 127.5, 117.4, 97.3, 81.3,
73.6, 73.2, 72.7, 71.6, 70.6, 68.0, 57.2. HRMS (FAB) calcd for
(C24H30O6Na)+ 437.1940, found 437.1940. Select IR peaks (neat):
3456 cm-1, 2913, 1497, 1454, 1367, 1088, 1043. [R]25 +10.0 (c
D
0.61, CH2Cl2).
Monosaccharide Acceptor 5. To a solution of 4 (9.15 g, 22.1
mmol) and Et3N (12 mL, 88 mmol) in CH2Cl2 (55 mL) was slowly
added TMSOTf (8.5 mL, 44 mmol). After 30 min, saturated
NaHCO3 was added, and the organic components were extracted
with CH2Cl2 (3×). The organic extracts were dried over MgSO4
and concentrated to dryness, and 5 (10.5 g, 98%) was isolated by
silica gel chromatography (9:1 hexanes/Et2O, 3:1 hexanes/Et2O).
1H NMR (CDCl3, 400 MHz): 7.47-7.28 ppm (m, 10 H), 5.94 (m,
1 H), 5.31 (m, 1 H), 5.23 (m, 1 H), 4.99 (d, J ) 1.5 Hz, 1 H), 4.79
(dd, J ) 12.4, 28.3 Hz, 2 H), 4.68 (s, 2 H), 4.25 (m, 1 H), 4.05-
3.97 (m, 2 H), 3.89 (dd J ) 1.9, 2.7 Hz, 1 H), 3.82-3.63 (m, 3 H),
3.46 (dd, J ) 3.1, 9.2 Hz, 1 H), 3.40 (s, 3 H), 0.14 (s, 9 H). 13C
NMR (CDCl3, 100 MHz): 138.7 ppm, 134.1, 128.2, 127.7, 127.6,
127.5, 127.3, 117.2, 97.5, 82.1, 73.9, 73.4, 73.3, 72.8, 69.9, 68.3,
67.9, 57.2, 0.5. HRMS (FAB) calcd for (C27H38O6SiNa)+ 509.2335,
found 509.2344. Select IR peaks (neat): 2953 cm-1, 2912, 1454,
[R]25 +60.7 (c 0.145, CH2Cl2).
D
Methyl Ether 3. A solution of 2 (39.7 g, 129 mmol) and n-Bu2-
SnO (33.7 g, 135 mmol) in MeOH (650 mL) was heated to reflux.
The reaction mixture was concentrated to dryness. The crude stannyl
ether and MeI (40 mL, 0.64 mol) were dissolved in DMF (650 mL)
(24) The purity was estimated from FAB-MS (see the text).
(25) The second step, i.e., H2/Pd(OH)2, in Scheme 11 effected depro-
tection of the benzyl group as well as reduction of the allyl group. However,
it should be noted that R-13, R-15, and R-17 contain 16, 24, and 32 benzyl
groups, respectively, and the increasing number of benzyl groups may also
contribute to the observed sharp decline in its efficiency.
(26) (a) Cheon, H. S.; Wang, Y.; Ma, J.; Kishi, Y. ChemBioChem., in
press. (b) Papaioannou, N.; Cheon, H.-S.; Kishi, Y., in preparation.
(27) Cheon, H.-S.; Kishi, Y., in preparation.
1248, 1104, 1060, 1028. [R]25 +29.6 (c 0.75, CH2Cl2).
D
Monosaccharide Donor 6d. A solution of 4 (9.72 g, 23.5 mmol),
benzoyl chloride (BzCl, 5.4 mL, 47 mmol), and DMAP (575 mg,
4.71 mmol) in pyr (50 mL) was heated to 65 °C for 1 h. Pyr was
removed in vacuo, and the residue was taken up in CH2Cl2, washed
(28) Complete spectroscopic data of this synthetic intermediate are given
in Supporting Information.
J. Org. Chem, Vol. 72, No. 6, 2007 1937

Hsu et al.
with saturated NH4Cl, and dried over MgSO4. The benzoate was
isolated by silica gel chromatography (3:1 hexanes/Et2O, 1:1
hexanes/Et2O).28
(m, 3 H), 3.31 (s, 3 H), 3.20 (s, 3 H). 13C NMR (CDCl3, 100
MHz): 165.6 ppm, 138.8, 138.6, 138.3, 134.0, 132.9, 130.4, 129.8,
128.3, 128.2, 128.1, 127.7, 127.4, 127.2, 117.4, 100.3, 97.0, 81.9,
79.3, 75.1, 74.4, 73.6, 73.3, 72.6, 72.4, 71.7, 71.6, 70.6, 70.3, 69.8,
68.2, 57.9, 56.7. HRMS (FAB) calcd for (C52H58O12Na)+ 897.3826,
found 897.3862. Select IR peaks (neat): 2924 cm-1, 1726, 1453,
A solution of the benzoate (12.2 g, 23.4 mmol), (Ph3P)3RhCl
(1.09 g, 5 mol %), and DABCO (400 mg, 15 mol %) in toluene-
EtOH-H2O (6:3:1, 150 mL) was heated overnight in a 100 °C oil
bath. Removal of the solvent yielded the enol-ether, which was
stirred with a solution of acetone/1 N HCl (9:1, 100 mL) for 2 h at
80 °C. Most of the solvent was removed in vacuo before adding
saturated NaHCO3 and extracting with CH2Cl2 (3×). The organic
extracts were dried over MgSO4. The lactol (9.64 g, 86% over two
steps) was isolated by silica gel chromatography (3:1 hexanes/Et2O,
3:2 hexanes/Et2O, 1:1 hexanes/Et2O, 1:3 hexanes-/Et2O).28
A solution of the lactol (9.64 g, 20.2 mmol), monomethyl
phthalate (7.26 g, 40.3 mmol), and DMAP (1.23 g, 10.1 mmol) in
CH2Cl2 (100 mL) was cooled to 0 °C. 1-(3-Dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDCI, 5.8 g, 30 mmol) was
added in portions to the reaction mixture. The ice-bath was removed,
and the reaction mixture was stirred overnight. The solvent was
removed in vacuo, and the crude mixture dissolved in EtOAc and
H2O. The organic layer was washed with H2O, saturated NaHCO3,
H2O, and saturated NaCl, then dried over MgSO4, and concentrated
to dryness. 6d (12.9 g, 100%, ca. 2.4:1 R/â mixture of anomers)
was isolated by silica gel chromatography (2:3 Et2O/hexanes, 3:2
Et2O/hexanes, 3:1 Et2O/hexanes, 4:1 Et2O/hexanes). 1H NMR
(CDCl3, 400 MHz): 8.04-7.11 ppm (m, 19 H), 6.48 (d, J ) 1.9 Hz,
1 H, major), 6.01 (s, 1 H, minor), 5.67 (dd, J ) 9.9, 9.9 Hz, 1 H,
major), 5.62 (dd, J ) 9.2, 9.3 Hz, 1 H, minor), 4.86 (s, 2 H, major),
4.83 (s, 2 H, minor), 4.50 (s, 2 H, major), 4.49 (s, 2 H, minor),
4.18-4.09 (m, 2 H), 3.94 (m, 1 H, minor), 3.84 (s, 3 H), 3.77-
3.55 (m, 3 H), 3.34 (s, 3 H, minor), 3.32 (s, 3 H, major). 13C NMR
(CDCl3, 100 MHz): 167.8 ppm, 167.3, 166.6, 166.1, 165.6, 165.5,
137.8, 133.1, 132.3, 131.6, 131.2, 131.1, 131.0, 129.9, 129.8, 129.4,
129.3, 129.0, 128.8, 128.4, 128.3, 128.2, 128.0, 127.8, 127.6, 127.4,
93.9, 93.7, 81.6, 78.7, 77.3, 77.0, 76.7, 75.4, 74.1, 73.6, 73.4, 73.0,
72.7, 72.5, 69.8, 69.0, 58.2, 58.0, 52.6. HRMS (FAB) calcd for
(C37H36O10Na)+ 663.2206, found 663.2191. Select IR peaks
(neat): 2915 cm-1, 1727, 1452, 1435, 1356, 1314, 1267, 1176,
1268, 1111, 1070, 1050, 1028. [R]25 +29.2 (c 0.67, CH2Cl2).
D
Disaccharide Donor 8. Following the deallylation procedure
given for the benzoate derived from 4, R-7d (4.05 g, 4.62 mmol)
was converted to the disaccharide lactol (3.12 g, 81%, solvent used
for silica gel chromatography: 1:1 Et2O/hexanes, 6:4 Et2O/
hexanes).28
Then, following the procedure described for formation of 6d,
disaccharide lactol (3.11 g, 3.72 mmol) was esterified to give 8
(3.64 g, 98%, solvent used for silica gel chromatography: 1:1 Et2O/
hexanes, 3:1 Et2O/hexanes). 1H NMR (CDCl3, 400 MHz): 8.05-
7.97 ppm (m, 2 H), 7.84-7.09 (m, 27 H), 6.47 (d, J ) 2.0 Hz, 1
H, major), 5.90 (br s, 1 H, minor), 5.59 (dd, J ) 9.5, 9.5 Hz, 1 H,
minor), 5.57 (dd, J ) 9.7, 9.8 Hz, 1 H, major), 5.36 (d, J ) 2.0
Hz, 1 H, major), 4.87-4.69 (m, 4 H), 4.58-4.40 (m, 4 H), 4.14-
3.95 (m, 3 H), 3.95-3.86 (m, 2 H), 3.86-3.82 (m, 3 H), 3.82-
3.75 (m, 1 H), 3.75-3.47 (m, 5 H), 3.37-3.32 (m, 4 H, minor),
3.29 (s, 3 H, major), 3.19 (br s, 3 H, major), 3.18 (br s, 3 H, minor).
13C NMR (CDCl3, 100 MHz): 167.3 ppm, 166.2, 165.5, 138.4,
138.1, 137.8, 132.9, 132.3, 131.6, 131.5, 131.1, 131.0, 130.1, 129.7,
129.3, 129.2, 129.0, 128.8, 128.3, 128.2, 128.1, 127.9, 127.8, 127.6,
127.5, 127.4, 127.3, 100.2, 93.9, 93.7, 84.2, 80.8, 79.2, 79.1, 76.5,
74.4, 74.2, 74.1, 73.5, 73.3, 72.6, 72.4, 72.3, 72.1, 71.5, 70.1, 70.0,
69.5, 58.0, 57.9, 56.8, 56.5, 52.7, 52.6. HRMS (FAB) calcd for
(C58H60O15Na)+ 1019.3830, found 1019.3845. Select IR peaks
(neat): 2925 cm-1, 1727, 1453, 1313, 1269, 1169, 1112, 1062,
1028. [R]25 +15.4 (c 0.70, CH2Cl2).
D
Disaccharide Acceptor 9. Disaccharide R-7d (4.47 g, 5.11
mmol) was stirred with methanolic NaOH (0.2 M, 50 mL) for 3.5 h.
The reaction mixture was concentrated to dryness, and the residue
was dissolved in EtOAc and washed with water, followed by
saturated NaCl. The organic layers were dried over MgSO4 and
concentrated to dryness. Purification of the crude product by silica
gel column chromatography (1:1 Et2O/hexanes, 3:2 Et2O/hexanes)
yielded the secondary alcohol (3.63 g, 92%).28
1110, 1068, 1039, 1028. [R]25 +2.9 (c 0.65, CH2Cl2).
D
Disaccharide R-7d. Precautions: for handling of anhydrous
AgClO4, see refs 29a,b.b Solid AgClO4 (310 mg, 1.50 mmol) in a
round-bottomed flask was azeotroped with toluene. Under an
atmosphere of nitrogen, AgClO4 was dissolved in ether (60 mL),
followed by the addition of SnCl4 solution (0.3 M, 5.0 mL of
solution, 1.50 mmol). The catalyst was generated by stirring this
mixture in the dark. After 1 h, it was cooled to 0 °C with an ice
bath. 6d (9.57 g, 14.9 mmol) and 5 (7.24 g, 14.9 mmol) were
premixed in a pear-shaped flask that had been dried overnight with
a P2O5 trap. When the catalyst was ready, the starting materials
were transferred to the reaction mixture with Et2O (240 mL). The
final concentration was 0.05 M relative to the glycosyl donor. The
reaction mixture was then placed in a 0 °C refrigerator, stirred for
36 h, and quenched by addition of saturated NaHCO3. The two
layers were separated. The aqueous layer was extracted with CH2-
Cl2 (3×), and the combined organic extracts were dried over MgSO4
and concentrated to dryness. Crude NMR showed an R/â ratio of
disaccharide to be ca. 17:1. Silica gel chromatography (7:3 hexanes/
Et2O, 1:1 hexanes/Et2O, 7:3 Et2O/hexanes) yielded R-7d (9.99 g,
76%). 1H NMR (CDCl3, 400 MHz): 8.02 ppm (m, 2 H), 7.56 (m,
1 H), 7.47-7.10 (m, 22 H), 5.91 (m, 1 H), 5.57 (dd, J ) 9.8, 9.8
Hz, 1 H), 5.34 (d, J ) 1.9 Hz, 1 H), 5.27 (m, 1 H), 5.19 (m, 1 H),
4.94 (d, J ) 1.7 Hz, 1 H), 4.75 (dd, J ) 12.6, 16.9 Hz, 2 H), 4.69
(s, 2 H), 4.58-4.40 (m, 4 H), 4.21 (m, 1 H), 4.05-3.88 (m, 5 H),
3.83-3.71 (m, 3 H), 3.68 (dd, J ) 2.9, 9.6 Hz, 1 H), 3.60-3.48
Following the procedure given for formation of 5, the secondary
alcohol (3.63 g, 4.70 mmol) was silylated to yield 9 (3.94 g, 99%,
solvent used for silica gel chromatography: 7:3 hexanes/Et2O, 1:1
1
hexanes/Et2O). H NMR (CDCl3, 400 MHz): 7.41-7.20 (m, 20
H), 5.90 (m, 1 H), 5.29-5.22 (m, 2 H), 5.18 (m, 1 H), 4.93 (d, J
) 1.9 Hz, 1 H), 4.74-4.45 (m, 8 H), 4.19 (m, 1 H), 4.00-3.82
(m, 5 H), 3.79 (dd, J ) 2.0, 3.1 Hz, 1 H), 3.76-3.67 (m, 3 H),
3.61-3.53 (m, 2 H), 3.47 (dd, J ) 3.1, 9.1 Hz, 1 H), 3.31 (s, 3 H),
3.30 (dd, J ) 3.0, 9.2 Hz, 1 H), 3.21 (s, 3 H), 0.08 (s, 9 H). 13C
NMR (CDCl3, 100 MHz): 138.9 ppm, 138.8, 138.7, 138.3, 133.9,
128.3, 128.2, 127.7, 127.6, 127.5, 127.2, 117.5, 100.3, 96.9, 81.9,
81.6, 77.2, 75.0, 74.3, 74.0, 73.5, 73.2, 73.1, 72.5, 72.2, 71.6, 70.4,
69.8, 68.1, 68.0, 57.0, 56.8, 0.5. HRMS (FAB) calcd for (C48H62O11-
SiNa)+ 865.3959, found 865.3978. Select IR peaks (neat): 2924
cm-1, 1454, 1248, 1111, 1047, 1028. [R]25 +32.3 (c 0.39, CH2-
D
Cl2).
Tetrasaccharide R-10. Following the procedure described for
formation of R-7d, 8 (4.07 g, 4.08 mmol) and 9 (3.44 g, 4.08 mmol)
were glycosidated at -20 °C for 36 h. The desired tetrasaccharide
R-10 (4.92 g, 76% with g98% purity) was isolated by HPLC (1:1
hexanes-MeOBu-t on an m-porasil column) and then size exclusion
HPLC (CHCl3). 1H NMR (CDCl3, 400 MHz): 8.05-7.93 ppm (m,
2 H), 7.62-7.51 (m, 1 H), 7.47-7.05 (m, 42 H), 5.89 (m, 1 H),
5.61 (dd, J ) 9.9, 9.9 Hz, 1 H), 5.37 (d, J ) 1.7 Hz, 1 H), 5.29 (d,
J ) 1.6 Hz, 1 H), 5.28 (d, J ) 1.8 Hz, 1 H), 5.24 (m, 1 H), 5.18
(m, 1 H), 4.93 (d, J ) 1.7 Hz, 1 H), 4.80-4.36 (m, 16 H), 4.18
(m, 1 H), 4.07-3.60 (m, 16 H), 3.58-3.43 (m, 4 H), 3.40 (dd, J )
3.1, 9.1 Hz, 1 H), 3.32-3.25 (m, 4 H), 3.25-3.13 (m, 11 H). 13C
NMR (CDCl3, 100 MHz): 165.5 ppm, 138.7, 138.6, 138.5, 138.4,
(29) (a) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals, 3rd ed.; Pergamon: New York, NY, 1988. (b) Encyclopedia of
Reagents for Organic Synthesis; Paquette, L. A., Ed.; John Wiley & Son:
New York, NY, 1995.
1938 J. Org. Chem., Vol. 72, No. 6, 2007

Synthetic 3-O-Methylmannose-Containing Polysaccharides
138.1, 138.0, 133.7, 132.8, 130.1, 129.7, 128.3, 128.2, 128.1, 127.6,
127.5, 127.4, 127.3, 127.2, 127.1, 117.5, 100.1, 99.7, 96.7, 81.6,
81.3, 79.1, 77.2, 75.3, 75.0, 74.8, 74.2, 74.0, 73.5, 73.4, 73.3, 73.2,
72.9, 72.7, 72.4, 72.3, 72.2, 71.8, 71.7, 71.4, 70.5, 70.0, 69.6, 68.1,
57.8, 56.6, 56.3. MS (FAB) calcd for (C94H106O22Na)+ 1609, found
1609. Select IR peaks (neat): 2921 cm-1, 1726, 1453, 1268, 1112,
NMR (CDCl3, 100 MHz): 165.6 ppm, 138.8, 138.5, 138.2, 138.1,
133.8, 132.8, 129.8, 128.3, 128.2, 128.1, 127.6, 127.5, 127.4, 127.3,
127.2, 117.5, 100.3, 99.8, 99.6, 96.8, 81.7, 81.4, 79.2, 75.2, 74.8,
74.1, 73.5, 73.4, 73.3, 73.1, 72.8, 72.5, 72.2, 71.8, 71.5, 70.6, 70.5,
70.1, 69.7, 68.1, 57.9, 56.6, 56.3. MS (FAB) calcd for (C178H202O42-
Na)+ 3037, found 3036. Select IR peaks (neat): 2923 cm-1, 1727,
1072, 1046, 1028. [R]25 +34.4 (c 0.34, CH2Cl2).
1453, 1269, 1114, 1074, 1047, 1028. [R]25 +38.8 (c 0.40, CH2-
D
D
Cl2).
Tetrasaccharide Donor 11. Following the deallylation procedure
given for the benzoate derived from 4, R-10 (1.15 g, 721 mmol)
was converted to the tetrasaccharide lactol (905 mg, 81%, solvent
used for silica gel chromatography: 2:1 Et2O/hexanes, 4:1 Et2O/
hexanes).28
Octasaccharide Acceptor 14. Following the procedure given
for hydrolysis of the benzoate of R-7d, R-13 (362 mg, 120 mmol)
was converted to the corresponding secondary alcohol (290 mg,
83%, solvent used for silica gel chromatography: 2:1 hexanes/
EtOAc, 3:2 hexanes/EtOAc, 1:1 hexanes/EtOAc).28 However, to
dissolve the starting material, an equal volume of THF and
methanolic NaOH solution was used for this case.
Then, following the procedure given for the formation of 6d,
the tetrasaccharide lactol (905 mg, 585 mmol) was esterified to
yield 11 (997 mg, 100%, solvent used for silica gel chromatogra-
phy: 7:3 Et2O/hexanes). 1H NMR (CDCl3, 400 MHz): 8.05-7.96
ppm (m, 2 H), 7.81-7.06 (m, 47 H), 6.46 (d, J ) 1.6 Hz, 1 H,
minor), 5.87 (s, 1 H), 5.63 (dd, J ) 9.9, 9.9 Hz, 1 H), 5.39 (s, 1
H), 5.32 (s, 1 H), 5.30 (s, 1 H), 4.87-4.67 (m, 6 H), 4.67-4.36
(m, 10 H), 4.10-3.90 (m, 7 H), 3.90-3.60 (m, 14 H), 3.60-3.23
(m, 8 H), 3.21 (s, 3 H), 3.18 (s, 3 H), 3.16 (s, 3 H). 13C NMR
(CDCl3, 100 MHz): 167.7 ppm, 165.6, 165.5, 138.7, 138.5, 138.4,
138.3, 138.1, 132.8, 132.2, 131.6, 131.5, 131.1, 130.9, 130.2, 129.7,
129.3, 129.1, 129.0, 128.7, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.6, 127.5, 127.3, 127.2, 127.1, 100.2, 99.7, 93.8, 93.6, 84.1,
81.3, 80.7, 79.1, 74.9, 74.8, 74.5, 74.3, 74.1, 73.5, 73.4, 73.2, 73.1,
73.0, 72.9, 72.5, 72.4, 72.2, 72.0, 71.9, 71.4, 70.4, 70.1, 70.0, 69.6,
57.9, 56.8, 56.5, 56.3, 52.6, 52.5. MS (FAB) calcd for (C100H108O25-
Na)+ 1731, found 1731. Select IR peaks (neat): 2925 cm-1, 1727,
Then, following the procedure described for formation of 5, the
octasaccharide secondary alcohol (286 mg, 98.1 mmol) was silylated
to furnish 14 (292 mg, 100%, solvent used for silica gel chroma-
tography: 2:1 hexanes/EtOAc, 3:2 hexanes/EtOAc). 1H NMR
(CDCl3, 500 MHz): 7.48-7.03 ppm (m, 80 H), 5.89 (m, 1 H),
5.37-5.32 (m, 2 H), 5.32-5.29 (m, 5 H), 5.28 (s, 1 H), 5.24 (m,
1 H), 5.18 (m, 1 H), 4.93 (d, J ) 1.5 Hz, 1 H), 4.75-4.63 (m, 9
H), 4.63-5.52 (m, 8 H), 4.52-4.44 (m, 8 H), 4.44-4.33 (m, 7 H),
4.18 (m, 1 H), 4.02-3.86 (m, 9 H), 3.86-3.68 (m, 21 H), 3.68-
3.52 (m, 9 H), 3.50-3.42 (m, 7 H), 3.39 (dd, J ) 2.7, 9.3 Hz, 1
H), 3.33 (dd, J ) 2.8, 9.3 Hz, 1 H), 3.23 (s, 3 H), 3.21 (s, 3 H),
3.20 (s, 3 H), 3.18 (s, 3 H), 3.16 (s, 3 H), 3.13 (s, 3 H), 3.12 (s, 6
H), 0.09 (s, 9 H). 13C NMR (CDCl3, 100 MHz): 139.0 ppm, 138.9,
138.6, 138.2, 133.9, 128.3, 128.2, 128.1, 127.6, 127.5, 127.4, 127.1,
117.3, 100.3, 99.8, 99.7, 97.0, 81.8, 81.7, 81.5, 75.2, 74.8, 74.6,
74.4, 74.2, 73.4, 72.7, 72.1, 71.9, 70.6, 70.5, 69.8, 68.2, 68.0, 56.9,
56.6, 56.4, 56.3, 0.5. MS (FAB) calcd for (C174H206O41SiNa)+ 3005,
found 3004. Select IR peaks (neat): 2925 cm-1, 1454, 1113, 1074,
1453, 1270, 1113, 1047, 1028. [R]25 +21.0 (c 0.40, CH2Cl2).
D
Tetrasaccharide Acceptor 12. Following the procedure given
for hydrolysis of the benzoate of R-7d, R-10 (1.04 g, 657 mmol)
was converted to the corresponding secondary alcohol (1.00 g).28
The crude product was used for the next step without purification.
Then, following the procedure given for formation of 5, the crude
alcohol (974 mg, 657 mmol) was silylated to yield 12 (864 mg,
85% for two steps, solvent used for silica gel chromatography: 6:4
hexanes/Et2O, 1:1 hexanes/Et2O, 7:3 Et2O/hexanes). 1H NMR
(CDCl3, 400 MHz): 7.42-7.15 ppm (m, 40 H), 5.95 (m, 1 H),
5.37 (s, 1 H), 5.34 (s, 1 H), 5.33 (s, 1 H), 5.30 (m, 1 H), 5.24 (m,
1 H), 4.99 (s, 1 H), 4.82-4.69 (m, 6 H), 4.69-4.42 (m, 10 H),
4.25 (m, 1 H), 4.09-3.90 (m, 7 H), 3.90-3.58 (m, 15 H), 3.55-
3.48 (m, 1 H), 3.48-3.38 (m, 2 H), 3.33 (s, 3 H), 3.27 (s, 3 H),
3.26 (s, 3 H), 3.23 (s, 3 H), 0.09 (s, 9 H). 13C NMR (CDCl3, 100
MHz): 138.6 ppm, 138.1, 128.3, 128.2, 127.7, 127.6, 127.5, 127.4,
127.3, 127.2, 127.1, 117.6, 100.2, 99.8, 99.7, 96.8, 81.7, 81.5, 81.3,
74.9, 74.6, 74.1, 73.8, 73.4, 73.3, 73.0, 72.7, 72.5, 72.4, 72.0, 71.8,
71.7, 70.4, 69.6, 68.1, 67.9, 57.0, 56.7, 56.5, 0.5. MS (FAB) calcd
for (C90H110O21SiNa)+ 1577, found 1577. Select IR peaks (neat):
1046, 1028. [R]25 +33.8 (c 0.13, CH2Cl2).
D
Dodecasaccharide R-15. Following the procedure given for the
synthesis of R-13, 11 (54.8 mg, 32.0 mmol) and 14 (95.4 mg, 32.0
mmol) were glycosidated at 0 °C for 8.5 h. The dodecasaccharide
R-15 (107 mg, 75% with 84% estimated purity23) was isolated by
JAI recycling HPLC. 1H NMR (CDCl3, 500 MHz): 8.03-7.97 ppm
(m, 2 H), 7.58-7.50 (m, 1 H), 7.47-7.03 (m, 122 H), 5.89 (m, 1
H), 5.62 (dd, J ) 9.9, 9.9 Hz, 1 H), 5.37 (d, J ) 1.6 Hz, 1 H),
5.36-5.33 (m, 8 H), 5.31 (s, 1 H), 5.30 (s, 1 H), 5.24 (m, 1 H),
5.18 (m, 1 H), 4.93 (d, J ) 1.6 Hz, 1 H), 4.78-4.65 (m, 14 H),
4.63-4.55 (m, 10 H), 4.55-4.44 (m, 12 H), 4.44-4.32 (m, 12 H),
4.18 (m, 1 H), 4.07-3.88 (m, 15 H), 3.88-3.71 (m, 31 H), 3.71-
3.57 (m, 12 H), 3.57-3.37 (m, 14 H), 3.23 (s, 3 H), 3.20 (s, 6 H),
3.18 (s, 3 H), 3.16 (s, 3 H), 3.13-3.04 (m, 21 H). MS (FAB) calcd
for (C262H298O62Na)+ 4462, found 4464.
Octasaccharide Donor 16. Following the deallylation procedure
given for the benzoate derived from 4, R-13 (221 mg, 73.2 mmol)
was converted to the octasaccharide lactol (167 mg, 77%, solvent
used for silica gel chromatography: 3:1 Et2O/hexanes, Et2O).28
Then, following the procedure given for preparation of 6d,
octasaccharide lactol (166 mg, 55.8 mmol) was esterified to yield
16 (165 mg, 94%, solvent used for silica gel chromatography: 98:2
CH2Cl2/MeOH). 1H NMR (CDCl3, 400 MHz): 8.05-7.95 ppm (m,
2 H), 7.82-7.50 (m, 5 H), 7.49-7.04 (m, 82 H), 6.46 (s, 1 H,
major), 5.87 (s, 1 H, minor), 5.63 (dd, J ) 9.9, 9.9 Hz, 1 H), 5.38
(s, 1 H), 5.35 (s, 4 H), 5.32 (s, 1 H), 5.31 (s, 1 H), 4.87-4.67 (m,
10 H), 4.67-4.55 (m, 7 H), 4.55-4.33 (m, 15 H), 4.12-3.90 (m,
11 H), 3.90-3.58 (m, 30 H), 3.58-3.32 (m, 8 H), 3.32-3.23 (m,
4 H, major), 3.21 (s, 3 H, minor), 3.20 (s, 3 H, minor), 3.19 (s, 3
H, major), 3.18 (s, 3 H, major), 3.17 (s, 3 H, minor), 3.16 (s, 3 H,
minor), 3.15 (s, 6 H, major), 3.12 (s, 6 H, major), 3.10 (s, 3 H,
major). 13C NMR (CDCl3, 100 MHz): 167.6 ppm, 167.2, 166.0,
165.5, 138.7, 138.4, 138.2, 138.1, 137.6, 132.8, 131.8, 131.6, 131.4,
131.1, 131.0, 130.1, 129.7, 129.2, 129.1, 129.0, 128.7, 128.3, 128.2,
128.0, 127.9, 127.8, 127.5, 127.4, 127.3, 127.1, 100.2, 99.8, 99.6,
93.8, 93.5, 84.1, 81.3, 80.8, 79.1, 74.9, 74.8, 74.7, 74.6, 74.5, 74.1,
2924 cm-1, 1454, 1112, 1074, 1046, 1028. [R]25 +35.8 (c 1.20,
D
CH2Cl2).
Octasaccharide R-13. Following the procedure given for
synthesis of R-7d, 11 (844 mg, 493 mmol) and 12 (766 mg, 493
mmol) were glycosidated at 0 °C for 9.5 h. The crude reaction
mixture was redissolved in CH2Cl2 (5.0 mL), followed by the
addition of Et3N (160 mL, 1.13 mmol) and TMSOTf (110 mL,
569 mmol). After aqueous workup (CH2Cl2 extractions (3×) from
NaHCO3 solution), silica gel chromatography (1:1 Et2O/hexanes,
3:2 Et2O/hexanes, 7:3 Et2O/hexanes) allowed recovery of unreacted
12 and isolation of desired octasaccharide R-13 (1.17 g, 79% with
90% estimated purity24). Further purification was done by JAI
1
recycling HPLC. H NMR (CDCl3, 400 MHz): 8.03-7.96 ppm
(m, 2 H), 7.59-7.52 (m, 1 H), 7.45-7.07 (m, 82m), 5.89 (m, 1
H), 5.62 (dd, J ) 9.9, 9.9 Hz, 1 H), 5.38 (s, 1 H), 5.37-5.32 (m,
4 H), 5.31 (s, 1 H), 5.29 (s, 1 H), 5.24 (m, 1 H), 5.18 (m, 1 H),
4.93 (d, J ) 1.5 Hz, 1 H), 4.81-4.65 (m, 10 H), 4.65-4.56 (m, 6
H), 4.56-4.33 (m, 16 H), 4.18 (m, 1 H), 4.08-3.88 (m, 11 H),
3.88-3.55 (m, 28 H), 3.55-3.36 (m, 9 H), 3.23 (s, 3 H), 3.20 (s,
6 H), 3.18 (s, 3 H), 3.16 (s, 3 H), 3.12 (s, 6 H), 3.10 (s, 3 H). 13C
J. Org. Chem, Vol. 72, No. 6, 2007 1939

Hsu et al.
74.0, 73.4, 73.3, 73.2, 73.1, 72.8, 72.5, 72.4, 72.1, 71.8, 71.4, 70.4,
70.2, 70.0, 69.6, 57.8, 56.7, 56.4, 56.3, 53.7, 52.5. MS (FAB) calcd
for (C184H204O45Na)+ 3159, found 3157. Select IR peaks (neat):
hydrolysis of the benzoate of R-7d, to yield the corresponding
secondary alcohol (48.5 mg, 78%, solvent for silica gel chroma-
tography: 2:1 hexanes/EtOAc, 55:45 hexanes/EtOAc).28
2924 cm-1, 1727, 1453, 1270, 1113, 1046, 1028. [R]25 +35.6 (c
Then, following the procedure given for sMMP 8-mer (18), the
secondary alcohol (48.5 mg, 11.2 mmol) was hydrogenated to
furnish sMMP 12-mer (19) (20.0 mg, 83%, solvent used for reverse-
phase silica gel chromatography: 4:1 H2O/MeOH, 3:2 H2O/MeOH).
Note that use of Celite should be avoided because it absorbed the
product too tightly. 1H NMR (D2O, 500 MHz): 5.23-5.20 (m, 11
H), 4.89 (d, J ) 1.5 Hz, 1 H), 4.22-4.20 (m, 1 H), 4.20-4.17 (m,
10 H), 4.16-4.14 (m, 1 H), 3.89-3.61 (m, 60 H), 3.54-3.44 (m,
2 H), 3.45 (br s, 10 × 3 H), 3.44 (br s, 2 × 3 H), 1.65-1.57 (m,
2 H), 0.91 (t, J ) 7.6 Hz, 3 H). 13C NMR (D2O, 100 MHz, 310
K): 101.6 ppm, 101.4, 99.6, 80.8, 79.9, 73.8, 73.4, 72.9, 72.4, 71.4,
69.8, 66.3, 65.6, 61.0, 56.3, 22.0, 9.9. MS (FAB) calcd for
D
0.23, CH2Cl2).
Hexadecasaccharide R-17. Following the glycosidation proce-
dure given for R-13, 16 (251 mg, 80.0 mmol) and 14 (238 mg,
80.0 mmol) were coupled at 0 °C for 9.5 h. The hexadecasaccharide
R-17 (345 mg, 74% with 76% estimated purity24) was isolated by
JAI recycling chromatography. 1H NMR (CDCl3, 500 MHz): 8.03-
7.95 ppm (m, 2 H), 7.57-7.49 (m, 1 H), 7.48-7.03 (m, 162 H),
5.88 (m, 1 H), 5.61 (dd, J ) 9.8, 9.9 Hz, 1 H), 5.37 (s, 1 H), 5.36-
5.32 (m, 12 H), 5.31 (s, 1 H), 5.29 (s, 1 H), 5.24 (m, 1 H), 5.18
(m, 1 H), 4.93 (s, 1 H), 4.79-4.64 (m, 17 H), 4.64-4.53 (m, 15
H), 4.53-4.32 (m, 32 H), 4.18 (m, 1 H), 4.07-3.88 (m, 18 H),
3.88-3.64 (m, 46 H), 3.64-3.55 (m, 14 H), 3.55-3.36 (m, 18 H),
3.23 (s, 3 H), 3.20 (s, 6 H), 3.18 (s, 3 H), 3.16 (s, 3 H), 3.14-3.05
(m, 33 H). 13C NMR (CDCl3, 125 MHz): 165.6 ppm, 138.9, 138.5,
129.8, 128.4, 128.2, 128.1, 127.7, 127.6, 127.5, 127.3, 127.1, 117.5,
100.3, 99.7, 81.8, 81.5, 79.2, 75.2, 74.7, 73.5, 73.4, 73.3, 72.6, 72.3,
71.9, 71.5, 70.6, 70.5, 68.2, 57.9, 56.3. MS (FAB) calcd for
(C346H394O82Na)+ 5888, found 5887. Select IR peaks (neat): 3366
cm-1, 2923, 1723, 1453, 1273, 1115, 1047, 1028. [R]25D +45.9 (c
0.14, CH2Cl2).
(C87H151O61)- 2173, found 2173. [a]25 +92.6 (c 0.47, H2O).
D
sMMP 16-mer (20). R-17 (122 mg, 20.9 mmol) was converted
to the corresponding secondary alcohol (105 mg, 87%), following
the procedure described for hydrolysis of the benzoate of R-7d,
except that an equal volume of THF and methanolic NaOH was
added to enhance the solubility of the starting material in the
reaction mixture. Solvent used for silica gel chromatography: 3:2
hexanes/EtOAc, 1:1 hexanes/EtOAc.28
Then, following the procedure, except for use of Celite, given
for the case of sMMP 8-mer (18), the secondary alcohol (28.0 mg,
4.86 mmol) was subjected to hydrogenolysis/hydrogenation to
provide sMMP 16-mer (20) (7.7 mg, 55%, solvent used for reverse-
phase silica gel chromatography: 4:1 H2O/MeOH, 3:2 H2O/MeOH).
1H NMR (D2O, 500 MHz): 5.23-5.20 (m, 15 H), 4.89 (br s, 1 H),
4.22-4.20 (m, 1 H), 4.20-4.17 (m, 14 H), 4.16-4.14 (m, 1 H),
3.90-3.61 (m, 80 H), 3.54-3.44 (m, 2 H), 3.45 (br s, 14 × 3 H),
3.44 (br s, 2 × 3 H), 1.65-1.57 (m, 2 H), 0.92 (t, J ) 7.6 Hz, 3
H). 13C NMR (D2O, 100 MHz, 310 K): 101.2 ppm, 99.3, 80.8,
80.6, 79.6, 73.6, 72.1, 71.0, 69.6, 66.0, 65.7, 65.3, 60.8, 56.0, 29.5,
21.8, 9.7. MS (FAB) calcd for (C115H199O81)- 2878, found 2878.
sMMP 8-mer (18). A solution of the octasaccharide secondary
alcohol (35.7 mg, 11.8 mmol, obtained in 83% by hydrolysis of
R-13 under the conditions given for R-15)28 in EtOH (1.0 mL) was
stirred overnight with a portion of 10% Pd(OH) on carbon (ca.
2
3 mg) under an H2 balloon. The reaction mixture was passed
through Celite to remove the catalyst, followed by removal of
solvents in vacuo. The deprotected compound was purified by
reverse-phase silica gel chromatography (4:1 H2O-MeOH) to give
18 (17.3 mg, 100%). 1H NMR (D2O, 500 MHz): 5.23-5.20 (m, 7
H), 4.89 (d, J ) 1.5 Hz, 1 H), 4.22-4.20 (m, 1 H), 4.20-4.17 (m,
6 H), 4.17-4.14 (m, 1 H), 3.89-3.61 (m, 40 H), 3.54-3.44 (m, 2
H), 3.45 (br s, 6 × 3 H), 3.43 (br s, 2 × 3 H), 1.64-1.57 (m, 2 H),
0.91 (t, J ) 7.6 Hz, 3 H). 13C NMR (D2O, 100 MHz, 310 K):
101.4 ppm, 101.2, 99.6, 81.1, 80.8, 79.9, 73.9, 72.9, 72.5, 72.4,
71.3, 69.8, 66.4, 66.3, 66.1, 65.6, 61.1, 56.4, 56.3, 22.1, 10.0. MS
(FAB) calcd for (C59H103O41)- 1467, found 1467. MS (FAB) calcd
for (C59H104O41Na)+ 1492, found 1491. [R]25D +88.2 (c 0.24, H2O).
sMMP 12-mer (19). Methanolic NaOH solution (0.5 M, 1.0 mL)
was added to R-15 (63.7 mg, 14.3 mmol) in a round-bottomed flask.
A small amount of CH2Cl2 was added dropwise to dissolve the
starting material. The reaction was worked-up as described for
[R]25 +95.7 (c 0.42, H2O).
D
Acknowledgment. Financial support from the National
Institutes of Health (NS 12108) is gratefully acknowledged.
Supporting Information Available: Includes the General
Method Section and spectroscopic data. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO061991N
1940 J. Org. Chem., Vol. 72, No. 6, 2007