Mannosazide methyl uronate donors. Glycosylating properties and use in the construction of β-ManNAcA-containing oligosaccharides

Article abstract of DOI:10.1021/jo101779v

Mannosazide methyl uronate donors equipped with a variety of anomeric leaving groups (β- and α-S-phenyl, β- and α-N- phenyltrifluoroacetimidates, hydroxyl, β-sulfoxide, and (R_s)- and (S_s)-α-sulfoxides) were subjected to activating co

Full text of DOI:10.1021/jo101779v

pubs.acs.org/joc
Mannosazide Methyl Uronate Donors. Glycosylating Properties and Use
in the Construction of β-ManNAcA-Containing Oligosaccharides
ꢀ
Marthe T. C. Walvoort, Gerrit Lodder, Herman S. Overkleeft, Jeroen D. C. Codee,* and
Gijsbert A. van der Marel*
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
jcodee@chem.leidenuniv.nl; marel_g@chem.leidenuniv.nl
Received September 16, 2010
Mannosazide methyl uronate donors equipped with a variety of anomeric leaving groups (β- and
R-S-phenyl, β- and R-N-phenyltrifluoroacetimidates, hydroxyl, β-sulfoxide, and (Rs)- and (Ss)-R-
sulfoxides) were subjected to activating conditions, and the results were monitored by H NMR.
1
While the S-phenyl and imidate donors all gave a conformational mixture of anomeric R-triflates, the
hemiacetal and β- and R-sulfoxides produced an oxosulfonium triflate and β- and R-sulfonium bis-
triflates, respectively. The β-S-phenyl mannosazide methyl uronate performed best in both activation
experiments and glycosylation studies and provided the 1,2-cis mannosidic linkage with excellent
selectivity. Consequently, an R-Glc-(1f4)-β-ManN3A-SPh disaccharide, constructed by the stereo-
selective glycosylation of a 6-O-Fmoc-protected glucoside and β-S-phenyl mannosazide methyl
uronate, was used as the repetitive donor building block in the synthesis of tri-, penta-, and
heptasaccharide fragments corresponding to the Micrococcus luteus teichuronic acid.
Introduction
bacterial glycans, ManNAcA is primarily β-1,3 or β-1,4 linked
to a wide variety of other hexapyranosides. The capsular
polysaccharide, teichuronic acid from Micrococcus luteus,
for example, is composed of alternating ManNAcA and
glucose residues, both linked in a 1,2-cis fashion (Figure 1).4
N-Acetyl-D-mannosaminuronic acid (ManNAcA) is a
common constituent of numerous bacterial acidic polysac-
charides. It is found in Gram-positive and Gram-negative
cell wall glycopolymers,1 bacterial (surface) antigens,2 and
the enterobacterial common antigen (ECA).3 Within these
(1) Haemophilus influenzae: Tsui, F.-P.; Schneerson, R.; Boykins,
R. A.; Karpas, A. B.; Egan, W. Carbohydr. Res. 1981, 97, 293–306.
Bacillus subtilis: Yoneyama, T.; Araki, Y.; Ito, E. Eur. J. Biochem. 1984,
141, 83–89.
(2) Escherichia coli: Tsui, F.-P.; Boykins, R. A.; Egan, W. Carbohydr.
Res. 1982, 102, 263–271. Staphylococcus aureus: Jones, C. Carbohydr. Res.
2005, 340, 1097–1106. Neisseria meningitidis: van der Kaaden, A.; Gerwig,
G. J.; Kamerling, J. P.; Vliegenthart, J. F. G.; Tiesjema, R. H. Eur. J.
Biochem. 1985, 152, 663–668. Michon, F.; Brisson, J. R.; Roy, R.; Ashton,
F. E.; Jennings, H. J. Biochemistry 1985, 24, 5592–5598.
FIGURE 1. Micrococcus luteus teichuronic acid displaying the
repetitive motif [f6)-R-D-Glcp-(1f4)-β-D-ManpNAcA-(1f].
(3) (a) Lugowski, C.; Romanowska, E.; Kenne, L.; Lindberg, B. Carbo-
hydr. Res. 1983, 118, 173–181. (b) Mannel, D.; Mayer, H. Eur. J. Biochem.
1978, 86, 361–370.
(4) (a) Perkins, H. R. Biochem. J. 1963, 86, 475–483. (b) Hase, S.;
Matsushima, Y. J. Biochem. (Tokyo) 1972, 72, 1117–1128. (c) Nasir-ud-Din;
Jeanloz, R. W. Carbohydr. Res. 1976, 47, 245–260.
7990 J. Org. Chem. 2010, 75, 7990–8002
Published on Web 11/04/2010
DOI: 10.1021/jo101779v
r
2010 American Chemical Society

Walvoort et al.
JOCFeatured Article
FIGURE 2. Mannosazide methyl uronate donors 1-7. The reported 4C1/1C4 ratios were determined by 1H NMR spectroscopy at -80 °C.
M. luteus has been implicated to play a role in recurrent
bacteremia,5 septic shock,6 and meningitis.7 Interestingly,
whereas the peptidoglycan part of the M. luteus cell wall
lacks immunomodulatory activity, its teichuronic acid com-
ponent induces the production of inflammatory cytokines.8
Additionally, it was shown that reduction of the carboxylic
acids to the primary alcohols led to elimination of the im-
munostimulating activity.8
To date, only a few research papers detail the synthesis of
ManNAcA-containing oligosaccharide fragments,9 and no
general protocol exists. We previously described the synthe-
sis of the β-mannosaminuronic acid-containing acidic tri-
saccharide, β-D-GlcpNAc-(1f4)-β-D-ManpNAcA-(1f3)-
R-L-GalNAcA(4-OAc), of the bacteriolytic complex of lyso-
amidase.10 The β-mannosamine linkage in this trimer was
constructed using a 4,6-O-benzylidene mannosazide thio-
glycoside11 following the pioneering work of Crich and co-
workers on β-mannoside synthesis.12 However, compared to the
2,3-O-benzyl-protected 4,6-O-benzylidene mannopyranoside,
the 2-azido-3-O-benzyl mannopyranoside showed reduced
β-stereoselectivity. As an alternative strategy, we disclosed that
appropriately derivatized donor mannuronates (ManA) can be
condensed with a variety of acceptor glycosides to produce 1,2-
cis ManA linkages with good efficiency and high stereo-
selectivity.13 In a recent preliminary report,14 we demonstrated
that 2-azido-2-deoxymannuronate (ManN3A) donors also have
the propensity to form β-glycosidic bonds. In this study, we
made the intriguing observation that activation of such man-
nosazide methyl uronates using the Ph2SO-Tf2O system12a,15,16
gives rise to the formation of a triflate intermediate which
preferentially adopts a 1C4 conformation, placing the anomeric
triflate in an equatorial position. We present here our in-depth
studies on the activation of a variety of ManN3A donors, the
nature of the activated species formed with emphasis on both
structural and conformational aspects, and their glycosylating
properties. We also present our results in the application of the
outcome of these studies in the first synthesis of a series of tri-,
penta-, and heptasaccharide fragments corresponding to the
Micrococcus luteus teichuronic acid.
Results and Discussion
In the first instance, we synthesized several ManN3A
donors and examined their behavior upon activation, their
reactivity, and their stereoselectivity in glycosylation re-
actions.17 The eight ManN3A donors used in this study are
β- and R-S-phenyl mannosides 1 and 2,18 β- and R-N-
phenyltrifluoroacetimidates 3 and 4,19 1-hydroxyl mannuron-
ate 5,20 β-sulfoxide 6, and the R-sulfoxides 7a/b (Figure 2).21
The sulfoxide moieties in 6 and 7a/b were obtained in diaste-
reomerically pure but undefined form22,23 (see the Supporting
Information for a full description of the synthesis of donors
(5) Von Eiff, C.; Kuhn, N.; Herrmann, M.; Weber, S.; Peters, G. Pediatr.
Infect. Dis. J. 1996, 15, 711–713.
(6) Albertson, D.; Natsios, G. A.; Gleckman, R. Arch. Intern. Med. 1978,
138, 487–488.
(7) Fosse, T.; Peloux, Y.; Granthil, C.; Toga, B.; Bertrando, J.; Sethian,
M. Infection 1985, 13, 280–281.
(8) Yang, S.; Sugawara, S.; Monodane, T.; Nishijima, M.; Adachi, Y.;
Akashi, S.; Miyake, K.; Hase, S.; Takada, H. Infect. Immun. 2001, 2025–
2030.
1
1-7). Interestingly, the H NMR spectra of the R-sulfoxides
(9) For the synthesis of an ECA fragment, see: Paulsen, H.; Lorentzen,
J. P. Carbohydr. Res. 1984, 133, C1–C4. For the synthesis of a Micrococcus
luteus fragment, see: Osa, Y.; Kaji, E.; Takahashi, K.; Hirooka, M.; Zen, S.;
Lichtenthaler, F. W. Chem. Lett. 1993, 1567–1570. For the synthesis of a
Haemophilus influenzae fragment, see: Classon, B.; Garegg, P. J.; Oscarson,
(14) Walvoort, M. T. C.; Lodder, G.; Mazurek, J.; Overkleeft, H. S.;
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Codee, J. D. C.; van der Marel, G. A. J. Am. Chem. Soc. 2009, 131, 12080–
12081.
(15) Crich, D.; Sun, S. J. Org. Chem. 1996, 61, 4506–4507.
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(16) Codee, J. D. C.; van den Bos, L. J.; Litjens, R. E. J. N.; Overkleeft,
H. S.; van Boeckel, C. A. A.; van Boom, J. H.; van der Marel, G. A.
Tetrahedron 2004, 60, 1057–1064.
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S.; Tiden, A.-K. Carbohydr. Res. 1991, 216, 187–196.
(10) Litjens, R. E. J. N.; den Heeten, R.; Timmer, M. S. M.; Overkleeft,
H. S.; van der Marel, G. A. Chem.;Eur. J. 2005, 11, 1010–1016.
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(17) (a) Zhu, X.; Schmidt, R. R. Angew. Chem., Int. Ed. 2009, 48, 2–37.
(b) Mydock, L. K.; Demchenko, A. V. Org. Biomol. Chem. 2010, 8, 497–510.
(c) Boltje, T. J.; Buskas, T.; Boons, G.-J. Nature Chem. 2009, 1, 611–622.
(11) (a) Litjens, R. E. J. N.; van den Bos, L. J.; Codee, J. D. C.; van den
Berg, R. J. B. H. N.; Overkleeft, H. S.; van der Marel, G. A. Eur. J. Org.
Chem. 2005, 918–924. (b) Litjens, R. E. J. N.; Leeuwenburgh, M. A.; van der
Marel, G. A.; van Boom, J. H. Tetrahedron Lett. 2001, 42, 8693–8696.
(12) (a) Crich, D.; Sun, S. Tetrahedron 1998, 54, 8321–8348. (b) Crich, D.;
Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020. (c) Crich, D.; Lim,
L. B. L. Org. React. 2004, 64, 115–251.
ꢀ
(18) Codee, J. D. C.; Litjens, R. E. J. N.; van den Bos, L. J.; Overkleeft,
H. S.; van der Marel, G. A. Chem. Soc. Rev. 2005, 34, 769–782.
(19) (a) Yu, B.; Tao, H. Tetrahedron Lett. 2001, 42, 2405–2407. (b) Yu, B.;
Sun, J. Chem. Commun. 2010, 46, 4668–4679.
(20) Gin, D. J. Carbohydr. Chem. 2002, 21, 645–665.
ꢀ
(13) Codee, J. D. C.; van den Bos, L. J.; de Jong, A. R.; Dinkelaar, J.;
Lodder, G.; Overkleeft, H. S.; van der Marel, G. A. J. Org. Chem. 2009, 74,
38–47.
(21) (a) Kahne, D.; Walker, S.; Cheng, Y.; van Engen, D. J. Am. Chem.
Soc. 1989, 111, 6881–6882. (b) Aversa, M. C.; Barattucci, A.; Bonaccorsi, P.
Tetrahedron 2008, 64, 7659–7683.
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FIGURE 3. Part of the 1H NMR spectra obtained after activation of donors 1-4 (A) at -80 °C, hemiacetal donor 5, (B) at -30 °C, β-sulfoxide
donor 6, (C) at -80 °C and R-sulfoxide donors 7a, (D) at -50 °C and 7b, and (E) at -80 °C.
1
7a/b show that both donors exist exclusively in the C4 con-
at -80 °C into the same mixture of anomeric R-triflates
I/I*.24 The 1H NMR spectrum of this conformational triflate
mixture is depicted in Figure 3A and shows that the equator-
ial anomeric 1C4 triflate I* prevails over its 4C1 counterpart I
(I*:I = 3:1). Structure I* arranges three substituents in
sterically unfavorable axial positions and does not benefit
from a stabilizing anomeric effect. This conformation is in line
with the structural preference of the related mannuronate
ester oxacarbenium ion, which preferentially adopts a 3H4 half
chair or closely related conformation.14,17b,25,26 Because the
anomeric carbon is quite electron depleted, the R-triflate I*
takes up a structure closely mimicking the structure of the 3H4-
like oxacarbenium ion, which is best stabilized by an equatorial
substituent at C-2 and by axial substituents at C-3, C-4, and
C-5. Treatment of the conformational mixture of anomeric
R-triflates I/I* with a 25-fold excess of MeOH-d4 at -80 °C
rapidly provided methyl mannoside 8 with high β-selectivity
(see Table 1, entries 1 and 3). The selective formation of the
β-linked products from the mannosaziduronic acid donors can
be explained by the SN2-like substitution on the R-triflate.
Alternatively, the selective attack of the 3H4-like oxacarbenium
1
formation. The H NMR spectra of the other R-configured
donors 2, 4, and 5 recorded at room temperature show coupling
constants for the signals belonging to the ring protons having a
1
4
value in between the typical values obtained for C4 and C1
chair protons. At -80 °C, interconversion of the 1C4 and 4C1
chairs was slowed down sufficiently to allow detection of
resonance sets for both conformers, and from these 1H NMR
spectra the conformer ratios were extracted, as reported in
Figure 2 (for NMR spectra, see the Supporting Information).
1
R-S-Phenyl mannoside 2 predominantly exists as a C4 chair,
whereas the two chair conformers are more evenly distributed
for the imidate 4 (4C1/1C4=1.3:1) and 1-hydroxyl mannuronate
5 (4C1/1C4=1:1.7).
Activation of the Donors. To investigate the behavior of the
ManN3A donors upon activation and subsequent glycosyla-
tion with MeOH-d4, a series of low-temperature NMR
experiments was conducted. We recently reported14 on the
activation of β-thiodonor 1 using Ph2SO-Tf2O as the acti-
vator system and β-imidate donor 3 using stoichiometric
TfOH and revealed that both donors were rapidly converted
(22) Oxidation of β-thio donor 1 yielded a single diastereomeric sulfoxide
6. On the other hand, oxidation of R-thio donor 2 resulted in a mixture of
diastereomeric R-sulfoxides 7a and 7b. Although readily separable, the
absolute configuration of the diastereomers, which were obtained as oils
after purification, could not be assigned. Empirical assignment of the
configuration based on axial R-sulfoxides as described by Crich et al. was
deemed unfeasible since the anomeric moiety is placed equatorially in donor
2. Crich, D.; Mataka, J.; Zakharov, L. N.; Rheingold, A. L.; Wink, D. J.
J. Am. Chem. Soc. 2002, 124, 6028–6036.
(25) (a) Lucero, C. G.; Woerpel, K. A. J. Org. Chem. 2006, 71, 2641–2647.
(b) Romero, J. A. C.; Tabacco, S. A.; Woerpel, K. A. J. Am. Chem. Soc. 2000,
122, 168–169. (c) Dinkelaar, J.; de Jong, A. R.; van Meer, R.; Somers, M.;
ꢀ
Lodder, G.; Overkleeft, H. S.; Codee, J. D. C.; van der Marel, G. A. J. Org.
ꢀ
Chem. 2009, 74, 4982–4991. (d) Codee, J. D. C.; de Jong, A. R.; Dinkelaar, J.;
Overkleeft, H. S.; van der Marel, G. A. Tetrahedron 2009, 65, 3780–3788.
(26) For recent reviews on oxacarbenium ions, see: (a) Walvoort,
M. T. C.; Dinkelaar, J.; van den Bos, L. J.; Lodder, G.; Overkleeft, H. S.;
ꢀ
Codee, J. D. C.; van der Marel, G. A. Carbohydr. Res. 2010, 345, 1252–1263.
(b) Smith, D. M.; Woerpel, K. A. Org. Biomol. Chem. 2006, 4, 1195–1201.
(23) The diastereoselective oxidation of β-thio glycosides has been re-
ꢀ
ported before: Khiar, N.; Fernandez, I.; Araujo, C. S.; Rodrı
Suarez, B.; Alvarez, E. J. Org. Chem. 2003, 68, 1433–1442.
(24) Anomeric triflates have been observed before; see, for example:
(a) Crich, D.; Sun, S. J. Am. Chem. Soc. 1997, 46, 11217–11223. (b) Crich,
D. J. Carbohydr. Chem. 2002, 21, 667–690.
ꢀ
´
guez, J.-A.;
(c) Satoh, H.; Hansen, H. S.; Manabe, S.; van Gunsteren, W. F.;
€
Hunenberger, P. H. J. Chem. Theory Comput. 2010, 6, 1783–1797. (d) Whitfield,
D. M. Adv. Carbohydr. Chem. Biochem. 2009, 62, 83–159. (e) Horenstein, N. A.
ꢀ
Adv. Phys. Org. Chem. 2006, 41, 275–314. (f) Bohe, L.; Crich, D. C. R. Chim.
2010, DOI: 10.1016/j.crci.2010.03.016.
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TABLE 1. Results of the Activation of Donors 1-7 and Coupling to MeOH-d4
entry
compd
leaving group
β-SPh
R-SPh
β-C(NPh)CF3
R-C(NPh)CF3
R-OH
β-S(O)Ph
R-S(O)Ph (R or S)
R-S(O)Ph (R or S)
T (°C)
-80
intermediates
I/I*
I/I*
I/I*
I/I*
R/β ratio of 8a
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7a
7b
1/5b
-80 f -40
-80
1/6b
1/>10b
1/>10b
0/1c
-80
-80 f þ10
II
-80
I/I*, III
IV-a (R or S)
I/I*, IV-b (R or S)
1/5d
nd
1/5e
-80 f -20
-80 f -60
aAs determined by NMR. bFull conversion of the activated species. cMixture of 5/8 ∼ 3/1. dMixture of 6/8 ∼ 2/3 . eMixture of 7b/8/2 ∼ 4/5/1.
ion from the β-face in an SN1-like process, can also account for
the observed selectivity. Both pathways can contribute to the
β-selectivity observed, and the amount of SN1 and SN2 char-
acter depends on the reactivity of the nucleophile and steric
interactions of the donor and acceptor at hand.14
Next, hemiacetal donor 5 was subjected to activating
conditions (1.3 equiv of Tf2O, 1.3 equiv of Ph2SO, 0.05 M
in DCM-d2). The donor was completely consumed at -40 °C
resulting in a single set of signals as displayed in Figure 3B.
The anomeric proton (δ=6.16 ppm) appeared as a doublet
with a coupling constant of 8.3 Hz, in analogy to the large
coupling constant (3JH1-H2=8.8 Hz) observed for the anom-
eric proton in equatorial triflate I*. The activated species
generated from donor 5 proved to be stable up to þ10 °C.
Given the anomeric chemical shift values reported by Garcia
and Gin29 for oxosulfonium triflates and the similarity be-
tween the 1H spectrum from activation of 5 and the resonance
set belonging to the equatorial triflate I*, the intermediate
formed upon activation of hemiacetal 5 was assigned oxo-
In similar activation experiments, donors 2, 4, 5, 6, and
7a/b were assessed, and the results of these experiments are
summarized in Figure 3 and Table 1. First, a mixture of
R-thiodonor 2 and Ph2SO (1.3 equiv) in DCM-d2 (0.05 M)
at -80 °C was treated with Tf2O (1.3 equiv), and a 1H NMR
spectrum was recorded. Upon activation, several new signals
appeared, indicating the formation of the conformational
mixture of R-triflates I/I*. However, unlike the rapid con-
sumption of β-thiodonor 1, R-thiodonor 2 remained present,
and a prolonged reaction time (∼1 h) at -80 °C did not lead
to more conversion.27 Raising the temperature to -40 °C
eventually gave complete conversion of donor 2 into the mix-
ture of R-triflates also observed after activation of β-donors 1
and 3. Above -40 °C decomposition was observed. Cooling
down to -80 °C and addition of MeOH-d4 to the activation
mixture of donor 2 generated mainly β-methyl mannopy-
ranoside 8, as expected (Table 1, entry 2).
To monitor the activation of R-imidate 4, a solution of do-
nor 4 in DCM-d2 (0.05 M) was treated with TfOH (1.3 equiv)
at -80 °C. As with β-imidate donor 3, compound 4 was
quickly consumed, and the spectrum obtained was identical
to the one displayed in Figure 3A and the one obtained from
activation of donor 3. Thus, both imidate donors produce
upon preactivation the same conformational mixture of
R-anomeric triflates. Addition of MeOH-d4 to the activation
mixture gave rapid conversion to the β-methyl mannopy-
ranoside 8 with excellent selectivity (Table 1, entry 4).28
1
sulfonium triflate structure II residing in the C4 chair con-
formation. Upon addition of MeOH-d4 (25equivat-80 °C) the
activated mixture of donor 5 remained unchanged, in con-
trast to the fast conversion of anomeric triflates I/I*. Only
after warming of the mixture to þ10 °C was full con-
sumption of intermediate II observed. Next to β-coupled
product 8, which was formed in 25% yield, regenerated
donor 5 was found as the main product (Table 1, entry 5).
When the β-sulfoxide donor 6 was treated with Tf2O at
-80 °C, the 1H NMR spectrum showed full consumption of
the donor, with the conformational mixture of R-triflates I/I* as
the major product alongside a second product (Figure 3C).
On the basis of the relatively small chemical shift of H-1 (δ=
5.22 ppm), the chemical shift of C-1 (δ=91.4 ppm), and the
activation experiments of the R-sulfoxides 7a/b (vide infra),
we presume that this latter species corresponds to the
β-sulfonium bistriflate species III.30 Addition of MeOH-d4
resulted in a mixture of products containing the methyl
mannoside product 8 (R:β = 1:5, ∼60%) and regenerated
donor 6.
(27) Although we previously established that thioglycosyl methyl
uronates require relatively high (pre)activation temperatures in a sulfonium-
based activation protocol (activation of the uronate donors generally
proceeds at -65 to -55 °C, as opposed to the activation of “non-oxidized”
thioglycosides which can be effected at -78 °C), in the ManN3A-case at
hand, the activation temperature seems to be largely dependent on the
anomeric configuration. Van den Bos, L. J.; Litjens, R. E. J. N.; van den
Berg, R. J. B. H. N.; Overkleeft, H. S.; van der Marel, G. A. Org. Lett. 2005, 7,
2007–2010.
Activation of R-sulfoxide diastereomer 7a (1.3 equiv of
Tf2O) at -80 °C led to the rapid formation of one predomi-
nant species (Figure 3D). However, the signals did not
correspond to the peaks assigned to the (conformational
mixture of) anomeric triflates I/I*. Since an overall down-
field shift was observed for the pyranosyl protons, the
(28) The difference in stereoselectivities between glycosylation of MeOH-
d4 with the thio- and the imidate donors may result from slight experimental
variations caused during the mixing of the NMR samples outside the
spectrometer. Furthermore, the reaction mixtures of the activated thioglyco-
sides contain different (sulfonium) species, generated upon expulsion of the
anomeric thiophenyl moiety, and unreacted diphenylsulfoxide, which poten-
tially affect the stereochemical outcome of the glycosylations.
(29) It has been shown that an anomeric triflate is instantaneously
converted to the oxosulfonium triflate species by the addition of diphenyl
sulfoxide, indicating that this is the more stable intermediate. Garcia, B. A.;
Gin, D. Y. J. Am. Chem. Soc. 2000, 122, 4269–4279.
(30) The existence of a sulfonium bistriflate species has been postulated
before (ref 24a).
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doublet assigned to H-1 at δ 5.38 ppm displayed a coupling
constant of JH1-H2=10.8 Hz, and the chemical shift of C-1
was indicative for an anomeric thio functionality (δ 86.2 ppm),
the activated species was considered to be the equatorial
R-anomeric sulfonium bistriflate IV-a.31 Because the stereo-
chemistry of the parent sulfoxide 7a could not be determined,
the stereochemistry of the sulfonium bistriflate cannot be
determined either. Prolonged reaction time and warming of
the reaction mixture to -20 °C did not lead to transforma-
tion of this species into the anomeric triflate I/I*. Treatment
of the activated mixture with MeOH-d4 resulted in a complex
mixture of compounds, which contained a substantial
amount of recovered donor 7a. Interestingly, activation of
the other R-sulfoxide diastereomer 7b in a similar NMR
experiment led to different intermediates. The R-triflates I/I*
were formed as well as a new species, which did not corre-
spond to the sulfonium bistriflate IV-a. On the basis of the
similarity of the 1H-resonances of this species and IV-a, and
the chemical shift of C-1 (δ 85.4 ppm), again indicative of an
anomeric thio group, we speculate that this species is the
other diastereomeric sulfonium bistriflate IV-b (Figure 3E).
Gradual warming of the reaction mixture to -60 °C led to
further conversion of IV-b into anomeric triflates I/I* (I/I*:
IVb ∼ 4:3). The addition of MeOH-d4 resulted in a mixture of
products containing methyl mannoside 8 (R:β=1:5, ∼50%),
together with regenerated donor 7b and R-thio mannuronate
2 (Table 1, entry 8).32
The activation experiments described above provide a
detailed picture of the behavior of donors 1-7 upon activa-
tion. The reactivity boundaries of the activation protocols
used and the influence of the anomeric configuration are
apparent. While β-thio donor 1 is rapidly activated at -80 °C,
its R-counterpart 2 requires a higher temperature (-40 °C) in
order to be fully consumed. The reactivity difference between
two anomers is often attributed to a stabilizing anomeric
effect in the R-anomer. However, both R- and β-mannoside 1
and 2 exist in a conformation in which the sulfur aglycon is
positioned equatorially, thereby lacking anomeric stabili-
zation.33 We therefore attribute the reactivity difference
between 1 and 2 to a difference in stability caused by the
(stereo)electronic repulsion between the substituents on C-1,
C-2, and the ring-oxygen (the destabilizing Δ2-effect).34 The
reactivity difference between the R- and β-thiomannosides 1
and 2 was not observed for the imidate anomers 3 and 4.
Under the influence of a stoichiometric amount of TfOH,
both donors were rapidly transformed into a mixture of
R-triflate conformers, which gave an identical β-selectivity in
the ensuing substitution by MeOD-d4. Hemiacetal donor 5
was fully converted to the relatively stable oxosulfonium
triflate II upon activation. Treatment of this activated inter-
mediate with a nucleophile did not result in effective glyco-
sylation. Instead, mainly hemiacetal 5 was regenerated. This
result shows that the oxosulfonium triflate is not easily
expelled from the mannuronate donor and that a competing
attack at either of the sulfonium centers in II can take place.
Although glycosyl sulfoxides are generally regarded to be
among the most powerful glycosyl donors, the results obtained
with the sulfoxide donors 6 and 7a,b show a reactivity limit
for the sulfoxide method. Because of the unreactivity of the
mannosaziduronic acid core, reactivity differences became
apparent not only between the R- and β-anomers but also
between the two different sulfoxide diastereomers, which
provided different reactive species upon Tf2O activation.35
Although the existence of pyranosyl sulfonium bistriflates
has been postulated before,24a such species have not been
experimentally observed, since they rapidly collapse to the
corresponding anomeric triflates.36
Glycosylations with Glucoside Acceptors. To assess the
glycosylating properties of mannosazide methyl uronates
with a glycosyl acceptor, the donors 1-4, which provided a
productive glycosylation with MeOH-d4 as described above,
were further examined.
First, β-thiodonor 1 was preactivated with the Ph2SO-
Tf2O reagent combination for 15 min during which time the
temperature was raised from -65 to -55 °C. Then acceptor 9
was added, and disaccharide 11 was produced in high yield
and selectivity (Table 2, entry 1). In contrast, when R-thio-
donor 2 was preactivated from -80 to -40 °C, as deduced
from the NMR experiments to be the optimal activation
temperature, and subsequently condensed with acceptor 9,
the yield of disaccharide 11 was significantly lower, while the
stereoselectivity remained intact (Table 2, entry 2). This poor
coupling efficiency may be attributed to the fact that the
preactivation temperature (-40 °C) is close to the tempera-
ture at which decomposition of the anomeric triflate sets in,
as observed in the NMR experiments. Optimization of the
glycosylation of donor 2 proved to be precarious; monitoring
of the activation progress was troublesome and slight adjust-
ments to the experimental procedure resulted in considerable
differences in glycosylation outcome. The best conditions
found involved activation of thiomannoside 2 with Ph2SO-
Tf2O for 15 min at -65 to -55 °C prior to addition of accep-
tor 9 and led to the stereoselective formation of disaccha-
ride 11 in 75% yield (Table 2, entry 3). The imidate donors 3
and 4 were coupled with acceptor 9 under the agency of a
catalytic amount of triflic acid (TfOH). The R-imidate 4
provided predominantly the β-linked disaccharide, whereas
the use of β-imidate 3 led to the formation of a substantial
amount of the R-linked disaccharide (Table 2, entries 5 and 4,
respectively). Since NMR analysis of imidate donors 3 and 4
showed that both form the same mixture of R-triflate inter-
mediates under preactivation conditions with a equimolar
amount of TfOH and that both provided excellent β-selectivity
in the glycosylation of MeOH-d4, the significant amount
of R-product 11 generated from β-imidate 3 must arise
from SN2-displacement of the anomeric imidate by the
(31) The chemical shift of the anomeric carbon atom in 13C NMR, the
recovery of unreacted sulfoxide donor and the reduction of the sulfoxide
moiety in donor 7b (ref 32). advocate against the intermediacy of an anomeric
sulfenate. Gildersleeve, J.; Pascal, R. A., Jr.; Kahne, D. J. Am. Chem. Soc.
1998, 120, 5961–5969.
(32) The formation of mannuronate 2 from donor 7b can be explained by
a Swern-like oxidation of methanol by the intermediate sulfonium bistriflate
IV-b.
(33) (a) Deslongchamps, P. Stereoelectronic Effects in Organic Chemistry;
Pergamon, New York, 1983; Chapter 6. (b) Juaristi, E.; Cuevas, G. Tetra-
hedron 1992, 48, 5019–5087.
(35) Different reactivities for sulfoxide diastereomers have been acknowl-
ꢁ
L.; Plusquellec, D. Tetrahedron Lett. 2000, 41, 5515–5519. (b) Khiar, N.;
Alonso, I.; Rodriguez, N.; Fernandez-Mayoralas, A.; Jimenez-Barbero, J.;
Nieto, O.; Cano, F.; Foces-Foces, C.; Martin-Lomas, M. Tetrahedron Lett.
1997, 38, 8267–8270.
edged before: (a) Ferrieres, V.; Joutel, J.; Boulch, R.; Roussel, M.; Toupet,
(34) (a) Reeves, R. E. J. Am. Chem. Soc. 1950, 72, 1499–1506. (b) Lii,
J.-H.; Chen, K.-H.; Allinger, N. L. J. Comput. Chem. 2003, 24, 1504–1513.
(36) Callam, C. S.; Gadikota, R. R.; Krein, D. M.; Lowary, T. L. J. Am.
Chem. Soc. 2003, 125, 13112–13119.
7994 J. Org. Chem. Vol. 75, No. 23, 2010

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TABLE 2. Condensations of the Mannosazide Methyl Uronate Donors 1-4 with Acceptors 9 and 10
entry
donor
acceptor
(pre)activation
yield (%) (R/β)
1
2
3
4
5
6
7
1
2
2
3
4
1
2
9
9
9
9
9
Ph2SO, Tf2O, -65 f -55 °C (15 min), then add 9
Ph2SO, Tf2O, -80 f -40 °C (1 h), then add 9
Ph2SO, Tf2O, -65 f -55 °C (15 min), then add 9
0.2 equiv of TfOH
0.2 equiv of TfOH
Ph2SO, Tf2O, -65 f -55 °C (15 min), then add 10
Ph2SO, Tf2O, -65 f -55 °C (15 min), then add 10
90 (1:7)
45 (1:5)
75 (1:6)
84 (1:2)
53 (1:5)
85 (0:1)a
58 (0:1)
10
10
aThe yield includes 45% of the β-linked disaccharide bearing one isopropylidene group on C-1 and C-2 because of cleavage of the C5,6-isopropylidene
functionality under the coupling conditions.
nucleophile, already present in the reaction mixture.37 Be-
cause the thiomannosides 1 and 2 performed best in terms of
yield and β-selectivity, these donors were further probed with
the secondary acceptor 1,2:3,4-diisopropylideneglucofuranose
(10). Under the optimal preactivation conditions, the condensa-
tions of 1 and 2 with 10 gave the β-linked dimer 12 as the
sole product (Table 2, entries 6 and 7). Also in this case, the
β-configured donor was shown to be superior to its R-linked
equivalent.
Oligosaccharide Assembly. Having established that man-
nosaziduronic acid donors provide highly β-selective con-
densations, the construction of an anionic M. luteus oligo-
saccharide containing the [f6)-R-D-Glcp-(1f4)-β-D-Man-
pNAcA-(1f] repetitive motif (Figure 1) was undertaken.
The alternating character of the Glc and ManNAcA building
blocks allows for a disaccharide block coupling strategy.
Guided by the excellent β-selectivities obtained with Man-
N3A β-thio donor 1, the Glc-ManN3A thiophenyl dimer 16
was selected to serve as an iterative building block (Scheme 1).
For the stereoselective installation of the R-gluco linkage
in compound 16, several strategies may be followed.38-42
For us,43 the best method turned out to be the protocol de-
scribed by Adinolfi and co-workers, who reported that a
glucosyl trifluoroimidate carrying a large C-6 dimethoxytri-
tyl protecting group is an effective R-glucosyl donor in
etherous solvents.42 We explored the use of glucose imidate
14 having the bulky Fmoc-protecting group44 at C-6. This
donor was readily obtained from 2,3,4-tri-O-benzyl-R/β-D-
glucopyranose (13)45 by regioselective N-(phenyl)trifluoro-
acetimidate46 formation and subsequent Fmoc installation
at the C-6 hydroxyl. Disaccharide 16 was efficiently pro-
duced by treating a mixture of donor 14 and acceptor 15 with
a catalytic amount of TfOH in diethyl ether (0.05 M) at -35
to -15 °C. The R-coupled product 16 was formed as the sole
product in 90% yield. To investigate how the R-glucosyl
appendage in 16 affects the glycosylation properties of the
ManN3A donor, disaccharide 16 was subjected to activation
conditions and the progress of the activation reaction was
monitored using low-temperature NMR spectroscopy as
described above. After addition of Tf2O at -80 °C, donor
16 was immediately consumed producing a conformational
mixture of anomeric triflates, in which the 1C4 chair product
dominates (1C4: 4C1=4: 1). The H-1 signal, characteristic of
the equatorial triflate, resides at δ 6.22 ppm with a coupling
constant of JH1-H2=8.8 Hz (C-1 δ 100.5 ppm), and the axial
triflate appeared as a singlet at δ 5.99 ppm. Addition of
MeOH-d4 to this mixture resulted in clean conversion to the
β-fused methyl disaccharide. Encouraged by this result, the
construction of M. luteus teichuronic acid fragments was
commenced. Thus, dimer 16 was activated (Ph2SO-Tf2O,
-65 °C to -55 °C for 15 min) and reacted with glucosyl
acceptor 9 at -60 °C to provide trisaccharide 17 as a single
stereoisomer in 65%. Liberation of the C600-OH was accom-
plished by treatment of compound 17 with a catalytic
amount of TBAF in THF to give trisaccharide acceptor 18
in quantitative yield. In the ensuing glycosylation event,
dimer 16 and trimer 18 were combined under analogous
conditions to provide the all-cis-linked product 19 as the sole
isomer in 42% yield. To improve the yield, the reaction
temperature and time were adjusted, and when 16 and 18
were condensed overnight at -80 °C, pentasaccharide 19 was
obtained in 65% yield.47
(37) Schmidt, R. R.; Hoffmann, M. Tetrahedron Lett. 1982, 23, 409–412.
(38) Lemieux, R. U.; Hendriks, K. B.; Stick, R. V.; James, K. J. Am.
Chem. Soc. 1975, 97, 4056–4062.
(39) (a) Hadd, M. J.; Gervay, J. Carbohydr. Res. 1999, 320, 61–69. (b) Lam,
S. N.; Gervay-Hague, J. Org. Lett. 2002, 4, 2039–2042.
(40) Kim, J.-H.; Yang, H.; Park, J.; Boons, G.-J. J. Am. Chem. Soc. 2005,
127, 12090–12097.
Removal of the Fmoc group in 19 with a catalytic amount
of TBAF in THF proceeded sluggishly to yield compound 20
in 83% yield after 3 days. The use of excess triethylamine in
pyridine improved both the yield (89%) and reaction time
(41) Crich, D.; de la Mora, M.; Vinod, A. U. J. Org. Chem. 2003, 68,
8142–8148.
(42) (a) Adinolfi, M.; Barone, G.; Iadonisi, A.; Shiattarella, M. Tetra-
hedron Lett. 2002, 43, 5573–5577. (b) Adinolfi, M.; Iadonisi, A.; Schiattar-
ella, M. Tetrahedron Lett. 2003, 44, 6479–6482.
(43) Initial attempts at achieving an R-selective glucosylation using 4,6-O-
benzylidene or 6-O-dimethoxytrityl thiophenyl glucosyl donors and various
ManN3A acceptors met with limited success.
ꢁ
(46) Adinolfi, M.; Iadonisi, A.; Ravida, A. Synlett 2006, 4, 583–586.
(44) Roussel, F.; Knerr, L.; Grathwohl, M.; Schmidt, R. R. Org. Lett.
2000, 5, 3043–3046.
(45) Zhang, G.-t.; Guo, Z.-w.; Hui, Y.-z. Synth. Commun. 1997, 27, 1907–
1917.
(47) Careful analysis of the crude glycosylation mixture revealed the
presence of de-Fmocylated oligosaccharide. A reduction of the amount of
pyridine to quench the glycosylation reaction did not prevent cleavage of the
Fmoc. The reported yields only include Fmoc-protected product.
J. Org. Chem. Vol. 75, No. 23, 2010 7995

Walvoort et al.
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SCHEME 1. Synthesis of M. luteus Tri-, Penta-, and Heptasaccharides 27, 29, and 31a
aReagents and conditions: (a) (i) CF3C(NPh)Cl, K2CO3, acetone; (ii) Fmoc-Cl, pyridine, DCM (14: 83% over two steps); (b) 15, TfOH, Et2O, -35 f
-15 °C (16: 90%); (c) Ph2SO, Tf2O, TTBP, DCM, -65 f -55 °C, then 9 (17: 65%); (d) TBAF, THF (18: 98%); (e) 16, Ph2SO, Tf2O, TTBP, DCM, -70
f -60 °C, then 18, -80 °C, o.n. (19: 65%); (f) Et3N, pyridine (20: 89%, 22: 78%); (g) 16, Ph2SO, Tf2O, TTBP, DCM, -70 f -55 °C, then 20, -80 °C,
2 days (21: 23%); (h) H2O2, aq KOH (23: 85%, 24: 83%, 25: 83%); (i) Na (s), liq NH3, THF, -60 °C (33: 70% over two steps); (j) Ac2O, NaHCO3, H2O/
THF (27: 43%, 29: 35%, 31: 14%, over two steps); (k) H2S, pyridine/H2O, 2 days.
(3 h) of this deprotection step. Finally, to construct heptasac-
charide 21, disaccharide donor 16 was activated and reacted
with pentasaccharide 20 over 2 days at -80 °C. Heptaman-
nuronate 21 was obtained in 23% yield, reflecting the lower
reactivity of the bulky pentasaccharide acceptor,47 alongside
40% of unreacted pentasaccharide 20. Cleavage of the Fmoc
group using TEA/pyridine proceeded uneventfully to give
heptasaccharide 22 in 78% yield.
Global deprotection of oligosaccharides 18, 20, and 22
started with saponification of the methyl esters. Reaction of
trisaccharide 18 with KOH in THF/H2O gave the desired
uronic acid 23 together with side products generated by
β-elimination in the ManN3A moiety. The use of a more
nucleophilic and less basic reagent mixture (H2O2 in aqueous
KOH) reduced the undesired β-elimination, and mannuronic
acid 23 was obtained in 85% yield. Application of these
conditions to substrates 20 and 22 delivered di- and triacid 24
and 25, respectively, in good yields. Simultaneous reduction
of the azide functionality and the benzyl ethers in trisaccha-
ride 23 with H2 and Pd/C proved to be troublesome and led
to an inseparable product mixture. A stepwise approach in
which the azide was transformed into the free amine using
H2S in pyridine/H2O prior to reduction of the benzyl groups
also failed because reduction of the azide was accompanied
by cyclization to provide lactam 32. Formation of this amide
probably results from attack of the free amine to the thiol
acid, generated from the carboxylic acid and H2S.48 In the
end, direct Birch reduction of trisaccharide 23 proved to be
the most efficient protocol, and anionic trisaccharide 27 was
obtained after acetylation of the free amine in 43%. When
pentasaccharide 24 was treated under similar conditions,
€
(48) Keller, M.; Blochl, E.; Wachtershauser, G.; Stetter, K. O. Nature
€
€
1994, 368, 836–838.
7996 J. Org. Chem. Vol. 75, No. 23, 2010

Walvoort et al.
JOCFeatured Article
exploited in the synthesis of M. luteus teichuronic acid
fragments. An R-stereoselective glycosylation between a
glucosyl N-phenyltrifluoro imidate and an S-phenyl manno-
saziduronic acid acceptor provided the key R-Glc-(1f4)-β-
ManN3A-SPh building block, which was used in the assem-
bly of tri-, penta-, and heptasaccharide fragments. Final
deprotection of the oligomers under Birch reduction condi-
tions, which was accompanied by partial fragmentation of
the oligosaccharide chain, yielded the anionic tri-, penta-,
and heptasaccharide M. luteus teichuronic acid fragments.
The results presented in this paper may facilitate the synthe-
sis of other complex (uronic acid-containing) oligosaccha-
rides. Moreover, this research illustrates the importance of a
comprehensive survey of the behavior in glycosylations when
unreactive carbohydrate moieties are the building blocks of
interest.
Experimental Section
General Procedure for the Low-Temperature NMR Experi-
ments. Ph2SO/Tf2O activation: A mixture of the donor (30 μmol)
and Ph2SO (39 μmol) was coevaporated with toluene (2ꢀ). The
residue was dissolved in DCM-d2 (0.6 mL) and transferred to an
NMR tube under an argon atmosphere. The tube was stoppered
and sealed. The NMR magnet was cooled to -80 °C, locked, and
shimmed. In an acetone bath (-80 °C), the sample was treated with
Tf2O (39 μmol), shaken three times, and placed back in the NMR
magnet. The first 1H spectrum was immediately recorded. Further
temperature changes were executed depending on the spectra
recorded, but always with multiples of 10 °C. Ultimately, the
sample was placed in the acetone bath (-80 °C), and MeOH-d4
(25 μL), which was used for its invisibility in 1H NMR, was added.
After the sample was shaken three times, it was placed back in the
FIGURE 4. HPAEC traces of the crude reaction mixture of the
Birch reduction of pentasaccharide 24 (A) and heptasaccharide 25 (B).
target pentamer 29 was formed in 35% yield. Unfortunately,
we observed that fragmentation of the oligosaccharide oc-
curred during the Birch reduction. High-performance anion
exchange chromatography (HPAEC, see Figure 4A) and
LC-MS indicated that a substantial amount of trisaccha-
ride 26 next to pentamer 28 was formed. Formation of the
trisaccharide cannot be explained by β-elimination of the
mannuronic acid residue but must have occurred via the un-
expected cleavage of the β-mannosyl glycosidic bond.49,50
Finally, heptamer 25 was subjected to the reduction condi-
tions, and after subsequent acetylation and purification tar-
get compound 31 was obtained. The reduction of the hep-
tamer was also accompanied by fragmentation, and HPAEC
analysis revealed the formation of zwitterionic tri- and
pentasaccharide 26 and 28, next to the desired product 30
(Figure 4B). Gel filtration (HW-40) of the product mixture
was hampered by poor separation of heptamer 30 from the
smaller fragments; however, pure 30 was obtained, which
yielded heptasaccharide 31 in 14% yield after N-acetylation.
1
NMR magnet at -80 °C and immediately a H spectrum was
recorded. Then the temperature was raised to rt, and a final H
1
spectrum was recorded. TfOH activation: The donor (39 μmol)
was coevaporated with dry toluene (2ꢀ), dissolved in DCM-d2
(0.6 mL), and transferred to an NMR tube under an argon atmo-
sphere. At -80 °C in an acetone bath TfOH (39 μmol) was added,
the sample was transferred to the precooled NMR magnet, and the
first 1H spectrum was immediately recorded. Further temperature
changes were executed depending on the spectra recorded, but
always with multiples of 10 °C. Ultimately, the sample was placed
in the acetone bath (-80 °C), and MeOH-d4 (25 μL) was added.
After the sample was shaken three times, it was placed back in the
Conclusion
1
NMR magnet at -80 °C and immediately a H spectrum was
recorded. The temperature was then raised to rt, and a final H
spectrum was recorded.
We have described a thorough evaluation of the glycosy-
lation properties of a series of mannosaziduronic methyl
ester donors. Depending on the anomeric leaving group and
the preactivation conditions, reactive intermediates with
various stabilities are formed: anomeric triflates from the
R- and β-S-phenyl and N-phenyltrifluoro imidates, an ox-
osulfonium triflate from the hemiacetal, and sulfonium
bistriflates from the R- and β-sulfoxides. Interestingly, the
intermediates formed from the sulfoxides, generally re-
garded to be very powerful glycosyl donors, did not provide
productive glycosylations. The high β-stereoselectivity and
good coupling efficiency of the β-S-phenyl ManN3A were
1
General Procedure for the Ph2SO/Tf2O-Mediated Glycosyla-
tions. A mixture of the donor (1 equiv), Ph2SO (1.3 equiv), and
TTBP (2.5 equiv) was coevaporated twice with toluene. While
the mixture was under an argon atmosphere, freshly distilled
DCM (0.05 M) was added, followed by the addition of activated
molecular sieves (3 A). The resulting mixture was stirred for
30 min at room temperature and cooled to the activation tem-
perature. Tf2O (1.3 equiv) was added in one portion, and the
activation progress was monitored by TLC analysis. The mix-
ture was then cooled to the indicated reaction temperature, and
a solution of the acceptor (0.3-0.5 M in DCM) was slowly
added via the wall of the flask. The mixture was allowed to warm
to 0 °C, after which Et3N or pyridine was added to quench the
reaction. Aqueous workup, passage of the residue through a
column of Sephadex LH-20 (eluted with DCM/MeOH, 1/1, v/v),
and purification using flash column chromatography (silica gel)
gave the coupled product.
(49) Birch reduction is commonly applied in the global deprotection of
oligosaccharides and has been used in the synthesis of β-mannans: Nitz, M.;
Purse, B. W.; Bundle, D. R. Org. Lett. 2000, 2, 2939–2942. For the use of
Birch reduction in a PSA glycopeptide, see: Dudkin, V. Y.; Miller, J. S.;
Danishefsky, S. J. J. Am. Chem. Soc. 2004, 126, 736–738.
(50) Seeberger and co-workers have previously noted the fragmentation
of a β-mannosazide-containing oligosaccharide under Birch reduction con-
General Procedure for the TfOH-Mediated Glycosylations. A
mixture of the donor (1 equiv) and the acceptor (1.5 equiv) was
€
ditions: Oberli, M. A.; Bindschadler, P.; Werz, D. B.; Seeberger, P. H. Org.
Lett. 2008, 10, 905–908.
J. Org. Chem. Vol. 75, No. 23, 2010 7997

Walvoort et al.
JOCFeatured Article
coevaporated with toluene (2ꢀ). While the mixture was under
an argon atmosphere, freshly distilled DCM (0.05 M) was
added, followed by the addition of activated molecular sieves
(3 A). The resulting mixture was stirred for 30 min at room
temperature and cooled to the activation temperature. TfOH
(0.2 equiv) was added, and the reaction mixture was warmed to
the desired temperature. The reaction was then quenched by the
addition of Et3N or pyridine. After aqueous workup, the
product was purified using Sephadex LH-20 (eluted with DCM/
MeOH, 1/1, v/v) and flash column chromatography (silica gel).
Methyl (Phenyl-4-O-[2,3,4-tri-O-benzyl-6-O-[9-fluorenylmethoxy-
carbonyl]-r-D-glucopyranosyl]-2-azido-3-O-benzyl-2-deoxy-1-thio-β-
D-mannopyranosyl uronate) (16). Imidate 14 (1.70 g, 2.02 mmol)
and acceptor 15 (1.89 g, 1.5 mmol) were coevaporated with dry
toluene (2ꢀ). Et2O (40 mL, dried over 4 A MS prior to use) was
added, and the mixture was cooled to -35 °C. TfOH (40 μL,
0.45 mmol) was added, and the mixture was allowed to warm
to -15 °C over 90 min. Pyridine (1 mL) was added, and the
mixture was diluted with EtOAc and washed with satd aq NaCl
(2ꢀ). The organic layer was dried over Na2SO4, concentrated in
vacuo, and purified using column chromatography (silica gel,
20% EtOAc in PE) to yield the title compound as a white foam
(1.44 g, 1.35 mmol, 90%): TLC Rf 0.47 (PE/EtOAc, 3/1, v/v);
[R]20D þ34.4 (c 1, DCM); IR (neat, cm-1) 725, 905, 1070, 1452,
1747, 2110; 1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC)
δ 7.74 (d, 2H, J=7.5 Hz, CHarom), 7.60 (dd, 2H, J=7.5, 11.1 Hz,
CHarom), 7.00-7.50 (m, 29H, CHarom), 5.31 (d, 1H, J=3.4 Hz,
H-10), 4.97 (d, 1H, J=10.8 Hz, CHH Bn), 4.89 (d, 1H, J=10.9
Hz, CHH Bn), 4.83 (d, 1H, J=10.8 Hz, CHH Bn), 4.72 (d, 1H,
J=0.9 Hz, H-1), 4.69 (d, 2H, J=6.4 Hz, CH2 Bn), 4.57-4.63
(m, 3H, CHH Bn, CH2 Bn), 4.32-4.46 (m, 5H, H-4, H-6, H-6,
CH2 Fmoc), 4.23 (t, 1H, J=7.2 Hz, CH Fmoc), 4.05 (d, 1H, J=
2.5 Hz, H-2), 3.94 (t, 1H, J=9.2 Hz, H-30), 3.86 (d, 1H, J=9.4
Hz, H-5), 3.76 (dd, 1H, J=3.6, 9.1 Hz, H-3), 3.73 (s, 3H, CH3
CO2Me), 3.65-3.72 (m, 1H, H-50), 3.58-3.64 (m, 1H, H-40),
3.52 (dd, 1H, J=3.5, 9.8 Hz, H-20); 13C NMR (CDCl3, 100 MHz,
HSQC) δ 167.4 (CdO CO2Me), 154.9 (CdO Fmoc), 143.4,
143.1, 141.2 (Cq Fmoc), 138.4, 137.9, 137.2 (Cq Bn), 133.9
(Cq SPh), 131.0, 128.5, 128.3, 127.9, 127.8, 127.6, 127.1, 125.1,
125.0, 120.0 (CHarom), 98.5 (C-10), 86.6 (C-1), 81.3 (C-3, C-30),
79.7 (C-20), 79.1 (C-5), 76.7 (C-40), 75.5, 75.1 (CH2 Bn), 74.8
(C-4), 73.0, 72.9 (CH2 Bn), 69.8 (CH2 Fmoc), 69.7 (C-50), 65.8
(C-60), 63.2 (C-2), 52.8 (CH3 CO2Me), 46.6 (CH Fmoc);
13C-GATED (100 MHz, CDCl3) δ 98.5 (JC1,H1 = 172 Hz, C-10),
86.6 (JC1,H1 = 154 Hz, C-1); HRMS [M þ Na]þ calcd for
C62H59N3O12SNa 1092.37117, found 1092.37178.
7.60 (t, 2H, J = 8.6 Hz, CHarom Fmoc), 7.10-7.50 (m, 39H,
CHarom), 5.28 (d, 1H, J=3.5 Hz, H-100), 4.99 (d, 1H, J=10.8 Hz,
CHH Bn), 4.98 (d, 1H, J=10.9 Hz, CHH Bn), 4.88 (d, 1H, J=
10.9 Hz, CHH Bn), 4.80-4.86 (m, 3H, CHH Bn, CH2 Bn), 4.77
(d, 2H, J=11.9 Hz, CHH Bn, CHH Bn), 4.47-4.70 (m, 7H, CH2
Bn, H-1), 4.26-4.45 (m, 6H, H-10, H-40, H-600, H-600, CH2
Fmoc), 4.23 (t, 1H, J=7.3 Hz, CH Fmoc), 4.04-4.10 (m, 1H,
H-6), 3.98 (t, 1H, J=9.2 Hz, H-3), 3.93 (t, 1H, J=9.3 Hz, H-300),
3.83 (d, 1H, J=8.6 Hz, H-50), 3.73-3.80 (m, 1H, H-5), 3.65-
3.73 (m, 1H, H-500), 3.68 (s, 3H, CH3 CO2Me), 3.56-3.65 (m,
3H, H-20, H-30, H-400), 3.52 (dd, 1H, J = 3.5, 9.8 Hz, H-200),
3.38-3.50 (m, 2H, H-2, H-6), 3.32 (t, 1H, J=9.4 Hz, H-4), 3.28
(s, 3H, CH3 OMe); 13C NMR (CDCl3, 100 MHz, HSQC) δ 168.0
(CdO CO2Me), 154.9 (CdO Fmoc), 143.4, 143.2, 141.2 (Cq
Fmoc), 138.7, 138.5, 138.2, 138.1, 137.9, 137.9, 137.5 (Cq Bn),
127.1-128.5 (CHarom Bn), 125.2, 125.1, 120.0 (CHarom Fmoc),
99.8 (C-10), 98.1 (C-100), 97.7 (C-1), 82.0 (C-3), 81.3 (C-300), 79.9
(C-2), 79.6 (C-200), 78.7 (C-400), 77.6 (C-4), 76.8 (C-30), 75.7, 75.6
(CH2 Bn), 75.2 (C-50), 75.1, 74.7 (CH2 Bn), 74.4 (C-40), 73.3,
72.9, 72.2 (CH2 Bn), 69.9 (CH2 Fmoc), 69.7 (C-5), 69.6 (C-500),
68.7 (C-6), 65.9 (C-600), 60.7 (C-20), 55.0 (OMe), 52.7 (CH3
CO2Me), 46.7 (CH Fmoc); 13C-GATED (CDCl3, 100 MHz) δ
0
00
0
0
00
00
99.8 (JC1 ,H1 = 162 Hz, C-1 ), 98.2 (JC1 ,H1 = 170 Hz, C-1 ),
97.7 (JC1,H1 =164 Hz, C-1); HRMS [M þ Na]þ calcd for C84-
H85N3O18Na 1446.57203, found 1446.57310.
Methyl 6-O-(Methyl 4-O-[2,3,4-tri-O-benzyl-r-D-glucopy-
ranosyl]-2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate)-
2,3,4-tri-O-benzyl-r-D-glucopyranoside (18). A solution of com-
pound 17 (1.26 g, 0.89 mmol) in THF (18 mL) was cooled to
0 °C under an argon atmosphere. TBAF (1 M solution in THF,
89 μL, 89 μmol) was added, and the reaction was stirred at 4 °C
for 24 h. The mixture was quenched with satd aq NaHCO3,
diluted with EtOAc, washed with satd aq NaCl (2ꢀ), dried over
Na2SO4, and concentrated in vacuo. Purification using flash
column chromatography (silica gel, 50% EtOAc in PE) afforded
the title product as a colorless oil (1.0 g, 0.87 mmol, 98%): TLC
Rf 0.50 (PE/EtOAc, 1/1, v/v); [R]20D þ42.5 (c 1, DCM); IR (neat,
cm-1) 696, 729, 1026, 1069, 1751, 2110, 2882; 1H NMR (CDCl3,
400 MHz, HH-COSY, HSQC) δ 7.20-7.40 (m, 35H, CHarom),
5.19 (d, 1H, J=3.6 Hz, H-100), 4.98 (d, 1H, J=10.2 Hz, CHH
Bn), 4.96 (d, 1H, J=9.3 Hz, CHH Bn), 4.85-4.90 (m, 3H, CHH
Bn, CH2 Bn), 4.80 (d, 1H, J=11.0 Hz, CHH Bn), 4.76 (d, 1H, J=
12.1 Hz, CHH Bn), 4.57-4.70 (m, 6H, CH2 Bn), 4.54 (s, 1H,
H-1), 4.76 (d, 1H, J=12.1 Hz, CHH Bn), 4.28 (s, 1H, H-10), 4.28
(t, 1H, J=8.4 Hz, H-40), 4.04-4.13 (m, 1H, H-6), 3.99 (t, 1H, J=
9.2 Hz, H-3), 3.91 (t, 1H, J=9.2 Hz, H-300), 3.83 (d, 1H, J=8.7
Hz, H-50), 3.73-3.80 (m, 2H, H-5, H-600), 3.68 (s, 3H, CH3
CO2Me), 3.64 (bd, 2H, J=3.4 Hz, H-20, H-600), 3.59 (dd, 1H, J=
3.6, 8.5 Hz, H-30), 3.40-3.56 (m, 5H, H-2, H-200, H-400, H-500,
H-6), 3.34 (t, 1H, J=9.4 Hz, H-4), 3.29 (s, 3H, CH3 OMe), 1.97
(bs, 1H, 600-OH); 13C NMR (CDCl3, 100 MHz, HSQC) δ 168.1
(CdO CO2Me), 138.5, 138.4, 138.1, 138.0, 137.9, 137.8, 137.4
(Cq Bn), 128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.4
(CHarom), 99.7 (C-10), 97.8 (C-1), 97.6 (C-100), 81.9 (C-300), 81.1
(C-3), 79.8, 79.6 (C-2, C-200), 78.7 (C-30), 77.4 (C-4), 77.1 (C-400),
75.6, 75.4 (CH2 Bn), 75.2 (C-50), 74.9, 74.5 (CH2 Bn), 73.9 (C-40),
73.2, 72.9, 72.3 (CH2 Bn), 72.0 (C-500), 69.5 (C-5), 68.6 (C-6),
Methyl 6-O-(Methyl 4-O-[2,3,4-tri-O-benzyl-6-O-[9-fluorenyl-
methoxycarbonyl]-r-D-glucopyranosyl]-2-azido-3-O-benzyl-2-
deoxy-β-D-mannopyranosyl uronate)-2,3,4-tri-O-benzyl-r-D-glu-
copyranoside (17). Disaccharide 16 (123 mg, 0.12 mmol), Ph2SO
(30 mg, 0.15 mmol), and TTBP (71 mg, 0.29 mmol) were co-
evaporated with dry toluene (2ꢀ), dissolved in freshly distilled
DCM (2.3 mL), and cooled to -65 °C. Tf2O (25 μL, 0.15 mmol)
was added, and the mixture was warmed to -55 °C during 15 min.
The reaction was cooled back to -60 °C, and a solution of
acceptor 9 (80 mg, 0.17 mmol, coevaporated twice with dry
toluene prior to use) in distilled DCM (1 mL) was slowly added.
The mixture was warmed to -40 °C over 1 h, quenched with
pyridine (0.2 mL), diluted with EtOAc (20 mL), and washed
with satd aq NaCl (2 ꢀ 30 mL). The organic fraction was dried
over Na2SO4, concentrated in vacuo, and purified by passing the
residue through a column of Sephadex LH-20 (eluted with
DCM/MeOH, 1/1, v/v) followed by column chromatography
(silica gel, 25% EtOAc in PE) to afford the title compound as a
colorless oil (107 mg, 75 μmol, 65%): TLC Rf 0.47 (PE/EtOAc,
61.4 (C-600), 60.9 (C-20), 54.9 (OMe), 52.5 (CH3 CO2Me);
13C-GATED (100 MHz, CDCl3) δ 99.7 (JC1 ,H1 =160 Hz, C-1 ),
0
0
0
00
00
00
97.8 (JC1,H1 = 169 Hz, C-1), 97.6 (JC1 ,H1 = 167 Hz, C-1 );
HRMS [M þ Na]þ calcd for C69H75N3O16Na 1224.50395,
found 1224.50511.
Methyl 6-O-(Methyl 4-O-[6-O-[methyl 4-O-(2,3,4-tri-O-ben-
zyl-6-O-[9-fluorenylmethoxycarbonyl]-r-D-glucopyranosyl)-2-azido-
3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate]-2,3,4-tri-O-benzyl-
r-D-glucopyranosyl]-2-azido-3-O-benzyl-2-deoxy-β-D-mannopy-
ranosyl uronate)-2,3,4-tri-O-benzyl-r-D-glucopyranoside (19). Disac-
charide 16 (214 mg, 0.2 mmol), Ph2SO (53 mg, 0.26 mmol), and
2/1, v/v); [R]20 þ39.8 (c 1, DCM); IR (neat, cm-1) 698, 739,
D
1028, 1072, 1257, 1749, 2110, 2910; 1H NMR (CDCl3, 400 MHz,
HH-COSY, HSQC) δ 7.74 (d, 2H, J=7.5 Hz, CHarom Fmoc),
7998 J. Org. Chem. Vol. 75, No. 23, 2010

Walvoort et al.
JOCFeatured Article
J = 9.1 Hz, H-3Glc, H-6Glc), 3.90 (bt, 2H, J = 9.5 Hz, H-3Glc,
TTBP (124 mg, 0.5 mmol) were coevaporated with dry toluene (2ꢀ),
dissolved in freshly distilled DCM (2.3 mL), and cooled to -70 °C.
Tf2O (37 μL, 0.22 mmol) was added, the reaction was allowed to
warm to -60 °C over 30 min and then cooled to -80 °C, and a
solution of acceptor 18 (172 mg, 0.14 mmol, coevaporated twice
with dry toluene prior to use) in distilled DCM (1 mL) was slowly
added. The reaction was allowed to stir at -80 °C overnight
(cryostat). Pyridine (0.2 mL) was added, and the mixture was
diluted with EtOAc, washed with satd aq NaCl (2ꢀ), dried over
Na2SO4, concentrated in vacuo, and purified by passing the residue
through a column of Sephadex LH-20 (eluted with DCM/MeOH,
1/1, v/v) to yield the title compound as a white foam (200 mg,
92 μmol, 65%): TLC Rf 0.26 (PE/EtOAc, 2/1, v/v); [R]20D þ36.4
(c 1, DCM); IR (neat, cm-1) 696, 727, 907, 1028, 1258, 1452, 1749,
2110; 1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC) δ 7.74 (d,
2H, J=7.6 Hz, CHarom), 7.60 (dd, 2H, J=7.5, 10.8 Hz, CHarom),
7.19-7.40 (m, 59H, CHarom), 5.35 (d, 1H, J=3.5 Hz, H-1Glc), 5.21
(d, 1H, J=3.5 Hz, H-1Glc), 4.99 (d, 1H, J=11.1 Hz, CHH Bn), 4.98
(d, 1H, J=10.9 Hz, CHH Bn), 4.96 (d, 1H, J=12.2 Hz, CHH Bn),
4.88 (d, 1H, J=10.7 Hz, CHH Bn), 4.74-4.86 (m, 7H, CH2 Bn),
H-3Glc), 3.85 (d, 1H, J = 8.2 Hz, H-5Man), 3.79 (bd, 2H, J =
9.2 Hz, H-2Man, H-5Man), 3.73-3.76 (m, 2H, H-5Glc, H-6Glc-OH),
3.69 (s, 3H, CH3 CO2Me), 3.64 (s, 3H, CH3 CO2Me), 3.54-3.63
(m, 6H, H-2Man, H-3Man, H-3Man H-5Glc, H-6Glc-OH, H-6Glc),
3.38-3.49 (m, 7H, H-2Glc, H-2Glc, H-2Glc, H-4Glc, H-4Glc
,
H-5Glc, H-6Glc), 3.32 (t, 1H, J = 9.4 Hz, H-4Glc), 3.26 (s, 3H,
CH3 OMe), 1.93 (s, 1H, 6-OHGlc); 13C NMR (CDCl3, 100 MHz,
HSQC) δ 168.2 (CdO CO2Me), 138.6, 138.5, 138.5, 138.3,
138.1, 138.1, 138.0, 138.0, 137.9, 137.5, 137.4 (Cq Bn), 128.4,
128.3, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.3 (CHarom),
99.7, 99.7 (C-1Man), 97.7, 97.6 (C-1Glc), 82.0, 81.4, 81.2 (C-3Glc),
79.8 (C-2Glc), 79.6, 79.5, 79.4 (C-2Glc, C-2Glc, C-3Man), 78.6 (C-
3
Man), 77.7, 77.1, 76.8 (C-4Glc), 75.7, 75.5, 75.5 (CH2 Bn), 75.4
(C-5Man), 75.0 (CH2 Bn), 74.9 (C-5Man), 74.7, 74.7 (CH2 Bn),
73.7, 73.5 (C-4Man), 73.3, 73.1, 72.8, 72.1, 72.1 (CH2 Bn), 72.0,
70.9 (C-5Glc), 68.7, 67.6 (C-6Glc), 61.6 (C-6Glc-OH), 61.1, 60.3
(C-2Man), 55.0 (OMe), 52.7, 52.6 (CH3 CO2Me); 13C-HMBC
(100 MHz, CDCl3) δ 99.7 (JC1,H1=160 Hz, C-1Man), 99.7 (JC1,H1
=160 Hz, C-1Man), 97.7 (JC1,H1=168 Hz, C-1Glc), 97.7 (JC1,H1
=
4.49-4.72 (m, 12H, CH2 Bn, H-1Glc), 4.26-4.44 (m, 8H, H-1Man
,
170 Hz, C-1Glc), 97.6 (JC1,H1 =170 Hz, C-1Glc); HRMS [M þ
NH4]þ calcd for C110H122N7O26 1956.84340, found 1956.84289.
Methyl 6-O-(Methyl 4-O-[6-O-[methyl 4-O-(6-O-[methyl
4-O-[2,3,4-tri-O-benzyl-6-O-(9-fluorenylmethoxycarbonyl)-r-D-
glucopyranosyl]-2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl
uronate]-2,3,4-tri-O-benzyl-r-D-glucopyranosyl)-2-azido-3-O-
benzyl-2-deoxy-β-D-mannopyranosyl uronate]-2,3,4-tri-O-benzyl-
r-D-glucopyranosyl]-2-azido-3-O-benzyl-2-deoxy-β-D-mannopyr-
anosyl uronate)-2,3,4-tri-O-benzyl-r-D-glucopyranoside (21).
Disaccharide 16 (230 mg, 0.22 mmol), Ph2SO (43 mg, 0.22 mmol),
and TTBP (53 mg, 0.22 mmol) were coevaporated with dry
toluene (2ꢀ), dissolved in freshly distilled DCM (1.4 mL), and
cooled to -70 °C. Tf2O (35 μL, 0.21 mmol) was added, the
reaction was allowed to warm to -55 °C in 15 min and cooled
to -80 °C, and a solution of acceptor 20 (139 mg, 72 μmol,
coevaporated twice with dry toluene prior to use) in distilled
DCM (1 mL) was slowly added. The reaction was allowed to stir
at -80 °C over two nights (cryostat). Then pyridine (0.02 mL)
was added, and the mixture was diluted with EtOAc, washed
with satd aq NaCl (2ꢀ), dried over Na2SO4, concentrated
in vacuo, and purified by passing the residue through a column
of Sephadex LH-20 (eluted with DCM/MeOH, 1/1, v/v), and
subsequent flash column chromatography (silica gel, 33%
EtOAc in PE) to yield the title compound as a colorless oil
(47 mg, 16.4 μmol, 23%). Acceptor 20 was recovered in 40%
H-1Man, H-4Man, H-4Man, H-6Glc, H-6Glc, CH2 Fmoc), 4.23 (t, 1H,
J=7.5 Hz, CH Fmoc), 4.10 (d, 1H, J=9.4 Hz, H-6Glc), 3.98 (t, 2H,
J=8.8 Hz, H-3Glc, H-6Glc), 3.87-3.94 (m, 2H, H-3Glc, H-3Glc), 3.85
(d, 1H, J=8.2 Hz, H-5Man), 3.82 (m, 1H, H-2Man), 3.79 (d, 1H, J=
9.3 Hz, H-5Man), 3.72-3.77 (m, 1H, H-5Glc), 3.69 (s, 3H, CH3
CO2Me), 3.64 (bs, 5H, H-3Man, H-5Glc, CH3 CO2Me), 3.54-3.62
(m, 5H, H-2Man, H-3Man, H-4Glc, H-5Glc, H-6Glc), 3.50 (dd, 1H, J=
3.7, 10.0 Hz, H-2Glc), 3.37-3.49 (m, 4H, H-2Glc, H-2Glc, H-4Glc
,
H-6Glc), 3.32 (t, 1H, J = 9.6 Hz, H-4Glc), 3.26 (CH3 OMe); 13C
NMR (CDCl3, 100 MHz, HSQC) δ 168.2, 168.0 (CdO CO2Me),
154.9 (CdO Fmoc), 143.4, 143.2, 141.2, 141.2 (Cq Fmoc), 138.6,
138.5, 138.5, 138.4, 138.2, 138.0, 138.0, 137.9, 137.9, 137.5, 137.4 (Cq
Bn), 128.4, 128.3, 127.8, 127.7, 127.5, 127.3, 127.1 (CHarom), 125.2,
125.1, 119.9 (CHarom Fmoc), 99.7, 99.7 (C-1Man), 97.9, 97.7, 97.7 (C-
1Glc), 82.0, 81.4, 81.3 (C-3Glc), 79.9, 79.6 (C-2Glc), 79.4, 79.4 (C-2Glc,
C-3Man), 78.6 (C-3Man), 77.7 (C-4Glc), 76.9, 76.7 (C-4Glc), 75.7, 75.5,
75.4 (CH2 Bn), 75.3 (C-5Man), 75.0 (CH2 Bn), 74.9 (C-5Man), 74.7,
74.7 (CH2 Bn), 74.0, 73.6 (C-4Man), 73.3, 73.1, 72.7, 72.1, 71.8 (CH2
Bn), 70.9 (C-5Glc), 69.8 (CH2 Fmoc), 69.6, 69.5 (C-5Glc), 68.7, 67.6,
65.8 (C-6Glc), 60.8, 60.3 (C-2Man), 55.0 (OMe), 52.6, 52.6 (CH3
CO2Me), 46.6 (CH Fmoc); 13C-HMBC (150 MHz, CDCl3) δ 99.7
(JC1,H1 =161 Hz, C-1Man), 99.7 (JC1,H1 = 160 Hz, C-1Man), 97.9
JC1,H1=171 Hz, C-1Glc), 97.7 (JC1,H1=171 Hz, C-1Glc), 97.7 (JC1,H1
=168 Hz, C-1Glc); HRMS [M þ NH4]þ calcd for C125H132N7O28
2179.91484, found 2179.91016.
Methyl 6-O-(Methyl 4-O-[6-O-[methyl 4-O-(2,3,4-tri-O-benzyl-
r-D-glucopyranosyl)-2-azido-3-O-benzyl-2-deoxy-β-D-manno-
pyranosyl uronate]-2,3,4-tri-O-benzyl-r-D-glucopyranosyl]-2-
azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate)-2,3,4-
tri-O-benzyl-r-D-glucopyranoside (20). A solution of compound
19 (133 mg, 62 μmol) in dry pyridine (1.3 mL) was treated with
Et3N (0.13 mL, 0.9 mmol) at rt. After 3 h, TLC analysis indi-
cated complete consumption of the starting material, and the
reaction was diluted with EtOAc (10 mL), washed with satd aq
NaCl (2ꢀ), dried over Na2SO4, and concentrated in vacuo. Puri-
fication using flash column chromatography (silica gel, 50%
EtOAc in PE) afforded the title compound as a colorless oil (106
yield: TLC Rf 0.37 (PE/EtOAc, 2/1, v/v); [R]20 þ33.6 (c 1,
D
DCM); IR (neat, cm-1) 698, 739, 1028, 1072, 1749, 2108, 2956;
1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC, tentatively
assigned based on 1H NMR of compound 19) δ 7.76 (d, 2H, J=
7.5 Hz, CHarom), 7.62 (dd, 2H, J = 7.5, 11.4 Hz, CHarom),
7.20-7.43 (m, 79H, CHarom), 5.38 (d, 1H, J=3.5 Hz, H-1Glc),
5.33 (d, 1H, J=3.4 Hz, H-1Glc), 5.22 (d, 1H, J=3.4 Hz, H-1Glc),
5.01 (d, 1H, J=10.8 Hz, CHH Bn), 4.99 (d, 1H, J=10.6 Hz,
CHH Bn), 4.97 (d, 1H, J=10.8 Hz, CHH Bn), 4.75-4.92 (m,
10H, CH2 Bn), 4.58-4.72 (m, 15H, CH2 Bn), 4.50-4.58 (m, 3H,
CH2 Bn, H-1Glc), 4.32-4.45 (m, 7H, H-1Man, H-6Glc, H-6Glc
H-6Glc, H-6Glc, CH2 Fmoc), 4.22-4.32 (m, 6H, H-1Man
,
,
H-1Man, H-4Man, H-4Man, H-4Man, CH Fmoc), 4.11 (d, 1H,
J = 9.8 Hz, H-6Glc), 3.90 (t, 2H, J = 9.1 Hz, H-3Glc, H-6Glc),
3.82-3.95 (m, 4H, H-3Glc, H-3Glc, H-3Glc, H-5Man), 3.73-3.82
(m, 5H, H-2Man, H-2Man, H-5Glc, H-5Man, H-5Man), 3.71 (s, 3H,
CH3 CO2Me), 3.70 (s, 3H, CH3 CO2Me), 3.64 (s, 3H, CH3
mg, 55 μmol, 89%): TLC Rf 0.73 (PE/EtOAc, 1/1, v/v); [R]20
D
þ35.9 (c 1, DCM); IR (neat, cm-1) 696, 733, 1026, 1070, 1751,
2108, 2880; 1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC) δ
7.19-7.37 (m, 55H, CHarom), 5.24 (d, 1H, J=3.6 Hz, H-1Glc),
5.21 (d, 1H, J=3.5 Hz, H-1Glc), 4.98 (d, 1H, J=7.4 Hz, CHH
Bn), 4.96 (d, 1H, J=7.5 Hz, CHH Bn), 4.74-4.89 (m, 7H, CH2
Bn), 4.70 (d, 1H, J = 12.0 Hz, CHH Bn), 4.54-4.68 (m, 10H,
CH2 Bn), 4.49-4.54 (m, 3H, CH2 Bn, H-1Glc), 4.34 (s, 1H,
H-1Man), 4.21-4.30 (m, 2H, H-4Man, H-4Man), 4.24 (s, 1H,
H-1Man), 4.10 (dd, 1H, J=1.1, 10.4 Hz, H-6Glc), 3.98 (bt, 2H,
CO2Me), 3.55-3.63 (m, 7H, H-2Man, H-3Man, H-3Man, H-3Man
,
,
H-4Glc, H-5Glc, H-6Glc), 3.39-3.55 (m, 8H, H-2Glc, H-2Glc
H-2Glc, H-2Glc, H-4Glc, H-4Glc, H-5Glc, H-6Glc), 3.33 (t, 1H,
J = 9.4 Hz, H-4Glc), 3.27 (s, 3H, CH3 OMe); 13C-APT NMR
(CDCl3, 100 MHz, HSQC, tentatively assigned based on 13C
NMR of compound 19) δ 168.2, 168.2, 168.0 (CdO CO2Me),
J. Org. Chem. Vol. 75, No. 23, 2010 7999

Walvoort et al.
JOCFeatured Article
154.9 (CdO Fmoc), 143.5, 143.2, 141.2, 141.2 (Cq Fmoc), 138.6,
138.6, 138.5, 138.5, 138.4, 138.2, 138.0, 138.0, 138.0, 137.9,
137.5, 137.5, 137.4 (Cq Bn), 128.4, 127.9, 127.8, 127.7, 127.6,
127.5, 127.4, 127.3, 127.2, 127.1 (CHarom), 125.2, 125.1, 120.0
(CHarom Fmoc), 99.8, 99.7, 99.7 (C-1Man), 97.9, 97.8, 97.7, 97.5
(C-1Glc), 82.0, 81.4, 81.3 (C-3Glc), 79.8, 79.6, 79.5, 79.4, 78.6
(C-2Glc, C-3Man), 77.7, 77.2, 76.9, 76.8 (C-4Glc), 75.7, 75.6, 75.5,
75.4 (CH2 Bn), 75.3, 75.2 (C-5Man), 75.1 (CH2 Bn), 75.0 (C-
(JC1,H1 = 172 Hz, C-1Glc); HRMS [M þ NH4]þ calcd for
C
151H165N10O36 2695.14160, found 2695.13146.
General Procedure for the KOOH-Mediated Saponification.
A mixture of KOH and H2O2 was freshly prepared: aq KOH
(0.5 M, 4.86 mL, 2.5 mmol) was added to H2O2 (50 wt % in H2O,
0.28 mL, 5 mmol). A solution of the methyl uronate (1 equiv) in
THF (0.05 M) was cooled to 0 °C, and the KOH-H2O2 solution
was dropwise added. The resulting mixture was stirred at rt until
full conversion of the starting material was indicated by TLC
analysis. When an emulsion was observed, THF was dropwise
added to obtain a clear solution. The reaction was quenched by
the addition of 1 M HCl until pH ∼6. Subsequently, the mixture
was partitioned between EtOAc and H2O, the organic layer was
washed with satd aq NaCl (2ꢀ), dried over Na2SO4, and
concentrated in vacuo. The product was obtained after passing
the residue through a column of Sephadex LH-20 (eluted with
DCM/MeOH, 1/1, v/v) to remove any eliminated side products.
Methyl 6-O-(4-O-[2,3,4-Tri-O-benzyl-r-D-glucopyranosyl]-2-
azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate)-2,3,4-
tri-O-benzyl-r-D-glucopyranoside (23). Compound 18 (76 mg,
63 μmol) was saponified using the general procedure (0.25 mL of
KOH-H2O2 solution) to produce the title compound as a
colorless oil (63 mg, 53 μmol, 85%): TLC Rf 0.38 (PE/EtOAc,
5Man), 74.7, 74.7, 74.6 (CH2 Bn), 74.0, 73.6 (C-4Man), 73.4 (CH2
Bn), 73.3 (C-4Man), 73.1, 72.8, 72.7, 72.1, 71.9, 71.7 (CH2 Bn),
70.9, 70.8 (C-5Glc), 69.9 (CH2 Fmoc), 69.6, 69.5 (C-5Glc), 68.7,
67.6, 67.6, 65.9 (C-6Glc), 60.9, 60.7, 60.3 (C-2Man), 55.0 (OMe), 52.7,
52.7, 52.6 (CH3 CO2Me), 46.7 (CH Fmoc); 13C-HMBC (150 MHz,
CDCl3) δ 99.8 (JC1,H1 =161 Hz, C-1Man), 99.7 (JC1,H1 =161 Hz,
C-1Man), 99.7 (JC1,H1 = 161 Hz, C-1Man), 97.9 (JC1,H1 = 172 Hz,
C-1Glc), 97.8 (JC1,H1 = 170 Hz, C-1Glc), 97.7 (JC1,H1 = 169 Hz,
C-1Glc), 97.5 (JC1,H1=171 Hz, C-1Glc); HRMS [M þ Na]þ calcd
for C166H171N9O38Na 2922.16508, found 2922.15435.
Methyl 6-O-(Methyl 4-O-[6-O-[methyl 4-O-(6-O-[methyl 4-O-
[2,3,4-tri-O-benzyl-r-D-glucopyranosyl]-2-azido-3-O-benzyl-2-deoxy-
β-D-mannopyranosyl uronate]-2,3,4-tri-O-benzyl-r-D-glucopyranosyl)-
2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate]-2,3,-
4-tri-O-benzyl-r-D-glucopyranosyl]-2-azido-3-O-benzyl-2-deoxy-
β-D-mannopyranosyl uronate)-2,3,4-tri-O-benzyl-r-D-glucopy-
ranoside (22). Compound 21 (48 mg, 16.5 μmol) was dissolved
in dry pyridine (1 mL) followed by the addition of Et3N (8 μL,
54 μmol), and the resulting solution was stirred at rt overnight.
The mixture was diluted with EtOAc and washed with satd
aq NaCl (3ꢀ). The combined aqueous layers were extracted
with EtOAc, and the combined organic layers were dried
over Na2SO4, filtered, and concentrated in vacuo. Purification
using flash column chromatography (silica gel, 40% EtOAc in
PE) yielded the title compound as a colorless oil (35 mg,
13 μmol, 78%): TLC Rf 0.30 (PE/EtOAc, 3/2, v/v); [R]20D þ35.6
(c 1, DCM); IR (neat, cm-1) 698, 1028, 1072, 1751, 2108, 2954;
1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC) δ 7.20-7.37
(m, 75H, CHarom), 5.31 (d, 1H, J=3.4 Hz, H-1Glc), 5.24 (d, 1H,
J=3.5 Hz, H-1Glc), 5.20 (d, 1H, J=3.4 Hz, H-1Glc), 4.98 (d, 1H,
J=10.7 Hz, CHH Bn), 4.96 (d, 2H, J=10.8 Hz, CHH Bn), 4.87
(d, 2H, J=10.8 Hz, CHH Bn), 4.79-4.84 (m, 4H, CH2 Bn), 4.77
(d, 2H, J=11.8 Hz, CHH Bn), 4.53-4.69 (m, 16H, CH2 Bn),
4.49-4.53 (m, 4H, CH2 Bn, H-1Glc), 4.33 (s, 1H, H-1Man), 4.27
(s, 1H, H-1Man), 4.22-4.27 (m, 3H, H-4Man, H-4Man, H-4Man),
4.21 (s, 1H, H-1Man), 4.09 (d, 1H, J=9.4 Hz, H-6Glc), 3.88-4.01
(m, 6H, H-3Glc, H-3Glc, H-3Glc, H-3Glc, H-6Glc, H-6Glc), 3.84 (d,
1/3, v/v þ 1% AcOH); [R]20D þ30.6 (c 1, DCM); IR (neat, cm-1
)
698, 1028, 1070, 1736, 2110, 2854, 2923; 1H NMR (CDCl3, 400
MHz, HH-COSY, HSQC) δ 7.18-7.36 (m, 35H, CHarom), 5.10
(d, 1H, J=3.5 Hz, H-100), 4.97 (d, 1H, J=10.9 Hz, CHH Bn),
4.96 (d, 1H, J=10.9 Hz, CHH Bn), 4.82-4.87 (m, 2H, CH2 Bn),
4.74-4.80 (m, 3H, CH2 Bn), 4.68 (d, 1H, J=11.9 Hz, CHH Bn),
4.57-4.63 (m, 4H, CH2 Bn), 4.52-4.57 (m, 3H, CH2 Bn, H-1),
4.47 (d, 1H, J=11.4 Hz, CHH Bn), 4.42 (s, 1H, H-10), 4.33 (t,
1H, J=7.0 Hz, H-40), 3.89-4.02 (m, 4H, H-3, H-300, H-50, H-6),
3.81 (app d, 1H, J = 10.2 Hz, H-600), 3.70-3.76 (m, 2H, H-5,
H-500), 3.59-3.67 (m, 4H, H-20, H-30, H-6, H-600), 3.43-3.52 (m,
3H, H-2, H-200, H-400), 3.37 (t, 1H, J=9.4 Hz, H-4), 3.28 (s, 3H,
CH3 OMe); 13C-APT NMR (CDCl3, 100 MHz, HSQC) δ 171.0
(CdO CO2H), 138.6, 138.5, 138.1, 137.9, 137,9, 137.8, 137.3 (Cq
Bn), 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5
(CHarom), 99.7 (C-10), 98.4 (C-100), 97.8 (C-1), 81.8, 81.2 (C-3,
C-300), 79.8, 79.7 (C-2, C-200), 77.4, 77.2, 77.1 (C-30, C-4, C-400),
75.7 (C-50), 75.6, 75.5 (CH2 Bn), 75.4 (C-40), 75.1, 74.6 (CH2 Bn),
73.3, 73.0, 72.6 (CH2 Bn), 72.0 (C-500), 69.4 (C-5), 69.2 (C-6),
61.4 (C-600), 59.9 (C-20), 55.2 (OMe); 13C-GATED (CDCl3, 100
MHz) δ 99.7 (JC1,H1 = 163 Hz, C-10), 98.4 (JC1,H1 = 171 Hz,
C-100), 97.8 (JC1,H1=170 Hz, C-1); HRMS [M þ NH4]þ calcd for
C68H77N4O16 1205.53291, found 1205.53387.
1H, J=8.2 Hz, H-5Man), 3.71-3.82 (m, 6H, H-2Man, H-2Man
H-5Man, H-5Man, H-5Glc, H-6Glc), 3.69 (s, 3H, CH3 CO2Me),
3.67 (s, 3H, CH3 CO2Me), 3.63 (bs, 4H, H-6Glc, CH3 CO2Me),
3.54-3.62 (m, 7H, H-2Man, H-3Man, H-3Man, H-3Man, H-5Glc
H-6Glc, H-6Glc), 3.38-3.53 (m, 10H, H-2Glc, H-2Glc, H-2Glc
,
Methyl 6-O-(4-O-[6-O-[4-O-(2,3,4-Tri-O-benzyl-r-D-glucopy-
ranosyl)-2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate]-
2,3,4-tri-O-benzyl-r-D-glucopyranosyl]-2-azido-3-O-benzyl-2-deoxy-
β-D-matnnopyranosyl uronate)-2,3,4-tri-O-benzyl-r-D-glucopy-
ranoside (24). Compound 20 (116 mg, 60 μmol) was saponified
using the general procedure (0.36 mL KOH-H2O2 solution) to
yield the title compound as a colorless oil (96 mg, 50 μmol, 83%):
,
,
H-2Glc, H-4Glc, H-4Glc, H-4Glc, H-5Glc, H-5Glc, H-6Glc), 3.32 (t,
1H, J=9.4 Hz, H-4Glc), 3.25 (s, 3H, CH3 OMe), 1.91 (bs, 1H,
6-OHGlc); 13C-APT NMR (CDCl3, 100 MHz, HSQC) δ 168.2,
168.2 (CdO CO2Me), 138.6, 138.6, 138.6, 138.5, 138.5, 138.4,
138.2, 138.1, 138.0, 138.0, 137.9 137.6, 137.5 137.4 (Cq Bn),
128.5, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
127.5, 127.4 127.3 (CHarom), 99.8, 99.7 99.7 (C-1Man), 97.8,
97.7, 97.7, 97.5 (C-1Glc), 82.0, 81.4, 81.2 (C-3Glc), 79.9, 79.6,
79.6, 79.5, 79.4 (C-2Glc, C-3Man), 78.6 (C-3Man), 77.7, 77.2, 77.2,
76.9 (C-4Glc), 75.7, 75.5, 75.6 (CH2 Bn), 75.4, 75.2 (C-5Man), 75.0
(CH2 Bn), 75.0 (C-5Man), 74.7, 74.7, 74.6 (CH2 Bn), 73.8, 73.6
(C-4Man), 73.4 (CH2 Bn), 73.3 (C-4Man), 73.1, 72.9, 72.1 (CH2
Bn), 72.0 (C-5Glc), 71.8 (CH2 Bn), 70.9, 70.7, 69.7 (C-5Glc), 68.7,
67.6, 67.6, 61.6 (C-6Glc), 61.1, 60.7, 60.3 (C-2Man), 55.0 (OMe),
52.7, 52.7 (CH3 CO2Me); 13C-HMBC (150 MHz, CDCl3) δ 99.8
(JC1,H1=162 Hz, C-1Man), 99.7 (JC1,H1=161 Hz, C-1Man), 99.7
(JC1,H1=160 Hz, C-1Man), 97.8 (JC1,H1=170 Hz, C-1Glc), 97.7
(JC1,H1 =171 Hz, C-1Glc), 97.7 (JC1,H1 =168 Hz, C-1Glc), 97.5
TLC Rf 0.60 (PE/EtOAc, 1/3, v/v þ 5% AcOH); [R]20 þ35.9
D
(c 1, DCM); IR (neat, cm-1) 696, 731, 1026, 1067, 1742, 2108,
2955; 1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC) δ
7.10-7.38 (m, 55H, CHarom), 5.37 (s, 1H, H-1Glc), 5.19 (s, 1H,
H-1Glc), 4.90-4.99 (m, 3H, CH2 Bn), 4.73-4.85 (m, 7H, CH2
Bn), 4.43-4.68 (m, 14H, CH2 Bn, H-1Glc, H-1Man), 4.36-4.42
(m, 1H, H-4Man), 4.33 (s, 1H, H-1Man), 4.25-4.32 (m, 1H,
H-4Man), 4.05 (app d, 1H, J=6.5 Hz, H-5Man), 3.82-3.43 (m,
9H, H-3Glc, H-3Glc, H-3Glc, H-5Glc, H-5Glc, H-5Glc, H-5Man
,
H-6Glc, H-6Glc), 3.70-3.76 (m, 2H, H-6Glc, H-6Glc), 3.63-3.67
(m, 1H, H-3Man), 3.52-3.63 (m, 5H, H-2Man, H-2Man, H-3Man
,
,
H-6Glc, H-6Glc), 3.41-3.52 (m, 4H, H-2Glc, H-2Glc, H-2Glc
H-4Glc), 3.33-3.37 (m, 1H, H-4Glc), 3.28 (bs, 4H, H-4Glc, CH3
OMe); 13C NMR (CDCl3, 150 MHz, HSQC) δ 172.0, 169.7
(CdO CO2H), 138.6, 138.5, 138.2, 138.1, 138.0, 137.4, 137.4
8000 J. Org. Chem. Vol. 75, No. 23, 2010

Walvoort et al.
JOCFeatured Article
(Cq Bn), 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6,
127.4 (CHarom), 99.8, 99.5 (C-1Man), 98.7, 97.7, 97.7 (C-1Glc),
81.9, 81.3 (C-3Glc), 79.8, 79.7, 79.7 (C-2Glc), 78.1, 77.9, 77.2, 75.6,
75.5, 75.4, 74.9, 74.6, 74.6, 74.3, 73.3, 73.1, 72.8, 72.7, 72.0, 71.7,
170 Hz, C-1), 99.1 (JC1,H1 =173 Hz, C-100); HRMS [M þ H]þ
calcd for C21H36NO17 574.1978, found 574.1975.
Methyl 6-O-(4-O-[6-O-[4-O-(r-D-Glucopyranosyl)-2-acetami-
do-2-deoxy-β-D-mannopyranosyl uronate]-r-D-glucopyranosyl]-
2-acetamido-2-deoxy-β-D-mannopyranosyl uronate)-r-D-gluco-
pyranoside (29). Compound 24 (63 mg, 33 μmol) was deprotected
using the general protocol for Birch reduction and subsequent
acetylation to yield compound 29 as a white amorphous solid (13.2
mg, 11.4 μmol, 35%): IR (neat, cm-1) 1034, 1369, 1603, 3285; 1H
NMR (D2O, 600 MHz, T=280K, HH-COSY, HSQC) δ 5.34 (d,
1H, J = 3.8 Hz, H-1Glc), 5.30 (d, 1H, J = 3.7 Hz, H-1Glc), 4.77
(s, 2H, H-1Man, H-1Man), 4.68 (d, 1H, J=3.6 Hz, H-1Glc), 4.38-
4.43 (m, 2H, H-2Man, H-2Man), 4.04 (d, 1H, J=10.7 Hz, H-6Glc),
3.99-4.02 (m, 2H, H-3Man, H-3Man), 3.93 (d, 1H, J = 10.8 Hz,
70.2, 69.5, 69.0, 68.4 (CH2 Bn, C-4Glc, C-5Glc, C-3Man, C-4Man
,
C-5Man), 63.9 (C-6Glc), 61.8 (C-6Glc), 60.3, 59.7 (C-2Man), 55.1
(OMe); 13C-HMBC (CDCl3, 150 MHz) δ 99.8 (JC1,H1=162 Hz,
C-1Man), 99.57 (JC1,H1=163 Hz, C-1Man), 98.7 (JC1,H1=169 Hz,
C-1Glc), 97.7 (JC1,H1 =173 Hz, C-1Glc), 97.7 (JC1,H1 =169 Hz,
C-1Glc); HRMS [M þ NH4]þ calcd for C108H118N7O26
1929.8155, found 1929.8157.
Methyl 6-O-(4-O-[6-O-[4-O-(6-O-[4-O-[2,3,4-Tri-O-benzyl-r-D-
glucopyranosyl]-2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl
uronate]-2,3,4-tri-O-benzyl-r-D-glucopyranosyl)-2-azido-3-O-benzyl-
2-deoxy-β-D-mannopyranosyl uronate]-2,3,4-tri-O-benzyl-r-
D-glucopyranosyl]-2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl
uronate)-2,3,4-tri-O-benzyl-r-D-glucopyranoside (25).Compound 22
(35 mg, 13 μmol) was saponified using the general procedure
(0.2 mL of KOH-H2O2 solution) to yield the title compound as a
colorless oil (29 mg, 10.8 μmol, 83%). The presence of three uronic
acid moieties resulted in such broadening of the NMR signals that
accurate assignment was impossible; however, the disappearance
of the CO2Me signals was confirmed: TLC Rf 0.65 (PE/EtOAc,
1/3, v/v þ 5% AcOH); IR (neat, cm-1) 698, 1028, 1607, 2112, 3414;
HRMS [M þ NH4]þ calcd for C148H159N10O36 2653.0946, found
2653.0844.
General Procedure for the Birch Reduction and Subsequent
Acetylation. THF was distilled over Na/benzophenone prior to
use. A three-necked 50-ml round-bottom flask was equipped
with a cooling condenser (-40 °C) and a bubbler and charged
with a solution of the oligosaccharide (1 equiv) in THF (0.1 M).
A glass stir bar and t-BuOH (16 equiv) were added, and the mixture
was cooled to -65 °C. A small piece of sodium was added, and
liquid ammonia was collected (1-2 mL) by passing ammonia gas
through the system. Extra sodium was added until the solution
remained dark blue in color. The resulting mixture was stirred for
30 min while the temperature was kept below -40 °C, quenched
with satd aq NH4Cl (∼1 mL), and warmed to rt. After evaporation
of the ammonia, the mixture was concentrated in vacuo and
desalted using size-exclusion chromatography (HW40, eluted with
Et3NHOAc). The crude zwitterionic oligosaccharide was redis-
solved in H2O/THF (0.01 M, 10/1, v/v). Ac2O (5 equiv per free
amine) was added, and the pH was adjusted to ∼9 by the addition
of solid NaHCO3. After being stirred for 1 h, the mixture was
neutralized by the addition of 1 M HCl. After concentration in
vacuo, the crude product was purified by size-exclusion chroma-
tography (HW40, eluted with Et3NHOAc).
H-6Glc), 3.78-3.85 (m, 5H, H-4Man, H-4Man, H-5Man, H-5Man
H-6Glc), 3.65-3.75 (m, 4H, H-5Glc, H-6Glc, H-6Glc, H-6Glc),
3.60-3.65 (m, 1H, H-5Glc), 3.53-3.60 (m, 4H, H-3Glc, H-3Glc
,
,
H-3Glc, H-5Glc), 3.45 (dd, 1H, J=3.8, 9.9 Hz, H-2Glc), 3.42 (dd, 1H,
J=3.9, 10.0 Hz, H-2Glc), 3.38 (dd, 1H, J=4.2, 9.7 Hz, H-2Glc),
3.31-3.37 (m, 2H, H-4Glc, H-4Glc), 3.30 (s, 3H, CH3 OMe), 3.28 (t,
1H, J=9.5 Hz, H-4Glc), 2.01 (s, 3H, CH3 NHAc), 2.00 (s, 3H, CH3
NHAc); 13C-APT NMR (D2O, 150 MHz, T= 280K, HSQC) δ
176.2, 176.2, 175.6, 175.2 (CdO NHAc, CO2H), 100.5, 100.5 (C-
1
Man), 99.8, 99.2, 99.2 (C-1Glc), 77.0, 76.9 (C-5Man), 74.4, 74.3 (C-
4Man), 73.7, 73.3, 73.2 (C-3Glc), 73.0, 73.0 (C-3Man), 72.5 (C-5Glc),
72.2, 72.1, 71.9 (C-2Glc), 71.5, 71.0 (C-5Glc), 70.1, 69.7 (C-4Glc), 69.6
(C-6Glc), 69.3 (C-4Glc), 68.7, 60.5 (C-6Glc), 55.6 (OMe), 54.1, 54.1
(C-2Man), 22.8, 22.7 (CH3 NHAc); 13C-HMBC (D2O, 150 MHz,
T=280 K) δ 100.5 (JC1,H1=163 Hz, C-1Man), 100.5 (JC1,H1=163
Hz, C-1Man), 99.8 (JC1,H1=171 Hz, C-1Glc), 99.2 (JC1,H1=176 Hz,
C-1Glc), 99.2 (JC1,H1=174 Hz, C-1); HRMS [M þ H]þ calcd for
C35H57N2O28 953.30924, found 953.31039.
Methyl 6-O-(4-O-[6-O-[4-O-(6-O-[4-O-[r-D-glucopyranosyl]-
2-acetamido-2-deoxy-β-D-mannopyranosyl uronate]-r-D-glucopyr-
anosyl)-2-acetamido-2-deoxy-β-D-mannopyranosyl uronate]-r-D-
glucopyranosyl]-2-acetamido-2-deoxy-β-D-mannopyranosyl uronate)-
r-D-glucopyranoside (31). Compound 25 (32 mg, 12 μmol) was
deprotected using the general protocol for Birch reduction
and subsequent acetylation to yield compound 31 as a white
1
amorphous solid (2.8 mg, 1.7 μmol, 14%): H NMR (D2O,
600 MHz, T=280 K, HH-COSY, HSQC) δ 5.35 (d, 1H, J=
3.9 Hz, H-1Glc), 5.33 (d, 1H, J=4.1 Hz, H-1Glc), 5.32 (d, 1H, J=
4.1 Hz, H-1Glc), 4.75 (s, 1H, H-1Man), 4.73 (s, 2H, H-1Man
H-1Man), 4.67 (d, 1H, J = 3.7 Hz, H-1Glc), 4.35-4.40 (m, 3H,
H-2Man, H-2Man, H-2Man), 3.96-4.05 (m, 4H, H-3Man, H-3Man
,
,
H-3Man, H-6Glc), 3.87-3.93 (m, 2H, H-6Glc, H-6Glc), 3.83-3.87
(m, 2H, H-6Glc, H-6Glc), 3.76-3.87 (m, 3H, H-4Man, H-4Man
,
,
Methyl 6-O-(4-O-[r-D-Glucopyranosyl]-2-acetamido-2-deoxy-
β-D-mannopyranosyl uronate)-r-D-glucopyranoside (27). Com-
pound 23 (99 mg, 84 μmol) was deprotected using the general
protocol for Birch reduction and subsequent acetylation to yield
compound 27 as a white amorphous solid (24.2 mg, 36 μmol,
H-4Man), 3.63-3.75 (m, 9H, H-5Man, H-5Man, H-5Man, H-5Glc
H-5Glc, H-5Glc, H-6Glc, H-6Glc, H-6Glc), 3.53-3.63 (m, 5H,
H-3Glc, H-3Glc, H-3Glc, H-3Glc, H-5Glc), 3.43-3.47 (m, 2H,
H-2Glc, H-2Glc), 3.40 (dd, 1H, J = 4.0, 10.0 Hz, H-2Glc),
3.34-3.39 (m, 3H, H-2Glc, H-4Glc, H-4Glc), 3.31-3.33 (m, 1H,
H-4Glc), 3.30 (s, 3H, CH3 OMe), 3.28 (t, 1H, J=9.5 Hz, H-4Glc),
2.00 (s, 6H, CH3 NHAc), 1.99 (s, 3H, CH3 NHAc); 13C-APT
NMR (D2O, 150 MHz, T=280 K, HSQC) δ 176.4, 176.3, 176.3,
176.2, 176.2 (CdO NHAc, CO2H), 100.5, 100.4 (C-1Man), 99.8
(C-1Glc), 98.9, 98.9, 98.8 (C-1Glc), 77.9, 77.8, 77.8 (C-5Man), 74.1,
73.9, 73.9 (C-4Man), 73.7, 73.4, 73.3, 73.2 (C-3Man, C-3Glc), 72.3
(C-5Glc), 72.3, 72.2, 71.9 (C-2Glc), 71.3, 71.3, 71.0 (C-5Glc), 70.1,
69.8 (C-4Glc), 69.5 (C-6Glc), 69.2, 69.1 (C-4Glc), 68.6, 68.6, 60.5
(C-6Glc), 55.6 (OMe), 54.5, 54.4, 54.4 (C-2Man), 22.8, 22.7, 22.7
(CH3 NHAc); HRMS [M þ Na]þ calcd for C49H77N3O39Na
1354.4026, found 1354.4035.
43%): IR (neat, cm-1) 619, 1132, 1406, 1558, 2340, 3298; H
1
NMR (D2O, 600 MHz, T=288 K, HH-COSY, HSQC) δ 5.34
(d, 1H, J=3.9 Hz, H-100), 4.75 (s, 1H, H-10), 4.68 (d, 1H, J=3.7
Hz, H-1), 4.39 (d, 1H, J = 4.1 Hz, H-20), 4.04 (d, 1H, J =
10.2 Hz, H-6), 4.00 (dd, 1H, J= 4.3, 9.6 Hz, H-30), 3.81 (t, 1H,
J=9.6 Hz, H-40), 3.66-3.74 (m, 5H, H-5, H-50, H-6, H-600, H-600),
3.58-3.63 (m, 2H, H-300, H-500), 3.56 (t, 1H, J=9.4 Hz, H-3), 3.46
(dd, 1H, J=3.8, 9.8 Hz, H-2), 3.42 (dd, 1H, J=3.9, 9.9 Hz, H-200),
3.33 (t, 1H, J=9.8 Hz, H-400), 3.31 (s, 3H, CH3 OMe), 3.29 (t, 1H,
J=9.4 Hz, H-4), 2.00 (s, 3H, CH3 NHAc); 13C-APT NMR (D2O,
150 MHz, T=288K, HSQC) δ 176.4, 176.2 (CdO NHAc, CO2H),
100.5 (C-10), 99.9 (C-1), 99.1 (C-100), 78.0 (C-50), 74.4 (C-40), 73.8
(C-3), 73.5 (C-300), 73.3 (C-30), 72.5 (C-500), 72.4 (C-200), 72.0 (C-2),
71.1 (C-5), 70.3 (C-4), 69.9 (C-400), 69.6 (C-6), 60.7 (C-600), 55.7
(OMe), 54.4 (C-20), 22.8 (CH3 NHAc); 13C-HMBC (D2O, 150
Methyl 6-O-(4-O-[r-D-Glucopyranosyl]-2-deoxy-β-D-manno-
pyranosylurono-6,2-lactam)-r-D-glucopyranoside (33). Com-
pound 23 (13.7 mg, 11.6 μmol) was dissolved in pyridine/H2O
(2 mL, 3/1, v/v), and the resulting solution was purged with H2S
for 10 min at rt. The three-necked flask was stoppered and
MHz, T=288K) δ 100.5 (JC1,H1=163 Hz, C-10), 99.9 (JC1,H1
=
J. Org. Chem. Vol. 75, No. 23, 2010 8001

Walvoort et al.
JOCFeatured Article
stirred overnight. Then the solution was again purged with H2S
for 10 min and stirred overnight, after which time the mixture
was transferred with toluene/EtOAc, concentrated in vacuo,
and co-concentrated with toluene (3ꢀ) to remove any traces of
pyridine/H2O. Product 32 was used crude in the next reaction step.
Analytical data are reported for the crude lactam intermediate 32:
IR (neat, cm-1) 698, 1028, 1070, 1454, 1705, 2855, 2922; 1H
NMR (CDCl3, 400 MHz, HH-COSY, HSQC) δ 6.34 (bs,
1H, NH), 4.89 (m, 1H, H-1), 4.55 (m, 1H, H-100), 4.54 (m, 1H,
H-10); 13C-APT NMR (CDCl3, 100 MHz, HSQC) δ 175.6
(CdO NHCO), 98.4 (C-1), 97.7 (C-100), 97.1 (C-10), 54.5 (C-20);
13C-HMBC (CDCl3, 150 MHz) δ 98.4 (JC1,H1=166 Hz, C-1), 97.7
(JC1,H1=169 Hz, C-100), 97.1 (JC1,H1=173 Hz, C-10); HRMS [M þ
NH4]þ calcd for C68H73N1O15 1161.53185, found 1161.53286.
Compound 32 was coevaporated with toluene (2ꢀ) and trans-
ferred to a three-necked flask using freshly distilled THF (3 mL).
t-BuOH (30 μL) was added, and the solution was cooled to -60 °C.
A piece of Na was added, and liquid NH3 (∼5 mL) was collected.
When the blue color disappeared an extra piece of Na was
added. The blue solution was stirred at -50 °C for 15 min and
quenched with AcOH. After evaporation of the NH3, the
solution was transferred with H2O and concentrated in vacuo.
Purification using gel filtration (HW-40, eluted with NH4-
HCO3) afforded the title compound as a white solid (4.2 mg, 8.1
μmol, 70% over two steps): 1H NMR (D2O, 600 MHz, T=290K,
HH-COSY, HSQC) δ 5.14 (d, 1H, J=0.7 Hz, H-10), 5.08 (d, 1H,
J=3.8 Hz, H-100), 4.71 (d, 1H, J=3.8 Hz, H-1), 4.44 (d, 1H, J=1.5
Hz, H-50), 4.01 (d, 1H, J=11.1 Hz, H-6), 3.92 (s, 2H, H-20, H-30),
3.69-3.82 (m, 6H, H-40, H-5, H-500, H-6, H-600, H-600), 3.67 (t, 1H,
J=9.6 Hz, H-300), 3.58 (t, 1H, J=9.4 Hz, H-3), 3.52 (dd, 1H, J=
3.8, 9.9 Hz, H-200), 3.49 (dd, 1H, J=3.8 Hz, H-2), 3.38 (t, 1H, J=9.5
Hz, H-400), 3.33 (s, 3H, OMe), 3.28 (t, 1H, J=9.4 Hz, H-4); 13C-
APT NMR (D2O, 150 MHz, T=290K, HSQC) δ 173.0 (CdO
CONH), 100.0 (C-1), 99.3 (C-100), 98.7 (C-10), 81.9 (C-40), 76.0
(C-50), 73.8 (C-3), 73.6 (C-300), 73.2 (C-500), 72.1 (C-200),
72.0 (C-2), 71.5 (C-5), 71.2 (C-30), 70.6 (C-4), 70.1 (C-400),
68.6 (C-6), 61.1 (C-600), 56.7 (C-20), 55.8 (OMe); 13C-HMBC
(D2O, 150 MHz, T = 290 K) δ 100.0 (JC1,H1 = 170 Hz, C-1),
99.3 (JC1,H1 = 170 Hz, C-100), 98.7 (JC1,H1 = 175 Hz, C-10);51-53
HRMS [M þ NH4]þ calcd for C19H35N2O15 531.20319, found
531.20313.
Acknowledgment. We thank C. Erkelens and F. Lefeber
for their assistance with executing the NMR experiments.
This research was supported by Top Institute Pharma and
The Netherlands Organization of Scientific Research
(NWO, Vidi grant).
(51) The conformational restriction of the lactam in mannopyranoside 33
forces the ring in a boat-like conformation, resulting in a parallel orientation
of the axial C1-H1 bond with respect to the ring oxygen lone pair.52 The
large JC1,H1 coupling constant of 175 Hz of compound 33 is analogous to the
coupling constant observed with β-mannofuranosides (JC1,H1 =175 Hz),53
which also place the C1-H1 bond parallel to the ring oxygen lone pair.
(52) Kalinowski, H.-O.; Berger, S.; Braun, S. Carbon-13 NMR Spectros-
copy; John Wiley & Sons: New York, 1988; pp 508-509.
Supporting Information Available: Experimental data for
donors 1-7, spectroscopic data of all compounds, and NMR
spectra of the low-temperature experiments. This material is
available free of charge via the Internet at http://pubs.acs.org.
(53) Cyr, N.; Perlin, A. S. Can. J. Chem. 1979, 57, 2504–2511.
8002 J. Org. Chem. Vol. 75, No. 23, 2010