Studies towards a conjugate vaccine for anthrax: Synthesis of the tetrasaccharide side chain of the Bacillus anthracis exosporium

Article abstract of DOI:10.1002/hlca.200690106

The first synthesis of β-L-glycoside 17 of the tetrasaccharide β-Ant-(1 → 3)-α-L-Rhap-(1 → 3)-α-L-Rhap-(1 → 2)-L-Rhap is described (Schemes 1-3). Its spacer can be functionalized to make it amenable to conjugation to proteins by different conjugation meth

Full text of DOI:10.1002/hlca.200690106

Helvetica Chimica Acta – Vol. 89 (2006)
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Studies towards a Conjugate Vaccine for Anthrax: Synthesis of the
Tetrasaccharide Side Chain of the Bacillus anthracis Exosporium
by Roberto Adamo, Rina Saksena, and Pavol Kovácˇ*1)
NIDDK, LMC, National Institutes of Health, 8 Center Drive, Bethesda, MD 20892-0815, USA
(phone: +1301-496-3569; fax: +1301-402-0589; e-mail: kpn@helix.nih.gov)
The first synthesis of b-L-glycoside 17 of the tetrasaccharide b-Ant-(1 ! 3)-a-L-Rhap-(1 ! 3)-a-L-
Rhap-(1 ! 2)-L-Rhap is described (Schemes 1–3). Its spacer can be functionalized to make it amenable
to conjugation to proteins by different conjugation methods. The synthesis was performed in a stepwise
manner starting from the aglycon-bearing terminal saccharide with thioglycosides as glycosyl donors. To
attach the upstream terminal anthrose residue, the assembled linker-equipped trisaccharide was glycosy-
lated with ethyl 4-azido-3-O-benzyl-2-O-(bromoacetyl)-4,6-dideoxy-1-thio-b-D-glucopyranoside (11).
Further functionalization of the tetrasaccharide thus obtained, followed by deprotection gave the target
substance 17. Synthesis of substructures of 17 equipped with the same spacer, namely b-L-Rhap-1-O-
(CH2)5
A
COOMe (22), and a-L-Rhap-(1 ! 3)-a-
L-Rhap-(1 ! 2)-b-L-Rhap-1-O-(CH2)5
AHCTREUNG
Introduction. – Bacillus anthracis, the causative agent of anthrax, is a spore-forming
pathogen. One of the components of its exosporium is a collagen-like protein [1] whose
carbohydrate portion is formed by the tetrasaccharide b-Ant-(1 ! 3)-a-L-Rhap-(1 !
3)-a-L-Rhap-(1 ! 2)-L-Rhap (Fig.) [2]. Anthrose (=4,6-dideoxy-4-[(3-hydroxy-3-
methyl-1-oxobutyl)amino]-2-O-methyl-D-glucopyranose), which forms the upstream
terminal residue of the tetrasaccharide, was first discovered as a component of the exo-
sporium glycoprotein. In connection with our attempts to develop a conjugate vaccine
for anthrax, we have recently synthesized this rare sugar, as well as its methyl glycosides
[3]. Our approach to the vaccine is based on the premise that spores could be destroyed
by antibodies specific for components on their surface. Such antibodies should be pos-
sible to elicit by immunization with, for example, immunogens prepared from the
above-mentioned tetrasaccharide. Within a project aimed at immunogenic neoglyco-
conjugates, we have prepared tetrasaccharide glycoside 17, whose aglycone makes it
amenable to conjugation with suitable carriers. Here we report full details of the syn-
1
thesis of 17, together with complete assignment of H- and 13C-NMR spectra, that
were not included in our preliminary report [4].
It has been proposed [2] that the tetrasaccharide is most probably attached to the
glycoprotein through an N-acetyl-D-galactosamine linker, but the configuration of
the glycosidic linkage providing that attachment to the linker is unknown. A prerequi-
site for obtaining a potent immunogen for anti exosporium antibodies from the spacer-
This work was presented at the 13th Eurocarb, August 22–26, 2005, Bratislava, Slovakia.
1
)
© 2006 Verlag Helvetica Chimica Acta AG, Zürich

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Helvetica Chimica Acta – Vol. 89 (2006)
Figure. Structure of the tetrasaccharide side chain of the collagen-like glycoprotein of the exosporium
of Bacillus anthracis
equipped tetrasaccharide is that its anomeric configuration in the glycoconjugate is the
same as in the exosporium glycoprotein. With that information unavailable, conjugates
made from both a- and b-linked tetrasaccharides will have to be prepared and tested
for their immunogenicity. Described below is the first synthesis of tetrasaccharide 17
whose spacer is attached to the downstream b-L-rhamnosyl terminus (Schemes 1–3).
Preparation of three structural fragments of 17, namely the linker-equipped b-glyco-
sides 21–23 (Scheme 4) is also described. These compounds will be used in binding
and inhibition studies with anti exosporium antibodies. An independent synthesis of
the tetrasaccharide equipped with a different, a-linked spacer has been published [5].
Results and Discussion. – Immune response to antigens is controlled by interaction
of antigenic determinants with receptors of the complex immune system. With short-
chain antigens, such as synthetic oligosaccharides, that interaction can be, presumably,
facilitated when the oligosaccharide has sufficient translational and rotational freedom
to access active sites on immunologically active cells. That can be achieved by linking
the oligosaccharide antigen to a carrier through a flexible spacer moiety. Tetrasacchar-
ide 17, whose stepwise synthesis is presented here, is equipped with a b-L-linked C6-
chain spacer attached to the downstream terminal L-rhamnosyl residue. Depending
on chemistry of conjugation, that spacer may become further extended when it is func-
tionalized for conjugation. The stepwise synthesis of 17 started from the downstream
rhamnose residue. In this way, fragments of 17, which will be needed in connection
with the development of the vaccine, were easily accessible. The initial glycosyl
acceptor 5 was synthesized from the triflate 4 of benzyl 6-hydroxyhexanoate (3) [6]
and the stannylidene acetal 2 of 3,4-di-O-benzyl-L-rhamnose (1) [7] (Scheme 1). The
formation of the b-L-rhamnopyranosyl linkage was stereospecific [8][9], as manifested
1
[10] in the H-coupled 13C-NMR spectrum of the sole glycoside formed (J(C(1),Hꢀ
C(1))=156.4 Hz). To introduce the spacer, we preferred the use of benzyl ester 3 to
the more commonly employed analogous methyl ester [11] since triflation of 3 (! 4)
could be monitored by TLC with detection by UV light. The required, known [6] benzyl

Helvetica Chimica Acta – Vol. 89 (2006)
1077
Scheme 1
a) Bu2
N
N
ester 3 was more conveniently made from commercially available e-caprolactone
(=oxepan-2-one) by transesterification with NaOBn in BnOH. The fully assigned
1H-NMR data of 3 agreed with those reported [6], albeit not analyzed completely.
Side reactions that occurred during subsequent triflation under standard conditions
(Tf2O, pyridine, not reported in the Exper. Part) were eliminated by the use of solid
G
K2CO3 as a base [12]. In this way, pure (NMR) [4] triflate 4 was obtained from 3 in vir-
tually theoretical yield. The compound is unstable, and should be freshly prepared
before use.
Stepwise extension of the oligosaccharide chain, to give rhamnotrioside 9 was
effected by repeated NIS (N-iodosuccinimide)/AgOTf-mediated glycosylation [13]
with methyl 3-O-acetyl-2,4-di-O-benzyl-1-thio-a-L-rhamnopyranoside (6) [7], first of
5 (! 7) and then of the product of deacetylation of 7, alcohol 8 (! 9) (Scheme 2).
The a-L-linked products were obtained in high yield, as shown by the typical [10]
large J(C(1),HꢀC(1)). Complete, unambiguous assignments of the NMR spectra of 6
were now made by 2-D correlation spectroscopy (see Exper. Part).
Positioning anthrose at the upstream terminus of the desired tetrasaccharide 17
required formation of the b-D-glucosidic linkage. Because of the presence of the 2-
O-methyl group in the b-D-glucosyl residue of 17, formation of such a linkage in a
highly stereoselective manner could be problematic with a glycosyl donor derived
from anthrose. Therefore, after deacetylation of 9, the glycosyl acceptor 10 formed
was glycosylated with the glucosyl donor 11 [14], obtained by 2-O-bromoacetylation
of methyl 4-azido-3-O-benzyl-4,6-dideoxy-b-D-glucopyranoside [3] to give the fully
protected tetrasaccharide 12 (Scheme 2). The general utility of glycosyl donor 11 is
due to the presence of a protecting at group O-2 that can be selectively removed in
the presence of other acyl groups. As such, it can also be used in alternate strategies
towards analogs of 17 that involve acyl-protected intermediates.

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Helvetica Chimica Acta – Vol. 89 (2006)
Scheme 2
a) 5, NIS, AgOTf, CH2Cl2. b) MeONa, MeOH. c) 6, NIS, AgOTf, CH2Cl2. d) NIS, AgOTf, CH2Cl2.
Transformation of 12 into the target tetrasaccharide 17 was accomplished as shown
in Scheme 32). Accordingly, the protected tetrasaccharide 12 was debromoacetylated,
2
)
The aglycon spacer (CH2)5COOMe is numbered with doubly primed locants. The sugar residue bear-
A
ing the aglycon spacer is denoted by the Roman numeral I and the remaining sugar residues sequen-
tially by II–IV, as required. The terminal butanamido group is numbered with primed locants.

Helvetica Chimica Acta – Vol. 89 (2006)
1079
Scheme 3
a) MeONa, MeOH. b) MeI, Me2
C
and the formed alcohol 13 was methylated [15] (! 14). Selective reduction of the azido
function in 14 with hydrogen sulfide [16] gave amine 15, whose treatment with 3-hy-

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Helvetica Chimica Acta – Vol. 89 (2006)
droxy-3-methylbutanoic acid in the presence of HATU (=O-(1H-7-azabenzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) yielded butanamido derivative
16. Finally, hydrogenolytic debenzylation over 5% Pd/C catalyst gave the target tetra-
saccharide 17 equipped with a spacer that makes it amenable to conjugation to proteins.
Future characterization of immunogenic epitopes in tetrasaccharide 17 and its a-L-
glycoside analog [14] by carbohydrate microarray analysis will require fragments that
mimic partial structures of both tetrasaccharides. Two such compounds, glycosides 22
and 23, were obtained by catalytic debenzylation of intermediates 8 and 10 of the syn-
thesis of 17, respectively (Scheme 4).
Scheme 4
a) Bu2SnO, MeOH. b) 4, CsF, DMF. c) MeONa, MeOH. d) H2, Pd/C, 2-methoxyethanol.
A

Helvetica Chimica Acta – Vol. 89 (2006)
1081
The b-L-rhamnopyranoside 21 was conveniently synthesized from benzyl ester 20,
obtained in turn from L-rhamnose (18) and triflate 4 by b-rhamnosylation via 1,2-O-
stannylation [8][9], a method which does not require protection of the OH groups
(Scheme 4). To insure an efficient use of the unstable triflate 4, the reaction leading
to 21 was carried out with excess stannylene acetal 19. In view of the experimental sim-
plicity of this method, we deem the relatively low yield (38%) of 20 satisfactory. Com-
pound 20 was the only glycoside formed, and its b-L-configuration was confirmed by 1H-
coupled 13C-NMR spectrum (J(C(1),HꢀC(1))=155.6 Hz). The desired methyl ester 21
was readily obtained from 20 by base-catalyzed transesterification.
The 1H- and 13C-NMR spectra of 17 (Tables 1 and 2), which were interpreted with
the aid of two-dimensional techniques, fully confirmed the structure of the tetrasac-
charide. Noteworthy in the 1H-NMR spectrum ((D6)DMSO) is the remarkable similar-
ity of chemical shifts for signals associated with the anthrose residue of 17 with the cor-
responding resonances found in the spectrum of the reducing tetrasaccharide isolated
from Bacillus anthracis [2] exosporium. Also, in addition to the resonances of HꢀC(1I)
to HꢀC(1IV)2), the 1H-NMR spectrum ((D6)DMSO) of 17 showed eight signals in the
Table 1. 1H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] for Tetrasaccharide 17a)
Moietyb) HꢀC(1) or
CH2(1’’)2)
HꢀC(2), CH2(2’’) HꢀC(3) or
HꢀC(4) or
CH2(4’’)2)
HꢀC(5) or
CH2(6’’)2)
HꢀC(6)
or CH2(2’)
CH2(3’’)2)
Ic)
4.641
4.002
3.691
3.400d)
3.400
ca. 1.301d)
(J(1,2)=0.9) (J(2,3)=3.1)
(J(3,4)=9.4)
3.333d)
3.902
4.393d)
5.005
ca. 3.755d)
2.091d)
3.508
2.091
4.213
1.134d)
1.249
IIe)
4.186
(J(1,2)=1.8 (J(2,3)=3.3)
4.906
(J(3,4)=9.8) (J=10.0)
(J(5,6)=6.3)
1.028
(J(5,6)=6.2)
ca. 1.315d)
ca. 3.755d)
3.615d)
3.281d)
3.939
3.876
3.628
3.561
3.270
(J(1,2)=1.5
5.028
IIIf)
IVg)
4.285
4.002d)
3.620
(J(1,2)=1.7 (J(2,3)=3.3)
(J=10.0)
3.404d)
4.894
3.879d)
ca. 3.755d)
1.129d)
(J(1,2)=1.4
4.740
3.135
3.543
3.636
1.223
(J(5,6)=6.1)
1.067
(J(1,2)=8.0 (J(2,3)=9.2)
4.576 2.850
(J=10.0)
3.275d)
(J=10.0)
ca. 3.400d)
(J(1,2)=7.9 (J(2,3)=8.9)
Spacerh) 3.840, 3.670 1.612
3.695, 3.333 1.480
(J(5,6)=6.2)
1.422
1.313
1.612
1.480
2.400 (J=7.5)
2.287 (J=7.5)
Amido
groupi)
2.472, 2.446
2.208
a) Measured at 600 MHz at 208. Data in the first and second row for each moiety refer to measurements in
D
2O and (D6)DMSO, respectively. b) For notation of the sugar residues, see Footnote 2. c) d ((D6)DMSO)
G
5.030 (d, J=4.2, OHꢀC(3)); 4.864 (br. d, J=4.7, OHꢀC(4)). d) Coupling constant not determined. e) d
f
((D6)DMSO) 4.844 (d, J=6.4, OHꢀC(4)); 4.643 (d, J=4.7, OHꢀC(2)). ) d ((D6)DMSO) 4.704 (d,
g
J=5.3, OHꢀC(4)); 4.511 (d, J=4.5, OHꢀC(2)). ) d (D2
G
J(NH,4)=9.1, NH); 4.997 (d, J=6.1, OHꢀCH(3)); 3.525 (s, MeO). h) d (D2
i
((D6)DMSO) 3.572 (s, COOMe). ) d (D2
OHꢀC(3’)); 1.158, 1.146 (2s, 2 Me).
O) ca. 1.309, ca. 1.300 (2s, 2 Me). d ((D6)DMSO) 4.831 (s,

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Helvetica Chimica Acta – Vol. 89 (2006)
Table 2. 13C-NMR Chemical Shifts [ppm] for Tetrasaccharide 17a)
Moietyb) C(1), C(1’’), or C(2), C(2’’), or C(3), C(3’’), or C(4) or
C(5) or
C(6) or
C(1’)2)
C(2’)2)
C(3’)2)
C(4’’)2)
C(5’’)2)
C(6’’)2)
I
102.336
99.733
79.615
75.304
72.781
70.196
72.606
69.634
86.030
84.348
26.858
29.024
51.687
48.645
76.053
73.803
80.864
79.966
82.392
79.585
75.620
72.749
27.728
25.224
72.990
68.674
75.150c)
72.320e)
74.099
71.527
73.912
71.633
59.345
59.389
31.451
24.424
74.979c)
72.132e)
71.547
68.099
72.047
68.187
73.536
70.315
36.393
33.261
19.455d)
18.043
19.492
17.678
19.424d)
17.678
19.806
18.153
176.830
171.401
II
103.995
100.756
104.929
101.476
106.431
103.479
III
IVf)
Spacerg) 72.486
68.414
Amido 180.399
grouph) 173.445
a) Measured at 150 MHz at 208. Data in the first and second row for each moiety refer to measurements in
2O and (D6)DMSO, respectively. b) For the notation of the sugar residues, see Footnote 2. c) The assign-
D
ACHTREUNG
ment can be reversed. d) The assignment can be reversed. e) The assignment can be reversed.
f) MeOꢀC(2): d (D2
N
A
51.213. h) Me: d (D2
ACHTREUNG
anomeric region whose presence in the spectrum of their material was not reported by
Daubenspeck et al. [2]. Two-dimensional NMR spectra of 17 showed that these reso-
1
nances, which did not produce cross-peaks in heteronuclear H,13C contour maps,
arose from the eight OH groups present in the molecule. The attachment to their
1
respective C-atoms could be individually assigned by homonuclear H,1H correlation
spectroscopy.
This research was supported by the Intramural Research Program of the NIH, NIDDK.
Experimental Part
General. Pd/C Catalyst (5%; ESCAT 103) was a product of Engelhard Industries. HATU (=O-1H-7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) was purchased from Applied
Biosystems. Solns. in org. solvents were dried with anh. Na2SO4, and evaporated at 408/2 kPa. TLC:
for reaction monitoring, silica gel 60 coated glass slides. Column chromatography (CC): silica gel, Com-
biFlash Companion Chromatograph (Isco, Inc.); solvent mixtures less polar than those used for TLC
were used at the onset of separation. Optical rotations: unless stated otherwise at r.t. in CHCl3; Per-
kin-Elmer-341 automatic polarimeter. NMR Spectra2): Varian-Gemini or Varian-Mercury spectrometers
at 300 (1H) and 75 MHz (13C); Bruker-Avance-600 spectrometer at 600 (1H) and 150 MHz (13C); chemical
shifts d in ppm and coupling constants J in Hz; assignments by homonuclear and heteronuclear 2-D cor-
relation spectroscopy, run with the software supplied with the spectrometers. MS: liquid chromatography
electron-spray ionization (ESI-MS) with Hewlett-Packard-1100-MSD spectrometer; gas chromatogra-
phy/electron impact ionization (GC/EI-MS) with Hewlett-Packard-5898A spectrometer. Microanalyses:
some compounds tenaciously retained traces of solvents, despite exhaustive drying, and anal. figures for
C could not be obtained within ꢁ0.4%; their structures were established from the mode of synthesis and
NMR and MS data.

Helvetica Chimica Acta – Vol. 89 (2006)
1083
3,4-Di-O-benzyl-6-deoxy-L-mannopyranose (1). H2O (0.1 ml) was added to a stirred soln. of methyl
3,4-di-O-benzyl-6-deoxy-1-thio-a-L-mannopyranoside [17] (4 g, 10.7 mmol) in acetone (15 ml), followed
by NIS (2.9 g, 12.8 mmol). After 1 h (TLC (hexane/AcOEt 3 :2) monitoring), the mixture was evapo-
1
rated, and the residue subjected to CC (toluene/AcOEt 4 :1): 2.61 g (85%) of 1. H-NMR: in accord
with that of independently synthesized material [7]. 13C-NMR (75 MHz, CDCl3): 93.68 (C(1) (a));
93.55 (C(1) (b)); 81.50 (C(3) (b)); 79.89 (C(4) (a)); 79.53 (C(3) (a)); 79.43 (C(4) (b)); 75.38 (PhCH2
(b)); 75.36 (PhCH2 (b)); 72.14 (PhCH2 (a)); 72.05 (PhCH2 (b)); 71.16 (C(5) (a)); 68.88 (C(2) (b));
68.60 (C(2) (a)); 67.48 (C(5) (a)); 17.94 (C(6) (b)); 17.89 (C(6) (a)).
Benzyl 6-Hydroxyhexanoate (3). e-Caprolactone (29 g, 20 ml) was added to a soln. of Na (1 g) in
BnOH (250 ml), and the mixture was stirred at r.t. overnight. After neutralization with Amberlite IR-
120, the filtrate was concentrated, and the residue subjected to CC (CH2Cl2/MeOH 100 :1). The product
thus obtained was distilled at 1458/1.3·10ꢀ2 Torr: pure (NMR, TLC) 3 (22 g, 39%). 1H-NMR (300 MHz,
CDCl3): 5.10 (s, PhCH2); 3.57 (t, J=6.5, CH2(6)); 2.50 (br. s, OHꢀC(6)); 2.35 (t, J=7.5, CH2(2));
1.71–1.61 (m, CH2(3)); 1.59–1.50 (m, CH2(5)); 1.42–1.34 (m, CH2(4)). 13C-NMR (75 MHz, CDCl3):
173.44 (C=O); 65.94 (PhCH2); 62.10 (C(6)); 34.00 (C(2)); 32.02 (C(5)); 25.06 (C(4); 24.43 (C(3)).
HR-ESI-MS: 245.1154 ([M+Na]+; calc. 245.1151).
6-(Benzyloxy)-6-oxohexyl 3,4-Di-O-benzyl-6-deoxy-b-L-mannopyranoside (5). Trifluoromethanesul-
fonic acid anhydride (1.95 ml, 11.52 mmol) was added at ꢀ208 to a stirred mixture of 3 (2.13 g, 9.6 mmol)
and finely powdered K2CO3 (1.6 g, 11.52 mmol) in CH2Cl2 (20 ml). After 1 h at ꢀ208 (TLC (hexane/
AcOEt 4 :1) monitoring), the mixture was filtered and washed quickly with ice-cold aq. NaHCO3 soln.
and the org. phase dried and concentrated at 15–208: unstable benzyl 6-{[(trifluoromethyl)sulfonyl]-
oxy}hexanoate (4; quant.), sufficiently pure for the next step. 1H-NMR (600 MHz, CDCl3): 5.13 (s,
PhCH2); 4.51 (t, J=7.3, CH2(6)); 2.39 (t, J=7.3, CH2(2)); 1.85–1.81 (m, CH2(5)); 1.73–1.67 (m,
CH2(3)); 1.48–1.44 (m, CH2(4)). 13C-NMR (150 MHz, CDCl3): 173.15 (C=O); 118.61 (q, CF3SO2);
77.16 (C(6)); 66.33 (PhCH2); 33.84 (C(2)); 28.93 (C(5)); 24.60 (C(4); 24.11 (C(3)). GC/EI-MS: 354
(M+; calc. 354).
A mixture of 1 (1.1 g, 3.2 mmol) and dibutyloxostannane (0.95 g, 3.8 mmol) in dry toluene (5 ml) was
stirred for 2 h under reflux in a Soxhlet apparatus. After addition of CsF (0.57 g, 3.8 mmol), the mixture
was concentrated at 508/0.1 Torr. Molecular sieves (3 Å, 1 g) and MeCN (15 ml) were added to the res-
idue containing stannylene acetal 2, the suspension was cooled to 08, and triflate 4 (3.4 g, 9.6 mmol) was
added slowly with vigorous stirring, which was continued for 2 h at r.t. (TLC (toluene/acetone 4 :1) mon-
itoring). The mixture was filtered through a Celite pad, the filtrate concentrated, and the residue chroma-
1
tographed (toluene/acetone 6 :1): 5 (0.82 g, 52%). [a]D =+11.0 (c=0.6, CHCl3). H-NMR (600 MHz,
CDCl3): 5.11 (s, PhCH2OCO); 4.94 (d, J=10.9, 1 H, PhCH); 4.76 (d, J=11.9, 1 H, PhCH); 4.67 (d,
G
J=11.9, 1 H, PhCH); 4.64 (d, J=10.9, 1 H, PhCH); 4.35 (d, J(1,2)=1.0, HꢀC(1)); 4.09 (br. s, Hꢀ
C(2)); 3.89, 3.88 (2t, J=6.5, 1 H, HaꢀC(1’’)); 3.55–3.50 (m, HꢀC(4), HꢀC(3)); 3.47, 3.45 (2t, J=6.5, 1
H, HbꢀC(1’’)); 3.33–3.28 (m, HꢀC(5)); 2.41 (br. s, OHꢀC(2)); 2.36 (t, J=7.5, CH2(5’’)); 1.69–1.60
(m, CH2(4’’), CH2(2’’)); 1.41–1.36 (m, CH2(3’’)); 1.33 (d, J(5,6)=6.2, Me(6)). 13C-NMR (150 MHz,
CDCl3): 173.40 (C=O); 95.53 (J(C,H)=156.4, C(1)); 81.35 (C(3)); 79.61 (C(4)); 75.43 (PhCH2); 71.37
(C(5)); 71.27 (PhCH2); 69.30 (C(1’’)); 68.44 (C(2)); 66.05 (PhCH2OCO); 34.10 (C(5’’)); 29.10 (C(2’’));
25.51 (C(3’’)); 24.60 (C(4’’)); 17.81 (C(6)). HR-ESI-MS: 571.2676 ([M+Na]+; calc. 571.2672). Anal.
calc. for C33H40O7 (548.67): C 72.24, H 7.35; found: C 72.13, H 7.38.
Methyl 3-O-Acetyl-2,4-di-O-benzyl-6-deoxy-1-thio-a-L-mannopyranoside (6) was prepared as descri-
1
bed [18]. H-NMR (300 MHz, CDCl3): 5.16–5.12 (m, 2 H; incl. br. s at 5.15, HꢀC(1)); 5.13 (dd, J(2,
3)=3.3, J(3,4)=9.6, HꢀC(3)); 4.68 (d, J=12.2, 1 H, PhCH); 4.56 (d, J=11.4, 1 H, PhCH); 4.53 (d,
J=11.4, 1 H, PhCH); 4.50 (d, J=12.2, 1 H, PhCH); 4.13–4.04 (m, HꢀC(5)); 3.95 (dd, J(1,2)=1.9, Hꢀ
C(2)); 3.66 (t, J=9.5, HꢀC(4)); 2.10 (s, MeS); 1.95 (s, MeCO); 1.34 (d, J(5,6)=6.3, Me(6)). 13C-NMR
(75 MHz, CDCl3): 170.04 (C=O); 82.94 (C(1)); 79.21 (C(4)); 77.30 (C(2)); 74.84 (PhCH2); 73.78
(C(3)); 72.42 (PhCH2); 68.04 (C(5)); 20.97 (MeCO); 17.96 (C(6)); 13.64 (MeS).
6-(Benzyloxy)-6-oxohexyl 2-O-[3-O-Acetyl-2,4-di-O-benzyl-6-deoxy-a-L-mannopyranosyl]-3,4-di-
O-benzyl-6-deoxy-b-L-mannopyranoside (7). A mixture of 5 (2.36 g, 4.37 mmol), 6 (2.2 g, 5.25 mmol),
and 4-Å molecular sieves (1.35 g) in CH2Cl2 (30 ml) was stirred for 15 min at r.t. The mixture was cooled
to 08, and NIS (1.39 g, 6.16 mmol) was added, followed by solid AgOTf (0.45 g, 1.76 mmol). The mixture

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turned immediately red, and stirring was continued for 30 min at r.t. when TLC (hexane/AcOEt 3 :2)
showed that the reaction was complete. The mixture was filtered through a Celite pad into a separatory
funnel containing aq. NaHCO3 and Na2S2O3 soln., and the product was extracted with CH2Cl2. The com-
bined org. phase was dried and concentrated and the residue subjected to CC (hexane/AcOEt 20 :1 !
7:1): 3.23 g (82%) of 7. [a]D =+38.5 (c=0.8, CHCl3). 1H-NMR (600 MHz, CDCl3): 5.27 (dd, J(2,
3)=3.5, J(3,4)=9.5, HꢀC(3II)); 5.14 (d, J(1,2)=1.6, HꢀC(1II)); 5.09 (s, PhCH2
C
6-Methoxy-6-oxohexyl
3,4-Di-O-benzyl-2-O-(2,4-di-O-benzyl-6-deoxy-a-L-mannopyranosyl)-6-
deoxy-b-L-mannopyranoside (8). Methanolic 0.1M MeONa was added to a soln. of 7 (3.35 g, 3.65
mmol) in MeOH (50 ml) until the soln. became strongly basic. After ca. 5 h (TLC (hexane/AcOEt
3 :2) monitoring), the mixture was neutralized with Amberlite IR-120 and filtered, the filtrate concen-
trated, and the residue subjected to CC (toluene/acetone 100 :1 ! 20 :1): 2.73 g (94%) of pure 8. Color-
less oil. [a]D =+13 (c=0.6, CHCl3). 1H-NMR (600 MHz, CDCl3): 5.26 (d, J(1,2)=1.2, HꢀC(1II));
4.92–4.63 (m, 6 H, PhCH); 4.42 (d, J=11.8, 1 H, PhCH); 4.33 (d, J(1,2)=0.9, HꢀC(1I)); 4.25–4.22
(m, 2 H, HꢀC(5II); incl. d at 4.23, J=11.8, PhCH); 4.20 (br. d, HꢀC(2I)); 4.07 (ddd, J(2,3)=3.7, J(3,
OH)=J(3,4)=9.1, HꢀC(3II)); 3.86, 3.85 (2t, J=6.7, 1 H, HaꢀC(1’’)); 3.79 (dd, HꢀC(2II)); 3.62 (s,
MeOCO); 3.51 (dd, J(2,3)=2.7, J(3,4)=9.3, HꢀC(3I)); 3.44 (t, J=9.2, HꢀC(4I)); 3.39–3.37 (2t,
J=6.9, 1 H, HbꢀC(1’’)); 3.31–3.27 (m, 2 H, HꢀC(5I); incl. t at 3.30, J=9.3, HꢀC(4I)); 2.33 (d, OHꢀ
C(3II)); 2.23 (t, J=7.4, CH2(5’’)); 1.62–1.56 (m, CH2(4’’), CH2(2’’)); 1.39–1.35 (m, CH2(3’’)); 1.33 (d,
J(5,6)=6.1, Me(6I)); 1.28 (d, J(5,6)=6.3, Me(6II)). 13C-NMR (150 MHz, CDCl3): 173.98 (C=O); 99.77
(C(1II)); 97.23 (C(1I)); 82.87 (C(3I)); 82.15 (C(4II)); 80.17 (C(4I)); 78.28 (C(2II); 75.32 (PhCH2); 74.38,
72.55 (PhCH2); 72.44 (C(2I)); 72.04 (PhCH2); 71.61 (C(5I)); 71.08 (C(3II)); 69.27 (C(1’’)); 66.88
(C(5II)); 51.25 (MeOCO); 33.78 (C(5’’)); 29.24 (C(2’’)); 25.57 (C(3’’)); 24.60 (C(4’’)); 17.79, 17.78
(C(6I), C(6II)). HR-ESI-MS: 799.4048 ([M+1]+; calc. 799.4057). Anal. calc. for C47H59O11 (798.96): C
70.65, H 7.32; found: C 70.52, H 7.41.
6-Methoxy-6-oxohexyl O-3-O-Acetyl-2,4-di-O-benzyl-6-deoxy-a-L-mannopyranosyl-(1 ! 3)-O-2,4-
di-O-benzyl-6-deoxy-a-L-mannopyranosyl-(1 ! 2)-3,4-di-O-benzyl-6-deoxy-b-L-mannopyranoside (9).
As described for 7, with 8 (2.65 g, 3.31 mmol), 6 (1.66 g, 3.98 mmol), 4-Å molecular sieves (1 g),
AgOTf (0.34 g, 1.32 mmol), and NIS (1.05 g, 4.63 mmol). After ca. 30 min (TLC (toluene/AcOEt 9 :1)
monitoring), the mixture was worked up as described for 7: 3.6 g (92%) of amorphous 9. [a]D =+21.9
(c=1.2, CHCl3). 1H-NMR (600 MHz, CDCl3): 5.28 (dd, J(2,3)=3.4, J(3,4)=9.7, HꢀC(3III)); 5.18 (d,
J(1,2)=1.7, HꢀC(1II)); 5.17 (d, J(1,2)=1.6, HꢀC(1III)); 4.88 (d, J=10.9, 1 H, PhCH); 4.81 (d, J=11.9,
1 H, PhCH); 4.68–4.65 (m, 3 H, PhCH); 4.61 (d, J=10.9, 1 H, PhCH); 4.58 (d, J=11.2, 1 H, PhCH);
4.48 (d, J=12.2, 1 H, PhCH); 4.43 (d, J=12.2, 1 H, PhCH); 4.36 (d, J=12.2, 1 H, PhCH); 4.34 (br. s,
HꢀC(1I)); 4.30–4.28 (m, HꢀC(5II)); 4.23 (dd, J(2,3)=3.0, J(3,4)=9.5, HꢀC(3II)); 4.18 (dd, J(1,
2)=0.8, J(2,3)=2.6, HꢀC(2I)); 4.13 (d, J=12.2, 1 H, PhCH); 3.90, 3.88 (2t, J=6.2, 1 H, HaꢀC(1’’));
3.83 (dd, J(2,3)=2.9, HꢀC(2II)); 3.82 (dd, J(2,3)=3.2, HꢀC(2III)); 3.81–3.77 (m, HꢀC(5III)); 3.63 (s,
MeOCO); 3.59 (t, Jꢂ9.8, HꢀC(4II), HꢀC(4III)); 3.50 (dd, J(3,4)=9.3, HꢀC(3I)); 3.41 (t, J=9.3, Hꢀ
C(4I)); 3.41, 3.39 (2t, J=6.2, 1 H, HbꢀC(1’’)); 3.32–3.27 (m, HꢀC(5I)); 2.27 (t, J=7.5, CH2(5’’)); 1.91
(s, MeCO); 1.65–1.59 (m, CH2(4’’), CH2(2’’)); 1.42–1.38 (m, CH2(3’’)); 1.32 (d, J(5,6)=6.1, Me(6I));
1.24 (d, J(5,6)=6.4, Me(6II)); 1.21 (d, J(5,6)=6.1, Me(6III)). 13C-NMR (150 MHz, CDCl3): 174.07,
170.07 (C=O); 99.90 (C(1I)); 99.17 (C(1III)); 98.07 (C(1II)); 82.72 (C(3I)); 80.68 (C(4II)); 80.29 (C(4I));
79.00 (C(4III)); 77.96 (C(2II); 77.84 (C(3II)); 76.86 (C(2III)); 75.41, 74.59, 74.36 (PhCH2); 73.53 (C(3III));

Helvetica Chimica Acta – Vol. 89 (2006)
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73.21 (C(2I)); 72.57, 72.51, 71.97 (PhCH2); 71.64 (C(5I)); 69.44 (C(1’’); 67.96 (C(5II)); 67.93 (C(5III)); 51.39
(MeOCO); 33.89 (C(5’’)); 29.33 (C(2’’)); 25.63 (C(3’’)); 24.69 (C(4’’)); 21.03 (MeCO); 17.91 (C(6III));
17.87 (C(6II)); 17.71 (C(6I)). HR-ESI-MS: 1189.5505 ([M+Na]+; calc. 1189.5501). Anal. calc. for
C69H82O16 (1167.38): C 70.99, H 7.08; found: C 70.96, H 7.24.
6-Methoxy-6-oxohexyl O-2,4-Di-O-benzyl-6-deoxy-a-L-mannopyranosyl-(1 ! 3)-O-2,4-di-O-ben-
zyl-6-deoxy-a-L-mannopyranosyl-(1 ! 2)-3,4-di-O-benzyl-6-deoxy-b-L-mannopyranoside (10). As de-
A
and concentrated and the residue subjected to CC (hexane/AcOEt 10 :1 ! 5 :1): 3.45 g (96%) of syrupy
10. [a]D =+13.7 (c=0.4, CHCl3). 1H-NMR (600 MHz, CDCl3): 5.22 (br. s, HꢀC(1III)); 5.18 (d, J(1,
2)=1.6, HꢀC(1II)); 4.89–4.39 (m, 10 H, PhCH); 4.34–4.25 (m, 3 H, HꢀC(5II), PhCH; incl. d at 4.34,
J(1,2)=0.8, HꢀC(1I)); 4.24 (dd, J(2,3)=2.9, J(3,4)=9.5, HꢀC(3II)); 4.17 (d, J(2,3)=2.6, HꢀC(2I));
4.03 (d, J=11.8, 1 H, PhCH); 3.93 (ddd, HꢀC(3III)); 3.90, 3.89 (2t, J=6.4, 1 H, HaꢀC(1’’)); 3.80 (dd,
J(1,2)=1.9, HꢀC(2II)); 3.72–3.68 (m, HꢀC(5III)); 3.67 (dd, J(1,2)=1.4, J(2,3)=3.5, HꢀC(2III)); 3.64
(s, MeOCO); 3.57 (t, J=9.5, HꢀC(4II)); 3.50 (dd, J(3,4)=9.2, HꢀC(3I)); 3.41 (t, J=9.2, HꢀC(4I));
3.42, 3.39 (2t, J=6.4, 1 H, HbꢀC(1’’)); 3.31–3.28 (m, HꢀC(5I)); 3.26 (t, J=9.3, HꢀC(4III)); 2.28 (t,
J=7.4, CH2(5’’)); 2.25 (d, J(3,OH)=10.1, OHꢀC(3III)); 1.65–1.60 (m, CH2(4’’), CH2(2’’)); 1.42–1.38
(m, CH2(3’’)); 1.32 (d, J(5,6)=6.1, Me(6I)); 1.25 (d, J(5,6)=6.2, Me(6II)); 1.21 (d, J(5,6)=6.3,
Me(6III)). 13C-NMR (150 MHz, CDCl3): 174.07 (C=O); 99.93 (C(1I)); 98.30 (C(1III)); 98.15 (C(1II));
82.69 (C(3I)); 82.17 (C(4III)); 80.99 (C(4II)); 80.41 (C(4I)); 79.18 (C(2III)); 78.06 (C(2II)); 77.00 (C(3II));
75.46, 74.74, 74.35 (PhCH2); 73.31 (C(2I)); 72.61, 72.32, 72.00 (PhCH2); 71.68 (C(3III)); 71.62 (C(5I));
69.50 (C(1’’)); 67.98 (C(5II)); 67.35 (C(5III)); 51.42 (MeOCO); 33.91 (C(5’’)); 29.33 (C(2’’)); 25.63
(C(3’’)); 24.71 (C(4’’)); 17.93 (C(6II)); 17.90, (C(6I)); 17.79 (C(6III)). HR-ESI-MS: 1147.5372
([M+Na]+; calc. 1147.5395). Anal. calc. for C67H80O15 (1125.34): C 71.51, H 7.17; found: C 71.56, H 7.16.
6-Methoxy-6-oxohexyl O-4-Azido-3-O-benzyl-4,6-dideoxy-b-D-glucopyranosyl-(1 ! 3)-O-2,4-di-O-
benzyl-6-deoxy-a-L-mannopyranosyl-(1 ! 3)-O-2,4-di-O-benzyl-6-deoxy-a-L-mannopyranosyl-(1 ! 2)-
3,4-di-O-benzyl-6-deoxy-b-L-mannopyranoside (13). As described for 9, with ethyl 4-azido-3-O-benzyl-2-
O-(bromoacetyl)-4,6-dideoxy-1-thio-b-D-glucopyranoside (11) [14] (0.72 g, 1.6 mmol), 10 (2 g, 1.78
mmol), NIS (0.5 g, 2.24 mmol), and AgOTf (0.16 g, 0.64 mmol). After the usual workup, chromatography
gave 1.72 g of 12 (66%). [a]D =+17.9 (c=1, CHCl3). 1H-NMR (600 MHz, CDCl3): 5.17 (d, J(1,2)=1.8,
HꢀC(1II)); 5.11 (br. s, HꢀC(1III)); 5.04 (dd, J(1,2)=8.0, J(2,3)=9.6, HꢀC(2IV)); 4.89–4.32 (m, 16 H,
14 PhCH; incl. d at 4.64, HꢀC(1IV) and s at 4.34, HꢀC(1I)); 4.32–4.28 (m, HꢀC(5II)); 4.19 (dd, J(2,
3)=3.1, J(3,4)=9.4, HꢀC(3II)); 4.16 (d, J(2,3)=2.5, HꢀC(2I)); 4.08 (dd, J(2,3)=3.0, J(3,4)=9.6, Hꢀ
C(3III)); 3.88, 3.87 (2t, J=6.5, 1 H, HaꢀC(1’’)); 3.86 (dd, HꢀC(2II)); 3.83–3.79 (m, 2 H, HꢀC(5III);
incl. dd at 3.82, J(1,2)=1.8, HꢀC(2III)); 3.63 (s, MeOCO); 3.55 (t, J=9.5, HꢀC(4III)); 3.52–3.49 (m,
HꢀC(4II), HꢀC(3I)); 3.43 (t, J=9.2, HꢀC(4I)); 3.41, 3.39 (2t, J=6.5, 1 H, HbꢀC(1’’)); 3.37 (t, J=9.4,
HꢀC(3IV)); 3.32, 3.30 (2d, J=12.6, CH2Br)); 3.31–3.28 (m, HꢀC(5I)); 3.12 (t, J=9.5, HꢀC(4IV));
A
3.02–2.90 (m, HꢀC(5IV)); 2.25 (t, J=7.8, CH2(5’’)); 1.62–1.58 (m, CH2(4’’), CH2(2’’)); 1.40–1.36 (m,
CH2(3’’)); 1.31 (d, J(5,6)=6.1, Me(6I)); 1.23 (d, J(5,6)=6.2, Me(6II)); 1.21 (d, J(5,6)=6.2, Me(6III));
1.10 (d, J(5,6)=6.1, Me(6IV)). 13C-NMR (150 MHz, CDCl3): 174.00, 165.53 (C=O); 100.59 (J(C,
H)=160.9, C(1IV)); 99.97 (br. s, J(C,H)=171.2, C(1III)); 98.84 (J(C,H)=152.3, C(1I)); 97.90 (br. s, J(C,
H)=169.9, C(1II)); 82.69 (C(3I)); 80.98 (C(3IV)); 82.42 (C(4III)); 80.37 (C(4II)); 80.29 (C(4I)); 79.14
(C(3III)); 78.59 (C(2III), C(3II)); 78.29 (C(2II)); 75.40, 75.04, 74.93, 74.51, 74.42 (PhCH2); 73.48 (br. s,
C(2I)); 73.15, 72.57, 71.70 (PhCH2); 71.63 (C(5I)); 70.51 (C(5IV)); 69.42 (C(1’’)); 68.14 (C(5III)); 67.72
(C(5II)); 67.63 (C(4IV)); 51.40 (MeOCO); 33.87 (C(5’’)); 29.31 (C(2’’)); 25.63 (C(3’’)); 25.22 (CH2Br);
24.70 (C(4’’)); 18.15 (C(6IV)); 17.86, 17.83, 17.79 (C(6I), C(6II), C(6III)). HR-ESI-MS: 1528.5813
([M+Na]+; calc. 1528.5719), 1506.5923 ([M+1]+; calc. 1506.5900).
As described for 6, the fully protected 12 (1.47 g, 1.39 mmol) was treated with 0.1M MeONa for 1 h
(TLC (toluene/acetone 9 :1) monitoring). CC (toluene/acetone 30 :1) gave amorphous 13 (1.2 g, 91%).
1
[a]D =+40 (c=0.4, CHCl3). H-NMR (600 MHz, CDCl3): 5.18 (d, J(1,2)=1.8, HꢀC(1II)); 5.10 (br. s,
HꢀC(1III)); 4.90–4.40 (m, 13 H, PhCH); 4.34–4.32 (m, 3 H, PhCH; incl. d at 4.34, J(1,2)=0.8, Hꢀ
C(1I), and d at 4.33, J(1,2)=8.0, HꢀC(1IV)); 4.27–4.22 (m, 1 H, HꢀC(5II)); 4.19–4.16 (m, HꢀC(3II),
HꢀC(2I)); 4.04 (dd, J(2,3)=3.1, J(3,4)=9.5, HꢀC(3III)); 3.89, 3.87 (2t, J=6.3, 1 H, HaꢀC(1’’));
3.84–3.83 (m, HꢀC(2II), HꢀC(2III)); 3.81–3.76 (m, HꢀC(5III)); 3.63 (s, MeOCO); 3.61 (t, J=9.6, Hꢀ

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Helvetica Chimica Acta – Vol. 89 (2006)
C(4III)); 3.52–3.48 (m, HꢀC(3I), HꢀC(2IV), HꢀC(4II)); 3.43 (t, J=9.2, HꢀC(4I)); 3.41, 3.39 (2t, J=6.3, 1
H, HbꢀC(1’’)); 3.32–3.27 (m, 2 H, HꢀC(5I); incl. t at 3.30, J=9.2, HꢀC(3IV)); 3.04 (t, J=9.2, HꢀC(4IV));
3.03–2.98 (m, HꢀC(5IV)); 2.70 (br. s, OHꢀC(2IV)); 2.25 (t, J=7.8, HꢀCH2(5’’)); 1.61–1.58 (m, 4 H,
CH2(4’’), CH2(2’’)); 1.38–1.33 (m, CH2(3’’)); 1.32 (d, J(5,6)=6.1, Me(6I)); 1.24 (d, J(5,6)=6.3,
Me(6III)); 1.20 (d, J(5,6)=6.2, Me(6II)); 1.14 (d, J(5,6)=5.8, Me(6IV)). 13C-NMR (150 MHz, CDCl3):
174.07 (C=O); 103.67 (C(1IV)); 99.89 (C(1I)); 99.77 (br. s, C(1III)); 97.88 (br. s, C(1II)); 82.72 (C(3I));
82.01 (C(3IV)); 80.65 (C(4III)); 80.44 (C(4II)); 80.40 (C(4I)); 80.08 (C(3III)); 78.31 (C(2II), C(2III)); 78.15
(C(3II)); 75.73 (C(2IV)); 75.47, 75.16, 74.75, 74.41 (PhCH2); 73.40 (br. s, C(2I)); 73.04 , 72.63, 71.78
(PhCH2); 71.68 (C(5I)); 70.61 (C(5IV)); 69.49 (C(1’’)); 68.28 (C(5III)); 67.76 (C(5II)); 67.00 (C(4IV));
51.40 (MeOCO); 33.87 (C(5’’)); 29.30 (C(2’’)); 25.61 (C(3’’)); 24.68 (C(4’’)); 18.46 (C(6IV)); 18.04
(C(6III)); 17.87 (C(6II)); 17.80 (C(6I)). HR-ESI-MS: 1408.6567 ([M+Na]+; calc. 1408.6508). Anal. calc.
for C80H95N3O18 (1386.62): C 69.29, H 6.91, N 3.03; found: C 69.44, H 7.04, N 3.05.
6-Methoxy-6-oxohexyl O-4-Azido-3-O-benzy-4,6-dideoxy-2-O-methyl-b-D-glucopyranosyl-(1 ! 3)-
O-2,4-di-O-benzyl-6-deoxy-a-L-mannopyranosyl-(1 ! 3)-O-2,4-di-O-benzyl-6-deoxy-a-L-mannopyrano-
syl-(1 ! 2)-3,4-di-O-benzyl-6-deoxy-b-L-mannopyranoside (14). A soln. of 13 (440 mg, 0.32 mmol) in 1,2-
dimethoxyethane (10 ml) was treated with MeI (18 ml, 288 mmol) and Ag2O (16.9 g, 65.8 mmol) in the
presence of a cat. amount of Me2S [15]. After stirring for 24 h at r.t. (TLC (toluene/acetone 10 :1) mon-
G
itoring), the mixture was filtered, the solids were washed with toluene, the filtrate was concentrated, and
the residue subjected to CC (toluene/acetone 1:1): syrupy 14 (400 mg, 90%). [a]D =+28.9 (c=0.4,
CHCl3). 1H-NMR (600 MHz, CDCl3): 5.16 (d, J(1,2)=1.7, HꢀC(1II)); 5.12 (br. s, HꢀC(1III));
4.96–4.36 (m, 15 H, 14 PhCH; incl. d at 4.58, J(1,2)=8.0, HꢀC(1IV)); 4.33 (d, J(1,2)=0.8, HꢀC(1I));
4.28–4.24 (m, HꢀC(5II)); 4.20 (dd, J(2,3)=3.1, J(3,4)=9.4, HꢀC(3II)); 4.16 (d, J(2,3)=2.3, HꢀC(2I));
4.10 (dd, J(2,3)=3.1, J(3,4)=9.5, HꢀC(3III)); 3.89, 3.88 (2t, J=6.9, 1 H, HaꢀC(1’’)); 3.87–3.80 (m, Hꢀ
C(2II), HꢀC(2III), HꢀC(5III)); 3.63 (s, 2 MeO); 3.61 (t, J=9.5, HꢀC(4III)); 3.54–3.49 (m, 2 H, Hꢀ
C(4II); incl. dd at 3.50, J(3,4)=9.3, HꢀC(3I)); 3.43 (t, J=9.2, HꢀC(4I)); 3.40, 3.38 (2t, J=6.9, 1 H,
HbꢀC(1’’)); 3.33–3.28 (m, 2 H, HꢀC(5I), incl. t at 3.31, J=9.2, HꢀC(3IV)); 3.11 (dd, J(2,3)=9.0, Hꢀ
C(2IV)); 3.02 (t, J=9.8, HꢀC(4IV)); 2.95–2.99 (m, HꢀC(5IV)); 2.26 (t, J=7.8, HꢀCH2(5’’)); 1.64–1.57
(m, CH2(4’’), CH2(2’’)); 1.41–1.36 (m, CH2(3’’)); 1.33 (d, J(5,6)=6.2, Me(6I)); 1.27 (d, J(5,6)=6.0,
Me(6III)); 1.21 (d, J(5,6)=6.2, Me(6II)); 1.07 (d, J(5,6)=5.9, Me(6IV)). 13C-NMR (150 MHz, CDCl3):
174.03 (C=O); 103.44 (C(1IV)); 100.03 (br. s, C(1III)); 99.84 (C(1I)); 98.04 (C(1II)); 84.66 (C(2IV));
82.68 (C(3I)); 82.59 (C(3IV)); 80.85 (C(4III)); 80.44 (C(4II)); 80.33 (C(4I)); 79.28 (C(2III)); 78.55
(C(3III)); 78.25 (C(2II)); 78.21 (C(3II)); 75.42, 75.32, 74.45 (PhCH2); 73.44 (br. s, C(2I)); 73.15, 72.53,
71.81 (PhCH2); 71.64 (C(5I)); 70.08 (C(5IV)); 69.41 (C(1’’)); 68.12 (C(5III)); 67.79 (C(5II)); 67.50
(C(4IV)); 60.04 (MeO); 51.37 (MeOCO); 33.87 (C(5’’)); 29.28 (C(2’’)); 25.60 (C(3’’)); 24.67 (C(4’’));
17.98 (C(6IV)); 17.92 (C(6III)); 17.83 (C(6II)); 17.76 (C(6IV)). HR-ESI-MS: 1422.6680 ([M+Na]+; calc.
1422.6665). Anal. calc. for C81H97N3O18 (1400.65): C 69.46, H 6.98, N 3.00; found: C 69.74, H 7.04, N 2.94.
6-Methoxy-6-oxohexyl O-3-O-Benzyl-4,6-dideoxy-4-[(3-hydroxy-3-methyl-1-oxobutyl)amino]-2-O-
methyl-b-D-glucopyranosyl-(1 ! 3)-O-2,4-di-O-benzyl-6-deoxy-a-L-mannopyranosyl-(1 ! 3)-O-2,4-di-
O-benzyl-6-deoxy-a-L-mannopyranosyl-(1 ! 2)-3,4-di-O-benzyl-6-deoxy-b-L-mannopyranoside
(16).
H2O (ca. 4 ml) was added to a mixture of 14 (440 mg, 0.31 mmol) and pyridine (16 ml) until slight turbid-
ity, followed by a few drops of pyridine until a clear soln. was formed. A slow stream of H2S gas was
passed through this soln. for 0.5 h. The mixture was stirred overnight at 408 (TLC (hexane/AcOEt
2 :1) monitoring). After concentration, the residue was subjected to CC (hexane/AcOEt 3 :1 ! 2 :5):
pure (TLC, NMR) 15 (310 mg, 72%). 1H-NMR (600 MHz, CDCl3): 5.15 (d, J(1,2)=1.7, HꢀC(1II));
5.13 (br. s, HꢀC(1III)); 4.98 (d, J=10.5, 1 H, PhCH); 4.97 (d, J=11.3, 1 H, PhCH); 4.88 (d, J=10.8, 1
H, PhCH); 4.71 (d, J=11.5, 1 H, PhCH); 4.68–4.37 (m, 11 H, 10 PhCH; incl. d at 4.67, J(1,2)ꢂ9.8,
HꢀC(1IV)); 4.32 (br. s, HꢀC(1I)); 4.27–4.23 (m, HꢀC(5II)); 4.19 (dd, J(2,3)=3.1, J(3,4)=9.2, Hꢀ
C(3II)); 4.15 (dd, J(2,3)=2.5, HꢀC(3III)); 4.12 (br. s, HꢀC(2I)); 3.90 (dd, J(1,2)=1.7, HꢀC(2III)); 3.88,
3.86 (2t, J=6.5, 1 H, HaꢀC(1’’)); 3.84 (dd, HꢀC(2II)); 3.83–3.79 (m, HꢀC(5III)); 3.65 (s, MeO); 3.63
(s, MeOCO); 3.62 (t, J=9.5, HꢀC(4III)); 3.54–3.51 (m, HꢀC(4II)); 3.49 (dd, J(2,3)=2.5, J(3,4)=9.4,
HꢀC(3I)); 3.42 (t, J=9.3, HꢀC(4I)); 3.39–3.37 (2t, J=6.5, 1 H, HbꢀC(1’’)); 3.30–3.26 (m, HꢀC(5I));
3.14–3.11 (m, HꢀC(2IV), HꢀC(3IV)); 3.05–3.01 (m, HꢀC(5IV)); 2.48–2.45 (m, HꢀC(4IV)); 2.26 (t,
J=7.8, CH2(5’’)); 1.65–1.58 (m, CH2(4’’), CH2(2’’)); 1.40–1.36 (m, CH2(3’’)); 1.31 (d, J(5,6)=6.1,

Helvetica Chimica Acta – Vol. 89 (2006)
1087
Me(6I)); 1.26 (d, J(5,6)=6.2, Me(6III)); 1.20 (d, J(5,6)=6.2, Me(6II)); 1.03 (d, J(5,6)=6.1, Me(6IV)). 13C-
NMR (150 MHz, CDCl3): 174.12 (C=O); 103.75 (C(1IV)); 100.17 (br. s, C(1III)); 99.87 (C(1I)); 98.13
(C(1II)); 85.28 (C(3IV); 83.95 (C(2II)); 82.70 (C(3I)); 80.99 (C(4III)); 80.50 (C(4II)); 80.38 (C(4I)); 79.44
(C(2III)); 78.25 (C(2II)); 78.17 (C(3III); 78.10 (C(3II)); 75.46, 74.95, 74.52, 74.43 (PhCH2); 73.52 (br. s,
C(2I)); 73.20, 72.55 (PhCH2); 72.36 (C(5IV)); 71.86 (PhCH2); 71.66 (C(5I)); 69.42 (C(1’’)); 68.16
(C(5III)); 67.86 (C(5II)); 60.41 (MeO); 58.04 (C(4IV)); 51.43 (MeOCO); 33.90 (C(5’’)); 29.31 (C(2’’));
25.63 (C(3’’)); 24.68 (C(4’’)); 17.99 (C6IV)); 17.94 (C(6III)); 17.86 (C(6I)); 17.75 (C(6II)). HR-ESI-MS:
1374.6906 ([M+1]+; calc. 1374.6900).
HATU (83 mg, 0.22 mmol) followed by iPr2
NEt (0.05 ml, 0.27 mmol) was added to a soln. of 15 (205
G
mg, 0.15 mmol) and 3-hydroxy-3-methylbutanoic acid (23 mg, 0.22 mmol) in CH2Cl2 (6 ml). The mixture
was stirred for 1 h at r.t., when the soln. became clear (TLC (CH2Cl2/AcOEt 4 :1) monitoring). After con-
centration, the residue was subjected to CC (CH2Cl2/MeOH 100 :1): syrupy 16 (195 mg, 89%).
[a]D =+14 (c=0.3, CHCl3). 1H-NMR (600 MHz, CDCl3): 5.35 (d, J(4,NH)=8.9, NH); 5.15 (d, J(1,
2)=1.7, HꢀC(1II)); 5.11 (br. s, HꢀC(1III)); 5.00–4.37 (m, 15 H, 14 PhCH; incl. d at 4.58, J(1,2)=8.0,
HꢀC(1IV)); 4.32 (d, J(1,2)=0.8, HꢀC(1I)); 4.27–4.23 (m, HꢀC(5II)); 4.19 (dd, J(2,3)=3.1, J(3,4)=9.4,
HꢀC(3II)); 4.15 (br. d, HꢀC(2I)); 4.11 (dd, J(2,3)=3.1, J(3,4)=9.5, HꢀC(3III)); 3.89 (dd, J(1,2)=1.8,
HꢀC(2III)); 3.88, 3.87 (2t, J=6.8, 1 H, HaꢀC(1’’)); 3.86–3.81 (m, 2 H, HꢀC(5III); incl. dd at 3.85, J(1,
2)=2.0, HꢀC(2II)); 3.65 (t, J=9.9, HꢀC(4IV)); 3.63 (s, MeOCO); 3.62 (s, MeO); 3.52–3.48 (m, 2 H,
HꢀC(4II); incl. t at 3.62, J=9.5, HꢀC(4III)); 3.49 (dd, J(2,3)=2.6, J(3,4)=9.3, HꢀC(3I)); 3.42 (t,
J=9.2, HꢀC(4I)); 3.39, 3.38 (2t, J=6.8, 1 H, HbꢀC(1’’)); 3.31–3.26 (m, 2 H, HꢀC(5I); incl. dd at ca.
3.28, HꢀC(3IV)); 3.16 (dd, J(2,3)=8.9, HꢀC(2IV)); 3.12–3.08 (m, HꢀC(5IV)); 2.26 (t, J=7.8, CH2(5’’));
2.19 (d, J=15.0, HaꢀC(1’)); 2.08 (d, J=15.0, HbꢀC(1’)); 1.63–1.57 (m, CH2(4’’), CH2(2’’)); 1.40–1.36
(m, CH2(3’’)); 1.32 (d, J(5,6)=6.1, Me(6I)); 1.27 (d, J(5,6)=6.3, Me(6III)); 1.21 (s, MeꢀC(3’)); 1.20 (d,
J(5,6)=6.3, Me(6II)); 1.18 (s, MeꢀC(3’)); 1.00 (d, J(5,6)=6.2, Me(6IV)). 13C-NMR (150 MHz, CDCl3):
174.10, 172.31 (C=O); 103.68 (C(1IV)); 100.15 (br. s, C(1III)); 99.86 (C(1I)); 98.08 (C(1II)); 84.82
(C(2IV); 82.69 (C(3I)); 80.82 (C(4III)); 80.44 (C(4II)); 80.36 (C(4I)); 80.03 (C(3IV)); 79.24 (C(2III)); 78.91
(C(3III); 78.23 (C(2II), C(3II)); 75.44 (PhCH2); 74.43 (2 PhCH2); 73.65, (PhCH2); 73.46 (br. s, C(2I));
73.27, 72.52, 71.83 (PhCH2); 71.66 (C(5I)); 70.78 (C(5I)); 69.42 (C(3’)); 69.39 (C(1’)); 68.15 (C(5III));
67.80 (C(5II)); 60.54 (MeO); 55.47 (C(4IV)); 51.43 (MeOCO); 47.68 (C(2’)); 33.92 (C(5’’)); 29.29
(C(2’’)); 29.28, 29.26 (Me2
C(3’)); 25.64 (C(3’’)); 24.71 (C(4’’)); 18.04 (C(6IV)); 17.96 (C(6III)); 17.84
U
(C(6I)); 17.78 (C(6II)). HR-ESI-MS: 1496.7227 ([M+Na]+; calc. 1496.7284). Anal. calc. for
C86H107NO20 (1474.76): C 70.04, H 7.31, N 0.95; found: C 69.83, H 7.31, N 0.85.
6-Methoxy-6-oxohexyl O-4,6-Dideoxy-4-[(3-hydroxy-3-methyl-1-oxobutyl)amino]-2-O-methyl-b-D-
glucopyranosyl-(1 ! 3)-O-6-deoxy-a-L-mannopyranoyl-(1 ! 3)-O-6-deoxy-a-L-mannopyranosyl-(1 ! 2)-
6-deoxy-b-L-mannopyranoside (17). A soln. of 16 (140 mg, 0.1 mmol) in 2-methoxyethanol (5 ml) was
added to a suspension of 5% Pd/C catalyst (140 mg) in the same solvent (5 ml), and the mixture was stir-
red overnight under H2 (TLC (CH2Cl2/MeOH 4 :1) monitoring: 1 product). After filtration through a
Celite pad, the filtrate was concentrated, and the residue subjected to CC (CH2Cl2/MeOH 4 :1). After
freeze-drying, 17 (75 mg, 94%) was obtained as a white amorphous solid. [a]D =ꢀ31 (c=0.4, H2O).
HR-ESI-MS: 866.3970 ([M+Na]+; calc. 866.3998). NMR: Tables 1 and 2.
6-Methoxy-6-oxohexyl 6-Deoxy-b-L-mannopyranoside (21). A mixture of 6-deoxy-L-mannose mono-
hydrate (1 g, 5.39 mmol) and dibutyloxostannane (1.3 g, 5.31 mmol) in dry MeOH (25 ml) was stirred for
2 h at 608 until a clear soln. was obtained. Toluene (5 ml) and CsF (1 g, 6.62 mmol) were added, and the
mixture was concentrated. The residue was dried at 508/0.3 kPa for 2 h, then dissolved in DMF (8 ml),
molecular sieves (3 Å, 0.75 g) were added, and the mixture was cooled to 08. Freshly prepared benzyl
6-{[(trifluoromethyl)sulfonyl]oxy}hexanoate (4; 1.3 g, 3.66 mmol) was added, and the mixture was stirred
at 08 for 2 h (TLC (CH2Cl2/MeOH 4 :1) monitoring of 4). The mixture was filtered and concentrated, and
the residue subjected to CC (CH2Cl2/acetone 4 :1): 0.52 g (38%) of 6-(benzyloxy)-6-oxohexyl 6-deoxy-b-
L-mannopyranoside (20). 1H-NMR (300 MHz, CHCl3): 5.11 (s, PhCH2
A
s, HꢀC(2)); 3.93, 3.90 (2t, J=6.4, 1 H, HaꢀC(1’’)); 3.53, 3.50 (2t, J=6.4, 1 H, HbꢀC(1’’)); 3.44–3.41 (m,
HꢀC(3), HꢀC(4)); 3.29–3.25 (m, HꢀC(5)); 2.52 (d, J(3,OH)=8.6, OHꢀC(3)); 2.48 (d, J(2,OH)=2.5,
OHꢀC(2)); 2.37 (t, J=7.2, CH2(5’’)); 2.33 (br. s, OHꢀC(4)); 1.73–1.58 (m, CH2(4’’), CH2(2’’));
1.45–1.35 (m, 5 H, CH2(3’’); incl. d at 1.36, J(5,6)=6.1, Me(6)). 13C-NMR (75 MHz, CHCl3): 175.82

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Helvetica Chimica Acta – Vol. 89 (2006)
(C=O); 99.46 (C(1)); 74.40 (C(3)); 73.62 (C(4)); 71.51 (C(5)); 70.83 (C(2)); 69.34 (PhCH2OCO); 66.18
(C(1’’)); 34.11 (C(5’’)); 29.03 (C(2’’)); 27.40 (C(3’’)); 24.62 (C(4’’)); 18.02 (C(6)). HR-ESI-MS: 391.1741
([M+Na]+; calc. 391.1733).
Ester 20 (0.5 g, 1.35 mmol) was dissolved in MeOH (25 ml), and the soln. was made strongly basic by
addition of 0.1M MeONa/MeOH. After stirring overnight at r.t. (TLC (CH2Cl2/acetone 4 :1) monitoring
of the UV-positive 20, formation of a single charring product) and usual workup, chromatography gave 21
1
(370 mg, 93%). M.p. 87–888 (from AcOEt). [a]D =+42.0 (c=0.3, CHCl3). H-NMR (600 MHz, CD3-
OD): 4.47 (d, J(1.2)=1.0, HꢀC(1)); 3.88–3.85 (m, HaꢀC(1’’)); 3.87 (br. d, HꢀC(2)); 3.67 (s,
MeOCO); 3.55, 3.53 (2t, J=6.7, 1 H, HbꢀC(1’’)); 3.42 (dd, J(2,3)=3.2, J(3,4)=9.4, HꢀC(3)); 3.39 (t,
J=9.3, HꢀC(4)); 3.26–3.22 (m, HꢀC(5)); 2.36 (t, J=7.4, CH2(5’’)); 1.68–1.62 (m, CH2(4’’), CH2(2’’));
1.46–1.41 (m, CH2(3’’)); 1.32 (d, J(5,6)=6.2, Me(6)). 13C-NMR (150 MHz, CD3
OD): 175.80 (C=O);
G
100.58 (J(C,H)=155.6)); 74.95 (C(3)); 73.84 (C(4)); 73.51 (C(5)); 72.49 (C(2)); 70.16 (C(1’’)); 34.66
(C(5’’)); 30.29 (C(2’’)); 26.59 (C(3’’)); 25.73 (C(4’’)); 17.98 (C(6)). HR-ESI-MS: 315.1411 ([M+Na]+;
calc. 315.1420). Anal. calc. for C13H24NO7 (292.33): C 53.41, H 8.28; found: C 53.11, H 8.27.
6-Methoxy-6-oxohexyl 6-Deoxy-2-O-[6-deoxy-a-L-mannopyranosyl]-b-L-mannopyranoside (22). As
described for 17, 8 (200 mg, 0.25 mmol) was treated with H2. The product was eluted from a small silica
gel column, mainly to remove residual catalyst. After freeze-drying, amorphous 22 (105 mg, 95%) was
1
obtained. White solid. [a]D =+66.6 (c=0.5, H2O). H-NMR (600 MHz, D2
G
C(1II)); 4.62 (d, J(1,2)=0.9, HꢀC(1I)); 4.17–4.13 (m, HꢀC(5II)); 4.08 (dd, J(2,3)=3.4, HꢀC(2II)); 4.00
(dd, J(2,3)=3.1, HꢀC(2I)); 3.85–3.81 (m, 2 H, HaꢀC(1’’); incl. dd at 3.84, J(3,4)=9.8, HꢀC(3II));
3.68–3.66 (m, 4 H, HꢀC(3I); incl. s at 3.68, MeOCO); 3.57, 3.55 (2t, J=6.5, 1 H, HbꢀC(1’’));
3.42–3.37 (m, 3 H, HꢀC(5I); incl. t at 3.41, J=9.8, HꢀC(4II) and t at 3.40, J=9.3, HꢀC(4I)); 2.40 (t,
J=7.5, CH2(5’’)); 1.64–1.57 (m, CH2(4’’), CH2(2’’)); 1.40–1.36 (m, CH2(3’’)); 1.29 (d, J(5,6)=5.9,
Me(6II)); 1.23 (d, J(5,6)=6.3, Me(6I)). 13C-NMR (150 MHz, D2
O): 181.75 (C=O); 105.55 (J(C,
A
H)=170.8, C(1II)); 103.94 (J(C,H)=158.4, C(1I)); 80.86 (C(2I)); 77.68 (C(3I)); 76.66 (C(5II)); 76.42,
76.38 (C(4I,II)); 74.49 (C(2II); 74.28 (C(3II)); 73.97 (C(1’’)); 72.80 (C(5I)); 56.39 (MeOCO); 37.92
(C(5’’)); 32.99 (C(2’’)); 29.73 (C(3’’)); 28.42 (C(4’’)); 21.03 (C(6I)); 21.01 (C(6II)). HR-ESI-MS:
461.1979 ([M+Na]+; calc. 461.1999).
6-Methoxy-6-oxohexyl O-6-Deoxy-a-L-mannopyranosyl-(1 ! 3)-O-6-deoxy-a-L-mannopyranosyl-(1
! 2)-6-deoxy-b-L-mannopyranoside (23). As described for 22, from 10 (110 mg, 0.1 mmol). Elution of
the crude product from a small silica gel column (CH2Cl2/MeOH 4 :1) and freeze-drying yielded 23
1
(55 mg, 96%). White amorphous solid. [a]D =+3.3 (c=0.4, H2O). H-NMR (600 MHz, D2
A
J(1,2)=1.7, HꢀC(1III)); 5.00 (d, J(1,2)=1.8, HꢀC(1II)); 4.63 (d, J(1,2)=0.9, HꢀC(1I)); 4.23–4.19 (m,
HꢀC(5II)); 4.18 (dd, J(2,3)=3.3, HꢀC(2III)); 4.07 (dd, J(2,3)=3.4, HꢀC(2II)); 4.00 (dd, J(2,3)=2.9,
HꢀC(2I)); 3.90 (dd, J(3,4)=9.8, HꢀC(3II)); 3.91–3.82 (m, HꢀC(3III), HꢀC(5III), HaꢀC(1’’));
3.70–3.67 (m, 4 H, HꢀC(3I); incl. s at 3.68, MeOCO); 3.58, 3.56 (2t, J=6.5, 1 H, HbꢀC(1’’)); 3.50 (t,
J=9.8, HꢀC(4II)); 3.46 (t, J=9.8, HꢀC(4III)); 3.42–3.39 (m, 2 H, HꢀC(5I); incl. t at 3.40, J=9.4, Hꢀ
C(4I)); 2.40 (t, J=7.5, CH2(5’’)); 1.65–1.58 (m, CH2(4’’), CH2(2’’)); 1.40–1.35 (m, CH2(3’’)); 1.31 (d,
J(5,6)=6.3, Me(6III)); 1.30 (d, J(5,6)=5.8, Me(6I)); 1.24 (d, J(5,6)=6.2, Me(6I)). 13C-NMR (150 MHz,
D
2O): 180.33 (C=O); 105.19 (J(C,H)=172.7, C(1III)); 103.94 (J(C,H)=173.8, C(1II)); 102.34 (J(C,
G
H)=155.0, C(1I)); 80.93 (C(3II)); 79.52 (C(2I)); 76.07 (C(3I)); 75.14 (C(5I)); 74.97 (C(4I)); 74.73
(C(4III)); 74.08 (C(4II)); 72.91 (C(2III)); 72.87 (C(2II)); 72.79 (C(3III)); 72.46 (C(1’’)); 71.84 (C(5III));
71.51 (C(5I)); 54.85 (MeOCO); 36.38 (C(5’’)); 31.44 (C(2’’)); 27.72 (C(3’’)); 26.85 (C(4’’)); 19.49
(C(6II)); 19.46 (C(6I)); 19.43 (C(6III)). HR-ESI-MS: 607.2599 ([M+Na]+; 607.2578).
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Received December, 30, 2005