Linear synthesis of the branched pentasaccharide repeats of O-antigens from Shigella flexneri 1a and 1b demonstrating the major steric hindrance associated with type-specific glucosylation

Article abstract of DOI:10.1039/c4ob01200c

Shigella flexneri serotypes 1b and 1a are Gram-negative enteroinvasive bacteria causing shigellosis in humans. The O-antigen from S. flexneri 1b is a {→2)-[3Ac/4Ac]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[2Ac]-α-l-Rhap-(1→3)-[α-d-Glcp-(1→4)]-β-d-GlcpNAc-(1→}_n branched polysaccharide ({_AcAB_AcC(E)D}_n). It is identical to that from S. flexneri 1a, except for the 2_C-acetate. A concise synthesis of the disaccharide ED, trisaccharides _AcC(E)D and C(E)D, tetrasaccharides B_AcC(E)D and BC(E)D, and pentasaccharides AB_AcC(E)D and ABC(E)D is described starting from a 2-N-acetyl-d-glucosaminide acceptor and using the imidate glycosylation chemistry. The E residue was efficiently introduced via a potent stereoselective [E + D] coupling. In contrast, harsh conditions and appropriate tuning of the donor were required for a high yielding [C + ED] glycosylation. Irrespective of the level of steric bulk at residue C, glycosylation at O-3_D of the ED acceptor generated a major change of conformation of the D residue within the obtained C(E)D trisaccharide, as attested by NMR data. Proper manipulation of the constrained C(E)D trisaccharide was necessary to proceed with the stepwise chain elongation at O-3_C of an acceptor having the 2_C-O-acetyl already in place. The protected intermediates went through a one- to three-step deprotection sequence to give the propyl glycoside targets, as portions of the O-antigens from both S. flexneri 1a and 1b. Protecting group removal was clearly associated with conformational relief, yielding oligosaccharides, for which NMR data were consistent with a ⁴C₁ conformation for the 3,4-di-O-glycosylated residue D, as in the native bacterial polymers.

Full text of DOI:10.1039/c4ob01200c

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Linear synthesis of the branched pentasaccharide
repeats of O-antigens from Shigella flexneri 1a
and 1b demonstrating the major steric hindrance
associated with type-specific glucosylation†
Cite this: Org. Biomol. Chem., 2014,
12, 7728
Jason M. Hargreaves,‡§a,b Yann Le Guen,‡a,b,c Catherine Guerreiro,a,b
Karine Descroix¶a,b and Laurence A. Mulard*a,b
Shigella flexneri serotypes 1b and 1a are Gram-negative enteroinvasive bacteria causing shigellosis in
humans. The O-antigen from S. flexneri 1b is a {→2)-[3Ac/4Ac]-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-[2Ac]-
α-L-Rhap-(1→3)-[α-D-Glcp-(1→4)]-β-D-GlcpNAc-(1→}n branched polysaccharide ({AcABAcC(E)D}n). It is
identical to that from S. flexneri 1a, except for the 2C-acetate. A concise synthesis of the disaccharide ED,
trisaccharides AcC(E)D and C(E)D, tetrasaccharides BAcC(E)D and BC(E)D, and pentasaccharides ABAcC(E)D
and ABC(E)D is described starting from a 2-N-acetyl-D-glucosaminide acceptor and using the imidate
glycosylation chemistry. The E residue was efficiently introduced via a potent stereoselective [E + D]
coupling. In contrast, harsh conditions and appropriate tuning of the donor were required for a high
yielding [C + ED] glycosylation. Irrespective of the level of steric bulk at residue C, glycosylation at O-3D
of the ED acceptor generated a major change of conformation of the D residue within the obtained C(E)D
trisaccharide, as attested by NMR data. Proper manipulation of the constrained C(E)D trisaccharide was
necessary to proceed with the stepwise chain elongation at O-3C of an acceptor having the 2C-O-acetyl
already in place. The protected intermediates went through a one- to three-step deprotection sequence
to give the propyl glycoside targets, as portions of the O-antigens from both S. flexneri 1a and 1b.
Protecting group removal was clearly associated with conformational relief, yielding oligosaccharides, for
which NMR data were consistent with a 4C1 conformation for the 3,4-di-O-glycosylated residue D, as in
the native bacterial polymers.
Received 11th June 2014,
Accepted 30th July 2014
DOI: 10.1039/c4ob01200c
www.rsc.org/obc
awareness of the burden of shigellosis in this population
boosted by the growing spread of resistant strains has accele-
Introduction
Shigella flexneri are Gram-negative enteroinvasive bacteria. As rated the search for novel vaccine strategies.2 The ongoing
the major agents of endemic shigellosis – an acute rectocolitis development of synthetic carbohydrate-based immunogens
otherwise known as bacillary dysentery – in humans, they re- targeting the most prevalent S. flexneri serotypes is part of this
present an important cause of diarrhoeal disease worldwide, effort.3 The strategy in place entails complete identification of
especially among children in developing countries.1 Renewed the protective carbohydrate epitopes, that is to say that the sac-
charide structures recognized by antibodies are protective
against infection, prior to any in vivo study.4 At least 17 S. flex-
neri serotypes and subtypes have been identified.5 They differ
by the chemical nature of the lipopolysaccharide (LPS)
embedded in their outer membrane, and in particular by the
structure of their O-antigen (O-Ag), which is the polysaccharide
component of their LPS. The latter contributes to virulence. It
aInstitut Pasteur, Unité de Chimie des Biomolécules, 28 rue du Dr Roux,
75724 Paris Cedex 15, France. E-mail: laurence.mulard@pasteur.fr;
Fax: +33 1 45 68 84 04; Tel: +33 1 40 61 38 20
bCNRS UMR3523, Institut Pasteur, 75015 Paris, France
cUniversité Paris Descartes Sorbonne Paris Cité, Institut Pasteur, 28 rue du Dr Roux,
75015 Paris, France
†Electronic supplementary information (ESI) available: The material includes is also a major target of the acquired immunity stimulated by
relevant NMR spectra (1H, DEPT, COSY and HSQC spectra) for all new
clinical infection.2
Although exceptions do exist, most S. flexneri O-Ags have a
common linear backbone defined by a tetrasaccharide unit
compounds. See DOI: 10.1039/c4ob01200c
‡These authors contributed equally.
§Present address: University of Calgary, 2500 University Drive N.W., Calgary,
comprising three L-rhamnose residues (A, B, C) and a N-acetyl-
D-glucosamine residue (D). Differences between S. flexneri
Alberta, Canada, T2N 1N4.
¶Present address: Arkema Saint-Auban, 04600 Saint-Auban, France.
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serotypes are mainly associated with the phage-encoded site repeating units of the O-Ags from SF1b and SF1a, albeit non-
selective modification of the ABCD unit with α-D-glucopyrano- O-acetylated at rhamnose A, respectively. The corresponding
syl residues (E) and/or O-acetyl groups (Ac).6,7 Moreover, 3A-O-acetyl oligosaccharides were not considered herein owing
several recently revised structures from S. flexneri O-Ags have to the known propensity for acetyl migration at all positions
outlined the frequent occurrence of non-stoichiometric within a terminal rhamnose residue.
O-acetylation in this family of bacterial polysaccharide antigens.5
Yet, the function of this substitution pattern is not clear. As
part of an ongoing study on S. flexneri O-Ag chemical diversity
in relation to antigenicity and protective epitope identification,
Results and discussion
we have previously synthesized panels of S. flexneri-related The 2C-acetate, 1,2-cis stereochemistry at the E–D linkage, and
oligosaccharides
α-D-glucosylated
at
either
OH-3A,8,9 3,4-di-O-glycosylation of the N-acetyl-D-glucosamine residue (D)
OH-4C,10,11 or OH-3B.12 Owing to their glucosylation pattern, occurring in pentasaccharide I were identified as key features
the corresponding synthetic oligosaccharides represent por- in the SF1b targets and as possible synthetic challenges. Both
tions from S. flexneri O-Ags featuring group factor 7,8, and type SF1b and SF3a O-Ags express group factor 6, which is associ-
factors II and V, respectively. Moreover, oligosaccharides ated with the 2C-Ac. Therefore, with regard to the first point,
related to the O-Ags from S. flexneri serotypes 2a (SF2a),13 3a the synthetic design was inspired from the successful strategy
(SF3a),14 and 6 (SF6)15,16 were designed so as to also encom- previously established for the synthesis of SF3a-specific oligo-
pass the O-acetylation patterns found in the natural polysac- saccharides.8,9,14 With a view to the early stage installation of
charides, when appropriate. In particular, O-acetylation at the 2C-O-acetyl group, the common precursor to rhamnoses A
position 2C, as found in SF3a, characterizes group factor 6.5
and B was protected at OH-2 with the orthogonal levulinoyl
Herein, we tackle for the first time the synthesis of frag- (Lev) ester, to ensure anchimeric assistance during glycosyla-
ments of the O-Ags from S. flexneri 1a (SF1a) and 1b (SF1b), tion. When considering the E–D linkage, a rapid literature
two relevant serotypes in the field.18–21 The two surface poly- survey evidenced that several syntheses of α-D-Glcp-(1→4)-β-D-
saccharides of interest have the α-D-Glcp-(1→4)-β-D-GlcpNAc GlcpNAc disaccharides have been reported. Original attempts
branching pattern in common (Fig. 1), a feature related to type by enzymatic routes23 were followed by the development of
factor I in the S. flexneri classification.5,22 On the one hand, diverse chemical pathways, which substantiated some limit-
the branched pentasaccharide
residue C is O-acetylated in a non-stoichiometric manner at benzyl
I
(
AcABAcC(E)D), whereby ations. For instance, a modest yield of the α-D-glucosylation of
2-acetamido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyrano-
position 2, defines the biological repeating unit of the O-Ag side was initially observed. It was tentatively explained by a
from SF1b.17 On the other hand, the branched pentasacchar- strong steric compression at the acceptor site and was success-
ide analogue II (AcABC(E)D), which has a free 2C-OH, reflects fully circumvented by the use of a 2-acetamido-3-O-acetyl-1,6-
the biological repeating unit of the O-Ag from SF1a.17 It is of anhydro-2-deoxy-β-D-glucopyranose acceptor.24 Yet, an excel-
note that residue A is O-acetylated at position 3 in a similar lent 84% yield of the α-D-glucosylation product of the afore-
non-stoichiometric extent in both the pentasaccharide repeats mentioned benzyl glucosaminide acceptor was subsequently
I and II.
reported using a 2,3,4,6-tetra-O-benzyl-D-glucopyranose precur-
In this context, we report the first synthesis of S. flexneri di- sor and different reaction conditions.25 In the same years, an
to pentasaccharides having the α-D-Glcp-(1→4)-β-D-GlcpNAc alternative route to α-D-Glcp-(1→4)-β-D-GlcpNAc glycosides was
(ED) moiety at their reducing end. All synthetic targets, includ- proposed in the context of SF1a. Azidonitration of maltal hexa-
ing the ED disaccharide, represent parts of the SF1b O-Ag. acetate, which has the α-(1→4)-linkage already in place, into
They are synthesized as their propyl glycosides. Moreover, the corresponding 2-azido-1-α/β-nitrate (47%) was the key
oligosaccharides synthesized with a free 2C-OH also exemplify step.26 More recently, the synthesis of two pentasaccharides
segments of the SF1a O-Ag. In particular, the pentasaccharides related to the O-Ag from SF1a, the aminopentyl glycosides of
ABAcC(E)D-Pr and ABC(E)D-Pr correspond to the biological segments C(E)DAB and BC(E)DA, was described.27 The con-
struction of the E–D linkage involved a sophisticated intra-
molecular glycosylation strategy. Thus, using appropriately
prearranged glycosides, the exemplified α-(1→4)-linked ED dis-
accharides were obtained stereospecifically, in a good 70%
yield. On the basis of this overview and convincing literature
data,25 we chose to reinvestigate a more straightforward route
to SF1a- and SF1b-related oligosaccharides. It features the
commercially available 2,3,4,6-tetra-O-benzyl-D-glucose as a
precursor to residue E, as already successfully exploited in the
synthesis of SF2a,28 SF3a8 and SF5a29 oligosaccharides. We
first examined the glycosylation of an orthogonally protected
Fig. 1 Structure of the biological repeating units of the O-Ags from
2-acetamido-2-deoxy-β-D-glucopyranoside acceptor (D) with easily
accessible 2,3,4,6-tetra-O-benzyl-D-glucopyranosyl donors, next
SF1b (top) and SF1a (bottom), AcABAcC(E)D (I) and AcABC(E)D (II),
respectively.17
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we examined the coupling of the corresponding ED acceptor stepwise transformation of allyl glycoside 1 into acceptor 4 was
with suitable rhamnosyl C donors, and finally the possible performed on a 5.0 g scale with an acceptable overall 71%
stepwise chain elongation strategies with rhamnoses B and A yield. We favoured this strategy for its robustness and crystal-
toward the propyl glycosides of pentasaccharides ABAcC(E)D line intermediates – 2 and 3 – when working on a 10 g scale.
and ABC(E)D.
In this case, the yield of the conversion of triol 1 into acceptor
4 reached 77%.
Glycosylation of acceptor 4 was first attempted with the
Synthesis of the ED-Pr disaccharide (11)
available perbenzylated thiophenyl glucopyranoside donor37
5
Despite possible interference of the 2-acetamido moiety
during glycosylation, allyl glycoside30 4 (Scheme 1), benzylated
at O-6 and orthogonally protected with an acetyl group at O-3,
was the precursor to the D reducing end residue common to
all targets. It was prepared from glucosamine hydrochloride
via the key triol31–34 1. Thus, regioselective protection of the
latter by treatment with benzaldehyde dimethyl acetal in the
presence of catalytic camphor-10-sulfonic acid (CSA) in MeCN
gave the partially protected35 2 (93%). Acetylation of the
remaining hydroxyl group gave the fully protected36 3 (89%)
and subsequent reductive regioselective opening of the benzy-
lidene acetal provided the 6-O-benzyl derivative30 4 (86%). The
in the presence of stoichiometric N-iodosuccinimide (NIS) in
combination with trifluoromethanesulfonic acid (TfOH) or tri-
methylsilyl trifluoromethanesulfonate (TMSOTf) as the promo-
ter. Glycosidic couplings were achieved in modest yields when
using a slight excess of donor 5 at 0 °C in 2 : 5 DCM–Et2O
(Table 1, entries 1 and 2). Under these conditions, large quan-
tities of acceptor 4 were recovered. An increase in the amount
of donor 5 to 5 equiv. resulted in an improved yield of the con-
densation (Table 1, entry 3). However, glycosylation at OH-4 of
acceptor 4 via this methodology proceeded with a maximum
observed α/β selectivity of 7 : 1. Changing donor 5 for the more
readily available glucopyranosyl trichloroacetimidate38 (TCA) 6
(Scheme 1) was the next step. The glycosylation was run in a
mixture of DCM and Et2O, the ratio of which (3 : 2 to 2 : 1) was
optimized to comply with solubility requirements on the one
hand and glycosylation stereoselectivity on the other hand.
Owing to the high donor propensity of TCA 6 as opposed to
the poor reactivity of acceptor 4, the inverse procedure39 was
applied. Pleasingly, proper tuning of the reaction conditions
(Table 1, entry 4) allowed isolation of the coupling products as
a 9 : 1 α/β mixture of diastereoisomers in a good 85% yield.
Subsequent treatment of the glycosylation product with metha-
nolic sodium methoxide gave the expected alcohol 10 in 70%
yield over two steps and the β-linked diastereoisomer 15 (7%
over two steps). This product distribution was in agreement
with the good α/β stereoselectivity of the coupling step. The
kinetics of deacetylation of the α-linked and β-linked disac-
charides differed with a faster reaction in the case of the
former. Attempts to exploit this difference were unsuccessful
and completion of the transesterification step for the two
isomers was necessary for a proper isolation of acceptor 10.
Moreover, partial conversion of donor 6 into the corres-
ponding N-glycoside40 8 could not be avoided during glycosyla-
tion. For that reason, conditions optimized for TCA 6 were
applied to the more recently disclosed N-phenyltrifluoroaceti-
midoyl (NPTFA) analogue41 (7). Advantageously, the reaction
was performed at room temperature in this case (Table 1, entry
5). These conditions were compatible with the use of an
enhanced Et2O–DCM ratio, in favour of the required α-D-gluco-
sylation. Moreover, while the separation of the α/β conden-
sation products was initially found problematic, optimized
coupling conditions using donor 7 allowed to achieve an excel-
Scheme 1 Synthesis of disaccharides 11 and 16. Reagents and con-
ditions: (a) PhCH(OMe)2, CSA, MeCN, rt, 93%; (b) Ac2O, Pyr, rt, 89%;
(c) Et3SiH, TfOH, DCM, 86%; (d) 5, NIS, DCM–Et2O, see Table 1; (e) 6,
TMSOTf, DCM–Et2O, 0 °C, 85% of 9/14 9 : 1; (f) 7, TMSOTf, DCM–Et2O, lent 76% yield of the pure α-linked disaccharide 9, as ascer-
rt, 76% for 9 and 11% for 14; (g) NaOMe, MeOH, rt, 97%; (h) route 1. H2,
Pd/C, 90% aq. EtOH, rt, 50% for 11 and 12% for 12 or route 2. H2, Pd/C,
HCl, 96% aq. EtOH, rt, 2 days, 70% for 11 or route 3. H2, Pd(OH)2/C, 96%
aq. EtOH, rt, 85% for 11; (i) Pd/C, 90% aq. EtOH, HCl, rt; ( j) MeONa,
MeOH, rt, 8% from 9/14 9 : 1; (k) H2, Pd/C, 90% aq. EtOH, HCl, rt, 67%
for 16 and 19% for 17. rt: room temperature.
tained by NMR analysis (NMR spectroscopic data for C-1E: δ =
97.3 ppm, 1JCH = 169.5 Hz). In this case, the β-linked isomer 14
1
(NMR spectroscopic data for C-1E: δ = 102.8 ppm, JCH = 162.7
Hz) was isolated in a meaningful 11% yield. These data
suggested that a good 9 : 1 α/β ratio was again reached at the
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Table 1 Attempts at α-D-glucosylation at OH-4 of acceptor 4
Entry
Donor (equiv.)
Promoter
Solvent DCM–Et2O
Temperature
9 and 14
α/β ratioc
1a
2a
3a
4b
5b
5 (1.25)
5 (1.25)
5 (5.0)
6 (1.3)
7 (1.2)
TfOH/NIS
TMSOTf/NIS
TMSOTf/NIS
TMSOTf
2 : 5
2 : 5
2 : 5
2 : 3 → 1 : 2
1 : 5 → 1 : 4
0 °C
0 °C
0 °C
−78 °C → 4 °C
rt
<51%
<44% (<81%)
<64%
85%
87%
7 : 1
7 : 1
7 : 1
9 : 1
9 : 1
TMSOTf
a Overestimated yields due to contamination with succinimide. b Use of the inverse procedure. c Calculated based on NMR data.
coupling step. Deacetylation of the major glycosylation
product 9 readily gave acceptor 10 (97%). Gratifyingly, despite
appearing somewhat less stereoselective than published pro-
cedures,25 appropriate control of the well-established imidate
chemistry42,43 allowed to reach a reproducible 70–73% isolated
yield of the α-linked disaccharide 10 over two steps. Whether
by means of crystalline TCA 6 or of the more stable NPTFA 7, it
provides a promising route with regard to multigram diastereo-
selective formation of an ED intermediate ready for chain
elongation.
Conventional hydrogenolysis of disaccharide 10, and conco-
mitant reduction of the allyl aglycon, provided the ED target
11 as its propyl glycoside (50%) following RP-MPLC purifi-
cation. This somewhat low yield was in part explained by the
isolation of a meaningful amount of the corresponding hemia-
cetal25 12 (12%), suggesting that partial degradation had
occurred. A similar, albeit more controlled, procedure was
Scheme 2 Synthesis of the C(E)D-Pr trisaccharides O-acetylated at
residue C or not. Reagents and conditions: (a) Ac2O, Pyr, rt, 91%;
(b) (i) [Ir(COD){PCH3(C6H5)2}2]+PF6−, H2, THF, rt, 2 h; (ii) I2, THF–H2O, rt,
1 h, 97%; (c) CCl3CN, DBU, DCE, rt, 1 h, 94%; (d) 10, toluene, see Table 2;
(e) H2, Pd/C, 96% aq. EtOH, HCl, rt, 2 days, 47% for 23/24/25 as a
4 : 5 : 1 mix, 23% for 26/27/28 as a complex mix; (f) MeONa, MeOH,
rt, 69%.
adopted to convert the β-linked disaccharide 15 to the propyl
glycoside isomer 16. While the yield of the expected 16
reached 67%, loss of the aglycon could not be avoided, and
hemiacetal 17 was also isolated (19%) following RP-MPLC
purification. Under the above conditions, the conversion of
disaccharide 10 into the ED-Pr disaccharide 11 was attained in
a good 70% yield.
Table 2 Attempts at α-L-rhamnosylation at OH-3D of acceptor 10
Synthesis of the AcC(E)D-Pr (23) and C(E)D-Pr (29)
trisaccharides
21
TMSOTf Reaction Yield of 22
Entry (equiv.) Temperature
(equiv.)
time (h)
(brsm)a
In an attempt to minimise the number of deprotection steps
to reach the target trisaccharides 23–25 and 29, the ortho-
gonally protected rhamnosyl donor 21, having an acetyl group
at position 2 and a para-methoxybenzyl (PMB) ether at position
3, was selected as a precursor to residue C (Scheme 2). It was
easily prepared in 8 steps from L-rhamnose monohydrate via
allyl 4-O-benzyl-3-O-para-methoxybenzyl-α-L-rhamnoside16 (18).
Acetylation of the remaining hydroxyl group gave the fully pro-
tected 19 (91%), which was converted to hemiacetal 20 follow-
ing a two-step anomeric deallylation procedure (97%). Finally,
the latter was transformed into the TCA donor 21 (94%) by
reaction with trichloroacetonitrile in the presence of catalytic
DBU (1,8-diazabicyclo[5.4.0]undec-7-ene).
1
2
3
4
2.1
1.3
1.5
1.5
−10 °C → rt
0.3
6
3
19
1.5
—
−10 °C → 40 °C 0.3
40% (68%)
43% (87%)
81% (90%)
40 °C
40 °C
0.1
0.3
a brsm: yield based on the recovered starting material 10.
performed at −10 °C to rt (Table 2, entry 1). It disappointingly
resulted in decomposition of the donor. Gratifyingly, a gradual
increase of the reaction temperature from −10 °C to 40 °C
allowed the isolation of the desired trisaccharide together with
some recovered acceptor (Table 2, entry 2). Setting the reaction
temperature to 40 °C, and diminishing the amount of promo-
ter (Table 2, entry 3) had no visible effect. Again, a large
amount of acceptor 10 was recovered. In contrast, performing
the condensation at 40 °C in the presence of 0.3 equiv.
of TMSOTf generated a rewarding 81% glycosylation yield
Attempts at the [ED + C] condensation used an established
methodology for coupling to rhamnose donors, somewhat
adapted to the inherent steric hindrance of the reactive centre.
Thus, TMSOTf-mediated glycosylation of a toluene solution
containing acceptor 10 and a two-fold excess of donor 21 was
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(Table 2, entry 4), while increasing the amount of donor led to
a diminished yield of trisaccharide 22 (not shown). Next, Pd/C-
mediated hydrogenolysis of the fully protected trisaccharide
intermediate 22 in a slightly acidic alcoholic medium furn-
ished the C(E)D-Pr target O-acetylated at rhamnose C (47%).
Owing to the propensity of acetyl moieties to migrate to vicinal
hydroxyl groups, the hydrogenolysis product was isolated fol-
lowing RP-MPLC purification as a 4 : 5 : 1 mixture of regio-
isomers 23, 24 and 25, O-acetylated at positions 2C, 3C, and 4C,
respectively (Scheme 2). As in the case of the hydrogenolysis of
disaccharides 10 and 15, degradation occurred during this
transformation. The corresponding hemiacetals 26–28 were
isolated (23%) as a complex mixture of α and β O-acetylated
regioisomers. As for disaccharides 10 and 15, it was hypo-
thesized that the cleavage of the allyl aglycon occurring during
the hydrogenolysis of trisaccharide 22 into 23 was caused by
an unfavourable competition between alkene reduction and
propen-1-yl hydrolysis. Gratifyingly however, there was no trace
of acetate loss. In order to investigate further the outcome of
the deprotection step yielding the desired AcC(E)D-Pr tri-
saccharide, an available fraction of mixed monoacetates 30 and
1
31 (3 : 2 based on H NMR data, see below, Scheme 3) was sub-
mitted to Pd/C hydrogenolysis using the above conditions. In
this case, the target trisaccharide was obtained in a good 74%
yield as a 4 : 5 : 1 mixture of regioisomers 23, 24 and 25, follow-
ing RP-MPLC purification. In support of our previous obser-
vation, there was no trace of any acetate loss in this case.
However, acetyl migration was still observed as expected. Inter-
estingly, the final distributions of the O-acetylated regio-
isomers 23–25 were identical whether starting from the fully
protected 22 or from a 3 : 2 mixture of alcohols 30 and 31,
suggesting that it is independent of the trisaccharide precur-
sor. Subsequent treatment of a sample of mixed monoacetates
23–25 under Zemplén conditions provided propyl glycoside 29
in a non-optimized 69% yield following RP-HPLC purification.
The isolation of hemiacetals upon conventional hydro-
genolysis of allyl glycoside precursors has no precedent in
our hands despite the frequent use of the Pd/C-induced
anomeric allyl to propyl conversion, whether in the case of
allyl rhamnosides,14,16 (allyl galactopyranosid)uronates,15 or
in the case of allyl glucosaminides.9 Puzzled by this pheno-
menon, we questioned the origin of the aglycon loss. It
could neither be explained by the quality of the catalyst nor by
any peculiar experimental feature. However, anomeric deallyla-
tion in the presence of the Pd/C catalyst in the acidic alcohol
medium has a precedent. For example, the high yielding selec-
tive Pd/C-mediated conversion in acidic methanol of an allyl
rhamnoside to the corresponding hemiacetal was reported.44
Referring to established procedures for Pd/C-mediated anome-
ric allyl cleavage, it is assumed that following Pd/C-induced
allyl conversion into a propen-1-yl, which often requires
forcing conditions,45,46 acid alcoholysis of the latter reveals the
hemiacetal.44,46,47 Nevertheless, partial loss of the allyl moiety
during isomerisation under neutral conditions, as observed
during the final deprotection of disaccharide 10, has been
occasionally reported.45 On that basis, our experimental
Scheme 3 Synthesis of tetrasaccharide 33 via a [B + C(E)D] coupling or
a [BC + ED] coupling. Reagents and conditions: (a) CAN, MeCN–H2O, rt;
(b) H2, Pd/C, HCl, 90% aq. EtOH, rt, 1 day, 74%; (c) MeONa, MeOH, 69%;
(d) TMSOTf, toluene, 55 °C, 58% over two steps; (e, f, g) see ref. 14; (h)
TMSOTf, toluene, 55 °C, 50% conversion at the most.
observations were tentatively explained by a competition
between the kinetics of the Pd/C-mediated hydrogenation of
the allyl moiety and the cleavage of a propenyl ether intermedi-
ate under the hydrogenolysis conditions. In the present case,
this competition strongly impaired the formation of the propyl
glycosides 11 and 16. To test this hypothesis, disaccharide 10
was subjected to conditions used for hydrogenolysis (Pd/C in
90% aq. EtOH, 1 M aq. HCl 0.1 equiv., overnight, rt) under a
saturated hydrogen atmosphere or an Ar atmosphere. The
former conditions led to the clean formation of the ED-Pr
(74%). In contrast, in the absence of hydrogen disaccharide 10
evolved into a complex mixture of products. HRMS and NMR
data demonstrated that the allyl moiety was strongly affected.
Furthermore, in support to our observations, HRMS data
(HRMS (ESI+): m/z 856.3798, calcd for C49H55NO11Na [M + Na]+
m/z 856.3723) clearly indicated the presence of detectable
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amounts of the corresponding hemiacetal 13. Undoubtedly,
Thus, chain elongation at OH-3D of disaccharide 10 was
the hemiacetal/glycoside ratio increased with time (not next attempted with the 3,4-di-O-acetyl-4-O-benzyl-α/β-L-rham-
described). As a general trend, under the conditions in use, nosyl trichloroacetimidate50 (40). Donor 40 does not feature
the dilution and reaction duration were identified as factors the required orthogonal set of protecting groups. However, it is
that were possibly interfering with allyl to propyl conversion. more stable and more readily available (3 steps, 82%) than its
Running the reaction in a buffered medium48 may prevent 3-O-PMB counterpart (4 steps, 72%) starting from allyl 4-O-
hydrolysis while retaining the efficiency of the reduction benzyl-α-L-rhamnoside,51 a common precursor in our labora-
process. Alternatively, the use of Pd(OH)2, which is known to tory. Moreover, an easy differentiation between the 3C-OH and
be more selective than Pd on charcoal, was considered to be a 2C-OH at the C(E)D trisaccharide level proceeded as antici-
relevant option. Satisfactorily, under conventional hydrogeno- pated (Scheme 4). To our satisfaction, the reaction conditions
lysis conditions where Pd/C was replaced by Pd(OH)2/C as the developed for the condensation of acceptor 10 and ortho-
catalyst, the model compound 10 was converted to disacchar- gonally protected donor 21 were successfully applied to the gly-
ide 11 in a very good 85% yield, while no traces of hemiacetal cosylation of the same acceptor and donor 40. Whether the
12 were detected.
condensation was performed on milligram or gram amounts
of the acceptor, trisaccharide 41 was isolated in an acceptable
81% yield. Conventional deacetylation gave diol 42 (99%),
which was in turn regioselectively acetylated at the axial
hydroxyl group to give acceptor 30. This two-step conversion of
Synthesis of the BAcC(E)D-Pr (47) and BC(E)D-Pr (48)
tetrasaccharides
Alternatively, oxidative removal of the PMB ether of the ortho- the fully protected 41 into the 2C-O-acetylated 30 (99% over
gonally protected trisaccharide 22 using ceric ammonium two steps, no purification) can be compared advantageously
nitrate (CAN) in MeCN–H2O enabled a fast and clean quanti- with the former strategy (Scheme 2), especially since tri-
tative unmasking of OH-3C, giving alcohol 30 as the sole regio- saccharide 30 here is free of any interfering contaminants.
isomer, as ascertained by NMR analysis of the crude material Having identified a feasible route to a C(E)D acceptor having
(Scheme 3). Owing to the propensity of acetyl groups occurring the 2C-acetate already in place, its condensation with donor 32
in cis-diol systems to migrate to the vicinal hydroxyl moiety was the next step (Table 3). Running the reaction at low temp-
during column chromatography, acceptor 30 was used without erature in diethyl ether in the presence of TMSOTf gave the
further purification in the reaction with the known 3,4-di-O-
benzyl donor9 32. Conventional TMSOTf-mediated glycosyla-
tion furnished tetrasaccharide 33 in an acceptable 58% yield
from the fully protected 22. Attempts to improve the yield of
this two step transformation were met with complete failure
(not described), and suggested poor reproducibility. Instead,
we placed more confidence in the [BC + ED] coupling reaction.
Thus, the rhamnosyl TCA 32 was reacted with allyl rhamno-
side49 34 to furnish rhamnobioside9 35, which in turn was
converted to the corresponding TCA donor 37, via hemiacetal
36, as described.14 In agreement with the best conditions
identified for the [C + ED] coupling, the condensation of the
latter with disaccharide 10 was set up in toluene at 55 °C in
the presence of 0.3 equiv. of TMSOTf. Despite these harsh con-
ditions, the reaction did not proceed to completion, showing
only a 50% conversion. Moreover, tetrasaccharide 33 could not
be isolated as a pure material. This route was therefore aban-
doned. Evidence for the poor reactivity of OH-3D in acceptor 10
was provided by the formation of the α-(1↔1)-β-linked tetra-
saccharide 38 (HRMS (ESI+): m/z 1445.5952, calcd for
C80H94O23Na [M + Na]+ m/z 1445.6136; NMR spectroscopic
1
data for C-1C and C-1C′: δ = 91.6 ppm, JCH = 161.9 Hz and
97.5 ppm, 1JCH = 175.2 Hz, respectively) arising as a major side
reaction from the glycosylation of hemiacetal 36 with TCA 37.
The corresponding α-(1↔1)-α-linked tetrasaccharide 39 (HRMS
(ESI+): m/z 1445.5952, calcd for C80H94O23Na [M + Na]+ m/z
Scheme 4 Synthesis of tetrasaccharides BAcC(E)D-Pr (46) and BC(E)
D-Pr (47) via a [B + C + (E)D] coupling. Reagents and conditions: (a)
TMSOTf, toluene, 50 °C, 81%; (b) MeONa, MeOH, rt, 99%; (c) (i) MeC
(OMe)3, PTSA, MeCN, rt; (ii) 80% aq. AcOH, rt, 99%; (d) See the table; (e)
H2NNH2·H2O, Pyr, AcOH, rt, 96%; (f) H2, Pd/C, HCl, 90% aq. EtOH, rt,
90%; (g) MeONa, MeOH, rt, 60%.
1445.6084; NMR spectroscopic data for both C-1C and C-1C′:
1
δ = 92.2 ppm, JCH = 173.5 Hz) was also isolated, albeit in a
minimal amount. These recurrent discouraging outcomes led
us to consider an alternative route.
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Table 3 Study on the condensation of acceptor 30 and donor 32
32
Promoter
Solvent/
33 (Corrected Side-products
Entry equiv. (0.2 equiv.) temperature yield)
(yield)
1
1.2
TMSOTf
Et2O/−15 °C 34%
43 (23%)
30/31 (29%)
a
2
3
1.2
1.3
TMSOTf
TMSOTf
Toluene/rt
Toluene/rt
81%
74%
—
43 (6%)
30/31 (11%)
—
44 (6%)
a
4
5
1.3
1.3
TBSOTf
TBSOTf
Toluene/rt
Toluene/rt
90%
83% (89%)
a The reaction was run on a small scale (150 mg of acceptor 30).
coupling product in 34% yield, while silylation of the acceptor
to give the unreactive trisaccharide 43 (23%) was identified as
a major side reaction (Table 3, entry 1). To our satisfaction,
substituting diethyl ether for toluene and performing the reac-
tion at room temperature resulted in an improved yield (81%)
of tetrasaccharide 33 (Table 3, entry 2). However, the robust-
ness of the conditions was questioned since large quantities of
the silylated acceptor 43 (6%), and of the remaining 30 in com-
bination with regioisomer 31 (11%) were isolated when
working on a larger scale (Table 3, entry 3). Gratifyingly, when
tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf)
was used as the promoter, the yield of tetrasaccharide 33
reached 90% (Table 3, entry 4). However, silylation of the
acceptor resulting in trisaccharide 44 (6%) could not be
avoided when working on gram amounts and the yield of the
condensation dropped to 83% (Table 3, entry 5). As in the case
of the trimethyl silyl derivative 43, the silylated 44 was isolated
as a single regioisomer. While the yield of the glycosylation
was thought to be acceptable, the fact that silylation at the
acceptor site occurs despite the use of room temperature and
of the bulky TBSOTf as a promoter suggests interference with
glycosylation at the acceptor site. Removal of the 2B-levulinoyl
ester of the glycosylation product 33 by reaction with hydrazine
hydrate in pyridine/AcOH was high yielding (96%). The result-
ing alcohol 45 could serve either as an intermediate for the
synthesis of BAcC(E)D-Pr and BC(E)D-Pr targets, or as an accep-
tor in the synthesis of pentasaccharide 51 (Scheme 5). In the
first place, conventional Pd/C-promoted benzyl ether hydroge-
nolysis and concomitant allyl reduction gave the propyl glyco-
side 46 in an excellent 90% yield after RP-MPLC purification.
As expected, the isolated 2C-acetate was stable in conventional
hydrogenolysis conditions. Moreover, although the same
hydrogenolysis conditions were used for the final deprotection
of trisaccharides 30/31 and tetrasaccharide 45, no trace of the
product of anomeric deallylation was detected in the latter
case. Subsequent treatment of the acetylated 46 with methano-
lic sodium methoxide gave the BC(E)D-Pr target 47 in an un-
optimized 60% yield.
Scheme 5 Synthesis of the target pentasaccharides 51 and 52.
Reagents and conditions: (a) TfOH, toluene, rt; (b) H2NNH2·H2O, Pyr,
AcOH, rt, 63% over two steps; (c) H2, Pd/C, HCl, 90% aq. EtOH, rt, 92%;
(d) MeONa, MeOH, rt, 85%.
except that trifluoromethanesulfonic acid (TfOH) replaced
TMSOTf in order to avoid any parasitic silylation at the reactive
hydroxyl group. The condensation evolved as desired. Never-
theless, contamination with hemiacetal 48 was observed
repeatedly and pentasaccharide 49 could not be isolated as a
pure material (not shown). Thus, the polluted glycosylation
product was treated under conventional delevulinoylation con-
ditions to give pentasaccharide 50, whose 2A hydroxyl group
was unmasked, in an acceptable 63% yield over two steps
(Scheme 5). Next, the one-step Pd/C-mediated benzyl ether
hydrogenolysis and concomitant allyl reduction gave the ABAcC
(E)D-Pr pentasaccharide (51) in an excellent 92% yield after
RP-MPLC purification. To our satisfaction, no trace of the
product of anomeric deallylation was detected at the penta-
saccharide stage. This outcome substantiates the result of the
hydrogenolysis of tetrasaccharide 45. However, the differences
in the behaviours of disaccharide 10 and trisaccharide 15 in
comparison with the performances of trisaccharides 30/31,
tetrasaccharide 45 and pentasaccharide 50, when submitted to
similar Pd/C mediated deprotection conditions, remain un-
explained. In particular, available data indicate that the change
in the conformation of the pyranoside ring of a 3,4-di-O-substi-
tuted residue D does not account for the disparity in the
results of the hydrogenolysis reactions. Last, treatment of the
2C-acetylated 51 with methanolic sodium methoxide resulted
in its smooth conversion to the corresponding ABC(E)D-Pr
target 52, which was isolated in a non-optimized 85% yield fol-
lowing RP-MPLC purification.
Synthesis of the ABAcC(E)D-Pr (51) and ABC(E)D-Pr (52)
pentasaccharides
The SF1a/SF1b-specific 3,4-di-O-glucosylation pattern of
N-acetyl-glucosamine: C(E)D
Alternatively, alcohol 45 underwent reaction with the rhamnosyl β- and α-Glycosides of 3,4-di-O-glycosyl-2-acetamido-2-deoxy-D-
TCA 32 under conditions adapted for the [B + C(E)D] coupling, glucopyranose have been synthesized in various instances,
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owing to the implication of such trisaccharides as components associated with this change in conformation. Removal of the
of carbohydrates of biological importance. They have been benzyl protecting groups resulted in conformational release
identified as part of plant N-glycans,52 glycosphingolipids at the vicinal glycosidic linkages, as attested by the J1,2 and
from parasites,53,54 lipopolysaccharides other than those from 1JCH values measured for trisaccharides 23 and 29 (Table 4,
S. flexneri,55,56 human milk oligosaccharides,57 and more gene- entries 8 and 9).
rally as key constituents of the human glycome, including
Non-chair conformations of 3,4-di-O-glycosyl-N-acetyl-D-glu-
Lewis determinants.58 The latter have generated the most cosamine have precedence. They are most often associated
attractiveness in terms of synthesis. As a general trend, no syn- with poor experimental outcomes.63 In complement to exten-
thetic difficulties associated with the 3,4-di-O-glycosylation of sive support from NMR spectroscopy analysis,64 and sub-
N-acetyl glucosamine have emerged, apart from observations sequent computational investigations,59 convincing evidence
related to the clearly established poor reactivity of the 4-OH for these unlikely conformations have more recently emerged
group in a N-acetyl-β-D-glucosaminide acceptor. This pheno- from structural data.65
menon was studied exhaustively and even tentatively explained
by computational analysis.59 It did not occur as a problem
herein, likely owing to the excellent reactivity of the involved
glucosyl donors. In contrast, α-L-rhamnosylation at OH-3D of
Conclusion
the ED acceptor 10 was found problematic in our hands. Reac- This study is part of a program aimed at a better understand-
tion temperatures of 50 °C at least were necessary for the glyco- ing of the molecular specificity of recognition of Shigella flex-
sylation to proceed. As a reward for our perseverance in the neri LPS by protective mAbs using synthetic fragments of the
quest for optimization, a good 81% yield was achieved in the O-Ags of interest. For the first time we have dealt with the syn-
presence of minor excess of donor (1.2 equiv. for TCA 40). The thesis of oligosaccharides specific for SF1a and SF1b. Except
need for such uncommon reaction temperatures is supported for the ED disaccharides, all novel oligosaccharides described
by previous reports of low fucosylation yields at the 3-OH herein have the 3,4-di-O-glycosyl-N-acetyl-β-D-glucosaminide
group of 4-O-glycosyl-N-acetyl-β-D-glucosaminide acceptors,60,61 moiety (C(E)D) in common. As previously observed in the
which were tentatively explained by steric hindrance at the course of the synthesis of Lewis determinants and analogues
reactive site. Moreover, analysis of the 1H and 13C NMR spectra thereof, our findings on protected intermediates encompass-
collected for trisaccharides 41 and 42 revealed some unconven- ing the C(E)D moiety clearly demonstrate that the N-acetyl-D-
tional features for several signals assigned to the N-acetyl-D- glucosamine ring (D) adopts a non-conventional confor-
glucosamine residue D (Table 4). In contrast to data character- mation. Conformational changes have occurred as a result of
izing the ED disaccharides 10, 11 and 15 (Table 4, entries 1–3), all successful ED to C(E)D conversions, independently of the
4
which were consistent with the expected C1 conformation for protecting groups at the rhamnose donor (C). It is hypo-
1
residue D, the coupling constants J1,2 and JCH measured for thesized that steric hindrance generated at OH-3D by the vicinal
N-acetyl-glucosamine D within the protected tri- to pentasac- tetra-O-benzyl-D-glucopyranosyl residue governs the glycosyla-
charides (Table 4, entries 4–7) strongly differed from those pre- tion outcomes. Nevertheless, conditions involving only a slight
dictable for a 2-N-acetyl-β-D-glucopyranoside.62 The measured excess (1.2 equiv.) of TCA donor 40 were found for a high yield-
values suggested that the successful α-L-rhamnosylation at the ing [C + ED] coupling. Satisfactorily, the change of confor-
3D-OH of disaccharide 10 had caused a major conformational mation of residue D did not interfere with subsequent chain
change at the D ring. It can be hypothesized that the glycosyla- elongation at OH-3C, with residues B and A, respectively.
tion reaction required heating to overcome the energy barrier Unfavourable kinetics led to partial aglycon cleavage upon
final protecting group removal at the di- and trisaccharide
level. To our satisfaction, no such side-reaction was observed
in the case of the tetra- and pentasaccharides. Besides, the
Table 4 Partial NMR data measured for residue D as part of the ED dis-
final deprotection step led to conformational release at the D
ring of all oligosaccharides bearing the C(E)D branching
pattern. The novel di- to pentasaccharides described herein –
compounds 11, 23–25, 29, 46, 47, 51, and 52 – will serve as
probes to clarify the structural requirements for SF1b and/or
SF1a O-Ag molecular recognition by protective mAbs.
accharides to ABC(E)D pentasaccharides (400 MHz, CDCl3 or D2O,
298 K)
Entry
Compound
J1,2 (Hz)
1JCH (Hz)
1
2
3
4
5
6
7
8
9
10
11
15
41
42
33
50
23
29
8.8a
8.4
163.8
162.5
162.8
170.7
169.5
171.4
170.7
163.9
162.9
8.3
3.7a
3.7a
3.2a
3.1a
7.4
Experimental section
General
7.8
Anhydrous (anhyd.) solvents – including toluene (Tol), dichloro-
methane (DCM), tetrahydrofuran (THF), N,N-dimethylform-
amide (DMF), methanol (MeOH), acetonitrile (MeCN), and
a The H-1 signal partially overlapped with signals from benzylic
protons. The J1,2 coupling constant was measured using Jres NMR
experiments.
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pyridine – were delivered on molecular sieves and used as (DCM–EtOAc 3 : 7) showed the conversion of the starting
received. Additional solvents cited in the text are abbreviated material (Rf 0.46) into a more polar product (Rf 0.20). The reac-
as cHex (cyclohexane), and EtOAc (ethyl acetate), in addition to tion was quenched by the addition of solid NaHCO3 (15.0 g)
acetone. Reactions requiring anhyd. conditions were run and MeOH (50 mL). The suspension was filtered over a pad of
under an argon (Ar) atmosphere, using dried glassware. 4 Å Celite, and the filtrate was concentrated under reduced
molecular sieves (4 Å MS) were activated before use by heating pressure. The residue was dissolved in EtOAc (300 mL),
under high vacuum. Analytical thin-layer chromatography washed with 5% aq. citric acid (150 mL), 5% aq. NaHCO3
(TLC) was performed with silica gel 60 F254, 0.25 mm pre- (150 mL) and brine (150 mL), then dried over anhyd. Na2SO4
coated TLC aluminium foil plates. Compounds were visualized and concentrated to dryness. The crude was purified by flash
using UV254 and/or orcinol (1 mg mL−1) in 10% aq. H2SO4 with chromatography (DCM–EtOAc 3 : 7 to 15 : 85) to give alcohol 4
charring. Flash column chromatography was carried out using (5.65 g, 86%) as a white foam. Compound 4 had 1H NMR
silica gel (particle size 40–63 µm). RP-HPLC purification was (CDCl3) δ 7.39–7.29 (m, 5H, HAr), 5.87 (m, 1H, CHvCH2), 5.73
carried out using a Kromasil 5 μm C18100 Å 10 × 250 mm (d, 1H, J2,NH = 9.2 Hz, NH), 5.27 (m, 1H, Jtrans = 17.1 Hz, Jgem
=
semi-preparative column. RP-MPLC purification was carried 1.5 Hz, CHvCH2), 5.18 (m, 1H, Jcis = 10.5 Hz, CHvCH2), 5.08
out using a NucleoPrep 20 µm C18 100 Å 26 × 313 cm semi-pre- (dd, 1H, J3,4 = 9.1 Hz, J2,3 = 10.6 Hz, H-3), 4.63 (d, 1H, J = 12.0
parative column). Unless stated otherwise, analytical RP-HPLC Hz, HBn), 4.59 (dpo, 1H, HBn), 4.56 (dpo, 1H, H-1), 4.34 (m, 1H,
of the final compounds (λ = 215 nm) was carried out using an
HAll), 4.08 (m, 1H, HAll), 3.97 (pdt, 1H, J1,2 = 8.7 Hz, H-2),
Aeris Peptide 3.6 µm C18 100 Å 2.1 × 100 mm analytical 3.83–3.78 (m, 2H, H-6a, H-6b), 3.74 (pt, 1H, J3,4 = J4,5 = 9.3 Hz,
column, eluting with a 0–20% linear gradient of MeCN in H-4), 3.56 (pdt, 1H, J4,5 = 8.9 Hz, J5,6a = 10.6 Hz, H-5), 3.14 (bs,
0.08% aq. TFA over 20 min at a flow rate of 0.3 mL min−1
.
1H, OH), 2.11 (s, 3H, CH3Ac), 1.96 (s, 3H, CH3NHAc); 13C NMR
NMR spectra were recorded at 303 K on a Bruker Avance (CDCl3) δ 172.0 (COAc), 170.2 (NHCO), 137.6 (CIVAr), 133.8
spectrometer equipped with a BBO probe at 400 MHz (1H) and (CHvCH2), 128.5–127.7 (5C, CAr), 117.4 (CHvCH2), 100.1
1
100 MHz (13C). Spectra were recorded in deuterated chloroform (C-1, JC,H = 160.5 Hz), 75.5 (C-3), 74.1 (C-5), 73.8 (CBn), 70.8
(CDCl3), deuterated dimethyl sulfoxide (DMSO-d6) and deuter- (C-4), 70.4 (C-6), 69.6 (CAll), 54.1 (C-2), 23.3 (CH3Ac), 21.0
ated water (D2O). Chemical shifts are reported in ppm (δ) rela- (CH3NHAc); HRMS (ESI+): m/z 416.1688 (calcd for C20H27NO7Na
tive to residual solvent peak CHCl3 in the case of CDCl3, HOD [M + Na]+ m/z 416.1788).
and DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) in the
Allyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-2-acet-
case of D2O, at 7.28/77.0, and 4.70/0.00 ppm for the 1H and amido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside (9)
13C spectra, respectively. Coupling constants are reported in and allyl 2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl-(1→4)-2-
hertz (Hz). Elucidations of chemical structures were based on acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside
1H, COSY, DEPT-135, HSQC, decoupled HSQC, 13C, decoupled (14). Route 1. 4 Å MS (162 mg) were added to a solution of
13C, HMBC, and Jres. Signals are reported as s (singlet), d acceptor 4 (100 mg, 253 µmol) and thioglycoside 5 (800 mg,
(doublet), t (triplet), dd (doublet of doublet), q (quadruplet), qt 1.27 mmol, 5.0 equiv.) in Et2O–DCM (2 : 5, 5.0 mL) at rt, under
(quintuplet), sex (sextuplet), dt (doublet of triplet), dq (doublet an atmosphere of Ar. The reaction mixture was cooled to 0 °C
of quadruplet), ddd (doublet of doublet of doublet), m (multi- and stirred at this temperature for 25 min, then NIS (341 mg,
plet). The signals can also be described as broad (prefix b), 152 mmol, 6.0 equiv.) and TMSOTf (23 µL, 126 µmol,
pseudo (prefix p), overlapped (suffixo) or partially overlapped 0.5 equiv.) were added. After stirring for 2.5 h at this tempera-
(suffixpo). Of the two magnetically non-equivalent geminal ture, TLC analyses revealed the conversion of acceptor 4
protons at C-6, the one resonating at lower field is denoted as (DCM–Me2CO 1 : 1, Rf 0.53) to one major new product
H-6a, and the one at higher field is denoted as H-6b. Inter- (DCM–Me2CO 1 : 1, Rf 0.77 and cHex–EtOAc 1 : 1, Rf 0.18). The
changeable assignments are marked with an asterisk. Sugar reaction was quenched with Et3N (500 µL) and the reaction
residues are lettered according to the lettering of the RU of the mixture was warmed to rt. The reaction mixture was filtered
SF1b O-Ag and identified by a subscript in the listing of signal over a pad of Celite and concentrated in vacuo to a yellow
assignments. HRMS spectra were recorded on a WATERS QTOF oil, which was purified by flash column chromatography
Micromass instrument in the positive-ion electrospray ionis- (cHex–EtOAc 4 : 1 → 1 : 1) to yield the coupling product as an
ation (ESI+) mode. Solutions were prepared using 1 : 1 MeCN– inseparable 7 : 1 α/β mixture (164 mg, 64%).
H2O containing 0.1% formic acid. In the case of sensitive com-
pounds, solutions were prepared using 1 : 1 MeOH–H2O to 4 (1.0 g, 2.54 mmol) in Et2O–DCM (3 : 2, 12.5 mL) at rt, under
which was added 10 mM ammonium acetate. an atmosphere of Ar. After 15 min of stirring, the reaction
Route 2. 4 Å MS (2.5 g) were added to a solution of acceptor
Allyl 2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyr- mixture was cooled to −78 °C, and then TMSOTf (140 µL,
anoside (4). To a solution of acetal36 3 (6.54 g, 16.7 mmol) in 0.76 mmol, 0.3 equiv.) was added. After an additional 30 min
anhyd. DCM (220 mL) containing activated 4 Å MS (12.0 g), of stirring, a solution of the TCA donor38 6 in a Et2O–DCM
stirred at −78 °C, were added dropwise first triethyl- (5 : 2, 7 mL) was added at a constant rate to the reaction
silane (8.3 mL, 52.0 mmol, 3.1 equiv.) over 5 min, and mixture at −78 °C, over a period of 35 min. After stirring for
then TMSOTf (3.1 mL, 35.0 mmol, 2.1 equiv.) over 12 min. 1 h at this temperature, the reaction mixture was allowed to
The mixture was stirred for 2 h at −78 °C. A TLC control reach 4 °C overnight. After this, TLC analyses revealed the
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conversion of acceptor 4 (DCM–Me2CO 1 : 1, Rf 0.53) and J = 12.2 Hz, HBn), 4.37 (dpo, 1H, J1,2 = 7.7 Hz, H-1E), 4.35
donor 6 (cHex–EtOAc 1 : 1, Rf 0.61) to one major new product (m, 1H, HAll), 4.08 (m, 1H, HAll), 4.03 (dddpo, 1H, J1,2 = 8.6 Hz,
(DCM–Me2CO 1 : 1, Rf 0.77 and cHex–EtOAc 1 : 1, Rf 0.18). The H-2D), 4.11–4.04 (pt, 1H, J4,5 = 9.2 Hz, H-4D), 3.81 (dd, 1H, J5,6a
reaction was quenched with Et3N (500 µL) and the reaction = 4.0 Hz, J6a,6b = 11.0 Hz, H-6aD), 3.78–3.67 (m, 3H, H-6aE,
mixture was warmed to rt. The reaction mixture was filtered H-6bE, H-6bD), 3.67 (pto, 1H, J4,5 = 9.6 Hz, H-4E), 3.53 (pt, 1H,
and concentrated in vacuo to a yellow oil, which was purified J2,3 = J3,4 = 9.2 Hz, H-3E), 3.48 (ddd, 1H, J5,6b = 1.9 Hz, H-5D),
by flash column chromatography (cHex–EtOAc 4 : 1 → DCM– 3.32 (ddpo, 1H, H-2E), 3.30 (mpo, 1H, H-5E), 2.01 (s, 3H, CH3),
Me2CO 7 : 3) to yield the coupling product as an inseparable 1.97 (s, 3H, CH3NHAc); 13C NMR (CDCl3) δ 171.7 (COAc), 170.0
9 : 1 α/β mixture (1.97 g, 85%).
(NHCO), 138.7–137.9 (5C, CIVAr), 133.9 (CHvCH2), 128.4–127.5
1
Route 3. Alcohol 4 (4.8 g, 12.2 mmol) was dissolved in (25C, CAr), 117.3 (CHvCH2), 102.8 (C-1E, JC,H = 162.7 Hz),
DCM–Et2O (1 : 5 180 mL) containing 4 Å MS (10.0 g). The sus- 100.2 (C-1D, JC,H = 161.0 Hz), 84.8 (C-3E), 82.5 (C-2E), 77.6
1
pension was stirred for 25 min at rt under an atmosphere of (C-4E), 75.5 (CBn), 75.3 (C-5D), 75.0 (CBn), 74.9 (C-4D), 74.8
Ar, and then TMSOTf (470 µL, 2.6 mmol, 0.2 equiv.) was (CBn), 74.4 (C-5E), 73.3, 73.2 (2C, CBn), 72.7 (C-3D), 69.5 (CAll),
added. After 5 min, donor41 7 (11.1 g, 15.6 mmol, 1.2 equiv.) 68.8 (C-6E), 67.8 (C-6D), 56.0 (C-2D), 23.4 (CH3NHAc), 20.7
in DCM–Et2O (1 : 3, 70 mL) was added dropwise over 50 min. (CH3Ac); HRMS (ESI+): m/z 938.4114 (calcd for C54H61NO12Na
The reaction was stirred for 2 h at rt. A TLC control (Tol–EtOAc [M + Na]+ m/z 938.4091).
5 : 5) showed the disappearance of the acceptor 4 (Rf 0.05) and
Allyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)-2-acet-
the presence of a less polar product (Rf 0.3). The reaction was amido-6-O-benzyl-2-deoxy-β-D-glucopyranoside (10). Methano-
quenched by the addition of Et3N (2 mL) and the suspension lic sodium methoxide (25% w/w, 350 µL, 1.53 mmol,
was filtered over a pad of Celite. Volatiles were removed under 0.3 equiv.) was added to a solution of allyl glycoside 9 (6.23 g,
reduced pressure and the residue was purified by flash chrom- 6.80 mmol) in anhyd. MeOH (100 mL), the solution was stirred
atography (Tol–EtOAc 7 : 3 to 65 : 35) to give in the order of at rt for 6 h, at which time a TLC control (Tol–EtOAc 5 : 5)
elution, first the α-linked disaccharide 9 (8.50 g, 76%) and showed the total conversion of the starting material (Rf 0.41)
then the β-linked isomer 14 (1.2 g, 11%) both as white foams. into a more polar product (Rf 0.29). The reaction was quenched
The α anomer 9 had 1H NMR (CDCl3) δ 7.37–7.27 (m, 23H, with Dowex H+ ion-exchange resin. Following filtration of the
HAr), 7.16–7.14 (m, 2H, HAr), 6.40 (d, 1H, J2,NH = 8.6 Hz, NH), resin and concentration to dryness, the crude was purified by
5.90 (m, 1H, CHvCH2), 5.30 (m, 1H, Jtrans = 17.2 Hz, Jgem
=
flash chromatography (Tol–EtOAc 55 : 45 to 3 : 7) to give
1.6 Hz, CHvCH2), 5.20 (m, 1H, Jcis = 10.4 Hz, CHvCH2), 5.08 alcohol 10 (5.77 g, 97%) as a white foam. Acceptor 10 had
(tpo, 1H, H-3D), 5.07 (dpo, 1H, H-1E), 4.90 (d, 1H, J = 10.9 Hz, 1H NMR (CDCl3) δ 7.39–7.24 (m, 23H, HAr), 7.20–7.16 (m, 2H,
H
Bn), 4.84 (dpo, 1H, HBn), 4.82 (dpo, 1H, J = 10.8 Hz, HBn), 4.74 HAr), 5.93 (m, 1H, CHvCH2), 5.84 (d, 1H, J2,NH = 7.0 Hz, NH),
(d, 1H, J = 11.8 Hz, HBn), 4.69 (d, 1H, HBn), 4.61 (d, 1H, J1,2 5.30 (m, 1H, Jtrans = 17.2 Hz, Jgem = 1.4 Hz, CHvCH2), 5.21 (m,
5.0 Hz, H-1D), 4.56 (dpo, 1H, HBn), 4.55 (dpo, 1H, J = 12.0 Hz, 1H, Jcis = 10.4 Hz, CHvCH2), 5.03 (d, 1H, J1,2 = 3.3 Hz, H-1E),
Bn), 4.49 (d, 2H, HBn), 4.38 (d, 1H, J = 12.2 Hz, HBn), 4.33 4.95–4.82 (m, 5H, J1,2 = 8.8 Hz, 4HBn, H-1D), 4.74 (d, 1H, J =
(m, 1H, HAll), 4.19 (ddd, 1H, J2,3 = 5.7 Hz, H-2D), 4.11–4.04 12.0 Hz, HBn), 4.58 (dpo, 1H, J = 12.0 Hz, HBn), 4.56 (do, 1H,
(m, 2H, HAll, H-4D), 3.94 (pt, 1H, J2,3 = J3,4 = 9.4 Hz, H-3E), Bn), 4.50 (dpo, 1H, J = 10.9 Hz, HBn), 4.48 (dpo, 1H, J =
3.87–3.79 (m, 4H, H-6aD, H-6bD, H-5D, H-5E), 3.68 (pto, 1H, 12.2 Hz, HBn), 4.43 (dpo, 1H, J = 12.2 Hz, HBn), 4.37 (mpo, 1H,
J3,4 = J4,5 = 9.2 Hz, H-4E), 3.64 (ddpo, 1H, J5,6a = 3.3 Hz, J6a,6b All), 4.20–4.10 (m, 2H, H-3D, HAll), 3.98 (pt, 1H, J2,3 = J3,4 = 9.4
=
H
H
=
H
11.0 Hz, H-6aE), 3.58 (dd, 1H, J1,2 = 3.4 Hz, J2,3 = 9.8 Hz, H-2E), Hz, H-3E), 3.86–3.78 (m, 2H, H-5E, H-6aD), 3.71 (pdd, 1H, J5,6b
3.48 (dd, 1H, J5,6b = 1.8 Hz, H-6bE), 1.98 (s, 3H, CH3), 1.83 = 4.5 Hz, J6a,6b = 10.5 Hz, H-6bD), 3.64–3.60 (m, 4H, H-4E, H-4D,
(s, 3H, CH3NHAc); 13C NMR (CDCl3) δ 170.3 (COAc), 169.7 H-5D, H-6aE), 3.56 (ddpo, 1H, H-2E), 3.51–3.45 (m, 2H, H-2D,
(NHCO), 138.6–137.4 (5C, CIVAr), 133.9 (CHvCH2), 128.7–127.6 H-6bE), 2.01 (s, 3H, CH3NHAc); 13C NMR (CDCl3) δ 170.8
1
(25C, CAr), 116.8 (CHvCH2), 99.2 (C-1D, JC,H = 164.7 Hz), 97.3 (NHCO), 138.5–137.1 (5C, CIVAr), 133.9 (CHvCH2), 128.6–127.5
1
1
(C-1E, JC,H = 169.5 Hz), 81.8 (C-3E), 79.5 (C-2E), 77.6 (C-4E), (25C, CAr), 117.6 (CHvCH2), 100.1 (C-1E, JC,H = 170.9 Hz),
1
75.7 (CBn), 75.0 (2C, CBn, C-5D), 73.9, 73.5, 73.2 (3C, CBn), 71.7 99.0 (C-1D, JC,H = 163.8 Hz), 82.2 (C-3E), 81.3 (C-3D), 79.3
(C-4D), 71.4 (C-5E), 70.6 (C-3D), 69.5 (C-6D), 69.1 (CAll), 68.2 (C-2E), 77.7 (C-4E), 75.7, 75.0 (2C, CBn), 74.5 (C-5D), 74.0, 73.5,
(C-6E), 51.1 (C-2D), 22.9 (CH3NHAc), 20.9 (CH3Ac); HRMS (ESI+): 73.2 (3C, CBn), 72.6 (C-4D), 71.4 (C-5E), 69.9 (CAll), 69.4 (C-6D),
m/z 938.4050 (calcd for C54H61NO12Na [M
+
Na]+ m/z 68.4 (C-6E), 57.1 (C-2D), 23.6 (CH3NHAc); HRMS (ESI+): m/z
896.3914 (calcd for C52H59NO11Na [M + Na]+ m/z 896.3686).
938.4091).
The β anomer 14 had 1H NMR (CDCl3) δ 7.35–7.27 (m, 23H,
Allyl 2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl-(1→4)-2-acet-
HAr), 7.18.16 (m, 2H, HAr), 5.88 (m, 1H, CHvCH2), 5.42 (d, 1H, amido-6-O-benzyl-2-deoxy-β-D-glucopyranoside (15). Methano-
J2,NH = 9.1 Hz, NH), 5.28 (m, 1H, Jtrans = 17.2 Hz, Jgem = 1.6 Hz, lic sodium methoxide (25% w/w, 203 µL, 0.90 mmol, 0.3
CHvCH2), 5.19 (m, 1H, Jcis = 10.4 Hz, CHvCH2), 5.11 (dd, 1H, equiv.) was added to a solution of a 9 : 1 mixture of allyl glyco-
J2,3 = 10.4 Hz, J3,4 = 9.0 Hz, H-3D), 4.89 (d, 1H, J = 11.0 Hz, sides 9 and 14 (2.89 g, 3.00 mmol) in anhyd. MeOH (22.5 mL),
H
Bn), 4.83–4.77 (m, 3H, HBn), 4.70 (d, 1H, J = 11.2 Hz, HBn), at 0 °C, under an atmosphere of Ar. The solution was stirred at rt
4.59 (d, 1H, J = 11.2 Hz, HBn), 4.69 (d, 1H, HBn), 4.57–4.51 (m, overnight, at which time TLC analysis (cHex–EtOAc 3 : 7) showed
3H, HBn, H-1D), 4.47 (dpo, 1H, J = 11.8 Hz, HBn), 4.45 (dpo, 1H, the total conversion of the starting material (Rf 0.48) into one
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major more polar product (Rf 0.28) and one minor more polar (0–20% linear gradient of CH3CN in 0.08% aq. TFA over
product (Rf 0.22). The reaction was quenched with Dowex H+ 20 min at a flow rate of 5.5 mL min−1) gave disaccharide 11
ion-exchange resin. Following filtration of the resin and con- (21 mg, 85%) as a white solid following repeated freeze-drying.
1
centration to dryness, the crude was purified by extensive flash
The propyl glycoside 11 had H NMR (D2O) δ 5.40 (d, 1H,
chromatography (cHex–EtOAc 3 : 7) to give first the above J1,2 = 3.7 Hz, H-1E), 4.51 (d, 1H, J1,2 = 8.4 Hz, H-1D), 3.92 (d,
described α-linked disaccharide 10 (2.18 g, 82%) as a white 1H, J6a,6b = 12.0 Hz, H-6aD), 3.85–3.66 (m, 7H, H-6aE, OCH2Pr
foam, then the β-linked analogue 15 (203 mg, 8%) as a solid. H-3D, H-6bD, H-6bE, H-2D, H-5E), 3.63 (ptpo, 2H, H-3E, H-4D),
The latter was subsequently crystallized from MeOH. The crys- 3.58–3.50 (m, 3H, H-2E, H-5D, OCH2Pr), 3.40 (pt, 1H, J3,4 = J4,5
,
=
1
talline material had mp=198 °C; H NMR (CDCl3) δ 7.36–7.24 9.6 Hz, H-4E), 2.01 (s, 3H, CH3NHAc), 1.53 (psex, 2H, J = 6.9 Hz,
(m, 23H, HAr), 7.17–7.15 (m, 2H, HAr), 5.93 (m, 1H, CHvCH2), CH2Pr), 0.85 (t, 3H, J = 7.4 Hz, CH3Pr); 13C NMR (D2O) δ 177.2
1
1
5.65 (d, 1H, J2,NH = 7.7 Hz, NH), 5.29 (m, 1H, Jtrans = 17.2 Hz, (NHCO), 103.6 (C-1D, JC,H = 162.5 Hz), 102.2 (C-1E, JC,H
=
Jgem = 1.4 Hz, CHvCH2), 5.21 (m, 1H, Jcis = 10.7 Hz, CHvCH2), 173.5 Hz), 79.5 (C-4D), 77.2 (C-5D*), 77.0 (C-3D), 75.5 (C-3E),
5.01 (d, 1H, J1,2 = 8.3 Hz, H-1D), 4.92 (d, 1H, J = 10.9 Hz, HBn), 75.4 (C-5E), 74.9 (OCH2Pr), 74.3 (C-2E*), 72.0 (C-4E), 63.4 (C-6D),
4.83 (dpo, 1H, HBn), 4.82 (do, 1H, HBn), 4.78 (dpo, 1H, HBn), 4.58 63.2 (C-6E), 58.2 (C-2D), 24.8, 24.7 (2C, CH3NHAc, CH2Pr), 12.2
(d, 1H, J = 12.0 Hz, HBn), 4.51 (do, 1H, J = 10.4 Hz, HBn), 4.50 (CH3Pr); HRMS (ESI+): m/z 448.1792 (calcd for C17H31NO11Na
(dpo, 1H, HBn), 4.42 (dpo, 1H, HBn), 4.40 (dpo, 1H, J = 10.7 Hz, [M + Na]+ m/z 448.1795); RP-HPLC (215 nm): Rt = 9.6 min.
H
H
Bn), 4.37 (do, 1H, J1,2 = 8.0 Hz, H-1E), 4.36–4.33 (m, 2H, HBn
All), 4.17 (pddpo, 1H, J2,3 = 7.9 Hz, J3,4 = 9.4 Hz, H-3D), 4.12 D2O) δ 5.39 (dpo, 1H, H-1E), 5.17 (d, 1H, J1,2 = 3.2 Hz, H-1D),
,
For hemiacetal 12, the α anomer had 1H NMR (partial,
(mpo, 1H, HAll), 3.72–3.55 (m, 8H, H-6aD, H-6bD, H-6aE, H-6bE, 3.99 (dd, 1H, J = 9.1 Hz, J = 10.3 Hz, H-3D), 3.93 (mo, 1H,
H-3E, H-4E, H-4D, H-5D), 3.50 (m, 1H, H-5E), 3.42 (m, 1H, J2,3 H-5D), 3.89 (mo, 1H, H-2D), 4.84–3.70 (m, 4H, H-6aD, H-6aE,
=
8.5 Hz, H-2E), 3.30 (ddd, 1H, H-2D), 2.02 (s, 3H, CH3NHAc); H-6bD, H-6bE), 3.72–3.62 (mo, H-5E, H-4D, H-3E), 3.57–3.53 (mo,
13C NMR (CDCl3) δ 170.6 (NHCO), 138.3–137.8 (5C, CIVAr), 1H, H-5E), 3.39 (pt, 1H, J3,4 = J4,5 = 9.6 Hz, H-4E), 2.01 (s, 3H,
1
134.1 (CHvCH2), 128.6–127.5 (25C, CAr), 117.6 (CHvCH2), CH3NHAc); 13C NMR (D2O) δ 177.2 (NHCO), 102.4 (C-1E, JC,H
=
103.3 (C-1E, 1JC,H = 162.2 Hz), 98.9 (C-1D, 1JC,H = 162.8 Hz), 84.6 171.6 Hz), 93.3 (C-1D, 1JC,H = 171.6 Hz), 80.1 (C-4D), 75.5 (C-3E),
(C-3E), 81.9 (C-2E), 81.5 (C-4D), 77.7 (C-4E), 75.8, 75.2, 75.0 (3C, 75.4 (C-5E), 74.4 (C-2E), 73.8 (C-3D), 72.8 (C-5D), 72.0 (C-4E),
C
Bn), 74.5 (C-5D), 74.4 (C-5E), 73.5, 73.2 (2C, CBn), 71.2 (C-3D), 63.3 (C-6E), 63.2 (C-6D), 56.6 (C-2D), 24.5 (CH3NHAc); HRMS
70.0 (CAll), 68.7 (2C, C-6D, C-6E), 58.0 (C-2D), 23.8 (CH3NHAc); (ESI+): m/z 406.1273 (calcd for C14H25NO11Na [M + Na]+ m/z
HRMS (ESI+): m/z 896.3940 (calcd for C52H59NO11Na [M + Na]+ 406.1325); RP-HPLC (215 nm): Rt = 0.9 min.
m/z 896.3986).
1
For hemiacetal 12, the β anomer had H NMR (D2O) δ 5.40
Propyl α-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-gluco- (dpo, 1H, H-1E), 4.71 (do, 1H, H-1D), 4.84–3.70 (m, 4H, H-6aD,
pyranoside (11) and α-D-glucopyranosyl-(1→4)-2-acetamido- H-6aE, H-6bD, H-6bE), 3.72–3.62 (mo, H-5E, H-4D, H-3E),
2-deoxy-α/β-D-glucopyranose (12). Route 1. Pd/C (670 mg) 3.57–3.53 (mo, 2H, H-2E, H-5E), 3.39 (pt, 1H, J3,4 = J4,5 = 9.6 Hz,
was added to a stirred solution of disaccharide 10 (584 mg, H-4E), 2.01 (s, 3H, CH3NHAc); 13C NMR (D2O) δ 177.4 (NHCO),
668 µmol) in 90% aq. EtOH (67 mL). The suspension was 102.2 (C-1E, 1JC,H = 172.7 Hz), 97.4 (C-1D, 1JC,H = 161.8 Hz), 79.4
stirred under an H2 atmosphere for 2 days at rt, and filtered (C-4D), 77.3 (C-5D*), 77.0 (C-3D), 75.5 (C-3E), 75.4 (C-5E), 74.3
over a pad of Celite. Evaporation of the volatiles, freeze-drying (C-2E), 72.0 (C-4E), 63.4 (C-6D), 63.2 (C-6E), 59.3 (C-2D), 24.8
and purification of the residue by RP-MPLC (0–50% linear (CH3NHAc); HRMS (ESI+): m/z 406.1273 (calcd for
gradient of 80% aq. MeCN over 60 min at a flow rate of 20 mL C14H25NO11Na [M + Na]+ m/z 406.1325); RP-HPLC (215 nm):
min−1) gave first the hemiacetal 12 (31 mg, 12%) as a 1.0 : 0.7 Rt = 0.9 min.
α/β mixture eluting at the solvent front, and then disaccharide
11 (141 mg, 50%). Both compounds were isolated as white β-D-glucopyranoside (16) and β-D-glucopyranosyl-(1→4)-2-acet-
solids following repeated freeze-drying. amido-2-deoxy-α/β-D-glucopyranose (17). Pd/C (300 mg) was
Propyl β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-
Route 2. Pd/C (50 mg) was added to a stirred solution of dis- added to a stirred solution of disaccharide 14 (298 mg, 341
accharide 10 (50 mg, 57 µmol) in 96% aq. EtOH (5 mL) con- µmol) in 90% aq. EtOH (33 mL) containing 1 M aq. HCl
taining 1 M aq. HCl (5 µL, 0.1 equiv.). The suspension was (38 µL, 0.1 equiv.). The suspension was stirred under an H2
stirred under an H2 atmosphere for 15 h, and filtered over a atmosphere for 2 days at rt. The suspension was filtered over a
pad of Celite. Evaporation of the volatiles, freeze-drying and pad of Celite. Evaporation of the volatiles, freeze-drying and
purification of the residue by RP-HPLC (0–20% linear gradient purification of the residue by RP-MPLC (0–40% linear gradient
of CH3CN in 0.08% aq. TFA over 20 min at a flow rate of of 80% aq. MeCN over 60 min at a flow rate of 20 mL min−1
5.5 mL min−1) gave disaccharide 11 (17 mg, 70%) as a white gave first hemiacetal 17 (25 mg, 19%) and then disaccharide
solid following repeated freeze-drying. 16 (89 mg, 67%), both as white fluffy powders following
)
Route 3. Pd(OH)2/C (50 mg) was added to a stirred solution repeated freeze-drying. The propyl glycoside 16 had 1H NMR
of disaccharide 10 (50 mg, 57 µmol) in 96% aq. EtOH (5 mL). (D2O) δ 4.51 (d, 2H, J1,2 = 8.5 Hz, H-1E, H-1D), 3.96 (dd, 1H,
The suspension was stirred under an H2 atmosphere for 16 h, J5,6a = 2.2 Hz, J6a,6b = 12.3 Hz, H-6aD), 3.88 (dd, 1H, J5,6a = 2.3
and filtered over a pad of Celite. Evaporation of the volatiles, Hz, J6a,6b = 12.4 Hz, H-6aE), 3.86–3.79 (m, 2H, H-6bD, OCH2Pr),
freeze-drying and purification of the residue by RP-HPLC 3.73–3.65 (m, 4H, H-6bE, H-2D, H-3D, H-4D), 3.59–3.44 (m, 4H,
7738 | Org. Biomol. Chem., 2014, 12, 7728–7749
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OCH2Pr, H-5E, H-3E, H-5E), 3.39 (pt, 1H, J3,4 = J4,5 = 9.4 Hz,
2-O-Acetyl-4-O-benzyl-3-O-para-methoxybenzyl-α/β-L-rhamno-
H-4E), 3.28 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.2 Hz, H-2E), 2.01 (s, pyranose (20). The [Ir] complex (560 mg, 66 µmol, 0.03 equiv.)
3H, CH3NHAc), 1.53 (pβsex, 2H, J = 7.3 Hz, CH2Pr), 0.85 (t, 3H, was dissolved in anhyd. THF (90 mL) and the solution was
J = 7.4 Hz, CH3Pr); 13C NMR (D2O) δ 177.2 (NHCO), 105.2 (C-1E, degassed repeatedly. Hydrogen was bubbled through the solu-
1
1JC,H = 163.1 Hz), 103.6 (C-1D, JC,H = 161.6 Hz), 81.5 (C-4D), tion for 15 min, resulting in a yellow coloration. The solution
78.7 (C-5E), 78.2 (C-3E), 77.4 (C-5D), 75.8 (C-2E), 75.1 (C-3D), was again degassed repeatedly, before being poured into a
75.0 (OCH2Pr), 72.1 (C-4E), 63.2 (C-6E), 62.7 (C-6D), 57.9 (C-2D), solution of allyl rhamnoside 19 (10.14 g, 22.0 mmol) in anhyd.
24.8 (CH3NHAc), 24.7 (CH2Pr), 12.2 (CH3Pr); HRMS (ESI+): m/z THF (90 mL). The mixture was stirred under Ar atmosphere at
448.1807 (calcd for C17H31NO11Na [M + Na]+ m/z 448.1795); rt for 2.5 h. Follow up by TLC (Tol–EtOAc 9 : 1) showed the con-
RP-HPLC (215 nm): Rt = 8.7 min.
version of the starting glycoside into a slightly less polar inter-
mediate. A solution of iodine (12.4 g, 49 mmol, 2.0 equiv.) in
Hemiacetal 17 had 1H NMR (D2O) δ 5.18 (d, 0.6H, J1,2
=
2.4 Hz, H-1Dα), 4.70 (d, 0.4H, H-1Dβ), 4.51 (2d, 1H, J1,2 = 7.9 Hz, THF–H2O (4 : 1, 80 mL) was added, and the mixture was stirred
H-1E), 3.98–3.92 (m, 1H, H-3Dα, H-6aDβ), 3.90–3.85 (m, 3.4H, for 2 h at rt. A TLC control (cHex–EtOAc 6 : 4) showed the con-
H-6aE, H-6aDβ, H-6bD, H-2Dα, H-3Dβ), 3.81 (dd, 0.4H, J6a,6b
=
version of the intermediate compound (Rf 0.68) to a more
12.5 Hz, J5,6b = 5.0 Hz, H-6bDβ), 3.73–3.65 (m, 3H, H-6bE, polar product (Rf 0.37). The reaction was quenched with 10%
H-2Dβ, H-4D, H-5Dα), 3.59–3.44 (m, 4H, H-5E, H-3E, H-5E), 3.57 aq. sodium bisulfate (100 mL). The reaction mixture was con-
(m, 0.4H, H-5Dα), 3.52–3.45 (m, 2H, H-3E, H-5E), 3.42–3.36 (m, centrated to 1/3rd of its volume and DCM (500 mL) was added.
1H, H-4E), 3.32–3.26 (m, 1H, H-2E), 2.02 (s, 3H, CH3NHAc); The organic layer was washed twice with water and twice with
1
13C NMR (D2O) δ 177.4, 177.1 (NHCO), 105.2 (C-1E, JC,H
=
=
brine, dried by passing through a phase separator filter and
concentrated to dryness. Column chromatography (cHex–
162.2 Hz), 97.5 (C-1Dβ
,
1JC,H = 161.7 Hz), 93.2 (C-1Dα
,
1JC,H
173.0 Hz), 81.8, 81.4 (C-5Dα), 78.6 (C-5E), 78.2 (C-3E), 77.4 EtOAc 7 : 3 to 1 : 1) of the crude material gave hemiacetal 20
(C-5Dβ), 75.8 (C-2E), 75.0 (C-4D), 72.9 (C-3Dα), 72.1 (C-4E), 71.9 (α/β 4 : 1) as a yellow oil (8.14 g, 97%). The α anomer had
(C-3Dβ), 63.2 (C-6E), 62.7 (C-6Dβ), 62.6 (C-6Dα), 59.0 (C-2Dβ), 56.5 1H NMR (CDCl3), δ 7.36–7.23 (m, 7H, HAr), 6.86 (m, 2H,
(C-2Dα), 24.9, 24.6 (CH3NHAc); HRMS (ESI+): m/z 406.1281
H
ArPMB), 5.41 (dd, 1H, J1,2 = 1.8 Hz, J2,3 = 3.3 Hz, H-2), 5.17 (d,
(calcd for C14H25NO11Na [M + Na]+ m/z 406.1325); RP-HPLC 1H, H-1), 4.93 (d, 1H, J = 11.0 Hz, HBn), 4.66 (d, 1H, J =
(215 nm): Rt = 1.1 min. 10.9 Hz, HPMB), 4.63 (d, 1H, HBn), 4.49 (d, 1H, HPMB),
Allyl 2-O-acetyl-4-O-benzyl-3-O-para-methoxybenzyl-α-L- 4.04–3.92 (m, 2H, H-3, H-5), 3.81 (s, 3H, CH3PMB), 3.44 (pt, 1H,
rhamnopyranoside (19). Alcohol16 18 (10.3 g, 25.1 mmol) was J3,4 = J4,5 = 9.4 Hz, H-4), 2.75 (bs, 1H, OH), 2.17 (s, 3H, CH3Ac),
stirred overnight in Ac2O–pyridine (1 : 1, 20 mL). TLC analysis 1.34 (d, 3H, J5,6 = 6.2 Hz, H-6). 13C NMR (CDCl3), δ 165.8 (CO),
(cHex–EtOAc 7/3) showed the total conversion of the starting 159.2 (CIVPMB), 138.5 (CIVBn), 130.3 (CIVPMB), 129.9, 129.6,
material into a less polar product (Rf 0.5). MeOH (40 mL) was 128.4, 128.3, 128.1, 127.6 (12C, 11CAr), 113.7 (2C, CArPMB), 92.5
added dropwise to the suspension and stirred at 0 °C. After (C-1), 80.0 (C-4), 77.2 (C-3), 75.3 (CBn), 71.3 (CPMB), 69.5 (C-2),
being stirred at rt for an additional 3 h, the reaction mixture 67.8 (C-5), 55.2 (CH3PMB), 21.2 (CH3Ac), 18.0 (C-6). HRMS
was concentrated under vacuum. Volatiles were coevaporated (ESI+): m/z 439.1730 (calcd for C23H28O7Na [M + Na]+: m/z
repeatedly with cyclohexane and toluene. The residue was dis- 439.1733).
solved in EtOAc and the organic phase was washed twice with
The β anomer had 1H NMR (partial, CDCl3), δ 7.36–7.23
5% aq. HCl, water, and satd aq. NaHCO3 and then dried over (m, 7H, HAr), 6.86 (m, 2H, HArPMB), 5.52 (dd, 1H, J1,2 = 1.2 Hz,
Na2SO4. The volatiles were evaporated. Column chromato- H-2), 4.92 (d, 1H, J = 10.9 Hz, HBn), 4.84 (d, 1H, H-1), 4.71 (d,
graphy (cHex–EtOAc 9 : 1 to 6 : 4) of the crude material gave the 1H, J = 10.8 Hz, HPMB), 4.46 (d, 1H, HPMB), 3.81 (s, 3H,
fully protected rhamnoside 19 as a yellow oil (10.26 g, 91%). CH3PMB), 3.64 (dd, 1H, J2,3 = 3.3 Hz, J3,4 = 9.0 Hz, H-3), 3.40
Allyl glycoside 19 had 1H NMR (CDCl3), 7.39–7.27 (m, 7H, HAr), (pt, 1H, J4,5 = 9.1 Hz, H-4), 2.52 (bs, 1H, OH), 2.24 (s, 3H,
6.88–6.85 (m, 2H, HArPMB), 5.90 (m, 1H, CHvAll), 5.40 (dd, 1H, CH3Ac), 1.38 (d, 3H, J5,6 = 6.0 Hz, H-6). 13C NMR (partial,
J1,2 = 1.8 Hz, J2,3 = 3.4 Hz, H-2), 5.29 (m, 1H, Jtrans = 17.2 Hz, CDCl3), δ 171.0 (CO), 159.4 (CIVPMB), 138.3 (CIVBn), 129.6,
Jgem = 1.6 Hz, vCH2All), 5.24 (m, 1H, Jcis = 10.4 Hz, vCH2All), 128.3, 127.6 (7C, 7CAr), 114.0 (2C, CArPMB), 92.9 (C-1), 79.7
4.93 (d, 1H, J = 10.9 Hz, HBn), 4.79 (d, 1H, H-1), 4.66 (d, 1H, J = (C-3), 79.1 (C-4), 77.2 (C-5), 75.2 (CBn), 71.3 (CPMB), 69.8 (C-2),
10.9 Hz, HPMB), 4.62 (d, 1H, HBn), 4.48 (d, 1H, HPMB), 4.17 55.2 (CH3PMB), 21.3 (CH3Ac), 18.2 (C-6). HRMS (ESI+): m/z
(m, 1H,
H
All), 4.01–3.96 (m, 2H, HAll
,
H-3), 3.81 (spo
,
439.1730 (calcd for C23H28O7Na [M + Na]+: m/z 439.1733).
2-O-Acetyl-4-O-benzyl-3-O-para-methoxybenzyl-α/β-L-rhamno-
3H, CH3PMB), 3.80 (dqpo, 1H, H-5), 3.44 (pt, 1H, J3,4 = J4,5
=
9.5 Hz, H-4), 2.17 (s, 3H, CH3Ac), 1.35 (d, 3H, J5,6 = 6.2 Hz, pyranosyl trichloroacetimidate (21). Hemiacetal 20 (8.14 g,
H-6); 13C NMR (CDCl3), 170.4 (CO), 159.3 (CIVPMB), 138.6 19.5 mmol) was dissolved in anhyd. DCE (30 mL). Trichloro-
(CIVBn), 133.6 (CHvAll), 130.2 (CIVPMB), 129.7, 128.3, 128.2, acetonitrile (10.7 mL, 107 mmol, 5.5 equiv.) and DBU (1.0 mL,
127.6 (7C, CAr), 117.5 (vCH2All), 113.9 (2C, CArPMB), 96.8 (C-1), 6.7 mmol, 0.34 equiv.) were added at −5 °C. The mixture was
80.1 (C-4), 77.8 (C-3), 75.4 (CBn), 71.4 (CPMB), 69.1 (C-2), 68.0 stirred under an Ar atmosphere for 3 h. Follow up by TLC
(CH2All), 67.8 (C-5), 55.2 (CH3PMB), 21.1 (CH3Ac), 18.2 (C-6); (cHex–EtOAc 7 : 3 + 1% Et3N) showed that the conversion of
HRMS (ESI+): m/z 479.2036 (calcd for C26H32O7Na [M + Na]+: the starting material (Rf 0.12) into a less polar product
m/z 479.2046).
(Rf 0.32) was completed. The mixture was concentrated to half
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1
of its volume and directly purified by flash chromatography (C-1C, JC,H = 172.4 Hz), 82.1 (C-3E), 79.7 (C-4C), 78.9 (C-2E),
(cHex–EtOAc 8 : 2 + 1% Et3N) to give TCA 21 (10.25 g, 94%) as a 77.7 (C-3C), 77.5 (C-4E), 75.9, 75.3, 75.0 (3C, CBn), 74.8 (C-5D),
brownish oil (α/β 4 : 1). The α anomer had 1H NMR (CDCl3), 74.4, 73.4, 73.1 (3C, CBn), 71.4 (C-5E), 71.1 (CBn), 70.9 (C-6D),
δ 8.67 (s, 1H, NH), 7.39–7.26 (m, 7H, HAr), 6.89 (m, 2H, 70.8 (C-3D), 69.4 (CAll), 68.9 (C-4D), 68.8 (C-5C), 68.2 (C-2C), 67.6
H
ArPMB), 6.19 (d, 1H, J1,2 = 1.9 Hz, H-1), 5.48 (dd, 1H, J2,3
=
(C-6E), 55.2 (CH3PMB), 46.5 (C-2D), 22.7 (CH3NHAc), 21.2 (CH3Ac),
3.3 Hz, H-2), 4.94 (d, 1H, J = 10.8 Hz, HBn), 4.68 (d, 1H, J = 10.9 18.2 (C-6C); HRMS (ESI+): m/z 1294.5577 (calcd for
Hz, HPMB), 4.64 (d, 1H, HBn), 4.53 (d, 1H, HPMB), 3.99 (ddpo
1H, H-3), 3.95 (dq, 1H, H-5), 3.81 (s, 3H, CH3PMB), 3.52 (pt, 1H,
J3,4 = J4,5 = 9.5 Hz, H-4), 2.21 (s, 3H, CH3Ac), 1.36 (d, 3H, J5,6
,
C75H85NO17Na [M + Na]+ m/z 1294.5715).
Propyl 2-O-acetyl-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyr-
anosyl-(1→4)]-2-acetamido-2-deoxy-β-D-glucopyranoside (23),
=
6.2 Hz, H-6); 13C NMR (CDCl3), δ 170.0 (CO), 160.1 (CvNH), propyl 3-O-acetyl-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyra-
159.4 (CIVPMB), 138.2 (CIVBn), 129.7 (CIVPMB), 129.9, 128.4, nosyl-(1→4)]-2-acetamido-2-deoxy-β-D-glucopyranoside (24),
128.1, 127.9 (7C, 7CAr), 113.9 (2C, CArPMB), 95.3 (C-1), 90.9 and propyl 4-O-acetyl-α-L-rhamnopyranosyl-(1→3)-[α-D-gluco-
(CCl3), 79.3 (C-4), 76.8 (C-3), 75.6 (CBn), 71.6 (CPMB), 70.8 (C-5), pyranosyl-(1→4)]-2-acetamido-2-deoxy-β-D-glucopyranoside
67.7 (C-2), 55.2 (CH3PMB), 21.0 (CH3Ac), 18.0 (C-6). HRMS (25). Route 1. To a stirred solution of trisaccharide 20 (281 mg,
(ESI+): m/z 582.0862 (calcd for C25H28NO7Na [M + Na]+: m/z 221 µmol) in 96% aq. EtOH (35 mL) containing 1 M aq. HCl
582.0829).
(38 µL) was added Pd/C (284 mg). The suspension was stirred
Allyl (2-O-acetyl-4-O-benzyl-3-O-para-methoxybenzyl-α-L- under an H2 atmosphere for 2 days at rt. The reaction mixture
rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyr- was filtered over a pad of Celite. Evaporation of the volatiles,
anosyl-(1→4)]-2-acetamido-6-O-benzyl-2-deoxy-β-D-glucopyra- freeze-drying and purification of the residue by RP-MPLC
noside (22). Activated 4 Å MS (418 mg) were added to a (0–40% linear gradient of 80% aq. MeCN over 60 min at a flow
solution of disaccharide 10 (167 mg, 0.19 mmol) and an rate of 20 mL min−1) gave, by order of elution, a complex
orthogonally protected donor 21 (161 mg, 0.29 mmol, 1.5 mixture of monoacetylated hemiacetals 26, 27 and 28 (29 mg,
equiv.) in toluene (2.9 mL). The suspension was stirred at rt, 23%) and then a 4 : 5 : 1 mixture of the acetylated propyl glyco-
under an atmosphere of Ar for 30 min, and then heated to sides 23, 24, and 25 (63 mg, 47%), all as white powders follow-
40 °C. Trimethylsilyl trifluoromethanesulfonate (10 µL, ing repeated freeze-drying.
0.06 mmol, 0.3 equiv.) was then added and the reaction
Route 2. To a stirred solution of trisaccharide 31/32
mixture was stirred for 1.5 h at this temperature. TLC analysis (150 mg, 130 µmol) in 90% aq. EtOH (7.0 mL) were added Pd/
(cHex–EtOAc 1 : 1) indicated the consumption of the acceptor C (213 mg) and 1 M aq. HCl (20 µL). The suspension was
10 (Rf 0.19) and the donor 19 (Rf 0.77), and the formation of stirred under a H2 atmosphere for a day at rt. After this time,
one new major product (Rf 0.57). The reaction was quenched MS analysis revealed a molecular weight corresponding to that
with Et3N (500 µL) and warmed to rt. The reaction mixture was of the target trisaccharide and the absence of any benzylated
filtered over a pad of Celite and the filtrate was concentrated intermediates. The reaction mixture was filtered over a pad
in vacuo to give a yellow oil. Purification of the crude by flash of Celite. Evaporation of the volatiles, freeze-drying and
column chromatography (cHex–EtOAc 4 : 1 to 1 : 1) gave trisac- purification of the residue by RP-MPLC (0–50% linear gradient
charide 22 (197 mg, 81%) as a white foam, followed by the of MeCN over 60 min at a flow rate of 20 mL min−1) gave
unreacted acceptor 10 (14 mg, 9%). The fully protected trisac- the deprotected material (59 mg, 74%) as a white powder
charide 22 had 1H NMR (CDCl3) δ 7.37–7.26 (m, 33H, NH, following repeated freeze-drying. NMR analysis indicated that
HAr), 6.80 (m, 2H, HArPMB), 5.99 (m, 1H, CHvCH2), 5.49 (dd, the isolated product was a 4 : 5 : 1 mix of 3 regioisomers,
1H, J1,2 = 1.8 Hz, J2,3 = 3.1 Hz, H-2C), 5.35 (m, 1H, Jtrans
17.3 Hz, Jgem = 1.7 Hz, CHvCH2), 5.23 (m, 1H, Jcis = 10.5 Hz,
=
corresponding to trisaccharides 23, 24 and 25 respectively.
The 2C-O-acetyl product 23 had 1H NMR (D2O) δ 5.42 (d,
CHvCH2), 5.07 (d, 1H, H-1C), 4.93 (dpo, 1H, HBn), 4.89 (bs, 2H, 1H, J1,2 = 3.9 Hz, H-1E), 5.11 (bs, 1H, H-1C), 5.06 (bs, 1H, H-2C),
Bn), 4.82 (do, 1H, H-1E), 4.81 (do, 1H, HBn), 4.80 (do, 1H, HBn), 4.55 (d, 1H, J1,2 = 7.4 Hz, H-1D), 4.07 (t, 1H, J2,3 = J3,4 = 7.2 Hz,
4.78 (do, 1H, J1,2 = 1.6 Hz, H-1D), 4.67 (dpo, 1H, J = 12.0 Hz, H-3D), 3.96–3.77 (m, 7H, H-4D, H-6aD, OCH2Pr, H-3C, H-5C,
Bn), 4.64 (dpo, 1H, HBn), 4.62 (dpo, 1H, J = 11.6 Hz, HBn), 4.55 H-6bD, H-6aE), 3.75 (ddpo, 1H, J5,6b = 4.5 Hz, J6a,6b = 12.8 Hz,
H
H
(do, 1H, HBn), 4.45 (d, 1H, J = 10.7 Hz, HBn), 4.35 (d, 1H, J = H-6bE), 3.70 (m, 1H, H-5D), 3.66–3.48 (m, 6H, H-5E, H-3E, H-2E,
10.5 Hz, HBn), 4.34 (bs, 2H, HBn), 4.33–4.29 (mo, 2H, H-2D, OCH2Pr), 3.46 (ptpo, 1H, J3,4 = J4,5 = 9.8 Hz, H-4C), 3.44 (pto,
H
H
All), 4.28 (dpo, 1H, J = 12.1 Hz, HBn), 4.09–3.90 (m, 6H, H-5D, 1H, J3,4 = J4,5 = 9.4 Hz, H-4E), 2.14 (s, 3H, CH3Ac), 1.99 (s, 3H,
All, H-3D, H-3E, H-4D, H-6aD), 3.82 (dd, 1H, J3,4 = 9.3 Hz, CH3NHAc), 1.54 (psex, 2H, J = 6.4 Hz, CH2Pr), 1.27 (dpo, 3H, J5,6
H-3C), 3.78–3.70 (m, 6H, CH3PMB, H-6bD, H-4E, H-5E), 3.67–3.60 = 5.6 Hz, H-6C), 0.85 (t, 3H, J = 7.4 Hz, CH3Pr); 13C NMR (D2O)
1
(m, 3H, H-5C, H-6aE, H-2E), 3.45 (pt, 1H, J3,4 = J4,5 = 9.4 Hz, δ 177.0 (NHCO), 175.8 (COAc), 103.5 (C-1D, JC,H = 163.9 Hz),
1
1
H-4C), 3.35 (bd, 1H, J6a,6b = 10.0 Hz, H-6bE), 2.22 (s, 3H, 100.3 (C-1C, JC,H = 171.6 Hz), 100.0 (C-1E, JC,H = 173.8 Hz),
CH3Ac), 1.76 (s, 3H, CH3NHAc), 1.33 (d, 3H, J5,6 = 6.2 Hz, H-6C); 81.4 (C-3D), 78.4 (C-5D), 75.4 (2C, C-5E, C-3E), 75.2 (C-2C), 74.8
13C NMR (CDCl3) δ 169.9 (2C, COAc, NHCO), 159.4 (CIVPMB), (2C, OCH2Pr, C-4C), 74.3 (C-4D), 73.8 (C-2E), 72.6 (C-5C), 71.9
138.5–137.2 (6C, CIVAr), 134.1 (CHvCH2), 130.0 (CIVPMB), (C-4E), 71.1 (C-3C), 63.9 (C-6D), 63.0 (C-6E), 56.9 (C-2D), 25.0
129.7–127.5 (32C, CAr), 116.8 (CHvCH2), 113.8 (2C, CHPMB), (CH3NHAc), 24.8 (CH2Pr), 23.1 (CH3Ac), 19.4 (C-6C), 12.3 (CH3Pr);
98.0 (C-1D, JC,H = 169.3 Hz), 97.0 (C-1E, JC,H = 170.6 Hz), 95.9 HRMS (ESI+): m/z 614.2626 (calcd for C25H44NO16 [M + H]+ m/z
1
1
7740 | Org. Biomol. Chem., 2014, 12, 7728–7749
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614.2660), HRMS (ESI+): m/z 636.2496 (calcd for C25H43NO16Na (C-1E28α*, C-1E28β*
,
1JC,H = 173.5 Hz), 99.8 (C-1E27α*, C-1E27β*
,
,
,
,
,
[M + Na]+ m/z 636.2479), HRMS (ESI+): m/z 1249.5182 (calcd for 1JC,H = 172.6 Hz), 99.2 (C-1C26α, 1JC,H = 170.7 Hz), 97.4 (C-1D26β
C50H86N2O32Na [2M + Na]+ m/z 1249.5061); RP-HPLC (215 nm): C-1D25β, C-1D28β
,
1JC,H = 167.5 Hz), 92.3 (C-1D26α, C-1D27α
1
Rt = 11.7 min.
C-1D28α, JC,H = 172.6 Hz), 63.8, 63.2, 63.0 (C-6Dα, C-6Dβ, C-6Eα
The 3C-O-acetyl product 24 had 1H NMR (D2O) (partial NMR) C-6Eβ), 58.2 (C-2Dβ), 54.8 (C-2Dα), 24.9, 24.8, 24.7 (CH3NHAcβ
δ 5.41 (d, 1H, J1,2 = 3.9 Hz, H-1E), 5.06 (bs, 1H, H-1C), 4.97 (dd, CH3NHAcα), 19.5, 19.4 (C-6Cα, C-6Cβ); HRMS (ESI+): m/z 594.1964
1H, J2,3 = 3.1 Hz, J3,4 = 9.9 Hz, H-3C), 4.54 (d, 1H, J1,2 = 7.8 Hz, (calcd for C22H37NO16Na [M + Na]+ m/z 594.2010); RP-HPLC
H-1D), 4.02 (bdd, 1H, J1,2 = 2.0 Hz, H-2C), 4.00 (t, 1H, J2,3 = J3,4
=
(215 nm): Rt = 1.97, 4.26 min.
7.7 Hz, H-3D), 3.91–3.77 (m, 6H, H-4D, H-6aD, H-5C, OCH2Pr
,
Propyl α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-
H-6bD), 3.75 (ddpo, 1H, J5,6b = 4.5 Hz, J6a,6b = 12.8 Hz, H-6aE), (1→4)]-2-acetamido-2-deoxy-β-D-glucopyranoside (29). To a
3.66–3.61 (m, 5H, H-5E, OCH2Pr, H-5D, H-4C, H-3E), 3.59 (ddpo stirred solution of trisaccharide 23, 24 and 25 (28 mg, 46
,
1H, H-2E), 3.43 (pto, 1H, J3,4 = J4,5 = 9.4 Hz, H-4E), 2.14 (s, 3H, µmol) in MeOH (5 mL) was added methanolic sodium methox-
CH3Ac), 2.02 (s, 3H, CH3NHAc), 1.54 (psex, 2H, J = 6.4 Hz, CH2Pr), ide (25% w/w, 12 µL). The solution was stirred at rt for 3 h.
1.28 (dpo, 3H, J5,6 = 5.8 Hz, H-6C), 0.85 (t, 3H, J = 7.4 Hz, CH3Pr); After this time, MS analysis of the reaction mixture revealed a
1
13C NMR (D2O) δ 176.6 (NHCO), 176.1 (COAc), 103.5 (C-1D, JC,H molecular weight corresponding to that of the target tetrasac-
1
1
= 163.9 Hz), 101.6 (C-1C, JC,H = 171.6 Hz), 99.9 (C-1E, JC,H
=
charide. The reaction solution was neutralized by addition of
173.8 Hz), 83.6 (C-3D), 77.9 (C-5D), 75.9 (C-3C),75.5 (C-4D), 75.4 Dowex H+ resin, and the suspension was filtered over a 0.2 µm
(2C, C-5E, C-3E), 74.9 (OCH2Pr), 73.9 (C-2E), 72.6 (C-5C), 72.2 filter. Evaporation of the volatiles, freeze-drying and purifi-
(C-4C), 71.9 (C-4E), 71.2 (C-2C), 63.7(C-6D), 63.0 (C-6E), 56.5 cation of the residue by RP-HPLC (0–20% linear gradient of
(C-2D), 24.8 (2C, CH3NHAc, CH2Pr), 22.9 (CH3Ac), 19.5 (C-6C), 12.3 CH3CN in 0.08% aq. TFA over 20 min at a flow rate of 5.5 mL
(CH3Pr); HRMS (ESI+): m/z 614.2626 (calcd for C25H44NO16 [M + min−1) gave the fully deprotected trisaccharide 29 (18 mg, 69%)
H]+ m/z 614.2660), HRMS (ESI+): m/z 636.2496 (calcd for as a white powder following repeated freeze-drying. Propyl glyco-
1
C25H43NO16Na [M + Na]+ m/z 636.2479), HRMS (ESI+): m/z side 29 had H NMR (D2O) δ 5.38 (d, 1H, J1,2 = 3.7 Hz, H-1E),
1249.5182 (calcd for C50H86N2O32Na [2M + Na]+ m/z 1249.5061); 5.05 (bs, 1H, H-1C), 4.57 (d, 1H, J1,2 = 7.8 Hz, H-1D), 4.00 (t, 1H,
RP-HPLC (215 nm): Rt = 11.7 min.
J2,3 = J3,4 = 7.3 Hz, H-3D), 3.92 (pt, 1H, J3,4 = J4,5 = 7.1 Hz, H-4D),
The 4C-O-acetyl product 25 had 1H NMR (D2O) (partial 3.90–3.87 (m, 3H, H-2C, H-2D, H-6aD), 3.85–3.73 (m, 5H, H-6aE,
NMR) δ 5.38 (d, 1H, J1,2 = 3.9 Hz, H-1E), 5.09 (bs, 1H, H-1C), OCH2Pr, H-6bD, H-6bE, H-5C), 3.69–3.64 (m, 4H, H-3C, H-5D,
4.85 (pt, 1H, J3,4 = J4,5 = 9.6 Hz, H-4C), 2.14 (s, 3H, CH3Ac), 2.02 H-5E, H-3E), 3.58 (ddpo, 1H, J2,3 = 9.9 Hz, H-2E), 3.53 (dt, 1H, J =
(s, 3H, CH3NHAc), 1.54 (psex, 2H, J = 6.4 Hz, CH2Pr), 1.15 (dpo
,
6.6 Hz, J = 9.6 Hz, OCH2Pr), 3.44 (pto, 1H, J3,4 = J4,5 = 9.4 Hz,
3H, J5,6 = 6.2 Hz, H-6C), 0.85 (t, 3H, J = 7.4 Hz, CH3Pr); 13C H-4E), 3.43 (pto, 1H, J3,4 = J4,5 = 9.5 Hz, H-4C), 2.03 (s, 3H,
NMR (D2O) (partial NMR) δ 176.7 (NHCO), 176.4 (COAc), 103.5 CH3NHAc), 1.54 (psex, 2H, J = 6.4 Hz, CH2Pr), 1.26 (d, 3H, J5,6
=
1
1
(C-1D, JC,H = 163.9 Hz), 102.6 (C-1C, JC,H = 170.3 Hz), 82.0 6.2 Hz, H-6C), 0.86 (t, 3H, J = 7.4 Hz, CH3Pr); 13C NMR (D2O)
1
(C-3D), 78.4 (C-5D), 75.1 (OCH2Pr), 73.9 (C-2E), 72.9 (C-5C), 70.8 δ 176.7 (NHCO), 103.5 (C-1D, JC,H = 162.9 Hz), 102.9 (C-1C,
1
(C-2C), 70.5 (C-3C), 63.8 (C-6D), 63.0 (C-6E), 56.8 (C-2D), 24.8 1JC,H = 169.1 Hz), 99.9 (C-1E, JC,H = 172.2 Hz), 82.1 (C-3D), 78.5
(CH2Pr), 23.1 (CH3Ac), 19.3 (C-6C), 12.3 (CH3Pr); HRMS (ESI+): (C-3C), 75.4 (C-3E*), 75.3 (C-5E*), 74.9 (C-4D), 74.8 (OCH2Pr), 74.6
m/z 614.2626 (calcd for C25H44NO16 [M + H]+ m/z 614.2660); (C-4C), 73.9 (C-2E), 73.1 (C-2C), 72.7 (C-5D), 72.6 (C-5C), 71.9
HRMS (ESI+): m/z 636.2496 (calcd for C25H43NO16Na [M + Na]+ (C-4E), 63.9 (C-6D), 63.0 (C-6E), 56.8 (C-2D), 24.8 (CH3NHAc), 24.7
m/z 636.2479); HRMS (ESI+): m/z 1249.5182 (calcd for (CH2Pr), 19.4 (C-6C), 12.3 (CH3Pr); HRMS (ESI+): m/z 572.2556
C50H86N2O32Na [2M + Na]+ m/z 1249.5061); RP-HPLC (215 nm): (calcd for C23H42NO15 [M + H]+ m/z 572.2554), HRMS (ESI+): m/z
Rt = 11.7 min.
594.2404 (calcd for C23H41NO15Na [M + Na]+ m/z 594.2374),
1
Regioisomers 26–28 had H NMR (D2O, partial) δ 5.45–5.40 HRMS (ESI+): m/z 1165.4824 (calcd for C46H82N2O30Na [2M +
(m, 1H, H-1E26α, H-1E27α, H-1E28α, H-1E26β, H-1E27β, H-1E28β), Na]+ m/z 1165.4850); RP-HPLC (215 nm): Rt = 8.6 min.
5.20 (d, 0.7H, J1,2 = 3.3 Hz, H-1D26α, H-1D27α, H-1D28α), 5.17 (d,
0.4H, J1,2 = 1.6 Hz, H-1C26α), 5.13–5.10 (m, 0.9H, H-2C26α
H-1C27α H-1C28α H-1C26β, H-1C28β), 5.08–5.06 (m, 0.3H, 6-O-benzyl-2-deoxy-β-D-glucopyranoside (30). Route 1. CAN
Allyl (2-O-acetyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-
[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-acetamido-
,
,
,
H-2C26β, H-1C27β), 4.98 (dd, 0.1H, J2,3 = 3.2 Hz, J3,4 = 9.8 Hz, (110 mg, 200 µmol, 4 equiv.) was added to a solution of
H-3C27β), 4.96 (dd, 0.25H, J2,3 = 3.1 Hz, J3,4 = 8.3 Hz, H-3C27α), the fully protected trisaccharide 22 (64 mg, 50 µmol) in
4.83 (dd, 0.2H, J3,4 = J4,5 = 9.7 Hz, H-4C28α), 4.76–4.72 (m, MeCN–H2O (10 : 1, 2.2 mL) at 0 °C, under an atmosphere of Ar.
0.34H, H-1D26β, H-1D27β, H-1D28β), 2.14, 2.05, 2.04, 2.02, 2.00 (5 After 3 h of stirring at this temperature, TLC analysis (cHex–
s, 6H, CH3Ac26
,
CH3Ac27
,
CH3Ac28
,
CH3NHAc26
,
CH3NHAc27
H-6C27
,
,
EtOAc 4 : 6) indicated the conversion of 22 (Rf 0.66) to a more
polar product (Rf 0.30). At this time, the reaction mixture was
CH3NHAc28), 1.29–1.24, 1.16–1.13 (m, 3H, H-6C26
,
H-6C28); 13C NMR (D2O) δ 176.9, 176.8, 176.1, 175.9 (NHCO26, diluted with DCM (30 mL) and washed with satd aq. NaHCO3
NHCO27, NHCO28, COAc26, COAc27, COAc28), 102.8 (C-1C26β*), (2 × 10 mL) and brine (10 mL). The organic layer was
1
1
102.5 (C-1C28α*, JC,H = 169.2 Hz), 101.9 (C-1C26β*, JC,H = 171.0 dried (Na2SO4), filtered and concentrated in vacuo to a pale
1
Hz), 101.5 (C-1C27α*, JC,H = 169.8 Hz), 100.5 (C-1E26α*, C-1E26β*
,
yellow residue (30, 58 mg), which was used without further
1JC,H = 172.9 Hz), 100.3 (C-1C27β* 1JC,H = 171.7 Hz), 100.1 purification.
,
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Route 2. Methanolic sodium methoxide (25% w/w, 400 µL, added to a solution of the disaccharide acceptor 10 (236 mg,
1.75 mmol, 0.4 equiv.) was added to allyl trisaccharide 41 270 µmol) and disaccharide donor14 37 (316 mg, 365 µmol,
(5.6 g, 4.69 mmol) in anhyd. MeOH (150 mL). The solution was 1.4 equiv.) in anhyd. Tol (6.0 mL), under an atmosphere of Ar.
stirred at rt for 2 h 30 min, at which time a TLC control (Tol– The reaction mixture was stirred at rt for 10 min, and then
EtOAc 5 : 5) showed the total conversion of the starting material TMSOTf (15 µL, 83 µmol, 0.3 equiv.) was added. The reac-
(Rf 0.7) into a more polar product (Rf 0.28). The reaction was tion mixture was heated to 55 °C and stirred at this tempera-
quenched with H+ Dowex resin. The suspension was filtered ture for 3 h. After this time, TLC analysis (Tol–EtOAc 6 : 4)
over a pad of Celite and the filtrate was concentrated to indicated the presence of a major new product (Rf 0.56) and
dryness under reduced pressure to give the crude diol 42 some remaining acceptor (Rf 0.21). Stirring at this temperature
(5.0 g, 97%) as a white foam. The material (5.0 g, 4.50 mmol) for an additional 3 h did not result in any improvement and
was dissolved in anhyd. MeCN (100 mL) under an atmosphere the reaction was quenched with Et3N. The reaction mixture
of Ar, then trimethyl orthoacetate (240 µL, 1.89 mmol, 2.0 equiv.) was filtered and the filtrate was concentrated in vacuo to a
and PTSA (21 mg, 0.11 mmol, 0.1 equiv.) were added. The yellow residue, which was purified by flash column chromato-
mixture was stirred at rt for 25 min, at which time a TLC control graphy (Tol–EtOAc 9 : 1 to 0 : 10) to give by the order of elution
(Tol–EtOAc 8 : 2) showed the total conversion of the starting the α-(1 ↔ 1)-α-linked dimer 39 (10 mg), the α-(1 ↔ 1)-β-linked
material (Rf 0.28) into a less polar product (Rf 0.78). 80% aq. dimer 38 (155 mg) both as colorless oils, and the somewhat
acetic acid (4.5 mL) was then added and the solution was contaminated tetrasaccharide 33 (160 mg), and finally the
stirred for 5 min at rt. A TLC control (Tol–EtOAc 8 : 2) showed unreacted acceptor 10 (126 mg, 50%), both as white foams.
the presence of a more polar product (Rf 0.52). The mixture Dimer 39 had Rf = 0.83 (Tol–EtOAc 6 : 4); 1H NMR (CDCl3)
was diluted with water (50 mL) and extracted with EtOAc δ 7.23–7.04 (m, 12H, HAr), 5.44 (dd, 2H, H-2B, H-2B′), 5.06 (dd,
(300 mL). The organic phase was washed with brine (150 mL), 2H, H-2C, H-2C′), 5.03 (d, 2H, J1,2 = 1.6 Hz, H-1B, H-1B′), 5.01 (d,
dried over anhyd. Na2SO4 and concentrated to dryness to give 1H, J1,2 = 1.6 Hz, H-1C, H-1C′), 4.90 (d, 2H, J = 11.0 Hz, HBn),
1
alcohol 30 (5.32 g, 99%) as a white foam. Acceptor 30 had H 4.82 (d, 2H, J = 11.0 Hz, HBn), 4.64 (d, 2H, J = 10.5 Hz, HBn),
NMR (CDCl3) δ 7.39–7.13 (m, 31H, NH, HAr), 5.92 (m, 1H, 4.61 (d, 2H, J = 11.0 Hz, HBn), 4.60 (d, 2H, J = 11.0 Hz, HBn),
CHvCH2), 5.29 (m, 1H, Jtrans = 17.3 Hz, Jgem = 1.7 Hz, 4.62 (do, 1H, J = 9.6 Hz, HBn), 4.43 (d, 2H, J = 11.3 Hz, HBn),
CHvCH2), 5.21–5.18 (m, 2H, H-2C, CHvCH2), 5.05 (d, 1H, J1,2 4.07 (dd, 2H, J2,3 = 3.5 Hz, J3,4 = 9.4 Hz, H-3C, H-3C′), 3.86 (dd,
= 1.4 Hz, H-1C), 4.89 (bs, 2H, HBn), 4.85 (do, 1H, HBn), 4.85 (do, 2H, J2,3 = 3.2 Hz, J3,4 = 9.2 Hz, H-3B, H-3B′), 3.82–3.73 (m, 4H,
1H, H-1E), 4.81 (dpo, 1H, HBn), 4.80 (dpo, 1H, HBn), 4.75 (do, H-5C, H-5C′, H-5B, H-5B′), 3.46 (pt, 2H, J4,5 = 9.5 Hz, H-4C,
1H, J1,2 = 1.5 Hz, H-1D), 4.72 (d, 1H, J = 11.2 Hz, HBn), 4.68 H-4C′), 3.39 (pt, 2H, J4,5 = 9.4 Hz, H-4B, H-4B′), 2.72–2.65 (m,
(dpo, 1H, J = 11.9 Hz, HBn), 4.57 (dpo, 1H, HBn), 4.48 (bs, 2H, 8H, HLev), 2.16 (s, 6H, 2 CH3Ac), 2.12 (s, 6H, 2 CH3Lev), 1.30 (do,
H
H
H
Bn), 4.47 (do, 1H, J = 10.8 Hz, HBn), 4.32 (dpo, 1H, J = 12.1 Hz, 6H, J5,6 = 6.2 Hz, H-6C, H-6C′), 1.27 (d, 6H, J5,6 = 6.3 Hz, H-6B,
Bn), 4.30–4.25 (mo, 2H, H-2D, HAll), 4.10–3.97 (m, 5H, H-5D, H-6B′); 13C NMR (CDCl3) δ 206.1 (2C, COLev), 171.7 (2C, CO2Lev),
All, H-3C, H-4D, H-3E), 3.93 (bd, 1H, J6a,6b = 9.0 Hz, H-6aD), 170.1 (2C, COAc), 138.5, 138.0, 137.9 (6C, CIVAr), 128.5–127.6
1
3.89 (bs, 1H, H-3D), 3.77–3.67 (m, 5H, H-4E, H-5E, H-6bD, H-5C, (30C, CAr), 99.6 (2C, C-1B, C-1B′, JC,H = 170.8 Hz), 92.2 (2C,
H-6aE), 3.62 (dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.7 Hz, H-2E), 3.42 C-1C, C-1C′, JC,H = 173.5 Hz), 79.9 (2C, C-4C, C-4C′), 79.8 (2C,
1
(bdpo, 1H, J6a,6b = 10.9 Hz, H-6bE), 3.38 (ptpo, 1H, J3,4 = J4,5
=
C-4B, C-4B′), 77.6 (2C, C-3B, C-3B′), 77.2 (2C, C-3C, C-3C′), 75.5,
9.3 Hz, H-4C), 2.21 (s, 3H, CH3Ac), 1.73 (s, 3H, CH3NHAc), 1.34 75.2 (4C, CBn), 71.9 (2C, C-2C, C-2C′), 71.5 (2C, CBn), 69.2 (2C,
(d, 3H, J5,6 = 6.3 Hz, H-6C); 13C NMR (CDCl3) δ 170.6 (COAc), C-2B, C-2B′), 68.7 (2C, C-5B, C-5B′), 68.6 (2C, C-5C, C-5C′), 38.1 (2C,
169.7 (NHCO), 138.4–137.2 (5C, CIVAr), 134.0 (CHvCH2), 129.9 CH2Lev), 29.8 (2C, CH3Lev), 28.2 (2C, CH2Lev), 21.0 (2C, CH3Ac),
(CIVAr), 128.8–127.5 (30C, CAr), 116.8 (CHvCH2), 97.9 (C-1D, 18.0 (2C, C-6B, C-6B′), 17.8 (2C, C-6C, C-6C′); HRMS (ESI+): m/z
1
1
1JC,H = 170.9 Hz), 96.9 (C-1E, JC,H = 170.3 Hz), 95.6 (C-1C, JC,H 1445.6136 (calcd for C92H105NO22Na [M + Na]+ m/z 1445.6084).
1
=
171.9 Hz), 82.1 (C-3E), 81.3 (C-4C), 79.0 (C-2E), 77.5
Dimer 38 had Rf = 0.60 (Tol–EtOAc 6 : 4); H NMR (CDCl3)
(C-4E), 75.8, 75.2, 75.0 (3C, CBn), 74.8 (C-5D), 74.3, 73.5, 73.2 δ 7.38–7.21 (m, 12H, HAr), 5.66 (bs, 1H, H-1C), 5.54 (dd, 1H, J1,2
=
(3C, CBn), 72.2 (C-2C), 71.4 (C-5E), 71.0 (C-4D), 70.8 (C-6D), 70.6 1.6 Hz, H-2B′), 5.45 (dd, 1H, J2,3 = 3.2 Hz, H-2B), 5.32 (dd, 1H,
(C-3C), 69.2 (CAll), 69.1 (C-3D), 68.4 (C-5C), 67.7 (C-6E), 46.3 H-2C′), 5.21 (d, 1H, J1,2 = 1.4 Hz, H-1C′), 5.15 (d, 1H, H-1B′), 5.09
(C-2D), 22.7 (CH3NHAc), 21.0 (CH3Ac), 18.2 (C-6C); HRMS (d, 1H, J1,2 = 1.6 Hz, H-1B), 4.90–4.82 (m, 4H, HBn), 4.72 (d, 1H,
(ESI+): m/z 1174.5121 (calcd for C67H77NO16Na [M + Na]+ m/z J = 10.9 Hz, HBn), 4.67–4.60 (m, 5H, HBn), 4.57 (d, 1H, J = 11.2
1174.5140).
Hz, HBn), 4.40 (d, 1H, J = 11.3 Hz, HBn), 4.28 (dd, 1H, J2,3 = 3.2
(2-O-Levulinoyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→ Hz, J3,4 = 9.5 Hz, H-3C′), 4.19 (dq, 1H, J4,5 = 9.5 Hz, J5,6 = 6.2 Hz,
3)-(2-O-acetyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→1)-(2-O- H-5C′), 4.02 (m, 1H, J = 1.6 Hz, H-2C), 3.95 (dd, 1H, J2,3 = 3.4
levulinoyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-2-O- Hz, J3,4 = 9.3 Hz, H-3B′), 3.88 (ddpo, 1H, H-3B), 3.87–3.82 (m,
acetyl-4-O-benzyl-β-L-rhamnopyranoside (38) and (2-O-levuli- 2H, H-3C, H-5C), 3.76 (dq, 1H, J4,5 = 9.4 Hz, J5,6 = 6.2 Hz, H-5B′),
noyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-(2-O-acetyl- 3.61 (pt, 1H, J3,4 = J4,5 = 9.4 Hz, H-4C), 3.49 (ptpo, 1H, J3,4 = J4,5
4-O-benzyl-α-L-rhamnopyranosyl)-(1→1)-(2-O-levulinoyl-3,4- = 9.4 Hz, H-4C′), 3.47 (mo, 1H, H-5B), 3.43 (ptpo, 1H, J4,5 = 9.2
di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-2-O-acetyl-4-O-benzyl- Hz, H-4B), 3.38 (ptpo, 1H, J3,4 = J4,5 = 9.4 Hz, H-4B′), 2.74–2.55
α-L-rhamnopyranoside (39). Activated 4 Å MS (300 mg) were (m, 8H, HLev), 2.17 (s, 3H, CH3Lev), 2.12 (s, 6H, 2 CH3Lev), 2.11
7742 | Org. Biomol. Chem., 2014, 12, 7728–7749
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(s, 3H, CH3Ac), 1.95 (s, 3H, CH3Ac), 1.36–1.25 (m, 12H, H-6B,
Allyl (4-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-
H-6B′, H-6C, H-6C′); 13C NMR (CDCl3) δ 206.0 (2C, COLev), 171.7, O-benzyl-α-D-glucopyranosyl-(1→4)]-2-acetamido-6-O-benzyl-
171.5 (2C, CO2Lev), 169.9, 168.9 (2C, COAc), 138.8, 138.7, 138.4, 2-deoxy-β-D-glucopyranoside (42). Methanolic sodium methox-
138.3, 138.0, 137.7 (6C, CIVAr), 129.0–127.4 (30C, CAr), 99.6 ide (25% w/w, 400 µL, 1.75 mmol, 0.4 equiv.) was added to
1
1
(C-1B, JC,H = 170.2 Hz), 99.5 (C-1B, JC,H = 172.7 Hz), 97.5 allyl trisaccharide 41 (5.6 g, 4.69 mmol) in anhyd. MeOH
1
1
(C-1C′, JC,H = 175.2 Hz), 91.6 (C-1C, JC,H = 161.9 Hz), 80.4 (150 mL). The solution was stirred at rt for 2 h 30 min, at which
(C-4C), 80.1 (C-4C′), 79.8 (C-4B′), 79.7 (C-4B), 78.5 (C-3B), 78.0 time a TLC control (Tol–EtOAc 5 : 5) showed the total conversion
(C-3B′), 77.7 (C-3C), 77.5 (C-3C′), 75.4, 75.3, 75.2 (3C, CBn), 74.8 of the starting material (Rf 0.7) into a more polar product
(2C, C-2C, CBn), 72.9 (C-5C), 72.2 (C-2C′), 71.6, 71.4 (2C, CBn), (Rf 0.28). The reaction was quenched with H+ Dowex resin. The
69.3 (C-2B), 69.1 (C-2B′), 68.6 (C-5B, C-5B′), 67.9 (C-5C′), 38.1, suspension was filtered over a pad of Celite and the filtrate was
38.0 (2C, CH2Lev), 29.8, 29.7 (2C, CH3Lev), 28.1, 28.0 (2C, concentrated to dryness under reduced pressure. Flash chrom-
CH2Lev), 21.0, 20.9 (2C, CH3Ac), 18.1, 18.0, 17.7 (4C, C-6B, C-6B′, atography of the crude material (Tol–EtOAc 6 : 4 to 3 : 7) gave
C-6C, C-6C′); HRMS (ESI+): m/z 1445.5952 (calcd for trisaccharide 42 (200 mg, 99%) as a white foam. The diol had
C80H94O23Na [M + Na]+ m/z 1445.6084).
1H NMR (CDCl3) δ 7.38–7.13 (m, 31H, NH, HAr), 5.88 (m, 1H,
Allyl (2,3-di-O-acetyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→ CHvCH2), 5.28 (m, 1H, Jtrans = 17.2 Hz, Jgem = 1.7 Hz,
3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-acet- CHvCH2), 5.21 (m, 1H, Jcis = 10.4 Hz, CHvCH2), 5.07 (d, 1H,
amido-6-O-benzyl-2-deoxy-β-D-glucopyranoside (41). Alcohol J1,2 = 1.8 Hz, H-1C), 4.89 (bs, 2H, HBn), 4.85 (dpo, 1H, H-1E),
10 (5.47 g, 6.26 mmol) and TCA 40 (3.63 g, 7.52 mmol, 1.2 4.82 (dpo, 1H, J = 10.7 Hz, HBn), 4.78 (do, 2H, HBn), 4.75 (do,
equiv.) were dissolved in anhyd. toluene (150 mL), and acti- 1H, J1,2 = 3.7 Hz, H-1D), 4.72 (dpo, 1H, HBn), 4.68 (dpo, 1H, J =
vated 4 Å MS (5.2 g) were then added. The suspension was 11.9 Hz, HBn), 4.57 (dpo, 1H, J = 12.1 Hz, HBn), 4.48 (dpo, 1H,
stirred for 15 min at rt under an atmosphere of Ar, and then J = 10.6 Hz, HBn), 4.47 (bs, 2H, HBn), 4.33 (dpo, 1H, HBn),
TMSOTf (200 µL, 1.10 mmol, 0.2 equiv.) was added. After stir- 4.30–4.24 (m, 2H, H-2D, HAll), 4.09–3.97 (m, 5H, H-5D, HAll
,
ring for 5 h at 50 °C, a TLC control (Tol–EtOAc 6 : 4) showed H-4D, H-3E, H-2C), 3.93–3.89 (m, 2H, H-3D, H-6aD), 3.84 (dd,
the disappearance of the acceptor 10 (Rf 0.21) and the presence 1H, J2,3 = 3.0 Hz, H-3C), 3.78–3.69 (m, 4H, H-5E, H-4E, H-6bD,
of a major less polar product (Rf 0.50). The reaction was H-5C), 3.67 (dpo, 1H, J5,6a = 1.9 Hz, J6a,6b = 10.5 Hz, H-6aE), 3.62
quenched by addition of Et3N (1 mL), and the suspension was (dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.7 Hz, H-2E), 3.37 (bdpo, 1H,
filtered over a pad of Celite. The volatiles were removed under H-6bE), 3.41 (ptpo, 1H, J3,4 = J4,5 = 8.9 Hz, H-4C), 2.82 (bs, 1H,
reduced pressure and the crude material was purified by flash OH), 2.47 (bs, 1H, OH), 1.74 (s, 3H, CH3NHAc), 1.34 (d, 3H, J5,6
chromatography (Tol–EtOAc 8 : 2 to 2 : 8) to give trisaccharide = 6.3 Hz, H-6C); 13C NMR (CDCl3) δ 169.9 (NHCO), 138.4–137.1
41 (5.6 g, 81%) as a white foam. The fully protected trisacchar- (6C, CIVAr), 133.9 (CHvCH2), 128.9–127.5 (30C, CAr), 116.9
1
1
ide 41 had 1H NMR (CDCl3) δ 7.39–7.11 (m, 31H, NH, HAr), (CHvCH2), 98.1 (C-1D, JC,H = 169.5 Hz), 97.6 (C-1C, JC,H
=
5.92 (m, 1H, CHvCH2), 5.37 (dd, 1H, J1,2 = 1.9 Hz, J2,3 = 3.3 169.8 Hz), 97.1 (C-1C, 1JC,H = 169.2 Hz), 82.1 (C-3E), 81.5 (C-4C),
Hz, H-2C), 5.31–5.25 (m, 2H, H-3C, CHvCH2), 5.21 (m, 1H, Jcis 79.1 (C-2E), 77.6 (C-4E), 75.8, 75.0 (2C, CBn), 74.9 (C-5D), 74.8,
= 10.5 Hz, Jgem = 1.6 Hz, CHvCH2), 5.07 (d, 1H, J1,2 = 1.8 Hz, 74.4, 73.5, 73.2 (4C, CBn), 71.4 (2C, C-3C, C-5E), 71.2 (C-4D),
H-1C), 4.89 (bs, 2H, HBn), 4.82 (dpo, 1H, H-1E), 4.80 (dpo, 1H, J = 70.8 (2C, C-6D, C-2C), 69.4 (C-3D), 69.2 (CAll), 68.5 (C-5C), 67.8
11.1 Hz, HBn), 4.78 (dpo, 1H, HBn), 4.76 (do, 1H, J1,2 = 3.7 Hz, (C-6E), 46.9 (C-2D), 22.6 (CH3NHAc), 18.2 (C-6C); HRMS (ESI+):
H-1D), 4.72 (d, 1H, J = 11.3 Hz, HBn), 4.66 (dpo, 1H, J = 12.1 Hz, m/z 1132.4995 (calcd for C65H75NO15Na [M
+
Na]+ m/z
H
Bn), 4.64 (dpo, 1H, HBn), 4.62 (dpo, 1H, J = 11.6 Hz, HBn), 4.56 1132.5034).
(dpo, 1H, J = 12.3 Hz, HBn), 4.53 (dpo, 1H, HBn), 4.46 (dpo, 1H,
Allyl (2-O-levulinoyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)-
H
Bn), 4.31–4.26 (mo, 2H, H-2D, HAll), 4.27 (do, 1H, HBn), (1→3)-(2-O-acetyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-
4.13–3.98 (m, 5H, H-5D, H-6aD, H-4D, HAll, H-3E), 3.94 (bs, 1H, [2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-acet-
H-3D), 3.83–3.74 (m, 4H, H-6bD, H-5C, H-4E, H-5E), 3.67 (bd, amido-6-O-benzyl-2-deoxy-β-D-glucopyranoside (33), allyl (2-O-
1H, J6a,6b = 10.5 Hz, H-6aE), 3.61 (dd, 1H, J1,2 = 3.7 Hz, J2,3 = 9.7 acetyl-4-O-benzyl-3-O-trimethylsilyl-α-L-rhamnopyranosyl)-(1→
Hz, H-2E), 3.54 (pt, 1H, J3,4 = J4,5 = 9.5 Hz, H-4C), 3.37 (bd, 1H, 3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→4)]-2-acet-
H-6bE), 2.19 (s, 3H, CH3Ac), 1.99 (s, 3H, CH3Ac), 1.76 (s, 3H, amido-6-O-benzyl-2-deoxy-β-D-glucopyranoside (43) and Allyl
CH3NHAc), 1.36 (d, 3H, J5,6 = 6.7 Hz, H-6C); 13C NMR (CDCl3) δ (2-O-acetyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-α-L-rhamno-
169.8 (NHCO), 169.6, 169.5 (2C, COAc), 138.7–137.3 (6C, CIVAr), pyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-
133.8 (CHvCH2), 129.0–127.3 (30C, CAr), 117.0 (CHvCH2), (1→4)]-2-acetamido-6-O-benzyl-2-deoxy-β-D-glucopyranoside
1
1
97.5 (C-1D, JC,H = 170.7 Hz), 96.7 (C-1E, JC,H = 169.5 Hz), 94.9 (44). Route 1. Activated MS 4 Å (159 mg) were added to a solu-
1
(C-1C, JC,H = 173.2 Hz), 82.1 (C-3E), 79.0 (C-2E), 78.6 (C-4C), tion of the crude alcohol 30 (obtained from the PMB-protected
77.5 (C-4E), 75.8, 75.0 (2C, CBn), 74.9 (2C, CBn, C-5D), 74.3, 73.4, precursor 22, see above) (46 mg, 40 µmol) and donor9 32
73.1 (3C, CBn), 71.7 (C-3C), 71.4 (C-5E), 70.8 (2C, C-6D, C-4D), (28 mg, 48 µmol, 1.2 equiv.) in anhyd. toluene (610 µL), under
69.8 (C-2C), 69.2 (CAll), 68.8 (C-5C), 68.2 (C-3D), 67.6 (C-6E), 45.8 an atmosphere of Ar. The reaction mixture was stirred at rt for
(C-2D), 22.7 (CH3NHAc), 20.9, 20.8 (2C, CH3Ac), 18.2 (C-6C); 30 min before being cooled to −10 °C. TMSOTf (0.7 µL, 4
HRMS (ESI+): m/z 1216.5172 (calcd for C75H85NO17Na [M + µmol, 0.1 equiv.) was then added, and the reaction mixture
Na]+ m/z 1216.5246).
was allowed to warm to rt while stirring for 18 h. After this
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time, TLC analysis (cHex–EtOAc 1 : 1) indicated the complete stirring for 3 h at rt, a TLC control (Tol–EtOAc 6 : 4) showed the
conversion of the acceptor 30 (Rf 0.17) and the donor 32 (Rf disappearance of the acceptor (Rf 0.23) and the presence of a
0.55) to a new product (Rf 0.42). The reaction was quenched major less polar product (Rf 0.56). The mixture was neutralized
with Et3N (100 µL). The reaction mixture was filtered and con- by addition of Et3N, and the suspension was filtered over a pad
centrated in vacuo to a yellow residue (60 mg), which was puri- of Celite. The volatiles were removed under reduced pressure
fied by flash column chromatography (Tol–EtOAc 8 : 2 to 4 : 6), and the crude material was purified by flash chromatography
to give tetrasaccharide 33 (36 mg, 58% over two steps) as a (Tol–EtOAc 8 : 2 to 6 : 4) to give first the silylated acceptor 44
pale yellow residue.
(300 mg, 6%) as a colorless oil, then tetrasaccharide 33 (4.54 g,
Route 2. Alcohol 30 (prepared from diacetate 41, see above) 83%) as a white foam. The tert-butyldimethylsilyl trisaccharide
(540 mg, 0.47 mmol) and TCA 32 (330 mg, 0.56 mmol, 1.2 44 had Rf = 0.66 (Tol–EtOAc 6 : 4); 1H NMR (CDCl3) δ 7.37–7.11
equiv.) were dissolved in anhyd. Et2O (20 mL), and activated (m, 31H, NH, HAr), 5.95 (m, 1H, CHvCH2), 5.31 (m, 1H, Jtrans
=
4 Å MS (1 g) were added. The suspension was stirred for 17.3 Hz, Jgem = 1.7 Hz, CHvCH2), 5.21–5.18 (m, 2H, H-2C,
25 min at rt, then cooled to −15 °C. TMSOTf (17 µL, 94 µmol, CHvCH2), 4.99 (d, 1H, J1,2 = 1.5 Hz, H-1C), 4.91 (dpo, 1H, HBn),
0.2 equiv.) was added. After stirring for 6 h at −15 °C, a TLC 4.89 (bs, 2H, HBn), 4.86 (do, 1H, J1,2 = 3.7 Hz, H-1E), 4.81 (dpo
,
control (Tol–EtOAc 6 : 4) showed the presence of the acceptor 1H, J = 11.0 Hz, HBn), 4.77 (dpo, 1H, HBn), 4.77 (bs, 1H, H-1D),
(Rf 0.23) and that of a major new product (Rf 0.56). In the 4.67 (d, 1H, J = 11.8 Hz, HBn), 4.62 (d, 1H, J = 11.2 Hz, HBn),
absence of any observed evolution, the suspension was filtered 4.57 (d, 1H, J = 12.1 Hz, HBn), 4.51 (dpo, 1H, J = 11.4 Hz, HBn),
over a pad of Celite following neutralization by addition of 4.47 (dpo, 1H, HBn), 4.45 (dpo, 1H, HBn), 4.30–4.26 (mo, 2H,
Et3N. The volatiles were removed under reduced pressure and H-2D, HAll), 4.29 (do, 1H, HBn), 4.10 (dddpo, 1H, J5,6a = 5.5 Hz,
the crude material was purified by flash chromatography (Tol– J4,5 = 8.8 Hz, H-5D), 4.05–3.98 (m, 5H, HAll, H-3C, H-3E, H-4D,
EtOAc 8 : 2 to 0 : 1) to give, by order of elution, first the sily- H-6aD), 3.91 (bs, 1H, H-3D), 3.81 (ddpo, 1H, J6a,6b = 8.9 Hz,
lated acceptor 43 (145 mg, 23%) as a colorless oil, and then H-6bD), 3.77–3.61 (m, 5H, H-4E, H-5E, H-5C, H-6aE, H-2E), 3.41
the tetrasaccharide 33 (254 mg, 34%) as a white foam, and (ptpo, 1H, J3,4 = J4,5 = 9.3 Hz, H-4C), 3.38 (bdpo, 1H, J6a,6b = 10.3
finally some remaining trisaccharide 30 (155 mg, 29%) as a Hz, H-6bE), 2.20 (s, 3H, CH3Ac), 1.72 (s, 3H, CH3NHAc), 1.29 (d,
white foam. The trimethylsilyl derivative 43 had Rf = 0.58 (Tol– 3H, J5,6 = 6.2 Hz, H-6C), 0.91 (bs, 9H, CH3tBuSi), 0.14 (bs, 3H,
1
EtOAc 6 : 4); H NMR (CDCl3) δ 7.37–7.12 (m, 31H, NH, HAr), SiCH3), 0.05 (bs, 3H, SiCH3); 13C NMR (CDCl3) δ 169.8 (2C,
5.95 (m, 1H, CHvCH2), 5.31 (m, 1H, Jtrans = 17.2 Hz, Jgem = 1.7 COAc, NHCO), 138.4–137.1 (6C, CIVAr), 134.0 (CHvCH2),
1
Hz, CHvCH2), 5.21–5.18 (m, 2H, H-2C, CHvCH2), 5.00 (d, 1H, 129.0–125.3 (30C, CAr), 116.9 (CHvCH2), 97.6 (C-1D, JC,H
=
J1,2 = 1.4 Hz, H-1C), 4.91 (dpo, 1H, HBn), 4.89 (bs, 2H, HBn), 4.86 170.6 Hz), 97.0 (C-1E, 1JC,H = 169.7 Hz), 95.6 (C-1C, 1JC,H = 171.1
(do, 1H, H-1E), 4.81 (dpo, 1H, J = 10.8 Hz, HBn), 4.78 (dpo, 1H, Hz), 82.1 (C-3E), 81.1 (C-4C), 79.0 (C-2E), 77.5 (C-4E), 75.9, 75.5,
H
Bn), 4.77 (bs, 1H, H-1D), 4.67 (d, 1H, J = 11.8 Hz, HBn), 4.62 75.0 (3C, CBn), 74.6 (C-5D), 74.3, 73.4, 73.0 (3C, CBn), 72.1
(d, 1H, J = 11.2 Hz, HBn), 4.56 (d, 1H, J = 12.1 Hz, HBn), (C-2C), 71.4 (C-5E), 71.1 (C-4D), 71.0 (2C, C-3C, C-6D), 69.4 (CAll),
4.53–4.46 (m, 3H, HBn), 4.31–4.26 (mo, 2H, H-2D, HAll), 4.30 68.8 (C-5C), 68.3 (C-3D), 67.7 (C-6E), 45.8 (C-2D), 25.8 (3C,
(do, 1H, HBn), 4.10 (ddpo, 1H, J4,5 = 8.7 Hz, H-5D), 4.06 (ddo, CH3tBuSi), 22.6 (CH3NHAc), 21.0 (CH3Ac), 18.1 (CIVSi), 17.8 (C-6C),
1H, J2,3 = 3.5 Hz, J3,4 = 9.2 Hz, H-3C), 4.05–3.98 (m, 4H, HAll
,
−4.5 (CH3Si), −4.8 (CH3Si); HRMS (ESI+): m/z 1266.5991 (calcd
for C73H92NO16Si [M + H]+ m/z 1266.6185), m/z 1288.5845
H-3E, H-4D, H-6aD), 3.91 (bs, 1H, H-3D), 3.81 (ddpo, 1H, J5,6b
=
5.6 Hz, J6a,6b = 9.0 Hz, H-6bD), 3.77–3.67 (m, 4H, H-4E, H-5E, (calcd for C73H91NO16SiNa [M + Na]+ m/z 1288.6005).
1
H-5C, H-6aE), 3.62 (dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.7 Hz, H-2E),
The coupling product 33 had: H NMR (CDCl3) δ 7.36–7.11
3.42–3.38 (m, 2H, H-6bE, H-4C), 2.21 (s, 3H, CH3Ac), 1.72 (s, (m, 41H, 40HAr, NH), 5.95 (m, 1H, CHvCH2), 5.42 (dd, 1H, J1,2
3H, CH3NHAc), 1.30 (d, 3H, J5,6 = 6.2 Hz, H-6C), 0.16 (bs, 9H, = 1.8 Hz, J2,3 = 3.2 Hz, H-2B), 5.31 (m, 1H, Jtrans = 17.3 Hz, Jgem
SiCH3); 13C NMR (CDCl3) δ 169.8 (COAc), 169.7 (NHCO), = 1.7 Hz, CHvCH2), 5.21–5.16 (m, 2H, H-2C, CHvCH2), 5.04
138.4–137.1 (6C, CIVAr), 134.0 (CHvCH2), 129.0–127.4 (30C, (d, 1H, J1,2 = 1.5 Hz, H-1C), 4.94 (d, 1H, H-1B), 4.90 (dpo, 1H, J =
CAr), 116.8 (CHvCH2), 97.7 (C-1D, 1JC,H = 170.9 Hz), 97.0 (C-1E, 11.3 Hz, HBn), 4.88 (bspo, 2H, HBn), 4.82 (do, 1H, H-1E), 4.81
1JC,H = 169.3 Hz), 95.7 (C-1C, 1JC,H = 170.8 Hz), 82.1 (C-3E), 81.1 (do, 1H, J = 11.4 Hz, HBn), 4.80 (do, 1H, J = 11.3 Hz, HBn), 4.77
(C-4C), 79.0 (C-2E), 77.5 (C-4E), 75.8, 75.5, 75.0 (3C, CBn), 74.7 (do, 1H, HBn), 4.76 (do, 1H, J1,2 = 3.2 Hz, H-1D), 4.66 (do, 1H, J =
(C-5D), 74.3, 73.5, 73.0 (3C, CBn), 72.2 (C-2C), 71.5 (C-5E), 71.2 10.2 Hz, HBn), 4.64 (do, 1H, HBn), 4.62 (do, 1H, J = 9.6 Hz, HBn),
(C-3C), 71.0 (2C, C-4D, C-6D), 69.4 (CAll), 68.7 (C-5C), 68.5 (C-3D), 4.59 (do, 1H, HBn), 4.55 (do, 1H, HBn), 4.54 (do, 1H, J = 11.6 Hz,
67.7 (C-6E), 45.9 (C-2D), 22.6 (CH3NHAc), 21.0 (CH3Ac), 18.1
(C-6C), 0.1 (3C, CH3Si); HRMS (ESI+): m/z 1224.5702 (calcd for
C70H86NO16Si [M + H]+ m/z 1224.5716), m/z 1224.5590 (calcd
for C70H85NO16SiNa [M + Na]+ m/z 1246.5535).
H
H
H
Bn), 4.49 (do, 1H, J = 11.4 Hz, HBn), 4.47 (do, 1H, J = 11.2 Hz,
Bn), 4.44 (do, 1H, J = 11.6 Hz, HBn), 4.30–4.24 (m, 2H, H-2D,
All), 4.26 (do, 1H, J = 12.0 Hz, HBn), 4.09–3.97 (m, 6H, H-6aD,
H-5D, HAll, H-3C, H-4D, H-3E), 3.89–3.83 (m, 3H, H-3D, H-3B,
Route 3. Alcohol 30 (4.0 g, 3.47 mmol) prepared from di- H-5B), 3.77–3.65 (m, 5H, H-6bD, H-5E, H-4E, H-5C, H-6aE), 3.60
acetate 41 and TCA 32 (2.7 g, 4.60 mmol, 1.3 equiv.) were dis- (dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.7 Hz, H-2E), 3.49 (pt, 1H, J3,4 = J4,5
solved in anhyd. Tol (150 mL), and activated 4 Å MS (12 g) = 9.4 Hz, H-4C), 3.40 (tpo, 1H, J3,4 = J4,5 = 9.4 Hz, H-4B), 3.36
were added. The suspension was stirred for 25 min at rt, and (bdpo, 1H, J6a,6b = 10.7 Hz, H-6bE), 2.63–2.61 (m, 4H, HLev),
then TBSOTf (170 µL, 0.74 mmol, 0.2 equiv.) was added. After 2.15 (s, 3H, CH3Ac), 2.12 (s, 3H, CH3Lev), 1.74 (s, 3H, CH3NHAc),
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1.29 (do, 3H, J5,6 = 6.0 Hz, H-6C), 1.28 (do, 3H, J5,6 = 6.2 Hz, (C-2B), 68.8 (C-5C), 68.2 (2C, C-3D, C-5B), 67.6 (C-6E), 45.8
H-6B); 13C NMR (CDCl3) δ 206.0 (COLev), 171.7 (CO2Lev), 169.7 (C-2D), 22.7 (CH3NHAc), 21.0 (CH3Ac), 18.1, 18.0 (2C, C-6B, C-6C);
(NHCO), 169.6 (COAc), 138.6–137.2 (8C, CIVAr), 134.0 HRMS (ESI+): m/z 1500.6599 (calcd for C87H99NO20Na [M +
(CHvCH2), 129.1–127.3 (40C, CAr), 116.9 (CHvCH2), 99.5 Na]+ m/z 1500.6658).
1
1
(C-1B, JC,H = 171.7 Hz), 97.6 (C-1D, JC,H = 171.4 Hz), 96.9
Propyl α-L-rhamnopyranosyl-(1→3)-(2-O-acetyl-α-L-rhamno-
1
1
(C-1E, JC,H = 170.7 Hz), 95.1 (C-1C, JC,H = 172.3 Hz), 82.1 pyranosyl)-(1→3)-[α-D-glucopyranosyl-(1→4)]-2-acetamido-2-
(C-3E), 80.4 (C-4C), 79.7 (C-4B), 79.0 (C-2E), 77.5 (2C, C-3B, deoxy-β-D-glucopyranoside (46). To a stirred solution of tetra-
C-4E), 76.4 (C-3C), 75.8, 75.5, 75.0 (3C, CBn), 74.9 (C-5D), 74.7, saccharide 45 (299 mg, 202 µmol) in 90% aq. EtOH (14.9 mL),
74.3, 73.5, 73.1 (4C, CBn), 71.6 (2C, C-2C, CBn), 71.4 (C-5E), 70.9 were added Pd/C (299 mg) and 1M aq. HCl (38 µL). The sus-
(C-6D), 70.7 (C-4D), 69.4 (C-2B), 69.3 (CAll), 68.8 (C-5C), 68.6 pension was stirred under an H2 atmosphere for a day at rt.
(C-3D), 68.5 (C-5B), 67.7 (C-6E), 46.0 (C-2D), 38.0 (CH2Lev), 29.8 After this time, MS analysis revealed a molecular weight corres-
(CH3Lev), 28.1 (CH2Lev), 22.7 (CH3NHAc), 21.0 (CH3Ac), 18.1 (2C, ponding to that of the target tetrasaccharide and the absence
C-6B, C-6C); HRMS (ESI+): m/z 1598.6995 (calcd for of any molecular weight corresponding to the benzylated inter-
C92H105NO22Na [M + Na]+ m/z 1598.7026).
mediates. The reaction mixture was filtered over a pad of
Allyl (3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-(2-O- Celite. Evaporation of the volatiles, freeze-drying and purifi-
acetyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra- cation of the residue by RP-MPLC (0–50% linear gradient of
O-benzyl-α-D-glucopyranosyl-(1→4)]-2-acetamido-6-O-benzyl- 80% aq. MeCN over 60 min at a flow rate of 20 mL min−1) gave
2-deoxy-β-D-glucopyranoside (45). To a solution of tetrasac- tetrasaccharide 46 (140 mg, 90%) as a white powder following
1
charide 33 (960 mg, 0.61 mmol) in anhyd. pyridine (30 mL) repeated freeze-drying. H NMR (D2O) δ 5.40 (d, 1H, J1,2 = 3.8
stirred at 0 °C under an Ar atmosphere were added dropwise Hz, H-1E), 5.15 (d, 1H, J2,3 = 2.5 Hz, H-2C), 5.13 (bs, 1H, H-1C),
AcOH (10 mL) and hydrazine monohydrate (215 µL, 4.98 (bs, 1H, H-1B), 4.53 (d, 1H, J1,2 = 7.6 Hz, H-1D), 4.06 (t, 1H,
4.42 mmol, 7.3 equiv.). The reaction mixture was stirred at rt J2,3 = J3,4 = 7.6 Hz, H-3D), 3.99–3.77 (m, 9H, H-2B, H-4D, H-3C,
for 90 min. A TLC control (Tol–EtOAc 6 : 4) showed the conver- H-6aD, H-2D, H-5C, OCH2Pr, H-6bD, H-6aE), 3.75 (ddpo, 1H, J5,6b
sion of the starting material (Rf 0.46) into a more polar = 4.6 Hz, J6a,6b = 12.4 Hz, H-6bE), 3.69–3.60 (m, 5H, H-5D, H-3B,
product (Rf 0.38). Following addition of DCM (80 mL) and H-3E, H-5E, H-5B), 3.59 (pto, 1H, J3,4 = J4,5 = 9.3 Hz, H-4C), 3.56
water (35 mL), the two layers were separated and the aq. one (ddpo, 1H, J2,3 = 9.8 Hz, H-2E), 3.50 (dt, 1H, J = 6.6 Hz, J = 12.8
was extracted twice with DCM. The combined organic extracts Hz, OCH2Pr), 3.43 (pto, 1H, J3,4 = J4,5 = 9.6 Hz, H-4E), 3.40 (pto,
were washed with 5% aq. citric acid (35 mL), and brine 1H, J3,4 = J4,5 = 9.7 Hz, H-4B), 2.16 (s, 3H, CH3Ac), 2.01 (s, 3H,
(100 mL), then dried over anhyd. Na2SO4 and concentrated to CH3NHAc), 1.52 (psex, 2H, J = 7.0 Hz, CH2Pr), 1.27 (d, 3H, J5,6
=
dryness. The residue was purified by flash chromatography 6.2 Hz, H-6C), 1.23 (d, 3H, J5,6 = 6.2 Hz, H-6B), 0.85 (t, 3H, J =
(Tol–EtOAc 8 : 2 to 0 : 1) to give alcohol 45 (0.86 g, 96%) as a 7.4 Hz, CH3Pr); 13C NMR (D2O) δ 176.9 (NHCO), 175.6 (COAc),
1
1
white foam. Tetrasaccharide 45 had 1H NMR (CDCl3) δ 104.6 (C-1B, JC,H = 169.4 Hz), 103.6 (C-1D, JC,H = 163.4 Hz),
1
1
7.37–7.12 (m, 41H, 40HAr, NH), 5.96 (m, 1H, CHvCH2), 5.33 99.8 (C-1E, JC,H = 173.4 Hz), 99.3 (C-1C, JC,H = 170.7 Hz), 82.7
(m, 1H, Jtrans = 17.2 Hz, Jgem = 1.7 Hz, CHvCH2), 5.23 (ddpo (C-3D), 78.7 (C-4D), 78.1 (C-5D), 75.5 (C-3E), 75.4 (C-5E), 74.8
,
1H, J1,2 = 1.8 Hz, J2,3 = 3.2 Hz, H-2C), 5.20 (mo, 1H, CHvCH2), (OCH2Pr), 74.7 (C-2C), 74.6 (C-4B), 74.5 (C-3C), 74.1 (C-4C), 73.9
5.06 (d, 1H, H-1C), 5.00 (d, 1H, J1,2 = 1.3 Hz, H-1B), 5.02–4.87 (C-2E), 72.7 (2C, C-2B, C-3B), 72.5 (C-5C), 72.0 (C-5B), 71.9
(m, 3H, HBn), 4.84 (do, 1H, H-1E), 4.81 (do, 2H, J = 10.9 Hz, (C-4E), 63.9 (C-6D), 63.1 (C-6E), 56.5 (C-2D), 25.1 (CH3NHAc), 24.8
H
Bn), 4.78 (bs, 1H, H-1D), 4.77 (do, 1H, J = 11.9 Hz, HBn), 4.73 (CH2Pr), 22.9 (CH3Ac), 19.5 (C-6C), 19.4 (C-6B), 12.3 (CH3Pr);
(d, 1H, J = 11.0 Hz, HBn), 4.70–4.57 (m, 5H, HBn), 4.56 (do, 1H, HRMS (ESI+): m/z 760.3248 (calcd for C31H53NO20 [M + H]+ m/z
J = 12.1 Hz, HBn), 4.54 (do, 1H, J = 11.6 Hz, HBn), 4.47 (do, 2H, 760.3239), HRMS (ESI+): m/z 782.3068 (calcd for C31H53NO20Na
H
Bn), 4.32–4.25 (m, 3H, H-2D, HBn, HAll), 4.11–3.98 (m, 6H, [M + Na]+ m/z 782.3058), HRMS (ESI+): m/z 1541.6234 (calcd for
H-6aD, H-5D, HAll, H-3C, H-4D, H-3E), 3.94 (dd, 1H, J2,3 = 3.2 Hz, C62H106N2O40Na [2M
H-2B), 3.90 (bs, 1H, H-3D), 3.82 (dqo, 1H, J4,5 = 9.5 Hz, H-5B), (215 nm): Rt = 12.8 min.
+
Na]+ m/z 1541.6219); RP-HPLC
3.80–3.66 (m, 6H, H-3B, H-6bD, H-5E, H-4E, H-5C, H-6aE), 3.61
Propyl α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-
(dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.7 Hz, H-2E), 3.47 (to, 1H, J3,4 = J4,5 (1→3)-[α-D-glucopyranosyl-(1→4)]-2-acetamido-2-deoxy-β-D-gluco-
= 9.4 Hz, H-4C), 3.46 (to, 1H, J3,4 = J4,5 = 9.3 Hz, H-4B), 3.36 (bd, pyranoside (47). To a stirred solution of slightly contami-
1H, J6a,6b = 10.4 Hz, H-6bE), 2.34 (bs, 1H, OH), 2.17 (s, 3H, nated tetrasaccharide 46 (82 mg, 108 µmol), issued from
CH3Ac), 1.74 (s, 3H, CH3NHAc), 1.31–1.29 (bd, 6H, J5,6 = 6.2 Hz, RP-HPLC purification, in MeOH (8 mL), was added methanolic
H-6B, H-6C); 13C NMR (CDCl3) δ 169.8 (NHCO), 169.7 (COAc), sodium methoxide to reach pH 10 (25% w/w, 25 µL). The solu-
138.5–137.2 (8C, CIVAr), 134.0 (CHvCH2), 128.9–127.4 (40C, tion was stirred at rt for 15 h. After this time, MS analysis
1
CAr), 116.9 (CHvCH2), 101.3 (C-1B, JC,H = 173.0 Hz), 97.6 revealed the presence of the target tetrasaccharide and the
1
1
(C-1D, JC,H = 168.7 Hz), 96.9 (C-1E, JC,H = 170.9 Hz), 95.0 absence of any starting 46. The reaction was neutralized by the
1
(C-1C, JC,H = 170.0 Hz), 82.1 (C-3E), 80.5 (C-4C), 79.7 (C-4B), addition of Dowex H+ resin, and the suspension was filtered
79.5 (C-3B), 78.9 (C-2E),77.5 (C-4E), 76.8 (C-3C), 75.9, 75.5 (2C, over a 0.2 µm filter. Evaporation of the volatiles, freeze-drying
CBn), 75.0 (2C, CBn), 74.7 (C-5D), 74.3, 73.5, 73.1, 72.1 (4C, CBn), and purification of the residue by RP-MPLC (0–50% linear gra-
71.8 (C-2C), 71.4 (C-5E), 70.9 (C-6D), 70.6 (C-4D), 69.3 (CAll), 69.1 dient of 80% aq. MeCN over 60 min at a flow rate of 20 mL
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min−1) gave tetrasaccharide 47 (47 mg, 60%) as a white powder H-2B, H-3A, H-3D), 3.80 (ddpo, 1H, J2,3 = 2.7 Hz, H-3B), 3.78–3.71
following repeated freeze-drying. The fully deprotected tetra- (m, 5H, H-5B, H-5A, H-6bD, H-5E, H-4E), 3.73–3.66 (m, 2H,
1
saccharide 47 had H NMR (D2O) δ 5.35 (d, 1H, J1,2 = 3.9 Hz, H-5C, H-6aE), 3.60 (dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.7 Hz, H-2E),
H-1E), 5.04 (dpo, 1H, J1,2 = 1.7 Hz, H-1C), 4.98 (dpo, 1H, J1,2 = 1.5 3.47 (to, 1H, J3,4 = J4,5 = 9.4 Hz, H-4C), 3.46 (to, 1H, J3,4 = J4,5
Hz, H-1B), 4.55 (d, 1H, J1,2 = 7.4 Hz, H-1D), 4.05 (t, 1H, J2,3 = J3,4 9.2 Hz, H-4B), 3.39–3.32 (m, 2H, H-4A, H-6bE), 2.77–2.65 (m,
= 6.7 Hz, H-3D), 4.02 (ddpo, 1H, J2,3 = 3.4 Hz, H-2B), 3.96 (bdpo 4H, HLev), 2.17 (s, 3H, CH3Lev), 2.14 (s, 3H, CH3Ac), 1.74 (bs,
=
,
1H, J2,3 = 3.6 Hz, H-2C), 3.94 (ptpo, J3,4 = J4,5 = 7.1 Hz, H-4D), 3H, CH3NHAc), 1.19 (d, 3H, J5,6 = 6.2 Hz, H-6B), 1.13 (d, 3H, J5,6
3.91 (ptpo, J2,3 = 7.0 Hz, H-2D), 3.86 (ddo, 1H, J5,6a = 7.9 Hz, = 6.5 Hz, H-6C), 1.09 (d, 3H, J5,6 = 6.2 Hz, H-6A); 13C NMR
J6a,6b = 11.8 Hz, H-6aD), 3.83–3.71 (m, 9H, H-6aE, H-5C, H-5B, (CDCl3) δ 206.1 (COLev), 171.7 (CO2Lev), 169.7 (NHCO), 169.6
OCH2Pr, H-3C, H-3B, H-6bD, H-6bE, H-5D), 3.66 (pt, 1H, J2,3
J3,4 = 9.6 Hz, H-3E), 3.62 (dddpo, 1H, J5,6a = 2.3 Hz, J5,6b = 4.6 (50C, CAr), 116.9 (CHvCH2), 100.9 (C-1B, JC,H = 171.2 Hz),
Hz, J4,5 = 10.0 Hz, H-5E), 3.57 (ddpo, 1H, J2,3 = 9.8 Hz, H-2E), 99.1 (C-1A, JC,H = 171.7 Hz), 97.5 (C-1D, JC,H = 170.6 Hz), 96.9
3.53 (pto, 1H, J3,4 = J4,5 = 9.6 Hz, H-4C), 3.50 (dt, 1H, J = 6.6 Hz, (C-1E, JC,H = 171.2 Hz), 95.2 (C-1C, JC,H = 172.9 Hz), 82.1
J = 9.6 Hz, OCH2Pr), 3.43 (pto, 1H, J3,4 = J4,5 = 9.7 Hz, H-4E), (C-3E), 80.2 (C-4C), 80.0 (C-4A), 79.7 (C-4B), 79.2 (C-3B), 79.0
3.42 (pto, 1H, J3,4 = J4,5 = 9.9 Hz, H-4B), 2.00 (s, 3H, CH3NHAc), (C-2E), 77.6 (C-3A), 77.5 (C-4E), 77.2 (C-3C), 75.8 (CBn), 75.7
1.53 (psex, 2H, J = 7.0 Hz, CH2Pr), 1.28 (d, 3H, J5,6 = 6.2 Hz, (C-2B), 75.3 (2C, CBn), 74.9, 74.7 (2C, CBn), 74.6 (C-5D), 74.3,
=
(COAc), 138.6–137.2 (10C, CIVAr), 134.0 (CHvCH2), 129.0–125.3
1
1
1
1
1
H-6C), 1.25 (d, 3H, J5,6 = 6.2 Hz, H-6B), 0.85 (t, 3H, J = 7.5 Hz, 73.4, 73.1, 72.2 (4C, CBn), 71.8 (C-2C), 71.6 (2C, CBn, C-5E), 70.8
1
CH3Pr); 13C NMR (D2O) δ 176.5 (NHCO), 104.7 (C-1B, JC,H
=
=
(2C, C-6D, C-4D), 69.3 (CAll), 69.2 (C-2A), 68.9 (C-5B), 68.7 (C-5C),
68.5 (C-3D), 68.3 (C-5A), 67.6 (C-6E), 46.0 (C-2D), 38.1 (CH2Lev),
1
1
172.4 Hz), 103.6 (C-1D, JC,H = 163.4 Hz), 101.6 (C-1C, JC,H
170.0 Hz), 99.6 (C-1E, 1JC,H = 173.0 Hz), 80.8 (C-3D), 79.9 (C-3C), 29.8 (CH3Lev), 28.2 (CH2Lev), 22.6 (CH3NHAc), 21.0 (CH3Ac), 18.1
78.7 (C-5D), 75.5 (C-3E), 75.4 (C-5E), 74.8 (OCH2Pr), 74.7 (C-4B), (2C, C-6B, C-6C), 17.9 (C-6A); HRMS (ESI+): m/z 1924.8542 (calcd
74.1 (C-4D), 73.8 (2C, C-2E, C-4C), 73.0 (C-2C), 72.8 (C-2B), 72.7 for C112H127NO26Na [M + Na]+ m/z 1924.8544); HRMS (ESI+):
(C-3B), 72.5 (C-5C), 71.9 (C-5B), 71.7 (C-4E), 64.1 (C-6D), 63.1 m/z 1902.8735 (calcd for C112H128NO26 [M + H]+ m/z 1903.8724);
(C-6E), 56.3 (C-2D), 24.9 (CH3NHAc), 24.8 (CH2Pr), 22.9 (CH3Ac), HRMS (ESI+): m/z 973.9128 (calcd for C112H127NO26Na2
19.4 (2C, C-6C, C-6B), 12.3 (CH3Pr); HRMS (ESI+): m/z 718.3145 [M + 2Na]+ m/z 973.9221).
(calcd for C29H52NO19 [M + H]+ m/z 718.3134); HRMS (ESI+):
m/z 740.2953 (calcd for C29H51NO19Na [M + Na]+ m/z 740.2953); O-benzyl-α-L-rhamnopyranosyl)-(1→3)-(2-O-acetyl-4-O-benzyl-
RP-HPLC (215 nm): Rt = 9.9 min. α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-gluco-
Allyl (3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4-di-
Allyl (2-O-levulinyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl)- pyranosyl-(1→4)]-2-acetamido-6-O-benzyl-2-deoxy-β-D-gluco-
(1→2)-(3,4-di-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-(2-O- pyranoside (50). To a solution of pentasaccharide 49 (400 mg,
acetyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-tetra- 0.21 mmol) in anhyd. pyridine (11 mL) stirred at rt under an
O-benzyl-α-D-glucopyranosyl-(1→4)]-2-acetamido-6-O-benzyl- atmosphere of Ar, were added dropwise AcOH (7 mL) and
2-deoxy-β-D-glucopyranoside (49). Alcohol 45 (350 mg, hydrazine monohydrate (70 µL, 1.44 mmol, 7.0 equiv.). The
0.24 mmol) and trichloroacetimidate 32 (210 mg, 0.36 mmol, reaction mixture was stirred at rt for 3 h. Following the
1.5 equiv.) were dissolved in anhyd. DCE (7 mL), and activated addition of DCM (35 mL) and water (35 mL), the two layers
4 Å MS (0.31 g) were added. The suspension was stirred for were separated and the aq. one was extracted twice with DCM
25 min at rt, and then TfOH (7 µL, 79 µmol, 0.3 equiv.) was (2 × 35 mL). The combined organic extracts were washed with
added. After stirring for 5 h at rt, a TLC control (Tol–EtOAc 5% aq. citric acid (75 mL), brine (75 mL), then dried over
6 : 4) showed the disappearance of the acceptor (Rf 0.29) and anhyd. Na2SO4 and concentrated to dryness. The residue was
the presence of a major less polar product (Rf 0.60). The purified by flash chromatography (Tol–EtOAc 8 : 2 to 0 : 1) to
mixture was neutralized by the addition of Et3N, and the sus- give product of delevulination 50 (0.27 g, 63% over 2 steps) as
1
pension was filtered over a pad of Celite. The volatiles were a white foam. Alcohol 50 had H NMR (CDCl3) δ 7.37–7.12 (m,
removed under reduced pressure and the crude material was 51H, 50 HAr, NH), 5.95 (m, 1H, CHvCH2), 5.32 (m, 1H, Jtrans
=
purified by flash chromatography (Tol–EtOAc 8 : 2 to 0 : 1) to 17.3 Hz, Jgem = 1.7 Hz, CHvCH2), 5.22–5.17 (m, 2H, H-2C,
give a 3 : 7 mixture of the hydrolyzed donor 48 and of the CHvCH2), 5.05 (dpo, 1H, J1,2 = 1.5 Hz, H-1A), 5.04 (dpo, 1H, J1,2
desired pentasaccharide 49 as a white foam. The coupling = 1.6 Hz, H-1C), 5.02 (dpo, 1H, J1,2 = 1.5 Hz, H-1B), 4.88 (bspo
,
1
product 49 had H NMR (CDCl3) δ 7.36–7.11 (m, 51H, 50 HAr, 2H, HBn), 4.87 (dpo, 1H, J = 10.7 Hz, HBn), 4.85 (dpo, 1H, J =
NH), 5.93 (m, 1H, CHvCH2), 5.50 (dd, 1H, J1,2 = 1.9 Hz, J2,3 11.1 Hz, HBn), 4.84 (do, 1H, J1,2 = 3.7 Hz, H-1E), 4.81 (do, 1H,
3.1 Hz, H-2A), 5.31 (m, 1H, Jtrans = 17.2 Hz, Jgem = 1.7 Hz, Bn), 4.79 (do, 1H, J = 11.6 Hz, HBn), 4.78 (do, 1H, J1,2 = 3.1 Hz,
CHvCH2), 5.19–5.16 (m, 2H, H-2C, CHvCH2), 5.03 (bs, 1H, H-1D), 4.77 (do, 1H, J = 11.0 Hz, HBn), 4.75 (d, 1H, J = 11.0 Hz,
H-1C), 4.99 (bs, 1H, H-1B), 4.92 (do, 1H, H-1A), 4.89–4.86 (m, Bn), 4.68–4.62 (m, 6H, HBn), 4.58 (do, 1H, J = 12.0 Hz, HBn),
4H, HBn), 4.83 (do, 1H, H-1E), 4.80 (dpo, 1H, J = 11.2 Hz, HBn), 4.54 (do, 1H, HBn), 4.52 (do, 1H, HBn), 4.47 (do, 1H, J = 10.9 Hz,
=
H
H
4.76–4.74 (m, 3H, H-1D, HBn), 4.70–4.62 (m, 4H, HBn),
4.60–4.49 (m, 5H, HBn), 4.47–4.42 (m, 3H, HBn), 4.30–4.23 (m,
H
H
Bn), 4.45 (do, 1H, J = 11.5 Hz, HBn), 4.31–4.24 (m, 2H, H-2D,
All), 4.27 (dpo, 1H, J = 12.1 Hz, HBn), 4.11 (ddpo, 1H, J1,2 = 1.8
2H, H-2D, HAll), 4.26 (do, 1H, J = 12.1 Hz, HBn), 4.09–3.97 (m, Hz, H-2A), 4.07–4.00 (m, 6H, H-5D, HAll, H-6aD, H-3C, H-4D,
6H, H-6aD, H-5D, HAll, H-3C, H-4D, H-3E), 3.95–3.86 (m, 3H, H-3E), 3.94 (ptpo, 1H, J1,2 = J2,3 = 2.2 Hz, H-2B), 3.89 (bs, 1H,
7746 | Org. Biomol. Chem., 2014, 12, 7728–7749
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H-3D), 3.82 (ddo, 2H, J2,3 = 3.6 Hz, J3,4 = 9.7 Hz, H-3A, H-3B), 81.9 (C-3D), 80.5 (C-2B), 78.4 (C-5D), 77.7 (C-3C), 75.5 (C-3E),
3.79–3.71 (m, 5H, H-5A, H-5B, H-6bD, H-5E, H-4E), 3.70–3.65 75.4 (C-5E), 74.8 (OCH2Pr), 74.7 (3C, C-4B, C-4A, C-4C), 74.6
(m, 2H, H-5C, H-6aE), 3.61 (dd, 1H, J1,2 = 3.6 Hz, J2,3 = 9.7 Hz, (C-2C), 73.9 (2C, C-4D, C-2E), 72.7 (2C, C-2A, C-3A), 72.5 (2C,
H-2E), 3.45 (tpo, 1H, J3,4 = J4,5 = 9.1 Hz, H-4C), 3.43 (to, 2H, J3,4
=
C-3B, C-5C), 72.1 (C-5B), 71.9 (C-4E), 71.8 (C-5A), 64.0 (C-6D),
J4,5 = 9.2 Hz, H-4A, H-4B), 3.35 (bd, 1H, J6a,6b = 10.4 Hz, H-6bE), 63.1 (C-6E), 56.2 (C-2D), 25.0 (CH3NHAc), 24.8 (CH2Pr), 22.9
2.16 (s, 3H, CH3Ac), 1.72 (s, 3H, CH3NHAc), 1.30 (d, 3H, J5,6 = 6.2 (CH3Ac), 19.5 (C-6C), 19.3 (2C, C-6B, C-6A), 12.4 (CH3Pr); HRMS
Hz, H-6B), 1.20 (d, 3H, J5,6 = 6.2 Hz, H-6C), 1.09 (d, 3H, J5,6
=
(ESI+): m/z 906.3813 (calcd for C37H63NO24 [M + H]+ m/z
6.2 Hz, H-6A); 13C NMR (CDCl3) δ 169.7 (NHCO), 169.6 (COAc), 906.3818), HRMS (ESI+): m/z 928.3572 (calcd for C37H63NO24
138.7–137.2 (10C, CIVAr), 134.0 (CHvCH2), 129.0–127.4 (50C, Na [M + Na]+ m/z 928.3638), HRMS (ESI+): m/z 1833.7378 (calcd
1
CAr), 116.9 (CHvCH2), 101.1 (C-1B, JC,H = 171.8 Hz), 100.6 for C37H63NO24 C37H63NO24 Na [2M + Na]+ m/z 1833.7412);
1
1
(C-1A, JC,H = 171.1 Hz), 97.5 (C-1D, JC,H = 170.7 Hz), 96.9 RP-HPLC (215 nm): Rt = 13.2 min.
1
1
(C-1E, JC,H = 172.5 Hz), 95.2 (C-1C, JC,H = 172.5 Hz), 82.1
Propyl α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-
(C-3E), 80.2 (C-4C), 80.1 (2C, C-4A, C-4B), 79.5 (C-3A), 79.2 (1→3)-α-L-rhamnopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→4)]-
(C-3B), 79.0 (C-2E), 77.5 (C-4E), 76.9 (C-3C), 75.8 (CBn), 75.6 2-acetamido-2-deoxy-β-D-glucopyranoside (52). To a stirred
(C-2B), 75.4, 75.3, 74.9, 74.7 (4C, CBn), 74.6 (C-5D), 74.3, 73.4, solution of pentasaccharide 51 (91 mg, 100 µmol) in MeOH
73.1, 72.4, 72.2 (5C, CBn), 71.9 (C-2C), 71.4 (C-5E), 70.9 (C-6D), (8 mL), was added methanolic sodium methoxide (25% w/w,
70.8 (C-4D), 69.3 (CAll), 68.9 (C-5B), 68.8 (2C, C-5C, C-2A), 68.7 25 µL). The solution was stirred at rt for 15 h. After this
(C-3D), 68.0 (C-5A), 67.6 (C-6E), 46.0 (C-2D), 22.6 (CH3NHAc), 21.0 time, MS analysis of the reaction mixture revealed a mole-
(CH3Ac), 18.1 (2C, C-6B, C-6C), 17.8 (C-6A); HRMS (ESI+): m/z cular weight corresponding to that of the target tetra-
1826.7903 (calcd for
1826.8176), HRMS (ESI+): m/z 924.9005 (calcd for of Dowex H+ resin, and the suspension was filtered over a 0.2 µm
107H119NO24Na2 [M + 2Na]2+ m/z 924.9037), HRMS (ESI+): m/z filter. Evaporation of the volatiles, freeze-drying and purification
921.8867 (calcd for C107H120NO24K [M + H + K]2+ m/z of the residue by RP-MPLC (0–50% linear gradient of 80% aq.
921.8997).
MeCN over 60 min at a flow rate of 20 mL min−1) gave penta-
C
107H119NO24Na [M
+
Na]+ m/z saccharide. The reaction solution was neutralized by addition
C
Propyl α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl- saccharide 52 (73 mg, 85%) as a white powder following
1
(1→3)-(2-O-acetyl-α-L-rhamnopyranosyl)-(1→3)-[α-D-gluco- repeated freeze-drying. The fully deprotected 52 had H NMR
pyranosyl-(1→4)]-2-acetamido-2-deoxy-β-D-glucopyranoside (D2O) δ 5.29 (d, 1H, J1,2 = 3.9 Hz, H-1E), 5.11 (d, 1H, J1,2 = 1.0
(51). To a stirred solution of pentasaccharide 50 (322 mg, 178 Hz, H-1B), 4.98 (d, 1H, J1,2 = 1.5 Hz, H-1C), 4.87 (d, 1H, J1,2
=
µmol) in 90% aq. EtOH (13.5 mL), were added Pd/C (300 mg) 1.5 Hz, H-1A), 4.49 (d, 1H, J1,2 = 7.4 Hz, H-1D), 3.99 (tpo, 1H, J2,3
and 1 M aq. HCl (33.5 µL). The suspension was stirred under a = J3,4 = 6.3 Hz, H-3D), 3.98 (ddo, 1H, J2,3 = 3.2 Hz, H-2A), 3.96
H2 atmosphere for a day at rt. After this time, MS analysis of (ddpo, 1H, J2,3 = 3.2 Hz, H-2B), 3.89 (ptpo, J3,4 = J4,5 = 6.5 Hz,
the reaction mixture revealed a molecular weight of corres- H-4D), 3.88 (ddo, 1H, J2,3 = 2.4 Hz, H-2C), 3.86–3.82 (m, 2H,
ponding to that of the target pentasaccharide. The reaction H-2D, H-3B), 3.80 (ddpo, 1H, J5,6a = 7.9 Hz, J6a,6b = 11.8 Hz,
mixture was filtered over a pad of Celite. Evaporation of the H-6aD), 3.78–3.65 (m, 8H, OCH2Pr, H-6aE, H-6bD, H-3A, H-6bE,
volatiles, freeze-drying and purification of the residue by H-5C, H-3C, H-5D, H-5B), 3.64 (dqpo, 1H, H-5A), 3.60 (ptpo, 1H,
RP-MPLC (0–50% linear gradient of 80% aq. MeCN over J2,3 = J3,4 = 9.6 Hz, H-3E), 3.54 (dddpo, 1H, J5,6a = 2.1 Hz, J5,6b
=
60 min at a flow rate of 20 mL min−1) gave pentasaccharide 51 4.6 Hz, J4,5 = 10.0 Hz, H-5E), 3.50 (ddpo, 1H, J2,3 = 9.8 Hz, H-2E),
(149 mg, 92%) as a white powder following repeated freeze- 3.48 (pto, 1H, J3,4 = J4,5 = 9.8 Hz, H-4C), 3.44 (dt, 1H, J = 6.6 Hz,
drying. Monoacetate 51 had 1H NMR (D2O) δ 5.38 (d, 1H, J1,2
=
J = 9.6 Hz, OCH2Pr), 3.39 (pto, 1H, J3,4 = J4,5 = 9.7 Hz, H-4B),
3.8 Hz, H-1E), 5.17 (d, 1H, J2,3 = 2.5 Hz, H-2C), 5.14 (bs, 1H, 3.36 (pto, 1H, J3,4 = J4,5 = 9.7 Hz, H-4E), 3.35 (pto, 1H, J3,4 = J4,5
H-1B), 5.12 (d, 1H, J1,2 = 1.4 Hz, H-1C), 4.93 (bs, 1H, H-1A), 4.54 = 9.7 Hz, H-4A), 1.93 (s, 3H, CH3NHAc), 1.46 (psex, 2H, J = 7.3
(d, 1H, J1,2 = 7.4 Hz, H-1D), 4.08 (t, 1H, J2,3 = J3,4 = 7.2 Hz, Hz, CH2Pr), 1.23 (d, 3H, J5,6 = 6.2 Hz, H-6B), 1.19 (dpo, 3H, J5,6
=
H-3D), 4.04 (dd, J1,2 = 1.6 Hz, J2,3 = 3.1 Hz, H-2A), 3.97–3.94 (m, 6.1 Hz, H-6C), 1.17 (dpo, 3H, J5,6 = 6.2 Hz, H-6A), 0.85 (t, 3H, J =
2H, H-2B, H-4D), 3.93 (ddo, 1H, J2,3 = 3.0 Hz, J3,4 = 9.2 Hz, 7.4 Hz, CH3Pr); 13C NMR (D2O) δ 176.4 (NHCO), 104.9 (C-1A,
1
H-3C), 3.92–3.76 (m, 9H, H-2D, H-6aD, H-5C, OCH2Pr, H-6bD, 1JC,H = 171.4 Hz), 103.6 (C-1D, JC,H = 162.2 Hz), 103.2 (C-1B,
1
1
H-6aE, H-6bE, H-3B, H-3A), 3.74–3.61 (m, 5H, H-5D, H-5A, H-3E, 1JC,H = 173.1 Hz), 101.5 (C-1C, JC,H = 170.2 Hz), 99.4 (C-1E, JC,
H-5B, H-5E), 3.60 (pto, 1H, J3,4 = J4,5 = 9.0 Hz, H-4C), 3.57 (ddpo
,
= 171.9 Hz), 80.6 (2C, C-2B, C-3D), 79.1 (C-3C), 78.7 (C-5D),
H
1H, J2,3 = 9.9 Hz, H-2E), 3.50 (dt, 1H, J = 6.2 Hz, J = 10.1 Hz, 75.4 (C-3E), 75.3 (C-5E), 74.7 (2C, C-4B, OCH2Pr), 74.6 (C-4A),
OCH2Pr), 3.43 (pto, 2H, J3,4 = J4,5 = 9.8 Hz, H-4B, H-4E), 3.41 74.4 (C-4C), 73.8 (C-2E), 73.3 (C-4D), 73.0 (C-2C), 72.6 (2C, C-2A,
(ptpo, 1H, J3,4 = J4,5 = 9.6 Hz, H-4A), 2.15 (s, 3H, CH3Ac), 1.99 (s, C-3A), 72.5 (C-5C), 72.4 (C-3B), 71.8 (2C, C-4E, C-5B), 71.7 (C-5A),
3H, CH3NHAc), 1.53 (psex, 2H, J = 6.9 Hz, CH2Pr), 1.30 (d, 3H, 64.0 (C-6D), 62.9 (C-6E), 56.2 (C-2D), 24.7 (2C, CH3NHAc, CH2Pr),
J5,6 = 6.2 Hz, H-6C), 1.27 (d, 3H, J5,6 = 6.1 Hz, H-6B), 1.24 (d, 19.4 (C-6B), 19.3 (2C, C-6C, C-6A), 12.2 (CH3Pr); HRMS (ESI+): m/
3H, J5,6 = 6.3 Hz, H-6A), 0.85 (t, 3H, J = 7.4 Hz, CH3Pr); 13C NMR z 451.6599 (calcd for C35H61NO23Na [M + H + Na]2+ m/z
1
(D2O) δ 176.7 (NHCO), 175.4 (COAc), 104.9 (C-1B, JC,H = 171.6 451.6675), HRMS (ESI+): m/z 864.3688 (calcd for C35H61NO23
1
1
Hz), 103.6 (C-1D, JC,H = 162.7 Hz), 103.2 (C-1B, JC,H = 172.3 [M + H]+ m/z 864.3713), HRMS (ESI+): m/z 886.3514 (calcd for
Hz), 99.6 (C-1E, JC,H = 172.3 Hz), 99.1 (C-1C, JC,H = 171.6 Hz), C35H60NO23Na [M + Na]+ m/z 886.3532), HRMS (ESI+): m/z
1
1
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Organic & Biomolecular Chemistry
1750.7321 (calcd for C70H120N2O46Na [2M
1750.6932); RP-HPLC (215 nm): Rt = 10.2 min.
+
Na]+ m/z 10 F. Belot, K. Wright, C. Costachel, A. Phalipon and
L. A. Mulard, J. Org. Chem., 2004, 69, 1060–1074.
11 F. Belot, C. Guerreiro, F. Baleux and L. A. Mulard, Chem. –
Eur. J., 2005, 11, 1625–1635.
12 M. J. Clement, A. Imberty, A. Phalipon, S. Perez,
C. Simenel, L. A. Mulard and M. Delepierre, J. Biol. Chem.,
Acknowledgements
We thank H. Sommerfelt, the P.I. of Entvac – the GLOBVAC
2003, 278, 47928–47936.
program – for stimulating discussions and F. Bonhomme 13 C. Gauthier, P. Chassagne, F. X. Theillet, C. Guerreiro,
(CNRS UMR 3523) for providing the HRMS spectra.
The research leading to these results has received funding
from the Institut Pasteur, the Research Council of Norway,
F. Thouron, F. Nato, M. Delepierre, P. J. Sansonetti,
A. Phalipon and L. A. Mulard, Org. Biomol. Chem., 2014, 12,
4218–4232.
GLOBVAC program, grant number 185872 (Entvac, Post- 14 J. Boutet, C. Guerreiro and L. A. Mulard, J. Org. Chem.,
doctoral fellowship to J. M. H.), the Ministère de l’Education 2009, 74, 2651–2670.
Nationale, de la Recherche et de la Technologie, France 15 P. Chassagne, C. Fontana, C. Guerreiro, C. Gauthier,
(MENRT, Ph.D. fellowship to Y. L. G.), the Agence Nationale
pour la Recherche, France, grant number ANR-05-Blanc-0022-
A. Phalipon, G. Widmalm and L. A. Mulard, Eur. J. Org.
Chem., 2013, 4085–4106.
01 (ANR, Post-doctoral fellowship to K. D.), and the European 16 P. Chassagne, L. Raibaut, C. Guerreiro and L. A. Mulard,
Commission Seventh Framework Program (FP7/2007–2013)
Tetrahedron, 2013, 69, 10337–10350.
under Grant agreement no. 261472-STOPENTERICS.
17 A. V. Perepelov, V. L. L’vov, B. Liu, S. N. Senchenkova,
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