Chitooligosaccharide Synthesis Using an Ionic Tag

Article abstract of DOI:10.1002/ejoc.201501476

An environmentally improved synthesis of the N-differentiated chitotetrasaccharide CO-IV-(NH₂), a key intermediate for the preparation of lipochitooligosaccharides and the TMG-chitotriomycin, is reported based on a chromatography-free ionic-liquid tagging approach. The method involves the glycosylation of ionic-liquid-tagged acceptors with thioglucosamine donors leading to the stereoselective formation of β-(1→4)-linked glucosamine-containing oligomers.

Full text of DOI:10.1002/ejoc.201501476

DOI: 10.1002/ejoc.201501476
Full Paper
Glycochemistry
Chitooligosaccharide Synthesis Using an Ionic Tag
Laura Gillard,[a] Anh-Tuan Tran,[a] François-Didier Boyer*[a,b] and Jean-Marie Beau*[a,c]
Abstract: An environmentally improved synthesis of the N-dif- ionic-liquid tagging approach. The method involves the glyc-
ferentiated chitotetrasaccharide CO-IV-(NH2), a key intermediate osylation of ionic-liquid-tagged acceptors with thioglucosamine
for the preparation of lipochitooligosaccharides and the TMG- donors leading to the stereoselective formation of ꢀ-(1→4)-
chitotriomycin, is reported based on a chromatography-free linked glucosamine-containing oligomers.
Introduction
The synthesis of oligosaccharides remains a major challenge
despite considerable advances in the past thirty years.[1] Oli-
gomers of N-acetylglucosamine are found in many natural pro-
ducts with interesting biological activities.[2] For example, the
Nod[3] and Myc factors[4] (lipochitooligosaccharides, LCOs), and
TMG-chitotriomycin[5] 1 (TMG = N,N,N-trimethylglucosamine) all
contain a sequence of 3 to 4 N-acetyl-D-glucosamine units cou-
pled to an N-substituted unit at the nonreducing terminus, all
linked by ꢀ-(1→4) linkages (chitin structure) (Figure 1). The syn-
thesis of these glycostructures requires the formation of ꢀ-
(1→4) glycosidic bonds between glucosamine units, which is
not always an easy task, due to the low reactivity of the 4-
hydroxy group of glucosamine derivatives,[6–8] and the selective
differentiation of the amino group of the nonreducing unit.[7a]
The classical chemical synthesis of oligosaccharides in solu-
tion requires laborious and very time-consuming purifications
by chromatography after each glycosylation step. Different
methods have been developed in glycochemistry to overcome
these problems: solid-phase organic synthesis,[9] synthesis on
soluble high-molecular-weight branched polymers,[10] the use
of fluorous soluble supports,[11] and the use of lipophilic pro-
tecting groups.[12] However, these methods have several draw-
backs: heterogeneous reaction conditions, low loading of sub-
strates, difficulties in analysis and purification, and expensive
Figure 1. Structures of selected lipo-chitooligosaccharides, the TMG-chitotrio-
mycin 1 and the targeted chitooligosaccharide 2.
supports. An alternative “greener” method based on the use of
ionic liquids (IL) has recently been developed to avoid the
above disadvantages.[13,14] This process, which involves grafting
an ionic tag onto one of the reactant molecules, produces mol-
ecules in high yields for the glycosylation step. The tagged mol-
ecules are soluble in polar organic solvents, but insoluble in less
polar solvents (e.g., diethyl ether, toluene). Thus, the reaction
products can be isolated by a simple phase separation without
further column chromatography, which makes it easier to carry
out glycosylation sequences. Notably, all compounds may be
fully characterized by the usual techniques, such as NMR spec-
troscopy and HRMS. This approach has previously been de-
scribed for the formation of oligosaccharides and glycopeptides
with ꢀ-(1→6), ꢀ-(1→2), α-(1→6), α-(1→2), α-(1→3), and α-
(1→4) glycosidic bonds bearing different ionic tags connected
to the donor at C-6[15] or C-4[16] by esters, and later connected
to the acceptor at the C-1 position through alkyl/benzyl
ether[17] linkages. In connection with our program to access
chitooligosaccharides,[18] including a selective biotransforma-
tion of oligomers,[18d] we present in this paper a synthesis of
the challenging chitotetrasaccharide CO-IV-(NH2) 2 (Figure 1)
using an ionic linker tag (ITag). This amine is a key intermedi-
ate[18b,19] in the formation of Nod/Myc factors and of TMG-chi-
totriomycin 1, all of which contain three ꢀ-(1→4) glycosidic
bonds.
[a] Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Univ. Paris-
Sud, Université Paris-Saclay,
1, av. de la Terrasse, 91198 Gif-sur-Yvette, France
E-mail: francois-didier.boyer@cnrs.fr
jean-marie.beau@cnrs.fr
http://www.icsn.cnrs-gif.fr/
[b] Institut Jean-Pierre Bourgin, Institut National de la Recherche
Agronomique (INRA), Paris Institute of Technology for Life, Food and
Environmental Sciences (AgroParisTech), Centre National de la Recherche
Scientifique (CNRS), Université Paris-Saclay,
RD10, 78026 Versailles, France
[c] Laboratoire de Synthèse de Biomolécules, Institut de Chimie Moléculaire et
des Matériaux, Univ. Paris-Sud, Université Paris-Saclay and CNRS,
91405 Orsay, France
Supporting information and ORCID(s) from the author(s) for this article
are available on the WWW under http://dx.doi.org/10.1002/
ejoc.201501476.
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Results and Discussion
14, bearing an C-4-OAc group, from donor 13 and acceptor 11
were not successful, even when a large excess of the promoter
was used (conditions B vs. A). After a work-up procedure involv-
ing simple organic solvent washes, an inseparable mixture of
ITagged acceptor 11 and ITagged disaccharidic product 14 was
obtained. This result might be explained by the lower reactivity
of 3-O-benzyl-2-deoxy-2-phthalimidoglucose derivatives as
glycosyl acceptors, as already observed in our group.[20,26]
Fortunately, we found that 3-O-acetyl donor 15, bearing an or-
thogonal 9-fluorenylmethoxycarbonyl (Fmoc) protecting group
at O-4, underwent glycosylation (Table 1, entry 3) to give disac-
charide 16 in excellent yield (92 %). The nature of the counter-
We chose to attach the ITag to the glycosyl acceptor with a
linker derived from 4 or 5 (Scheme 1) connected to the C-1
position by a stable benzyl glycoside linkage, and removable
by hydrogenolysis.[13b,17a,17c,20] Glycosylation of benzyl alcohol
4 with donor 3[18a] prepared from the nonmalodorous 2-
methyl-5-tert-butylphenylthiol (HSMbp),[21] followed by halide
displacement at the benzylic position provided ITagged glyc-
osyl acceptor precursor 6 in moderate yield (44 %).[22] This se-
quence was preferred over direct glycosylation of a benzyl alco-
hol already bearing the ITag[23] (viscous liquid) for practical rea-
sons, even though the overall yield was similar. The best results
were obtained using more reactive donors 7 and 8, which have
a free hydroxyl group at C-4, in glycosylation reactions with
benzyl alcohol acceptor 4. Under the conditions used, no oli-
gomerization was noticed. The resulting products were trans-
formed into hydrophobic ITagged glycosides 10a/10b and 11
in high overall yield (85–80 %) by treatment with potassium
hexafluorophosphate or potassium tetrafluoroborate. The
length of the alkyl chain connecting the ionic moiety to the
phenyl group dramatically influences the overall yield of the
ITagged glycosides (10a/10b vs. 9: 85 vs. 42 %); this was not
observed in a previous report.[17b]
Our initial approach was based on the use of a disaccharide
bearing a benzylidene moiety such as 12, which can provide a
C-4-OH disaccharide using either reductive or oxidative open-
ing. Under optimized coupling conditions, disaccharide 12 was
obtained in very good yield (80 %). However, ring opening gave
unsatisfactory results under reductive or oxidative conditions.
Reductive opening of the 4,6-O-acetal of 12 using the classical
Et3SiH/TFA (trifluoroacetic acid)[24] method gave an inseparable
mixture of the 4-OH derivative and the diol. Also, attempted
regioselective oxidative ring opening[25] was ineffective; disac-
charide 12 was recovered quantitatively after an extended pe-
riod of time in the presence of dimethyldioxirane. These results
–
–
anion (BF4 vs. PF6 ) had no effect on either the reactivity or
the yield of the glycosylation.[13b] For the following steps, PF6
–
was chosen for its hydrophobic character, which is greater than
–
that of the BF4 anion.[13a]
Selective removal of the Fmoc group by triethylamine gave
alcohol 17 in high yield (90 %; Table 1). Glycosylation of ITagged
alcohol 17 with an excess of disaccharide donor 18[18a] in the
presence of the NIS (N-iodosuccinimide)/TfOH promoter pro-
vided protected ITagged tetrasaccharide 19 in 83 % yield
(Scheme 2). In some experiments, a KPF6 treatment was used
to correct the triflate/hexafluorophosphate counterion mixture
resulting from the glycosylation step, which complicated the 1H
NMR spectra. This anion exchange was not necessary during
the preparation of disaccharide 16. The N-Phth groups in 19
were then replaced by N-acetyl groups using the standard two-
step procedure [(NH2CH2)2 then Ac2O] to give ITagged com-
pound 20 with three NHAc groups (Table 2, entry 1).
However, it was impossible to purify the crude product (i.e.,
20) by simple solvent washes. At this stage, HPLC-MS analysis
showed that the Cbz (benzyloxycarbonyl) group survived the
two steps. For comparison, the analogous tetrasaccharide lack-
ing an ITag (i.e., 23) gave, without complication, chitotetrasac-
charide 24 in 84 % yield, as reported previously[18a] (Table 2,
prompted us to change to a different approach involving an entry 3). Furthermore, monosaccharide 10a was transformed
orthogonal protecting group. Attempts to prepare disaccharide into ITagged N-acetyl glucosamine derivative 21, which was
Scheme 1. Synthesis of the ITagged-monosaccharides (Phth = phthalimido).
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Table 1. Synthesis of the ITagged-disaccharides.
[a] A = NIS (2.5 equiv.), TfOH (0.15 equiv.), –10 °C to room temp., 24 h. B = NIS (5 equiv.), TfOH (0.3 equiv.), –10 °C to room temp., 48 h. C = NIS (6 equiv.),
TfOH (0.3 equiv.), –10 °C to room temp., 18 h. [b] 50 % acceptor conversion yield given by 1H NMR spectroscopic analysis (see text).
Scheme 2. Synthesis of the ITagged-tetrasaccharide 19.
easily purified by simple organic solvent washes (82 % yield; wave heating[27] led to cleavage of the ionic tag, along with the
Table 2, entry 2). These results underline the particular and un- O-benzyl and N-benzyloxycarbonyl groups, accompanied by
favorable physicochemical behavior of ITagged tetrasaccharide partial de-O-acetylation. Removal of the remaining O-acetyl
20 that makes it difficult to purify by simple washings. The groups by methanolysis produced target chitooligosaccharide
crude product 20 was therefore used in the final deprotections. 2 in a yield of 13 % over four steps from protected intermediate
Cleavage of the benzyl-derived protecting groups, including 19, after a single chromatographic purification on silica gel
the ITag, was problematic, which is in contrast with results re- from the starting monosaccharide 7 (10 steps) (Table 2, entry 1).
ported recently for the preparation of gluco- and mannooligo- Amine 2 is the last intermediate in the synthesis of Nod/Myc
saccharides.[17a,17c,23] All attempts using hydrogenolysis under factors[18b] and TMG-chitotriomycin.[18b] In contrast, hydrogen-
H2 catalyzed by Pd/C or Pd black with various solvents in an olysis of ITagged monosaccharide 21 occurred without de-O-
acidic medium failed to remove the ionic tag, and complex mix- acetylation, and, after methanolysis, the expected N-acetyl
tures were formed in all cases. Only the very recently developed glucosamine (i.e., 22) was obtained in 67 % overall yield from
method of transfer hydrogenolysis using resin-supported ITagged precursor 10a (Table 2, entry 2), showing again the
ammonium formate and palladium on charcoal under micro- distinct behavior of ITagged acetamido oligomer 20.
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Table 2. The final removal of the phthalimido, benzyloxycarbonyl, benzyl, acetyl, and ITag groups.
[a] n.d.: not determined. [b] Overall yield from 19 (four steps). [c] See Gillard et al.[18a] Phth = phthalimido; Cbz = benzyloxycarbonyl; TBDPS = tert-butyldiphenyl-
silyl.
1.0 equiv.) and 4-(chloromethyl)benzyl alcohol
4 (33.5 mg,
Conclusions
0.22 mmol, 1.3 equiv.) were stirred in toluene (3.4 mL) with molecu-
lar sieves (4 Å; 50 mg) at room temperature for 1 h. NIS (45 mg,
0.20 mmol, 1.2 equiv.) was then added, and the suspension was
cooled to –30 °C. TfOH (2.9 μL, 0.03 mmol, 0.2 equiv.) was added,
and the reaction mixture was stirred for 3 h. The mixture was then
neutralized with triethylamine (3 drops). The mixture was diluted
with EtOAc (15 mL), and the molecular sieves were removed by
filtration. The filtrate was then washed with saturated aqueous
Na2S2O3 (20 mL), dried with Na2SO4, and concentrated under re-
duced pressure. The crude material was purified by chromatogra-
phy on silica gel (heptane/EtOAc, 1:0 to 6:4) to give 4-(chloro-
We have demonstrated that the ionic catch-and-release
oligosaccharide synthesis (ICROS) methodology[17a] can be ex-
tended to the construction of the ꢀ-(1→4) glycosidic bonds
found in chitooligosaccharides. Targets containing these linka-
ges are more demanding than those glucose or mannose oli-
gomers bearing ꢀ- or α-(1→6), and ꢀ- or α-(1→2) glycosidic
bonds whose synthesis has already been described by
ICROS.[15a–15c,16,17] The introduction of the ionic label at the
anomeric position of the oligosaccharide target is a versatile
approach that is compatible with the various protecting groups
used in this synthesis (acetyl, phthalimido, and benzyloxy-
carbonyl for N-protection; acetyl, benzyl, and 9-fluorenyl-
methoxycarbonyl for O-protection). Seven steps, including glyc-
osylations using thioglycosides, were carried out without purifi-
cation by chromatography; non-ITagged materials can be se-
lectively washed away with appropriate solvents. The difficulties
experienced during the last four steps of the synthesis (13 %
overall, 60 % per step) distinctly underline the unsuitable asso-
ciation of an oligo-chitin acetamido framework with a dialkyl-
imidazolium ionic liquid; the physicochemical properties of this
combination make purification by simple washing unfavorable.
The development of new IL-functional supports to overcome
this problem is currently in progress in our group.
methyl)benzyl 3-O-acetyl-6-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-
glucopyranoside (82.2 mg, 85 %) as an amorphous white solid.
4-(Chloromethyl)benzyl
3-O-acetyl-6-O-benzyl-2-deoxy-2-phthal-
imido-ꢀ- -glucopyranoside (117 mg, 0.19 mmol, 1.0 equiv.) was dis-
D
solved in CH3CN (2.5 mL), and N-methylimidazole (53 μL, 0.67 mmol,
3.6 equiv.) was added. The reaction mixture was stirred at 85 °C for
24 h. Then KPF6 (38 mg, 0.21 mmol, 1.1 equiv.) was added, and the
mixture was stirred at 85 °C for a further 24 h. The remaining KPF6
was removed by filtration, and the filtrate was concentrated under
reduced pressure. The residue was dissolved in dichloromethane
(20 mL), and HCl (1
M aq.; 3 mL) was added. The mixture was stirred
for 30 min. The layers were separated, and the aqueous layer was
extracted with CH2Cl2 (2 × 10 mL). The combined organic layers
were dried with Na2SO4, filtered, and concentrated under reduced
pressure. The residue was washed with Et2O (3 × 10 mL) and tolu-
ene (3 × 10 mL) to give compound 10a (144 mg, 100 %) as a beige
powder. [α]D20 = –30.3 (c = 1.0, CHCl3). 1H NMR (300 MHz, CDCl3):
δ = 8.49 (s, 1 H), 7.79–7.63 (m, 4 H), 7.34–7.20 (m, 5 H), 7.17 (t, J =
2.0 Hz, 1 H), 7.09 (t, J = 2.0 Hz, 1 H), 7.04 (s, 4 H), 5.61 (dd, J = 11.0,
J = 8.5 Hz, 1 H), 5.25 (d, J = 8.5 Hz, 1 H), 5.08 (s, 2 H), 4.72 (AB
system, J = 12.5 Hz, 1 H), 4.59 (AB system, J = 12.0 Hz, 1 H), 4.54
Experimental Section
General: See ref.[28]
4-(1-Methyl-3-methylimidazolium)benzyl 3-O-Acetyl-6-O-benz-
yl-2-deoxy-2-phthalimido-ꢀ-
D
-glucopyranoside
Hexafluoro- (AB system, J = 12.0 Hz, 1 H), 4.48 (AB system, J = 12.0 Hz, 1 H),
4.45–4.41 (br. s, 1 H), 4.18 (dd, J = 11.0, J = 8.5 Hz, 1 H), 3.92–3.68
phosphate (10a): Thioglucoside 7[18a] (100 mg, 0.17 mmol,
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(m, 4 H), 3.74 (s, 3 H), 1.81 (s, 3 H) ppm. 13C NMR (75 MHz, CDCl3):
δ = 171.1 (CO), 168.1 (C), 167.9 (C), 138.6 (C), 138.2 (C), 136.0 (CH),
134.8 (2 CH), 132.2 (C), 131.3 (2 C), 129.1 (2 CH), 129.0 (2 CH), 128.6
(2 CH), 127.9 (CH), 127.8 (2 CH), 123.9 (CH), 123.6 (2 CH), 122.1 (CH),
96.9 (CH), 75.2 (CH), 73.7 (CH2), 73.6 (CH), 70.3 (CH), 70.2 (CH2), 69.9
5 H), 3.83 (s, 3 H), 3.63–3.55 (m, 4 H), 3.50–3.44 (m, 2 H), 1.82 (s, 3
H), 1.75 (s, 3 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 170.2 (CO), 170.1
(CO), 167.9 (CO), 154.0 (C), 149.0 (C), 143.2 (C), 143.0 (C), 141.25 (C),
141.23 (C), 138.4 (C), 138.2 (C), 137.5 (C), 136.4 (CH), 134.5 (2 CH),
134.3 (2 CH), 131.2 (2 C), 131.3 (2 C), 129.3 (2 CH), 129.2 (2 CH),
129.0 (CH), 128.4 (3 CH), 128.2 (CH), 128.0 (CH), 127.9 (CH), 127.7
(CH), 127.6 (2 CH), 127.5 (CH), 127.4 (2 CH), 127.23 (CH), 127.20 (CH),
(CH2), 54.8 (CH), 53.0 (CH2), 36.3 (CH3), 20.8 (CH3) ppm. 19F NMR
1
(CDCl3): δ = –72.08 (d, JP, F = 712 Hz, PF6–) ppm. IR (film): ν = 3155,
˜
2873, 1775, 1734, 1712, 1386, 1231, 1043, 972, 722 cm–1. MS (ESI): 125.3 (CH), 125.09 (CH), 125.07 (CH), 123.7 (CH), 123.5 (2 CH), 122.0
m/z (%) = 626 (100) [M – PF6]+. HRMS (ESI-TOF): calcd. for
(CH), 120.07 (CH), 120.04 (CH), 97.0 (CH), 96.1 (CH), 74.4 (CH), 74.1
(CH), 73.4 (CH), 72.8 (CH2), 72.5 (CH), 70.9 (CH), 70.7 (CH), 70.2 (CH2),
69.7 (CH2), 68.4 (2 CH2), 67.7 (CH2), 55.1 (CH), 54.7 (CH), 53.1 (CH2),
C35H36N3O8 [M – PF6]+ 626.2502; found 626.2493.
2-Methyl-5-(tert-butyl)phenyl 3-O-Acetyl-6-O-benzyl-2-deoxy-4-
O-fluorenylmethyloxycarbonyl-2-phthalimido-1-thio-ꢀ-D-gluco-
46.5 (CH), 36.4 (CH3), 20.6 (CH3), 20.4 (CH3) ppm. 3 carbon resonan-
1
ces [N(CO)2] were missing. 19F NMR (CDCl3): δ = –72.37 (d, JP, F
=
pyranoside (15): Thioglucoside 7[18a] (710 mg, 1.18 mmol,
1.0 equiv.) and FmocCl (609 mg, 2.36 mmol, 2.0 equiv.) were stirred
in pyridine (6.1 mL) at room temperature for 4 h. The reaction mix-
ture was then filtered, and the volatiles were evaporated under re-
duced pressure. The crude product was purified by chromatography
on silica gel (heptane/EtOAc, 9:1 to 5:5) to give compound 15
712 Hz, PF6–) ppm. IR (film): ν = 2947, 1774, 1748, 1714, 1385, 1257,
˜
1224, 1045, 1029, 838, 720 cm–1. MS (ESI): m/z (%) = 1271 (100) [M
–
PF6]+. HRMS (ESI-TOF): calcd. for C73H67N4O17 [M PF6]+
–
1271.4501; found 1271.4564.
4-(1-Methyl-3-methylimidazolium)benzyl 3-O-Acetyl-6-O-benz-
-glucopyranosyl-(1→4)-3-O-
acetyl-6-O-benzyl-2-deoxy-2-phthalimido-ꢀ- -glucopyranoside
7.60–7.52 (m, 3 H), 7.45–7.37 (m, 2 H), 7.36–7.29 (m, 6 H), 7.27–7.20 Hexafluorophosphate (17): Fmoc-disaccharide 16 (60 mg,
(802 mg, 82 %) as a white powder. [α]D20 = –47.3 (c = 1.0, CHCl3). 1H yl-2-deoxy-2-phthalimido-ꢀ-
NMR (300 MHz, CDCl3): δ = 7.92–7.87 (m, 2 H), 7.80–7.74 (m, 4 H),
D
D
(m, 2 H), 7.10–7.05 (m, 1 H), 5.95 (dd, J = 10.5, J = 9.0 Hz, 1 H), 5.72
(d, J = 10.5 Hz, 1 H), 5.02 (dd, J = 10.0, J = 9.0 Hz, 1 H), 4.59 (AB
system, J = 12.0 Hz, 1 H), 4.53 (AB system, J = 12.0 Hz, 1 H), 4.46 (t,
J = 10.5 Hz, 1 H), 4.40–4.22 (m, 2 H), 4.21–4.13 (m, 1 H), 3.95 (td,
J = 10.0, J = 4.5 Hz, 1 H), 3.73 (d, J = 4.0 Hz, 2 H), 2.20 (s, 3 H), 1.81
0.04 mmol, 1.0 equiv.) and Et3N (0.6 mL, 4.45 mmol) were stirred in
dry dichloromethane (5.0 mL) at room temperature for 3 h. The
solvents were then evaporated, and then coevaporated with tolu-
ene (3 × 5 mL). The residue was washed with Et2O (3 × 5 mL) and
a mixture of CH2Cl2/n-pentane (4:1; 3 × 5 mL) to give compound
(s, 3 H), 1.27 (s, 9 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 170.1 (CO), 17 (46 mg, 90 %) as a yellow powder. [α]D20 = –6.7 (c = 0.5, CHCl3).
154.1 (C), 149.6 (C), 143.2 (C), 143.0 (C), 141.25 (C), 141.20 (C), 137.7
(C), 137.4 (C), 134.4 (CH), 134.3 (CH), 130.9 (CH), 130.0 (CH), 128.3 (2
1H NMR (300 MHz, CDCl3): δ = 8.65 (s, 1 H), 7.80–7.57 (m, 8 H), 7.28–
7.20 (m, 10 H), 7.10–6.95 (m, 6 H), 5.56–5.45 (m, 2 H), 5.37 (d, J =
CH), 127.93 (CH), 127.90 (CH), 127.7 (2 CH), 127.6 (CH), 127.23 (CH), 8.5 Hz, 1 H), 5.09 (s, 2 H), 5.00 (d, J = 8.5 Hz, 1 H), 4.62 (AB system,
127.20 (CH), 125.6 (CH), 125.14 (CH), 125.10 (C), 123.6 (2 CH), 120.05 J = 12.5 Hz, 1 H), 4.45 (AB system, J = 12.0 Hz, 2 H), 4.38 (AB system,
(CH), 120.02 (CH), 84.2 (CH), 73.7 (CH), 73.6 (CH2), 71.6 (CH), 70.3 J = 12.5 Hz, 1 H), 4.31 (dd, J = 18.0, J = 12.0 Hz, 2 H), 4.13 (dd, J =
(CH2), 69.0 (CH2), 54.0 (CH), 46.5 (CH), 34.4 (C), 31.2 (3 CH3), 20.4
(CH3), 20.3 (CH3) ppm. 2 carbon resonances CO and 2 quaternary
10.5, J = 8.5 Hz, 1 H), 4.06–3.98 (m, 2 H), 3.78 (s, 3 H), 3.74–3.60 (m,
3 H), 3.46–3.33 (m, 4 H), 1.81 (s, 3 H), 1.75 (s, 3 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 170.9 (CO), 170.0 (CO), 167.7 (CO), 138.4 (2 C),
138.1 (C), 137.4 (C), 136.4 (CH), 134.3 (4 CH), 131.9 (2 C), 131.4 (2
C), 129.2 (CH), 129.1 (CH), 128.51 (2 CH), 128.49 (2 CH), 128.3 (2 CH),
127.9 (2 CH), 127.6 (2 CH), 127.4 (2 CH), 123.5 (4 CH), 122.0 (CH),
121.0 (CH), 97.2 (CH), 96.2 (CH), 74.4 (CH), 74.3 (CH), 73.7 (CH), 73.6
(CH2), 73.3 (CH), 72.8 (CH2), 71.2 (CH), 71.1 (CH), 69.8 (2 CH2), 67.8
(CH2), 55.0 (CH), 54.9 (CH), 53.0 (CH2), 36.3 (CH3), 20.6 (CH3), 20.5
carbon resonances (phthalimido group) are missing. IR (film): ν =
˜
2960, 1780, 1753, 1717, 1450, 1383, 1254, 1218, 1065, 1037,
718 cm–1. MS (ESI): m/z (%) = 848 (100) [M + Na]+. HRMS (ESI-TOF):
calcd. for C49H47NO9SNa [M + Na]+ 848.2869; found 848.2861.
4-(1-Methyl-3-methylimidazolium)benzyl 3-O-Acetyl-6-O-benz-
yl-2-deoxy-4-O-fluorenylmethyloxycarbonyl-2-phthalimido-ꢀ-
glucopyranosyl-(1→4)-3-O-acetyl-6-O-benzyl-2-deoxy-2-phthal-
imido-ꢀ- -glucopyranoside Hexafluorophosphate (16): Donor
D-
(CH ) ppm. 3 carbon resonances [N(CO) ] were missing. IR (film): ν =
˜
3
2
D
2869, 1776, 1746, 1713, 1385, 1226, 1045, 973, 841, 720 cm–1. MS
(ESI): m/z (%) = 1049 (100) [M – PF6]+. HRMS (ESI-TOF): calcd. for
C58H57N4O15 [M – PF6]+ 1049.3820; found 1049.3839.
15 (160 mg, 0.19 mmol, 3.0 equiv.) and acceptor 10a (50 mg,
0.06 mmol, 1.0 equiv.) were mixed, and toluene (5 mL) was added.
The solvent was then evaporated, and the residue was dried in
vacuo for 1 h. Dichloromethane (1.5 mL) was then added under
argon. The solution was stirred with molecular sieves (4 Å; 40 mg)
at room temperature for 1 h. NIS (86 mg, 0.38 mmol, 6.0 equiv.) was
then added, and the suspension was cooled to –10 °C. TfOH (2.7 μL,
0.03 mmol, 0.6 equiv.) was added, and the reaction mixture was
4-(1-Methyl-3-methylimidazolium)benzyl 3,4,6-Tri-O-acetyl-2-
benzyloxycarbonylamino-2-deoxy-ꢀ-
O-acetyl-6-O-benzyl-2-deoxy-2-phthalimido-ꢀ-
syl-(1→4)-3-O-acetyl-6-O-benzyl-2-deoxy-2-phthalimido-ꢀ-
glucopyranosyl-(1→4)-3-O-acetyl-6-O-benzyl-2-deoxy-2-phthal-
D
-glucopyranosyl-(1→4)-3-
D
-glucopyrano-
D
-
stirred for 18 h at room temperature. The mixture was then diluted imido-ꢀ-D-glucopyranoside Hexafluorophosphate (19): Donor
with dichloromethane (15 mL), and neutralized with saturated
aqueous NaHCO3 (3 mL). The molecular sieves were removed by
filtration, and the filtrate was then washed with saturated aqueous
Na2S2O3 (2 × 3 mL), dried with Na2SO4, and concentrated under
reduced pressure. The residue was washed with Et2O (3 × 5 mL) and
18[18a] (108 mg, 0.10 mmol, 3.0 equiv.) and acceptor 17 (42 mg,
0.04 mmol, 1.0 equiv.) were mixed, and toluene (5 mL) was added.
The solvent was then evaporated, and the residue was dried in
vacuo for 1 h. Dichloromethane (1.0 mL) was then added under
argon. The solution was stirred with molecular sieves (4 Å; 70 mg)
a mixture of CH2Cl2/n-pentane (4:1; 3 × 5 mL) to give compound at room temperature for 1 h. The suspension was then cooled to
1
16 (85 mg, 92 %) as a yellow foam. [α]D20 = +6.3 (c = 1.8, CHCl3). H –40 °C, and NIS (53 mg, 0.23 mmol, 6.0 equiv.) and TfOH (2.0 μL,
NMR (300 MHz, CDCl3): δ = 8.73 (s, 1 H), 7.85–7.70 (m, 8 H), 7.52 (2
0.02 mmol, 0.6 equiv.) were added. The reaction mixture was stirred
H), 7.42–7.27 (m, 12 H), 7.18–7.01 (m, 10 H), 5.83 (dd, J = 10.5, J = at room temperature for 24 h. The mixture was diluted with di-
9.0 Hz, 1 H), 5.59 (dd, J = 11.0, J = 9.0 Hz, 1 H), 5.48 (d, J = 8.5 Hz,
1 H), 5.16 (s, 2 H), 5.07 (d, J = 8.7 Hz, 1 H), 4.69 (AB system, J =
12.5 Hz, 1 H), 4.53–4.41 (m, 5 H), 4.39–4.29 (m, 2 H), 4.27–4.10 (m,
chloromethane (15 mL), and neutralized with saturated aqueous
NaHCO3 (3 mL). The molecular sieves were removed by filtration
through Celite®. The filtrate was then washed with saturated aque-
Eur. J. Org. Chem. 2016, 1103–1109
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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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ous Na2S2O3 (2 × 3 mL), dried with Na2SO4, and concentrated under
reduced pressure. The residue was washed with Et2O (3 × 5 mL) and
due was purified by chromatography on silica gel (MeCN/H2O/
NH3(aq), 7:3:0.1) to give compound 2 (5 mg, 20 % from the prece-
a mixture of CH2Cl2/n-pentane (4:1; 3 × 5 mL) to give compound ding chitotetrasaccharide, α/ꢀ = 4:6) as a white solid. [α]D20 = –3.2
19 (60 mg, 83 %) as a yellow foam. [α]D20 = –19.3 (c = 0.2, CHCl3). (c = 0.6, MeOH). 1H NMR (500 MHz, D2O): δ = 5.23 (d, J = 2.0 Hz,
1H NMR (500 MHz, CD3CN): δ = 8.38 (s, 1 H), 7.97–7.80 (m, 12 H),
0.4 H), 4.69 (d, J = 8.0 Hz, 0.6 H), 4.66–4.61 (m, 2 H), 4.52 (d, J =
7.41–7.36 (m, 9 H), 7.34–7.30 (m, 6 H), 7.28–7.22 (m, 4 H), 7.15–7.08 8.0 Hz, 1 H), 3.97–3.41 (m, 23 H), 2.72 (t, J = 9.0 Hz, 1 H), 2.11 (s, 6
(m, 4 H), 7.07–7.04 (m, 2 H), 6.96 (t, J = 7.5 Hz, 1 H), 5.57–5.46 (m,
4 H), 5.26 (d, J = 8.5 Hz, 1 H), 5.21 (d, J = 8.0 Hz, 1 H), 5.15–5.07 (m,
4 H), 5.01 (AB system, J = 12.5 Hz, 1 H), 4.84 (t, J = 9.5 Hz, 1 H), 4.68
H), 2.09 (s, 3 H) ppm. IR (film): ν = 3276, 2925, 1635, 1563, 1377,
˜
1313, 1155, 1066, 1025, 946 cm–1. MS (ESI): m/z (%) = 789 (30) [M
+ H]+. HRMS (ESI-TOF): calcd. for C30H53N4O20 [M + H]+ 789.3253;
(d, J = 12.0 Hz, 1 H), 4.59–4.54 (m, 1 H), 4.47 (s, 2 H), 4.43 (d, J = found 789.3220. The spectroscopic data are in agreement with liter-
12.0 Hz, 2 H), 4.38 (br. s, 2 H), 4.34 (AB system, J = 12.0 Hz, 1 H),
4.23 (dd, J = 12.0 Hz, J = 2.0 Hz, 1 H), 4.02–3.96 (m, 3 H), 3.94–3.85
(m, 5 H), 3.78 (s, 3 H), 3.67–3.61 (m, 1 H), 3.55–3.45 (m, 4 H), 3.44–
3.41 (m, 1 H), 3.40–3.35 (m, 1 H), 3.35–3.25 (m, 3 H), 3.20–3.15 (m,
1 H), 1.85 (s, 3 H), 1.80 (s, 3 H), 1.78 (s, 3 H), 1.69 (s, 3 H) ppm. 2
methyl resonances were obscured. 13C NMR (75 MHz, CD3CN): δ =
171.31 (CO), 171.29 (CO), 171.25 (CO), 171.19 (CO), 171.1 (CO), 139.6
(C), 139.5 (C), 139.4 (C), 137.1 (C), 136.0–135.7 (CH and C), 134.2
(CH), 132.5–132.3 (C), 129.7–128.3 (CH), 125.1–124.15 (CH), 123.31
(CH), 123.30 (CH), 101.1 (CH), 98.2 (CH), 97.76 (CH), 97.72 (CH), 75.2
(CH), 75.1 (CH), 74.96 (CH), 74.92 (CH), 74.8 (2 CH), 74.4 (CH2), 74.3
(CH2), 74.1 (CH2), 72.3 (CH), 71.7 (CH), 71.4 (CH), 71.35 (CH), 71.3
(CH2), 69.65 (CH), 69.62 (CH), 69.0 (2 CH2), 69.85 (CH2), 69.81 (CH2),
67.3 (CH2), 56.2 (CH), 56.1 (2 CH), 55.8 (CH), 53.4 (CH2), 21.1 (2 CH3),
ature data.[18b]
Acknowledgments
The authors thank B. Vauzeilles for his support. The authors are
grateful to the Institut de Chimie des Substances Naturelles
(ICSN) (grant to L. G.), and to the French Agence Nationale de
la Recherche (ANR) (ANR-12-BS07-0022, Rosas 2013–2016, grant
to A.-T. T.). The CHARM3AT Labex program is also acknowledged
for its support.
Keywords: Synthetic methods · Carbohydrates ·
Glycosylation · Ionic liquids · Protecting groups
20.96–20.82 (4 CH3) ppm. 5 carbon resonances {[N(CO)2] and
1
(NHCO2)} were missing. 19F NMR (CDCl3): δ = –72.96 (d, JP, F
=
706 Hz, PF6–) ppm. IR (film): ν = 2940, 1776, 1745, 1715, 1595, 1386,
˜
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deoxy-ꢀ- -glucopyranosyl-(1→4)-2-acetamido-2-deoxy-2-
phthalimido-ꢀ- -glucopyranosyl-(1→4)-2-acetamido-2-deoxy-
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Eur. J. Org. Chem. 2016, 1103–1109
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Received: November 22, 2015
Published Online: January 29, 2016
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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim