Synthesis of the fungal lipo-chitooligosaccharide Myc-IV (C16:0, S), symbiotic signal of arbuscular mycorrhiza

Article abstract of DOI:10.1002/ejoc.201301015

A new synthesis of the fungal lipo-chitooligosaccharide Myc-IV (C16:0, S), which was recently reported to be a major symbiotic signalling molecule in arbuscular mycorrhiza, is described. Key steps include the oxidative cleavage of a 4,6-O-benzylidene acet

Full text of DOI:10.1002/ejoc.201301015

FULL PAPER
DOI: 10.1002/ejoc.201301015
Synthesis of the Fungal Lipo-Chitooligosaccharide Myc-IV (C16:0, S),
Symbiotic Signal of Arbuscular Mycorrhiza
Laura Gillard,[a] Arnaud Stévenin,[a] Isabelle Schmitz-Afonso,[a] Boris Vauzeilles,[a,b]
François-Didier Boyer,*[a,c] and Jean-Marie Beau*[a,b]
Keywords: Natural products / Carbohydrates / Glycosylation / Oxidation / Protecting groups
A new synthesis of the fungal lipo-chitooligosaccharide Myc-
IV (C16:0, S), which was recently reported to be a major sym-
biotic signalling molecule in arbuscular mycorrhiza, is de-
scribed. Key steps include the oxidative cleavage of a 4,6-O-
benzylidene acetal to prepare a disaccharidic glycosyl ac-
ceptor, and stereoselective glycosylations with 2-methyl-5-
tert-butylphenyl thioglycosyl donors.
trations as low as 0.1 pm. Forty years ago, these molecules
were characterized as seed-germination stimulants of
witchweeds (Striga spp) and broomrapes (Orobanche and
Phelipanche spp), parasitic plants from the Orobanchaceae
family.[5] More recently, it has been shown that strigolac-
tones act as a new class of hormones in regulating plant
architecture.[6] It was recently revealed that strigolactones
induce the overexpression of short-chain chitin oligomers
by AM fungi.[7]
Introduction
The association of arbuscular mycorrhizal (AM) fungi
(Glomeromycota group) with plant roots is the oldest and
ecologically most important symbiotic relationship between
higher plants and microorganisms.[1] It is more than 400
million years old; the rhizobia–legume endosymbiosis ap-
peared only 60 million years ago.[2] Inside plant-root cells,
these fungi transport rare or poorly soluble mineral nutri-
ents such as phosphorus, copper, and zinc from the soil to
the plant, which in turn supplies carbohydrates to the fun-
gus. The plant benefits from its fungal partner by improved
nutrition, water control, and disease resistance, all of which
could be beneficial to low-input sustainable agriculture. The
fungus depends on its host plant for carbon, and its devel-
opment is strictly under the control of the plant. The AM
fungi endosymbiosis starts with an exchange of chemical
signals between the root and the microsymbiont, priming
both partners for the subsequent association. Each partner
produces chemical mediators. The signals produced by the
host plant were identified recently.[3,4] They are strigolac-
tones, carotenoid lactones secreted by plant roots, which
rapidly stimulate the development of AM fungi at concen-
By analogy with the rhizobial Nod factors, which induce
molecular responses in the host root, the Myc factors were
proposed early as compounds released in the rhizosphere
by AM fungi.[8–10] These are diffusible factors that are rec-
ognized by plant hosts and are necessary for the establish-
ment of a successful mycorrhizal association. Myc factors,
like Nod factors, activate calcium spiking.[11] This is consis-
tent with the hypothesis of biologists that the “Myc” signal-
ling pathways, which are older, could have been used by
nitrogen-fixing bacteria for their recognition by plants (the
“Nod” signalling pathways). Nod factors are recognized by
receptor-like kinases that contain sugar-binding lysine-mo-
tif domains.[12] Analogous AM-specific receptors most
probably also exist but need to be characterized. As the
chitin backbone of the Nod factor molecule is more typical
of fungi than bacteria, it was proposed earlier that the dif-
fusible AM factors could be Nod-factor-like mo-
lecules or chitin oligomers.[9,13] Myc factors (11 compounds
isolated to date, Figure 1) were finally structurally charac-
terized in 2011, having been isolated from Glomus intrarad-
ices (Glomeromycota), and were found to have structures
very similar to the Nod factors.[14] The Myc factors were
purified from exudates from germinating spores of the AM
fungus. The biologically active compounds characterized by
mass spectrometry (MS) by Dénarié et al.[14] consist of a
mixture of sulfated and non-sulfated lipo-chitooligosac-
charides (LCOs) present at a concentration between 10–5
[a] Centre de Recherche de Gif, Institut de Chimie des Substances
Naturelles, UPR2301 Centre National de la Recherche
Scientifique (CNRS),
1 avenue 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 de Chimie Moléculaire et des Matériaux, UMR 8182
CNRS – Université Paris-Sud,
91405 Orsay, France
E-mail: jean-marie.beau@u-psud.fr
http://www.icmmo.u-psud.fr/
[c] Institut Jean-Pierre Bourgin, UMR1318 Institut National de la
Recherche Agronomique (INRA) – AgroParisTech,
Route de Saint-Cyr (RD 10), 78026 Versailles, France
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301015.
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Synthesis of a Fungal Lipo-Chitooligosaccharide
Figure 1. Molecular signalling molecules involved in the initiation of arbuscular mycorrhizal (AM) symbiosis. a) Myc factor structures
isolated to date from Glomus intraradices. b) Two examples of strigolactone structures. c) Structures of most of the Nod factors.
and 10–11 m in the medium. As for Nod factors, they share in the arbuscular mycorrhizal symbiosis. LCO Myc factors
a common backbone of four or five (β1–4)-linked N-acetyl- could also be used to stimulate root system development,
glucosamine units, N-acylated at the non-reducing end unit which is essential to improve water and mineral uptake, es-
with a common fatty acid group, such as stearic (C18:0), pecially in parts of the world where soil fertility and water
oleic (C18:1), or palmitic (C16:0) acid. The only O-substitu- availability are poor. These remarkable biological activities
tion is the O-sulfation at the C-6 position of the monosac- prompted us to develop a new chemical synthesis of Myc
charide residue at the reducing end of the molecule. The factors using new tools in oligosaccharide chemistry and
synthesis of the major Myc factors (i.e., 3, 3S, 5, and 5S) learning from past syntheses[26] of Nod factors. Thus we
has been performed to unambiguously prove the structures considered: (i) selection of a suitable glycosylation pro-
established by MS. This synthesis of LCOs 3, 3S, 5, and 5S cedure able to cleanly and efficiently produce the difficult
was accomplished using recombinant Escherichia coli (β1–4) glycosidic bonds between two 2-acetamido-2-deoxy-
strains coexpressing the nodBC genes (encoding the chi- d-glucopyranosyl units; (ii) choice of an oligomerization
tooligosacchahe nodH gene (encoding the chito-oligosac- strategy that would provide the oligosaccharide with the
charide sulfotransferase) from Sinorhizobium meliloti, as de- highest efficacy; (iii) selective differentiation of identical
scribed by Samain et al.[15,16] This straightforward approach functional groups, e.g., primary or secondary hydroxy and
produced a mixture of chito tetrasaccharides and pentasac- amino groups present in the chitin fragment.
charides.[14a] The characterized Myc factors have simpler
structures than the Nod factors. Some of them had already
been isolated as Nod factors [compound 1S is NodRm-IV
Results and Discussion
(S), 2S is produced by Rhizobium meliloti[17,18] and Rhizo-
bium sp. GRH2[19] (as is 5 in minor amounts)], and had
Our new synthesis of an LCO used a method developed
been synthesized by various groups in recent decades chem- in our group for the formation of 4-OH-acceptors. The oxi-
ically[20–23] or by genetically engineered bacteria.[24] An- dative cleavage of 4,6-O-benzylidene acetals of various gly-
other one of them (compound 3S) had been synthesized as copyranosides was carried out with dimethyldioxirane
an “unnatural” Nod factor.[21] The crucial role of Nod fac- (DMDO),[27] and regioselectivity was ensured by placement
tors in an agronomically and ecologically important symbi- of a suitable protecting group at the C-3 position. Similarly
osis stimulated synthetic interest soon after their discovery to the strategy of Hui et al.,[23] we chose a blockwise synthe-
in 1990. Because these molecules are produced by rhizobia sis for the preparation of tetrasaccharide 10 from two pre-
or AM fungi as mixtures, and only in minute quantities, formed disaccharidic moieties 11a and 12 (Scheme 1). As
chemical synthesis greatly facilitates biochemical and physi- direct glycosylation with N-acetylglucosamine derivatives is
ological studies of the sensing mechanisms involved in Nod difficult,[28] N-protected derivatives were used, and selective
and Myc factor signalling.[25,14b]
differentiation of the amino groups was achieved by using
Myc factors or their analogues could be used to stimulate a benzyloxycarbonyl (Z) protecting group for the non-re-
mycorrhization for a broad range of applications in agricul- ducing terminal residue and orthogonal phthalimido (Phth)
ture with higher specificity than the strigolactones (see protecting groups for the other amino groups. The TBDPS
above),[3,4,6] which are also involved as chemical mediators (tert-butyldiphenylsilyl) protecting group at the C-6 posi-
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Scheme 1. Retrosynthetic analysis of sulfated glycolipid 3S Myc-IV (C16:0, S).
tion of the reducing terminal unit was chosen to be stable TFA (trifluoroacetic acid), Et3SiH/cat. Cu(OTf)2,
over several steps of the synthesis. A glycosylation between NaBH3CN (excess)/trimethylsilyl chloride (excess)].[33] At
α-imidate donor 13 and acceptor 14 would give disacchar- best, a poor yield (22%) of benzyl derivative 20a was ob-
ide 11a with the nitrogen atoms on the two sugar units dif- tained, probably due to the incompatibility of the reducing
ferentiated. The other glycosylation reactions, i.e., the syn- reagents with the ester and/or silyl groups of substrate 18[34]
thesis of disaccharide 12 and tetrasaccharide 10 would be (Scheme 2).
carried out using thioglycoside donors.
Oxidative ring opening of 18 and 19 was studied as an
The synthesis of Myc (C16:0, S) 3S started with the for- alternative route to an acceptor that could be used in the
mation of monomers 13, 14, 15, 16, and 17,[29] which were next glycosylation (Table 1). We have previously shown that
efficiently obtained by known procedures from commer- the regioselective oxidative cleavage of the 4,6-O-benzyl-
cially available d-glucosamine hydrochloride. Glycosylation idene protection of monosaccharides is governed by the na-
of α-imidate donor 13 with acceptor 14 in toluene at –78 °C ture of the hydroxy protecting group at the C-3 position.[27]
resulted in an efficient (76%) 1,2-trans glycosylation, as al- In this case, an acetate group provided a good selectivity
ready observed with an α-imidate donor bearing O-benzyl (4:1) in favour of the 4-alcohol. When this method was ap-
protecting groups in our total synthesis of NodRm-IV S in plied to disaccharides 18 and 19, the expected 4-OH com-
1994.[22] Lower yields were observed when dichloromethane pounds were formed in moderate yields (48–57%), along
was used as solvent (23–38%), or when the reaction was with traces of the 6-OH derivatives (21 and 24). For the
carried out at 0 °C in toluene (68%). We observed the for- reaction with 18 (Table 1, entry 1), we detected traces of
mation of the unreactive oxazolidinone A[30] under these diol 22. We also observed, when 19 was used as starting
glycosylation conditions, resulting from participation by the material, the formation of 4-OH derivative 25 resulting
NHZ group. The reactivity of the non-malodorous 2- from oxidation of the benzyl group at the anomeric posi-
methyl-5-tert-butylphenylthio group[31,32] (SMbp) was suf- tion. However, the 4-methoxyaryl group favoured the oxi-
ficient to allow the coupling of 15 (16) with acceptor 17 dation by DMDO at the benzylic position, and so a better
using NIS-TfOH as promoters without affecting the yield for the oxidative ring opening was obtained with com-
TBDPS group, providing disaccharides 18 and 19 in high pound 19 (57%) than with 18 (48%).
yields (97 and 81%, respectively). Reductive opening of the
The crucial 2 + 2 coupling between disaccharide acceptor
4,6-O-arylidene acetal of disaccharide 18 proved to be inef- 20b and donor 11a was first performed using NIS-TfOH as
ficient with any of the classical methods attempted [Et3SiH/ promoter (Table 2, entry 1) to give tetrasaccharide 26 in low
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Synthesis of a Fungal Lipo-Chitooligosaccharide
Scheme 2. Formation of disaccharides 11a, 18, and 19. a) 14 (1 equiv.), 13 (2 equiv.), BF3·OEt2 (0.5 equiv.), toluene, 7 h, –78 °C to room
temp. b) 17 (1 equiv.), 15 or 16 (1.5–1.7 equiv.), NIS (2.5 equiv.), TfOH (0.15 equiv.), MS 4 Å, CH2Cl2, 1 h 30 min, –10 °C. c) Et3SiH
(2 equiv.), Cu(OTf)2 (0.2 equiv.), CH2Cl2/CH3CN, 3 h, room temp.
Table 1. Regioselective oxidative cleavage of the 4,6-O-benzylidene group of disaccharides by DMDO.
Entry
Starting material
Product[a] (yield [%])[b]
By-products[a] (yield [%])[b]
1
2
18
19
20b (48)
23 (57)
21 (Ͻ5), 22 (Ͻ5)
24 (Ͻ5); 25 (10)
[a] Conditions: DMDO (ca. 0.1 m in acetone; 5 equiv.), 96 h, 5 °C. [b] Yield after silica gel chromatography. MBz = 4-methoxybenzoyl.
yield (36%). The stereochemistry of the product was proved Using the same sequence, the deprotection of 27 unfortu-
1
from its H NMR spectrum. Simply stirring acceptor 20b nately led to a mixture of products due to the difficult
(or 23) and donor 11a with molecular sieves (MS 4 Å) for aminolysis of the 4-methoxybenzoyl (MBz) group. Hence,
Ͼ1 h instead of 10 min before the addition of the promoters the synthesis was continued with tetrasaccharide 28. Depro-
increased the yield of tetrasaccharide 26 (or 27) to 46% (or tection of the TBDPS group using NH4F in MeOH under
48%) (Table 2, entries 2 and 5). When sulfoxide 11b[35] was reflux conditions smoothly gave the 6-OH tetrasaccharide,
used as donor, together with the appropriate promoters, tet- which was then easily sulfated by the SO3·pyridine complex,
rasaccharide 26 was not obtained efficiently (Table 2, en- using the standard procedure, to give compound 29. Final
try 4). Finally, as for the formation of disaccharide 11a, methanolysis of the O-acetate groups, followed by hydro-
changing the solvent of the glycosylation from CH2Cl2 to genolysis of the Z-group, gave the free chito-oligosaccharide
toluene resulted in an increase of the yield of tetrasacchar- (CO) 30.[23a] After chromatography on silica gel, this com-
ide 26 to 61% (Table 2, entry 3).[36]
pound was subjected to selective N-acylation using an ex-
The N-Phth groups in 26 were then selectively replaced cess of palmitoyl chloride 31[37] by heating (40 °C) in the
by N-acetyl groups in the presence of the NHZ group using presence of NaHCO3. The target compound, Myc (C16:0,
a two-step procedure [(NH2CH2)2 then Ac2O] to give com- S) 3S, was purified by chromatography on silica gel (ethyl
pound 28 with three NHAc groups, which was difficult to acetate/methanol/water, 5:2:1 as eluent) and gel filtration
separate from N,NЈ-diacetyl ethylenediamine (Scheme 3). through a Sephadex G-25 column. The regioselectivity of
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Table 2. Final glycosylation for the synthesis of the tetrasaccharidic the final acylation was unambiguously proved by tandem
unit.
mass spectrometry (MS/MS) data, as described for natural
3S.[14a] No β-elimination of the fatty acyl chain was ob-
served, as would be expected for O-acyl groups,[14a,38] which
conclusively showed that the N-acyl derivative had been ob-
tained. The fragmentation of the LCOs by MS/MS was
studied extensively, and the results obtained for 3S are con-
sistent with the MS/MS data for LCOs (32,[22] 33,[39] and
34[40]) previously synthesized in our group (Figure 2 and
Supporting Information). In all cases, we observed, by MS/
MS from the parent molecular ion, the fragment ions: [M –
H2O], 0,2A4, Y3, [Y3 – H2O], [0,2A4 – (non reducing terminal
residue)], and Y2.[41]
Entry Donor Acceptor Donor/
acceptor
Conditions[a]
Product
(yield [%])[e]
1
2
3
4
5
11a
11a
11a
11b
11a
20b
20b
20b
20b
23
1.5:1
2:1
1:1.3
1:1.1
1:1.1
A[a]
B[b]
C[c]
D[d]
B[b]
26 (36)
26 (46)
26 (61)
–
Figure 2. Diagnostic fragmentations by tandem mass spectrometry
for sulfated CO (30) and LCOs (32–34 and 3S).
27 (48)
[a] A: CH2Cl2, MS 4 Å (10 min, room temp.), then NIS (2.5 equiv.),
TfOH (0.15 equiv.), 1 h, –10 °C to room temp. [b] B: CH2Cl2, MS
4 Å (Ն1 h, room temp.), then NIS (2.5 equiv.), TfOH (0.15 equiv.),
1.5 h, –10 °C to room temp. [c] C: toluene, MS 4 Å (Ն1 h, room
temp.), then NIS (1.2 equiv.), TfOH (0.2 equiv.), 3 h, –30 °C.
[d] D: CH2Cl2, MS 4 Å (Ն1 h, room temp.), then Tf2O (1.2 equiv.),
TTBP (2,4,6-tri-tert-butylpyrimidine; 2 equiv.), 1.5 h, –78 °C to
–30 °C. [e] Yield after silica gel chromatography.
Conclusions
We have achieved the synthesis of lipo-chitooligosacchar-
ide 3S using robust glycosylation procedures (α-imidate and
β-thioglycoside donors), combined with new protecting
groups for this class of compound, without relying on azido
Scheme 3. Final deprotections and functionalizations. a) (NH2CH2)2 (150 equiv.), EtOH, 12 h, 60 °C; b) Ac2O (275 equiv.), pyridine, 48 h,
room temp.; c) NH4F (0.5 m in MeOH; 5 equiv.), 12 h, reflux; d) (i) SO3·pyridine (3 equiv.), DMF, 48 h, r.t. (ii) Dowex® 50 (Na+),
MeOH. e) MeONa (1 m in MeOH; 2 equiv.), MeOH, 12 d, room temp.; f) H2, Pd (10% on C), EtOAc/H2O/EtOH (1:1:0.1), 16 h, room
temp.; g) 31 (6 equiv.), NaHCO3, DMF/THF/H2O (5:3:2), 48 h.
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Synthesis of a Fungal Lipo-Chitooligosaccharide
phase was washed with Na2S2O3 (saturated aq.), dried with
Na2SO4, and concentrated under reduced pressure. The crude ma-
terial was purified by chromatography on silica gel (toluene/acet-
one, 98:2 to 96:4) to give disaccharide 18 (1.57 g, 97 %) as an
amorphous white solid. [α]2D5 = –0.5 (c = 1.0, CHCl3). 1H NMR
(500 MHz, CDCl3): δ = 7.82–7.57 (m, 12 H, Ar), 7.46–7.29 (m, 11
H, Ar), 7.16 (d, J = 7.0 Hz, 1 H, Ar), 7.04 (t, J = 7.0 Hz, 2 H, Ar),
6.95 (d, J = 7.3 Hz, 2 H, Ar), 5.81 (t, JA-3,A-2 = JB-3,B-4 = 9.2 Hz,
1 H, 3A-H), 5.70–5.65 (m, 1 H, 3B-H), 5.66 (d, JA-1,A-2 = 8.0 Hz,
1 H, 1A-H), 5.48 (s, 1 H, CHPh), 5.22 (d, JB-1,B-2 = 8.2 Hz, 1 H,
chemistry to differentiate the amino groups. The disac-
charide acceptor was prepared by a dimethyldioxirane-me-
diated oxidative ring opening of 4,6-O-benzylidene acetals,
as recently reported by our group. New syntheses of LCO
analogues are currently in progress in our laboratory, and
these analogues could be valuable in improving mycor-
rhization or nodulation for plants of interest.
1B-H), 4.63 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.37 (dd, JA-6,A-6Ј
=
Experimental Section
10.4, JA-6,A-5 = 4.6 Hz, 1 H, 6A-H), 4.28 (d, J = 12.0 Hz, 1 H,
CH2Ph), 4.20 (dd, JB-2,B-3 = 10.7, JB-2,B-1 = 8.2 Hz, 1 H, 2B-H),
4.19–4.13 (m, 2 H, 2A-H, 4B-H), 3.79 (br. d, JB-6,B-6Ј = 11.5 Hz, 1
General Remarks: See Supporting Information.
(2-Methyl-5-tert-butylphenyl) (3,4,6-Tri-O-acetyl-2-benzyloxycarb-
H, 6B-H), 3.75–3.65 (m, 2 H, 4A-H, 6ЈA-H), 3.63 (dd, JA-5,A-4
=
onylamino-2-deoxy-β-
benzyl-2-deoxy-2-phthalimido-1-thio-β-
D
-glucopyranosyl)-(1Ǟ4)-3-O-acetyl-6-O-
-glucopyranoside (11a): Ac-
9.5, JA-5,A-6 = 4.6 Hz, 1 H, 5A-H), 3.58 (dd, JB-6Ј,B-6 = 11.5,
JB-6Ј,B-5 = 4.0 Hz, 1 H, 6ЈB-H), 3.41 (br. dd, JB-5,B-4 = 9.5, JB-5,B-6Ј
= 4.0 Hz, 1 H, 5B-H), 1.94 (s, 3 H, Ac), 1.84 (s, 3 H, Ac), 1.06 (s,
9 H, tBu) ppm. 13C NMR (75 MHz, CDCl3): δ = 170.2 (C, COMe),
169.9 (C, COMe), 167.9 [4 C, 2N(CO)2], 137.2 (C, Ar), 137.0 (C,
Ar), 136.3 (2 CH, Ar), 136.1 (2 CH, Ar), 134.4 (4 CH, Ar), 133.8
(C, Ar), 133.4 (C, Ar), 131.8 (2 C, Ar), 131.4 (2 C, Ar), 129.9 (2
CH, Ar), 129.4 (CH, Ar), 128.5 (2 CH, Ar), 128.3 (CH, Ar), 127.9
(2 CH, Ar), 127.8 (CH, Ar), 127.7 (2 CH, Ar), 127.6 (2 CH, Ar),
126.5 (2 CH, Ar), 126.4 (CH, Ar), 123.8 (2 CH, Ar), 123.6 (2 CH,
Ar), 101.9 (CH, CHPh), 97.5 (CH, C-1A), 96.7 (CH, C-1B), 79.5
(CH, C-4A), 75.3 (CH, C-5B), 74.2 (CH, C-4B), 71.8 (CH, C-3B),
70.5 (CH2, CH2Ph), 69.9 (CH, C-3A), 68.9 (CH2, C-6A), 66.3 (CH,
C-5A), 62.6 (CH2, C-6B), 55.9 (CH, C-2A), 55.3 (CH, C-2B), 27.1
(3 CH3, tBu), 21.1 (CH3, Ac), 20.7 (CH3, Ac), 19.7 (C, tBu) ppm.
D
ceptor 14[29] (300 mg, 0.49 mmol, 1.0 equiv.) and donor 13[29]
(571 mg, 0.98 mmol, 2.0 equiv.) were cooled in toluene (3.6 mL) to
–78 °C. BF3·OEt2 (30 μL, 0.245 mmol, 0.5 equiv.) was added drop-
wise to this solution. The reaction mixture was stirred at this tem-
perature for 2 h, then it was warmed slowly to room temperature
over a period of 5 h, and then neutralized with triethylamine
(200 μL). The volatiles were evaporated under reduced pressure,
and the crude material was purified by chromatography on silica
gel (heptane/EtOAc, 8:2 to 1:1) to give disaccharide 11a (380 mg,
76%) as an amorphous white solid. [α]2D5 = +5.3 (c = 1.0, CHCl3).
1H NMR (300 MHz, CD3CN): δ = 7.94–7.82 (m, 4 H, Ar), 7.50
(d, J = 2.1 Hz, 1 H, Ar), 7.45–7.32 (m, 9 H, Ar), 7.29 (dd, J = 7.9,
J = 2.1 Hz, 1 H, Ar), 7.25–7.18 (m, 1 H, Ar), 7.13 (d, J = 7.9 Hz,
1 H, Ar), 5.62 (m, 1 H, 3B-H), 5.56 (d, JB-1,B-2 = 10.5 Hz, 1 H, 1B-
H), 5.52–5.45 (m, 1 H, NH), 5.19–5.02 (m, 3 H, 3A-H, OCH2Ph),
4.91 (t, JA-4,A-3 = JA-4,A-5 = 10.0 Hz, 1 H, 4A-H), 4.68–4.51 (m, 3
H, 1A-H, CH2Ph), 4.36–4.18 (m, 2 H, 2B-H, 6A-H), 4.06–3.91 (m,
2 H, 4B-H, 6ЈA-H), 3.69–3.56 (m, 3 H, 6B-H, 6ЈB-H, 5A-H), 3.56–
3.36 (m, 2 H, 5B-H, 2A-H), 2.18 (s, 3 H, Me), 2.00 (s, 3 H, Ac),
1.98 (s, 3 H, Ac), 1.92 (s, 3 H, Ac), 1.86 (s, 3 H, Ac), 1.26 (s, 9 H,
tBu) ppm. 13C NMR (75 MHz, CD3CN): δ = 171.6 (2 C, COMe),
171.4 (C, COMe), 170.9 (C, COMe), 163.9 [2 C, N(CO)2], 157.2
(C, NHCO), 139.9 (C, Ar), 138.5 (C, Ar), 138.4 (C, Ar), 136.3 (CH,
Ar), 136.2 (CH, Ar), 132.8 (C, Ar), 132.5 (C, Ar), 131.7 (CH, Ar),
131.5 (CH, Ar), 129.9 (3 CH, Ar), 129.7 (C, Ar), 129.4 (3 CH, Ar),
129.1 (3 CH, Ar), 127.0 (2 CH, Ar), 125.0 (CH, Ar), 124.6 (CH,
Ar), 122.3 (C, Ar), 101.7 (CH, C-1A), 85.4 (CH, C-1B), 79.9 (CH,
C-5B), 76.3 (CH, C-4B), 74.1 (CH2, CH2Ph), 73.4 (CH, C-3A or
3B-H), 73.0 (CH, C-3A or C-3B), 72.7 (CH, C-5A), 70.0 (CH, C-
4A), 69.6 (CH2, C-6B), 67.6 (CH2, OCH2Ph), 63.1 (CH2, C-6A),
57.5 (CH, C-2A), 55.5 (CH, C-2B), 35.4 (C, tBu), 31.9 (3 CH3,
tBu), 21.3 (3 CH3, Ac), 21.2 (CH3, Ac), 20.8 (CH3, Me) ppm. IR
IR (film): ν = 2955, 2911, 2852, 1779, 1747, 1720, 1387 cm–1. MS
˜
(ESI): m/z (%) = 1123 (35) [M + Na]+. HRMS (ESI): calcd. for
C62H60N2NaO15Si [M + Na]+ 1123.3661; found 1123.3715.
Benzyl (3-O-Acetyl-6-O-benzoyl-2-deoxy-2-phthalimido-β-
pyranosyl)-(1Ǟ4)-3-O-acetyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-
2-phthalimido-β- -glucopyranoside (20b): Benzylidene acetal 18
D-gluco-
D
(485 mg, 0.44 mmol) was added to a solution of freshly distilled
dimethyldioxirane[42] in acetone (22 mL, ≈ 0.1 m), and the mixture
was stirred at 5 °C for 96 h. The crude material was purified by
chromatography on silica gel (heptane/EtOAc, 9:1 to 8:3) to give
alcohol 20b (240 mg, 48%) as an amorphous white solid. [α]2D5
=
1
+4.9 (c = 1.0, CHCl3). H NMR (500 MHz, CDCl3): δ = 8.06 (d,
J = 6.0 Hz, 2 H, Ar), 7.84–7.61 (m, 13 H, Ar), 7.57 (t, J = 7.0 Hz,
1 H, Ar), 7.49–7.40 (m, 5 H, Ar), 7.39–7.27 (m, 3 H, Ar), 7.12–
7.01 (m, 3 H, Ar), 6.96 (d, J = 7.0 Hz, 1 H, Ar), 5.71–5.62 (m, 2
H, 3A-H, 3B-H), 5.60 (d, JA-1,A-2 = 8.2 Hz, 1 H, 1A-H), 5.20 (d,
JB-1,B-2 = 8.5 Hz, 1 H, 1B-H), 4.73 (dd, JA-6,A-6Ј = 12.0, JA-6,A-5
=
3.4 Hz, 1 H, 6A-H), 4.67–4.57 (m, 2 H, 6ЈA-H, CH2Ph), 4.32 (t,
JB-4,B-3 = JB-4,B-5 = 9.5 Hz, 1 H, 4B-H), 4.27 (d, J = 12.0 Hz, 1 H,
CH2Ph), 4.23 (dd, JB-2,B-3 = 10.7, JB-2,B-1 = 8.5 Hz, 1 H, 2B-H),
4.13 (dd, JA-2,A-3 = 11.0, JA-2,A-1 = 8.2 Hz, 1 H, 2A-H), 3.81 (br. d,
JB-6,B-6Ј = 11.0 Hz, 1 H, 6B-H), 3.73–3.63 (m, 3 H, 4A-H, 5A-H,
(film): ν = 3030, 2960, 2874, 2355, 1778, 1715, 1610 cm–1. MS
˜
(ESI): m/z (%) = 1047 (100) [M + Na]+. HRMS (ESI): calcd. for
C54H60N2NaO16S [M + Na]+ 1047.3687; found 1047.3645.
Benzyl (3-O-Acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-
glucopyranosyl)-(1Ǟ4)-3-O-acetyl-6-O-(tert-butyldiphenylsilyl)-2-de- 6ЈB-H), 3.36 (br. d, JB-5,B-4 = 9.5 Hz, 1 H, 5B-H), 3.12 (d, JOH,A-4
oxy-2-phthalimido-β-
D
-glucopyranoside (18): Acceptor 17[29] (1.00 g,
= 4.0 Hz, 1 H, OH), 1.88 (s, 3 H, Ac), 1.83 (s, 3 H, Ac), 1.06 (s, 9
H, tBu) ppm. 13C NMR (75 MHz, CDCl3): δ = 171.3 (C, COMe),
170.3 (C, COMe), 168.1 [2 C, N(CO)2], 167.8 (C, PhCO), 167.5 [2
C, N(CO)2], 137.3 (2 C, Ar), 136.4 (2 CH, Ar), 136.1 (2 CH, Ar),
134.4 (2 CH, Ar), 133.7 (2 CH, Ar), 131.5 (3 C, Ar), 130.1 (2 CH,
Ar), 129.9 (CH, Ar), 129.8 (CH, Ar), 129.6 (C, Ar), 128.8 (2 CH,
Ar), 128.3 (2 CH, Ar), 127.9 (2 CH, Ar), 127.7 (4 CH, Ar), 127.6
(2 CH, Ar), 123.8 (2 CH, Ar), 123.7 (2 C, Ar), 123.6 (2 CH, Ar),
96.9 (CH, C-1A), 96.7 (CH, C-1B), 75.4 (CH, C-5B), 74.5 (CH, C-
5A), 73.3 (2 CH, C-3A, C-4B), 71.1 (CH, C-3B), 70.5 (CH2,
1.47 mmol, 1.00 equiv.) and donor 15[29] (1.30 g, 2.21 mmol,
1.50 equiv.) were stirred with molecular sieves (4 Å; 0.8 g) in
CH2Cl2 (5 mL) at room temperature for 30 min. NIS (826 mg,
3.67 mmol, 2.50 equiv.) was then added, and the suspension was
cooled to –10 °C. TfOH (19 μL, 0.22 mmol, 0.15 equiv.) was added,
and then the reaction mixture was allowed to warm to room tem-
perature and stirred for 1.5 h. The molecular sieves were removed
by filtration, and the filtrate was poured into NaHCO3 (saturated
aq.). The mixture was extracted with CH2Cl2, and the organic
Eur. J. Org. Chem. 2013, 7382–7390
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7387

F.-D. Boyer, J.-M. Beau et al.
FULL PAPER
CH2Ph), 70.0 (CH, C-4A), 63.7 (CH2, C-6A), 62.4 (CH2, C-6B),
55.4 (CH, C-2B), 55.1 (CH, C-2A), 27.1 (3 CH3, tBu), 20.9 (CH3,
1223, 1025, 720, 698 cm–1. MS (ESI): m/z (%) = 1983 (70) [M +
Na]+. HRMS (ESI): calcd. for C105H104N4NaO32Si [M + Na]+
1983.6301; found 1983.6265.
Ac), 20.8 (CH , Ac), 19.7 (C, tBu) ppm. IR (film): ν = 2935, 1777,
˜
3
1745, 1715, 1385, 1274, 1226, 1070, 1040, 719, 701 cm–1. MS (ESI):
m/z (%) = 1139 (100) [M + Na]+. HRMS (ESI): calcd. for
C62H60N2NaO16Si [M + Na]+ 1139.3610; found 1139.3616.
Benzyl (3,4,6-Tri-O-acetyl-2-benzyloxycarbonylamino-2-deoxy-β-
glucopyranosyl)-(1Ǟ4)-(2-acetamido-3-O-acetyl-6-O-benzyl-2-
deoxy-β- -glucopyranosyl)-(1Ǟ4)-(2-acetamido-3,6-di-O-acetyl-
2-deoxy-β- -glucopyranosyl)-(1Ǟ4)-2-acetamido-3-O-acetyl-6-O-
glucopyranosyl)-(1Ǟ4)-(3-O-acetyl-6-O-benzyl-2-deoxy-2-phthal- (tert-butyldiphenylsilyl)-2-deoxy-β- -glucopyranoside (28): Tetrasac-
imido-β- -glucopyranosyl)-(1Ǟ4)-(3-O-acetyl-6-O-benzoyl-2-deoxy- charide 26 (448 mg, 0.228 mmol, 1.0 equiv.) and ethylenediamine
2-phthalimido-β-
butyldiphenylsilyl)-2-deoxy-2-phthalimido-β-
Acceptor 20b (120 mg, 0.107 mmol, 1.30 equiv.) and donor 11
D-
D
Benzyl (3,4,6-Tri-O-acetyl-2-benzyloxycarbonylamino-2-deoxy-β-
D-
D
D
D
D
-glucopyranosyl)-(1Ǟ4)-3-O-acetyl-6-O-(tert-
-glucopyranoside (26):
(2.29 mL, 34 mmol, 150 equiv.) were stirred in ethanol (39 mL) at
60 °C for 12 h. The volatiles were evaporated under reduced pres-
sure, and the crude product was used in the next step without puri-
D
(84 mg, 0.082 mmol, 1.00 equiv.) were stirred with molecular sieves fication. The residue was stirred with acetic anhydride (5.9 mL,
(4 Å; 80 mg) in anhydrous toluene (1.6 mL) at room temperature
for 1 h. NIS (23 mg, 0.102 mmol, 1.20 equiv.) was then added, and
the suspension was cooled to –30 °C. TfOH (1.5 μL, 0.016 mmol,
0.20 equiv.) was added, and the reaction mixture was stirred at
–30 °C for 3 h. Et3N was added, and the molecular sieves were
removed by filtration. The filtrate was washed with Na2S2O3 (satu-
rated aq.), dried with Na2SO4, and concentrated under reduced
pressure. The crude material was purified by chromatography on
silica gel (toluene/acetone, 95:5 to 88:12) to give tetrasaccharide 26
63 mmol, 275 equiv.) in pyridine (71 mL) at room temperature for
2 d. The volatiles were evaporated under reduced pressure, and the
crude product was purified by chromatography on silica gel
(CH2Cl2/MeOH, 1:0 to 96:4) to give 28 (330 mg, 84 %) as an
amorphous white solid. [α]2D5 = –33.8 (c = 1.0, CHCl3). H NMR
1
(500 MHz, CD3CN): δ = 7.84–7.74 (m, 4 H, Ph), 7.52–7.26 (m, 21
H, Ph), 6.41–6.33 (m, 2 H, NH, NH), 5.92 (d, JNH,D-2 or C-2
=
9.5 Hz, 1 H, NH), 5.13–4.94 (m, 6 H, 3B-H, 3C-H, 3D-H, NH,
OCH2Ph), 4.88 (t, JA-4,A-3 = JA-4,A-5 = 9.8 Hz, 1 H, 4A-H), 4.77 (d,
J = 12.0 Hz, 1 H, CH2Ph), 4.67 (d, JC-1,C-2 or D-1,D-2 = 8.2 Hz, 1 H,
(98 mg, 61%) as an amorphous white solid. [α]2D5 = +3.7 (c = 0.3,
1
CHCl3). H NMR (600 MHz, CD3CN): δ = 7.92 (d, J = 7.7 Hz, 2 1C-H or 1D-H), 4.62–4.43 (m, 7 H, 1A-H, 3A-H, 1B-H, 1C-H or
H, Ar), 7.88–7.63 (m, 16 H, Ar), 7.58 (t, J = 7.7 Hz, 1 H, Ar),
1D-H, CH2Ph, CH2Ph), 4.35–4.25 (m, 2 H, 6A-H, 6C-H), 4.17 (dd,
7.53–7.18 (m, 20 H, Ar), 7.09 (t, J = 7.3 Hz, 1 H, Ar), 7.01 (t, J = JC-6Ј,C-6 = 12.2, JC-6,C-5 = 5.5 Hz, 1 H, 6ЈC-H), 4.04–3.93 (m, 3 H,
7.3 Hz, 1 H, Ar), 6.93 (d, J = 7.7 Hz, 1 H, Ar), 5.65 (t, JC-3,C-2
JC-3,C-4 = 10.3 Hz, 1 H, 3C-H) (see Figure 3), 5.58 (t, JD-3,D-2
JD-3,D-4 = 10.0 Hz, 1 H, 3D-H), 5.51 (br. t, JB-3,B-2 = JB-3,B-4
=
=
=
6ЈA-H, 4D-H, 6D-H), 3.92–3.81 (m, 3 H, 2B-H, 4B-H, 6ЈD-H),
3.73 (t, JC-4,C-3 = JC-4,C-5 = 9.2 Hz, 1 H, 4C-H), 3.69–3.55 (m, 4 H,
6B-H, 6ЈB-H, 2C-H, 2D-H), 3.55–3.41 (m, 5 H, 2A-H, 5A-H, 5B-
H, 5C-H, 5D-H), 2.05 (s, 3 H, Ac), 2.01 (s, 6 H, Ac), 1.98 (s, 6 H,
Ac), 1.89 (s, 6 H, Ac), 1.84 (s, 3 H, Ac), 1.82 (s, 3 H, Ac), 1.65 (s,
10.0 Hz, 1 H, 3B-H), 5.47–5.42 (m, 2 H, 1C-H, NH), 5.33 (d,
JB-1,B-2 = 8.1 Hz, 1 H, 1B-H), 5.13–4.98 (m, 4 H, 3A-H, 1D-H,
OCH2Ph), 4.85 (t, JA-4,A-3 = 9.5 Hz, 1 H, 4A-H), 4.62–4.49 (m, 5 3 H, Ac), 1.11 (s, 9 H, tBu) ppm. 13C NMR (75 MHz, CD3CN): δ
H, 1A-H, 6C-H, CH2Ph, CH2Ph), 4.28 (d, J = 12.5 Hz, 1 H,
CH2Ph), 4.24 (dd, JA-6,A-6Ј = 12.5, JA-6,A-5 = 4.4 Hz, 1 H, 6A-H), (C, COMe), 171.2 (2 C, COMe), 171.1 (C, COMe), 170.9 (C,
4.19 (t, JD-4,D-3 = JD-4,D-5 = 10.0 Hz, 1 H, 4D-H), 4.11–3.87 (m, 7 COMe), 157.2 (C, NHCO), 139.8 (C, Ph), 139.2 (C, Ph), 138.5 (C,
= 172.0 (C, COMe), 171.6 (3 C, COMe), 171.5 (C, COMe), 171.4
H, 6ЈA-H, 2B-H, 4B-H, 2C-H, 4C-H, 6ЈC-H, 2D-H), 3.74–3.59 (m, Ph), 137.2 (CH, Ph), 137.0 (CH, Ph), 135.2 (C, Ph), 134.4 (C, Ph),
5 H, 6B-H, 6ЈB-H, 5C-H, 6D-H, 6ЈD-H), 3.56–3.45 (m, 2 H, 5A- 132.6 (CH, Ph), 131.4 (CH, Ph), 131.3 (CH, Ph), 129.9 (3 CH, Ph),
H, 5B-H), 3.39–3.31 (m, 2 H, 2A-H, 5D-H), 1.95 (s, 3 H, Ac), 1.93
(s, 3 H, Ac), 1.86 (s, 3 H, Ac), 1.83 (s, 3 H, Ac), 1.80 (s, 3 H, Ac),
1.63 (s, 3 H, Ac), 0.91 (s, 9 H, tBu) ppm. 13C NMR (75 MHz,
CD3CN): δ = 171.6 (C, COMe), 171.5 (3 C, COMe), 171.4 (C,
129.8 (CH, Ph), 129.7 (2 CH, Ph), 129.4 (CH, Ph), 129.3 (2 CH,
Ph), 129.1 (7 CH, Ph), 129.0 (CH, Ph), 128.9 (2 CH, Ph), 128.3
(CH, Ph), 102.1 (CH, C-1A), 101.5 (CH, C-1B), 101.2 (2 CH, C-
1C, C-1D), 77.3 (CH, C-4C), 76.6 (CH, C-5D), 76.0 (CH, C-4B),
COMe), 171.3 (C, COMe), 170.9 (C, COPh), 169.1 (C, NCO), 75.3 (2 CH, C-4D, C-5B or C-5C), 74.3, 74.1, 74.0, 73.9 (4 CH, C-
168.9 [2 C, N(CO)2], 168.8 (C, NCO), 166.7 [2 C, N(CO)2], 157.2 3B, C-3C, C-3D, C-5B or C-5C), 73.8 (CH2, CH2Ph), 73.6 (CH,
(C, NHCO), 139.8–124.5 (42CH, 12 C, Ar), 101.4 (CH, C-1A), 98.7 C-3A), 72.6 (CH, C-5A), 71.5 (CH2, CH2Ph), 69.9 (CH, C-4A),
(CH, C-1B), 98.0 (2 CH, C-1C, C-1D), 76.9 (CH, C-4C), 76.3 (CH,
C-4B), 76.0 (CH, C-5D), 75.7 (CH, C-5B), 74.6 (CH, C-4D), 74.1
69.4 (CH2, C-6B), 67.5 (CH2, OCH2Ph), 63.9 (CH2, C-6D), 63.7
(CH2, C-6C), 63.0 (CH2, C-6A), 57.5 (CH, C-2A), 55.7 (2 CH, C-
(CH, C-5C), 73.9 (CH2, CH2Ph), 73.5 (CH, C-3A), 72.7 (CH, C- 2C, C-2D), 54.9 (CH, C-2B), 27.7 (3 CH3, tBu), 23.6 (2 CH3, Ac),
5A), 72.0 (CH, C-3B), 71.8 (CH, C-3C), 71.7 (CH2, CH2Ph), 71.5 23.5 (CH3, Ac), 21.6 (3 CH3, Ac), 21.5 (CH3, Ac), 21.3 (2 CH3,
(CH, C-3D), 69.9 (CH, C-4A), 69.3 (CH2, C-6B), 67.6 (CH2,
Ac), 21.2 (CH , Ac), 20.4 (C, tBu) ppm. IR (film): ν = 3292, 2939,
˜
3
OCH2Ph), 64.2 (CH2, C-6C), 63.5 (CH2, C-6D), 63.0 (CH2, C-6A), 1743, 1655, 1534, 1428, 1368, 1225, 1030, 740, 698 cm–1. MS
57.6 (CH, C-2A), 56.5 (CH, C-2B), 56.3 (2 CH, C-2C, C-2D), 27.6 (MALDI): m/z (%) = 1657 (100) [M + Na]+. HRMS (MALDI):
(3 CH3, tBu), 21.4 (CH3, Ac), 21.3 (3 CH3, Ac), 21.2 (2 CH3, Ac), calcd. for C82H102N4NaO29Si [M + Na]+ 1657.6297; found
20.4 (C, tBu) ppm. IR (film): ν = 2947, 1777, 1744, 1713, 1384,
1657.6324.
˜
Benzyl (3,4,6-Tri-O-acetyl-2-benzyloxycarbonylamino-2-deoxy-β-D-
glucopyranosyl)-(1Ǟ4)-(2-acetamido-3-O-acetyl-6-O-benzyl-2-
deoxy-β- -glucopyranosyl)-(1Ǟ4)-(2-acetamido-3,6-di-O-acetyl-
2-deoxy-β- -glucopyranosyl)-(1Ǟ4)-2-acetamido-3-O-acetyl-2-
deoxy-6-O-sodium Sulfonato-β- -glucopyranoside (29): Compound
D
D
D
28 (310 mg, 0.189 mmol, 1.0 equiv.) and NH4F (0.5 m in MeOH;
1.89 mL, 0.948 mmol, 5.0 equiv.) were stirred at 70 °C for 12 h. The
volatiles were removed under reduced pressure. The crude material
was purified by chromatography on silica gel (CH2Cl2/MeOH, 1:0
to 96:4) to give benzyl (3,4,6-tri-O-acetyl-2-benzyloxycarbonylamino-
Figure 3. Numbering for compound 26 and the following tetra-
mers.
7388
www.eurjoc.org
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 7382–7390

Synthesis of a Fungal Lipo-Chitooligosaccharide
2-deoxy-β-d-glucopyranosyl)-(1Ǟ4)-(2-acetamido-3-O-acetyl-6-O-
benzyl-2-deoxy-β-d-glucopyranosyl)-(1Ǟ4)-(2-acetamido-3,6-di-
After 48 h, the volatiles were removed under reduced pressure, and
the crude product was purified by chromatography on silica gel
O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1Ǟ4)-2-acet- (EtOAc/MeOH/H2O, 8:2:1 to 6/2:1) and then by Sephadex G-25
amido-3-O-acetyl-2-deoxy-β-d-glucopyranoside (191 mg, 72%) as
(H2O). Myc-IV (C16:0, S) 3S (6.8 mg, 44%) was obtained after
an amorphous white solid. 1H NMR (500 MHz, CD3CN): δ =
lyophilisation as a white powder. [α]2D5 = –4.1 (c = 0.26, MeOH).
7.45–7.29 (m, 15 H), 6.52 (d, J = 9.5 Hz, 1 H), 6.38–6.31 (m, 2 H), 1H NMR (600 MHz, CD3OD) partial data: δ = 5.05 (d, J = 3.0 Hz,
5.35 (d, J = 9.0 Hz, 1 H), 5.13–4.94 (m, 6 H), 4.88 (t, J = 9.8 Hz,
0.6 H, 1D-Hα), 4.61 (d, J = 8.5 Hz, 1 H, 1-Hβ), 4.54 (d, J = 8.5 Hz,
1 H), 4.84 (d, J = 12.1 Hz, 1 H), 4.64–4.44 (m, 7 H), 4.33 (dd, J = 0.4 H, 1D-Hβ), 4.50 (d, J = 8.5 Hz, 1 H, 1-Hβ), 4.46 (d, J = 8.5 Hz,
12.0, J = 1.1 Hz, 1 H), 4.29 (dd, J = 12.4, J = 4.4 Hz, 1 H), 4.16
(dd, J = 12.0, J = 4.6 Hz, 1 H), 3.96 (dd, J = 12.4, J = 2.0 Hz, 1
H), 3.88–3.55 (m, 11 H), 3.54–3.48 (m, 1 H), 3.45–3.32 (m, 3 H),
2.97 (br. t, J = 5.2 Hz, 1 H), 2.08 (s, 3 H), 2.01 (s, 3 H), 1.98 (s, 3
H), 1.97 (s, 3 H), 1.96 (s, 6 H), 1.89 (s, 3 H), 1.85 (s, 3 H), 1.83 (s,
3 H), 1.82 (s, 3 H) ppm. MS (ESI): m/z (%) = 1419 (100) [M +
1 H, 1-Hβ), 4.06 (br. d, J = 9.5 Hz, 1 H, 6-H), 3.98 (br. d, J =
9.5 Hz, 1 H, 6-H), 1.95, 1.89, 1.87 (3 s, 9 H, 3 NHAc), 1.55–1.47
[m, 2 H, NH(CO)CH2], 1.24–1.15 (m, 26 H, 13 CH2), 0.80 (t, J =
7.0 Hz, CH3) ppm. MS (ESI): m/z (%) = 1105 (30) [M – Na]–.
HRMS (ESI): calcd. for C46H81N4O24S [M – Na]– 1105.4961;
found 1105.5002. For the MS/MS data, see Supporting Infor-
Na]+. HRMS (ESI): calcd. for C66H84N4NaO29 [M + Na]+ mation.
1419.5119; found 1419.5115.
Supporting Information (see footnote on the first page of this arti-
cle): General methods, experimental procedures, characterization
This alcohol was stirred with SO3·pyridine (65 mg, 0.408 mmol,
3.0 equiv.) in DMF (10 mL) at room temperature for 2 d. The reac-
tion mixture was concentrated under reduced pressure. The pyrid-
inium salt was exchanged to the sodium salt by eluting over
Dowex® (Na+ form) ion-exchange resin, and the volatiles were re-
moved under reduced pressure. The crude product was purified by
chromatography on silica gel (EtOAc/iPrOH/H2O/Et3N,
8.8:0.8:0.4:0.1 to 7.6:1.6:0.8:0.1) to give 29 (150 mg, 74 %) as a
1
data and copies of H and 13C NMR spectra of new compounds.
Acknowledgments
The authors thank R. Beau for her comments on the manuscript
and J. Crouhy for technical assistance. The ICSN – Centre National
de la Recherche Scientifique (CNRS), the Institut Universitaire de
France (IUF), and the Institut National de la Recherche Agrono-
mique (INRA) are thanked for their financial support of this study.
white amorphous solid. [α]2D5 = –28.4 (c = 0.35, CHCl3). H NMR
1
(500 MHz, CD3CN): δ = 7.40–7.32 (m, 15 H), 6.61–6.51 (m, 2 H),
6.45 (dd, J = 11.0, J = 10.0 Hz, 1 H), 6.37 (dd, J = 7.5, J = 6.5 Hz,
1 H), 5.08–4.99 (m, 5 H), 4.87–4.79 (m, 3 H), 4.61–4.54 (m, 5 H),
4.32–4.26 (m, 3 H), 4.10–4.07 (m, 2 H), 3.97–3.92 (m, 2 H), 3.85–
3.76 (m, 4 H), 3.61–3.57 (m, 3 H), 3.53–3.48 (m, 3 H), 3.46–3.42
(m, 2 H), 3.38–3.34 (m, 2 H), 2.06–1.85 (10s, 30 H) ppm. IR (film):
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ν = 3303, 2926, 1741, 1651, 1539, 1428, 1369, 1226, 1032, 741,
˜
824–827.
699 cm–1. MS (ESI): m/z (%) = 1475 (100) [M – Na]–. HRMS (ESI):
[4] A. Besserer, V. Puech-Pages, P. Kiefer, V. Gomez-Roldan, A.
Jauneau, S. Roy, J. C. Portais, C. Roux, G. Bécard, N. Sejalon-
Delmas, PLoS Biol. 2006, 4, 1239–1247.
[5] a) C. E. Cook, L. P. Whichard, B. Turner, M. E. Wall, Science
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calcd. for C66H83N4O32S [M – Na]– 1475.4711; found 1475.4711.
(2-Acetamido-2-deoxy-β-
deoxy-2-phthalimido-β- -glucopyranosyl)-(1Ǟ4)-2-acetamido-6-O-
sodium Sulfonato-2-deoxy- -glucopyranose (30): Compound 29
D-glucopyranosyl)-(1Ǟ4)-(2-acetamido-2-
D
D
(75 mg, 0.0500 mmol, 1.0 equiv.) was stirred with NaOMe (1 m in
MeOH; 100 μL, 2.0 equiv.) in MeOH (1.4 mL) at room temperature
for 12 d. The solvent was removed under reduced pressure. The
residue was dissolved in a mixture of EtOAc/H2O/EtOH (1:1:0.1),
and Pd(10% on C; 75 mg) was added. The reaction mixture was
stirred for 16 h under H2. The catalyst was then removed by fil-
tration, and rinsed with EtOAc, H2O, and MeOH. The solvents
were removed from the filtrate under reduced pressure, and the
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+182 (c = 1.8, H2O) [ref.[23a] +3.8 (c = 0.1, H2O)]. 1H NMR
(500 MHz, D2O): δ = 4.97 (d, J = 3 Hz, 0.5 H, 1D-Hα), 4.65 (d, J
= 8.5 Hz, 1 H, 1-Hβ), 4.51 (d, J = 8.5 Hz, 0.5 H, 1D-Hβ), 4.44–
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Eur. J. Org. Chem. 2013, 7382–7390
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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7389

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Received: July 9, 2013
Published Online: September 19, 2013
7390
www.eurjoc.org
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 7382–7390