Synthesis of lipo-chitooligosaccharide analogues and their interaction with LYR3, a high affinity binding protein for Nod factors and Myc-LCOs

Article abstract of DOI:10.1039/c7ob01201b

Lipo-chitotetrasaccharide analogues where one central GlcNAc residue was replaced by a triazole unit have been synthesized from a derivative obtained by chitin depolymerization and a functionalized N-acetyl-glucosamine via the copper-catalyzed azide-alkyn

Full text of DOI:10.1039/c7ob01201b

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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ARTICLE
Synthesis of lipo-chitooligosaccharide analogues and evaluation of
their ability to interact with the LysM receptor-like kinase LYR3, a
high affinity binding protein for Nod factors and Myc-LCOs
Received 00th January 20xx,
Accepted 00th January 20xx
a
a,b
c
c
a
Nathan Berthelot, Antoine Brossay, Virginie Gasciolli, Jean-Jacques Bono, Aurélie Baron,
a,b
a
a,d
a,b
DOI: 10.1039/x0xx00000x
Jean-Marie Beau, Dominique Urban, François-Didier Boyer, * Boris Vauzeilles *
www.rsc.org/
Lipo-chitotetrasaccharide analogues where one central GlcNAc residue was replaced by a triazole unit have been
synthesized from a derivative obtained by chitin depolymerization and a functionalized N-acetyl-glucosamine via the
Copper-catalyzed Azide-Alkyne Cycloaddition. Their evaluation in a binding assay using LYR3, a putative lipo-
chitooligosaccharide receptor in Medicago truncatula, shows a complete loss of binding.
produces chemical mediators. The signals produced by the
2
host plant were identified respectively as strigolactones and
Introduction
3, 4
flavonoids
metabolites stimulate the synthesis and secretion of lipo-
chitooligosaccharide (LCO) symbiotic signals called
for AM and RL endosymbiosis. These plant
The arbuscular mycorrhizal (AM) and the rhizobia-legume (RL)
symbioses are the two major ecologically important symbiotic
1
relationships between higher plants and micro-organisms.
5
6
respectively, Nod factors and Myc LCOs for RL and AM
symbioses. These diffusible factors perceived by plant hosts
are necessary for the establishment of a successful association.
They are present in the medium as mixtures in very small
quantities. The Myc-LCOs share a common backbone of three
The AM symbiosis is formed between fungi (Glomeromycota
group) and a wide range of plants, including angiosperms,
gymnosperms, pteridophytes, and some bryophytes and
liverworts. Inside plant root cells, these AM fungi exchange
rare or poorly soluble mineral nutrients such as phosphorus,
copper and zinc from the soil to the plant. The plant benefits
from its fungal partner by improved nutrition, water uptake
and disease resistance. The RL symbiosis is essentially
restricted to members of the Fabaceae (legume family) and a
variety of phylogenetically diverse gram-negative bacteria,
termed rhizobia. The RL symbiosis leads to the development of
root nodules in which the bacteria fix dinitrogen to the benefit
of host plants. In these two symbiotic relationships, the plant
supplies the microorganism with energy in the form of
photosynthetic products. Both of these symbioses could be
improved in low-input sustainable agriculture. The AM and RL
endosymbioses are initiated with an exchange of chemical
signals between the root and the microsymbiont, priming both
partners for the subsequent association. Each partner
or four β-1,4-linked N-acetylglucosamine units, N-acylated at
the non-reducing end unit by a common fatty acid group such
as stearic (C18:0), oleic (C18:1) and palmitic (C16:0) acids. The
only observed O-substitution was the optional O-sulfation at
6
the C-6 position of the reducing end unit of the molecule. The
LCOs 1, 2, 1S, 2S (Figure 1) which were identified as Myc-LCOs
have simpler structures than those of the Nod factors which
harbor different chemical substitutions on the oligosaccharidic
moiety, depending on the rhizobia and the importantly
7
impacting host range.
LCOs are biologically active on plant roots at very low
concentrations indicating that they are perceived by specific
receptors. Nod factors are recognized by receptor-like kinases
that contain sugar binding lysin-motif (LysM) in their
8
-15
extracellular domains, called LysM-RLKs.
intervene in the establishment of the AM symbiosis.
[LysM domain-containing receptor-like kinase]-related 3) is
LysM-RLKs also
1
6-18
LYR3
(
the only protein identified so far in Medicago truncatula and in
other legumes as a LysM-RLK exhibiting a high affinity (in the
1
nanomolar range) for both Nod factors and Myc-LCOs.
3, 15
However its biological function remains unknown. Binding
studies have shown that the affinity of LCOs for LYR3 increases
1
3
as the number of GlcNAc units increases and, together with
molecular docking and site directed mutagenesis approaches,
established the critical role played by the LysM3 domain in
1
5
LCO binding.
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involved the selective differentiation of the amino groups from
The synthesis of natural LCOs and analogues demonstrated the a disaccharide readily obtained from chitin by chemoselective
2
importance of the fatty acid chain for the biological activities N-transacylation of a peracetylated chitobiose, subsequent
9
1
2, 19-23
29
and possible variations without loss of bioactivity.
Modifications of the chitin backbone were rarely described stereoselective synthesis of a glycosyl azide. The N-acetyl
and often the effect on biological activity and/or binding has monosaccharide was prepared from the -benzyl glycoside
-glucosamine by standard sequence of
analogues with a modified chitin backbone in order to obtain protecting groups manipulations. The tert-butyldiphenylsilyl
protection using the tert-butyloxycarbonyl (Boc) group, and a
7
β
2
4-26
not yet been reported.
Therefore we synthesized new LCO of N-acetyl-
D
a
better insights into structure-activity relationships.
(TBDPS) protecting group at the C-6 position was chosen for its
being sufficiently stable over several steps of the synthesis as
2
8
Our synthetic work was based on novel tools in demonstrated in our recent LCO synthesis.
7-
oligosaccharide chemistry and the lessons of past syntheses:
2
3
2
(
i) selection of a suitable method to produce cleanly and
efficiently the difficult 1,4 β-glycosidic bonds between two 2-
acetamido-2-deoxy- -glucopyranosyl units, (ii) choice of a
D
strategy that would provide the oligosaccharide analogue with
the highest efficacy, (iii) selective differentiation of identical
functional groups, e.g., primary or secondary hydroxyl and
amino groups present in the chitin fragment.
The LCO analogues 3, 3S, 4 and 4S, possessing a triazole ring
instead of a GlcNAc unit, corresponding to Myc-LCOs 1, 1S, 2,
2S
(Figure 1) were targeted. Previous examples reporting
oligosaccharidic analogues with a triazole unit led in numerous
3
3-35
cases to similar activities to natural structures.
analogues possessing triazole ring instead of
monosaccharide unit were designed in accordance to the
In addition,
a
a
efficiency
of
the
Copper-catalysed
(CuAAC) recently
Azide-Alkyne
3
6,
37
Cycloaddition
reviewed
in
3
8, 39
glycochemistry.
Scheme 1 Retrosynthetic analysis for the synthesis of LCO analogues.
The synthesis of the monosaccharide
formation of 10 by an efficient -selective glycosylation (93%)
of benzyl alcohol with the commercially available or easily
7 started with the
β
4
0
synthesized in bulk quantity, peracetylated
β
-GlcNAc donor
, in the presence of Fe(III) triflate (Scheme 2). Removal of
the O-acetyl groups of 10 subsequent 4,6-O-
isopropylidenation and benzylation at O-3 furnished the
4
1
9
,
4
2
derivative 13
, soluble in the usual organic solvents and easily
Figure 1 Natural LCOs (
1
,
1S,
2
,
2S) and the targeted LCO analogues (
3
,
3S
,
4
,
4S).
purified by chromatography on silica gel in an 87% overall
yield. The 4,6-O-benzylidene derivatives already reported,
analogous to 12 and 13 suffered from a lower solubility in
Results and discussion
4
3
organic solvents. The cleavage of the 4,6-O-isopropylidene
group in acidic medium at room temperature and O-6 selective
protection with the TBDPS group provided 4-alcohol 15 in a
high yield (82% yield over 2 steps). The propargylation of O-4
under optimized conditions furnished the expected compound
The targeted analogues 3, 3S, 4 and 4S were obtained by N-
selective acylation at the non-reducing end of the deprotected
CO analogue derived from
the preparation of the tetrasaccharidic analogue
reaction from one preformed disaccharidic moiety
functionalized monosaccharide (Scheme 1). Our strategy
avoided glycosylation of a glycosyl acceptor to form 1,4 β-
glycosidic bonds. Instead, the preparation of the disaccharide
5
. We chose a block synthesis for
by a CuAAC
and
5
6
8
(66%) with minor amounts of the 4,6-di-O-propargylated
7
derivative. The presence of this by-product resulted from the
deprotection of the TBDPS group in the propargylation
6
2
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conditions. The synthesis of the monosaccharide
completed by the deprotection of the TBDPS group with NH
to produce the derivative , from peracetylated -GlcNAc , in salt (30 mol%), the tetrasaccharide analogue
steps with a 46% overall yield. 96% yield after purification on silica gel. From compound 5,
7
was materials
6
and
7. After 2 hours and in the presence of
4
F
substantial amounts of sodium ascorbate (60 mol%) and Cu(II)
was obtained in
7
β
9
5
8
deacetylation, de-O-benzylation and removal of the tert-
butyloxycarbonyl group by standard conditions provided the
CO analogue 19 possessing the free amino group at the non-
reducing end. Alternatively, the 6-OH tetrasaccharide analogue
OAc
O
OR
O
OBn
NHAc
0: R = Ac
1: R = H
AcO
AcO
a)
3%
RO
RO
OAc
NHAc
9
9
1
3
5 was sulfated by the SO ꢀtriethylamine complex and the
b)
1
deprotection sequence furnished the sulfated chito-
tetrasaccharide analogue 20 in an 87% overall yield. It is
interesting to note that the removal of the tert-
butyloxycarbonyl group was performed without cleavage of
the sulfate group in a mixture of trifluoroacetic acid and water
OR1
e)
8%
c)
O
O
2
O
O
R O
BnO
OBn
8
OBn
NHAc
4: R1 = R2 = H
15: R1 = TBDPS, R2 = H
: R1 = TBDPS, R2 = propargyl
: R1 = H, R2 = propargyl
RO
NHAc
12: R = H
13: R = Bn
4
6
1
at 0 °C. Final selective acylation of 19 and 20 respectively
d)
7%
2 steps)
f) 94%
g) 66%
2
with the N-hydroxysuccinimide ester of palmitic acid 21 or
9
8
(
29
oleic acid 22 afforded the targeted LCO analogues 3, 3S, 4, 4S
8
§
h) 92%
7
in yields ranging from 67% to quantitative with good purities
without purification by chromatography.
6
+
7
Scheme 2 Synthesis of the 4-O-propargylated monosaccharide
Fe(OTf) , CH Cl , r.t. ; (b) CH ONa, CH OH, r.t. ; (c) 2,2-dimethoxypropane, PTSA,
DMF, 40 °C, 250 mbar ; (d) BnBr, BaO, Ba(OH) 8H O, DMF, r.t. ; (e) 50%
trifluoroacetic acid in H O, CH Cl , r.t. ; (f) TBDPSCl, pyridine, CH Cl , r.t. ; (g)
7. (a) BnOH,
3
2
2
3
3
2
ꢀ
2
a) 96%
2
2
2
2
2
propargyl bromide, NaH, DMF, r.t. ; (h) NH
§
The oxazoline 16 was prepared as recently reported from
4 3
F, CH OH, 70 °C.
OAc
O
NHAc
N
NHAc
OBn
2
9
AcO
O
N
BnO
O
chitin via the peracetylated
α
glycosyl donor 16 was coupled with TMSN
-chitobiose (Scheme 3). This
in the presence of
AcO
AcO
N
O
O
3
NHBoc
OR1
4
4
OAc
TBAF via an hypervalent azidosilicate intermediate, providing
the glycosyl azide 17 with the amino groups differentiated by
orthogonal protections (Boc and TFA) in 82% yield. The same
b)
7%
: R1 = H
18: R1 = SO -, Na+
3
5
8
c,d,e)
6%
c,e,d)
4
5
reaction with promoters as CuCl
29-38%). The removing of N- and O-acetate groups and TFA
group with LiOH and reacetylation with Ac O furnished the
in a 78% overall yield. The
was accomplished from 16 in 3
2
or CuBr
2
gave lower yields
7
quantitative
(
2
OH
O
peracetylated glycosyl azide
synthesis of the disaccharide
steps and a 64% overall yield.
6
NHAc
O
N
NHAc
OH
O
N
HO
O
HO
O
HO
HO
N
6
NH3+
TFA-
OR1
OH
F
3
C
1
9: R1 = H
20: R1 = SO -Na+
OAc
O
O
O
N
3
O
AcO
O
AcO
AcO
N
O
R2
O
N
f)
Ac
OAc
Boc
O
2
2
1: R2 = C15
2: R2 = C17
H
H
31
33
16
a) 82%
3
: R1 = H ; R2 = C16:0: quantitative
3S: R1 = SO -, Na+ ; R2 = C16:0: 95%
OAc
NHR2
3
O
AcO
O
4: R
4S: R
1
= H ; R
= SO3
2
= C18:1(9Z): 67%
AcO
AcO
N
3
O
1
-
, Na
+
; R
2
= C18:1 (9Z): 67%
R1
N
OAc
Boc
1
6
7: R1 = Ac, R2 = TFA
: R1 = H, R2 = Ac
b,c)
7
Scheme 4 Synthesis of the LCO analogues (
and disaccharide . (a) CuSO 5H
3
,
3S
,
4
,
4S) from the monosaccharide
O (30 mol%), sodium ascorbate (60 mol%),
7
6
4
ꢀ
2
8%
CH Cl /t-BuOH/H O, 14 h, r.t. ; (b) SO3ꢀNEt3 (5 equiv.), DMF, 15 h, r.t. ; (c)
2
2
2
CH
3
OH/H
2
O/NEt
3
, 70 °C, 18 h ; (d) H
2
, Pd/C (10%), EtOAc/EtOH/H
2
O, r.t., 15 h ; (e)
O (95:5), 0 °C, 2 h ; (f) 21 or 22 (1.2 equiv.), Et N (1.8
trifluoroacetic acid /H
equiv.), DMSO, 60 °C, 24 h. TFA = trifluoroacetyl.
2
3
Scheme 3 Synthesis of the disaccharide
H
3
6. (a) TMSN , TBAF, THF, 65 °C ; (b) LiOH,
2
O/CH OH, r.t. ; (c) Ac O, pyridine, r.t. TFA = trifluoroacetyl.
2
3
Coupling of the glycosyl azide
monosaccharide was accomplished by CuAAC (Scheme 4).
This reaction was performed in mixture of
6 and the 4-O-propargyl-
The ability of LCO analogues 3, 3S, 4 and 4S to bind LYR3 was
35
determined by a radioligand binding assay using [ S]-NodRt
7
a
factor (23*) as described in the experimental section. As
dichloromethane/tert-butanol/water, to solubilize the starting
shown in Figure 2, none of the analogues, used at a
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concentration of 2 µM, were able to compete the binding of
Materials and methods. All reagents and chemicals were
3
the [ S]-NodRt in comparison to the reference LCO (23). purchased from commercial suppliers and used without
5
Previous work established that high affinity binding to LYR3 further purification. Reactions were generally run under inert
1
3
required a LCO with at least three sugar residues.
atmosphere (argon), by using standard techniques for
Furthermore molecular modeling and docking proposed that manipulating air-sensitive compounds. All the glassware was
three sugar units, starting from the reducing end, were stored in the oven before using. Chromatography was
1
m silica. H NMR
engaged in the predicted LCO binding site located in the LysM3 performed using packed cartridge or 40-63
5
ꢀ
domain, the reducing end pointing to the solvent. Therefore, spectra were recorded on a Bruker 300 or 500 MHz
1
13
the substitution of the third sugar unit by the triazole ring instrument. C NMR spectra were recorded on the same
should not allow accommodation of the chitin backbone, instruments at 75 or 125 MHz. Chemical shifts δ are expressed
resulting in a dramatic decrease of binding and an affinity in parts per million, using the residual protonated solvent as an
1
probably in the micromolar range as was observed for a LCO internal standard. For H NMR spectra, data are reported as
1
3
with 2 sugar units.
follows: chemical shift, multiplicity (s = singlet, d = doublet, t =
triplet, q = quadruplet, m = multiplet, brs = broad singulet, dt =
doublet of triplets), coupling constant (in Hz) and integration.
1
1
1
13
Interpretations were obtained using H- H COSY, H- C HSQC
and HMBC experiments. High-resolution mass spectra were
obtained with a Waters Acquity UPLC (by direct injection or
with a BEH C18 2.1 × 50 mm, 1.7 μm column) combined with a
Diode Array Detector (DAD) and a Waters LCT Premier XE mass
instrument (electrospray ionization with a time-of-flight (ToF)
analyzer). IR spectra were recorded on a PerkinElmer
Spectrum 100 FT-IR spectrometer. Melting points were
determined using a Büchi B-540 apparatus. Optical rotations
were determined using an Anton Paar MCP 300 polarimeter
with a 1 dm-long cell and data are reported as follows:
2
2
1
5
0
.E+04
.E+04
.E+04
.E+03
.E+00
temperature
-1
2
[
α]
and solvent.
The preparation and characterization details of
intermediate compounds 10-14 16 21-22 are included within
D
(in 10 deg.cm /g), concentration (c in g/100 mL)
,
,
e
the ESI†.
Benzyl 2-acetamido-3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-
deoxy- -glucopyranoside (15). To a solution of diol 14 (500
mg, 1.25 mmol, 1.0 equiv.) in CH Cl /pyridine (2.5 mL, 1:1.5)
was added tert-butyldiphenylsilyl chloride (500 L, 1.92 mmol,
.5 equiv.) under an argon atmosphere. The reaction mixture
was stirred at room temperature for 18 hours. The reaction
β
-D
2
2
ꢀ
1
3
5
Figure 2 Competitive inhibition of [ S]-NodRt factor (23*) binding to LYR3 by
LCO analogues ( 3S, 4,
4S) and the reference LCO 23. Membrane extracts were was quenched with CH OH (10 mL) and the mixture
3
35
3
,
incubated as described in the experimental section, with 0.5 nM [ S]-NodRt
either in the absence or presence of 2 µM unlabelled ligands. Results are means
S.E.M. dpm = disintegrations per minute.
concentrated. The residue was purified by silica gel column
chromatography (CH Cl /CH OH 100:0 to 95:5) to give silylated
product 15 (749 mg, 94%) as a white foam. R 0.65
). H NMR
: 7.73-7.21 (m, 20H, CH arom.); 5.42 (d, 1H,
±
2
2
3
f
2
0
1
(CH
2
Cl
2
/CH
3
OH 95:5). [
α
]
D
= −31.40 (c = 0.50, CHCl
3
Conclusions
(300 MHz, CDCl3)
δ
2
J
NH,2 7.9 Hz, NH); 4.83 (d, 1H, J1,2 8.2 Hz, H-1); 4.81 (d, 1H, JHCH
2
1.9 Hz, CHHPh-1); 4.80 (d, 1H, JHCH 11.7 Hz, CHHPh-3); 4.69
The synthesis of tetrameric LCO analogues possessing a
triazole ring instead of the third GlcNAc unit from the non-
reducing end was accomplished by a high yielding strategy
avoiding glycosylation of 2-acetamido-2-deoxy-glycosyl
acceptors. The synthesized analogues revealed the importance
of the third GlcNAc unit of LCOs from the non-reducing end for
binding to the LysM-RLK LYR3. The synthesis of other
analogues exhibiting additional variations of the chitin
backbone is currently in progress to decipher the respective
role of the different sugar units in high affinity binding.
1
2
2
(
d, 1H, JHCH 11.7 Hz, CHHPh-3); 4.51 (d, 1H, JHCH 11.9 Hz,
CHHPh-1); 3.95 (dd, 1H, J2,3 10.1, J3,4 8.6 Hz, H-3); 3.92 (d, 2H,
5,6 4.8 Hz, H-6); 3.74 (dd, 1H, J4,5 9.6, J3,4 8.6 Hz, H-4); 3.45 (dd,
H, J4,5 9.6, J5,6 4.8 Hz, H-5); 3.40 (ddd, 1H, J2,3 10.1, J1,2 8.2,
NH,2 7.9 Hz, H-2); 2.82 (s, 1H, OH); 1.85 (s, 3H, Ac); 1.06 (s, 9H,
J
1
J
t
13
Bu). C NMR (125 MHz, CDCl
3
)
δ
: 170.6 (COCH
3
); 138.8-128.0
Ph-3);
Ph-1); 65.1 (C-6); 57.1 (C-2); 27.0 (C(CH );
). HRMS (ESI ): calculated for
(C arom.); 99.1 (C-1); 80.8 (C-3); 74.8 (C-5); 74.4 (CH
2
7
2
C
3.6 (C-4); 70.8 (CH
2
3 3
)
+
3.8 (COCH ); 19.5 (C(CH )
3
3 3
+
+
-1
38 6
H46NO Si 640.3089 [M+H ]; found 640.3099. IR: υ (cm )
Experimental section
=3400-3100, 2931, 2858, 1655, 1428, 1113, 1065, 739, 701.
4
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Benzyl 2-acetamido-3-O-benzyl-6-O-tert-butyldiphenylsilyl-2- (CH
deoxy-4-O-propargyl- -glucopyranoside (8). To a solution of (COCH
alcohol 15 (400 mg, 0.63 mmol, 1.0 equiv.) and propargyl [M+H ]; found 440.2094. IR:
bromide (91 L, 80% in toluene, 0.81 mmol, 1.3 equiv.) in DMF 1121, 1090, 1027, 742, 730, 696.
3 mL) was added sodium hydride (28 mg, 60% oil dispersion, [3,4,6-tri-O-Acetyl-2-(N-acetyl-tert-butyloxycarbonylamino)-
.69 mmol, 1.1 equiv.) under argon atmosphere. The reaction 2-deoxy-
-glucopyranosyl]-(1→4)-3,6-di-O-acetyl-2-deoxy-
mixture was stirred at room temperature for 20 min. The 2-trifluoroacetamido- -glucopyranosyl azide (17). To a
reaction was quenched with CH OH (10 mL) and the mixture solution of oxazoline 16 (325 mg, 0.42 mmol, 1.0 equiv.) in dry
concentrated. The residue was purified by silica gel column THF (3.6 mL) was added trimethylsilyl azide (78 L, 0.59 mmol,
chromatography (CH Cl /methyl tert-butyl ether 100:0 to 93:7) 1.4 equiv.) and tetrabutylammonium fluoride (590 L, 1 mol/L
to give product (424 mg, 66%) as a white amorphous solid. R in THF, 0.59 mmol, 1.4 equiv.). The reaction mixture was
.20 (CH Cl /CH ). H stirred at 65°C, under argon atmosphere, for 15 hours. The
NMR (300 MHz, CDCl : 7.78-7.65, 7.47-7.20 (m, 20H, CH mixture was concentrated and the residue was purified by
arom.); 5.38 (d, 1H, JNH,2 7.7 Hz, NH); 4.85 (d, 1H, JHCH 11.7 Hz, silica gel column chromatography (cyclohexane/EtOAc 6:4) to
2
Ph-1); 61.2 (C-6); 59.3 (CH
2
CCH-4); 55.5 (C-2); 22.1
+
+
β
-D
3
). HRMS (ESI ): calculated for C25
H30NO
6
440.2068
+
-1
υ
(cm ) = 3290, 1647, 1555, 1374,
ꢀ
(
0
β-D
β
-D
3
ꢀ
2
2
ꢀ
8
f
2
D
0
1
0
2
2
3
OH 99:1). [
α
]
= +1.50 (c = 1.00, CHCl
3
3
) δ
2
2
CHHPh-1); 4.85 (d, 1H, JHCH 11.4 Hz, CHHPh-3); 4.74 (d, 1H, J1,2 give azide 17 (282 mg, 82%) as a yellow foam. R
20
f
0.50
2
.8 Hz, H-1); 4.64 (d, 1H, JHCH 11.4 Hz, CHHPh-3); 4.52 (d, 1H, (C
1
7
6
H
12/EtOAc 1:1). [
α
]
D
=
−51.20 (c = 1.00, CHCl ). H NMR
3
2
2
HCH 11.7 Hz, CHHPh-1); 4.43 (dd, 1H, JHCH 15.3, JHCCCH 2.4 Hz, (500 MHz, CD CN, 70°C)
4
A
A
A
: 7.48 (d, 1H, JNH ,2 9.2 Hz, NH );
J
3
δ
2
4
CHHCCH-4); 4.37 (dd, 1H, JHCH 15.3, JHCCCH 2.4 Hz, CHHCCH-4); 5.64 (dd, 1H, J
B
B
B
B
,4 8.9 Hz, H-3 ); 5.31 (d, 1H, J
B
B B
,2
2
,3 10.4, J
3
1
2
B
.00 (dd, 1H, J6a,6b 11.3, J5,6a 2.2 Hz, H-6a); 3.96 (dd, 1H, J2,3 7.7 Hz, H-1 ); 5.17 (dd, 1H, J
A
A
A
A
A
,4 9.0 Hz, H-3 ); 4.95
4
9
6
9
2
,3 10.4, J
3
2
.9, J3,4 8.4 Hz, H-3); 3.93 (dd, 1H, J6a,6b 11.3, J5,6b 4.0 Hz, H- (dd, 1H, J
B
B
B
B
,4 8.9 Hz, H-4 ); 4.93 (d, 1H, J
A
B
A A
4
,5 10.1, J
3
2
1
,2 9.5 Hz,
A
A
b); 3.70 (dd, 1H, J4,5 9.2, J3,4 8.4 Hz, H-4); 3.52 (ddd, 1H, J2,3 H-1 ); 4.44 (dd, 1H, J6a ,6b 12.2, J
A
A
A
,6a 2.0 Hz, H-6a ); 4.32 (dd,
5
2
B
B
B
B
B
2
B
B
.9, J1,2 7.8, JNH,2 7.7 Hz, H-2); 3.39 (ddd, 1H, J4,5 9.2, J5,6b 4.0, 1H, J6a ,6b 12.3, J
5
,6a 4.7 Hz, H-6a ); 4.07 (dd, 1H, J6a ,6b
4
5,6a 2.2 Hz, H-5); 2.35 (t, 1H, JHCCCH 2.4 Hz, CHHCCH-4); 1.85 (s, 12.3, J
B
B
B
2
A
A
A
A
J
5
,6b 2.7 Hz, H-6b ); 4.00 (dd, 1H, J6a ,6b 12.2, J
5
,6b 5.3
t 13
H, Ac); 1.05 (s, 9H, Bu). C NMR (125 MHz, CDCl
A
: 170.5 Hz, H-6b ); 3.92 (dd, 1H, J
A
A
A A
A
,4 9.0 Hz, H-4 ); 3.82
A
,2 9.5, JNH ,2 9.2 Hz, H-2 ); 3.79 (ddd,
3
3
)
δ
); 138.5-127.9 (C arom.); 99.0 (C-1); 80.5 (C-3); 80.0 (ddd, 1H, J
4
,5 9.8, J
3
A
A
A
A
A
A
(
(
(
(
COCH
3
2
,3 10.4, J
1
B
B
B
B
B
B
B
CH
CH
2
2 4 5 5
CCH-4); 78.4 (C-4); 75.7 (C-5); 74.9 (CH Ph-3); 74.8 1H, J ,5 10.1, J ,6a 4.7, J ,6b 2.7 Hz, H-5 ); 3.75 (ddd, 1H,
A
A
A
A
A
A
A
2
CCH-4); 70.4 (CH
2
Ph-1); 63.1 (C-6); 59.8 (CH
); 19.6 (C(CH ). HRMS (ESI ): Ac); 1.56 (s, 9H, Boc). C NMR (125 MHz, CD
Si 678.3245 [M+H ]; found 678.3265. 175.0 (COCH NAc); 171.6-170.8 (5 COCH OAc); 158.7 (q, JC,F
(cm ) = 3287, 2931, 2858, 1654, 1563, 1113, 1074, 1056, 37.6 Hz, COCF ); 100.6 (C-1 ); 88.6 (C-1 ); 86.2 (C(CH ); 76.6
(C-5 ); 76.0 (C-4 ); 73.4 (C-3 ); 73.1 (C-5 ); 71.8 (C-3 ); 71.3 (C-
2
CCH-4); 56.8
4 5 5
J ,5 9.8, J ,6b 5.3, J ,6a 2.0 Hz, H-5 ); 2.33-1.93 (6 s, 18H, 6
+
13
C-2); 27.0 (C(CH
3
)
3
); 23.8 (COCH
3
3
)
3
3
CN, 70°C) δ:
2
+
+
calculated for C41
H
48NO
6
3
3
-1
B
A
IR:
39, 700.
Benzyl 2-acetamido-3-O-benzyl-2-deoxy-4-O-propargyl-
glucopyranoside (7). To silyl derivative (80 mg, 0.12 mmol, 27.6 (COCH
.0 equiv.) was added NH F solution (1.2 mL, 0.5 mol/L in calculated for C31
CH OH, 0.60 mmol, 5.0 equiv.). The reaction mixture was 836.2428. IR:
stirred at 60°C for 4 hours. The mixture was then concentrated [3,4,6-tri-O-Acetyl-2-(tert-butyloxycarbonylamino)-2-deoxy-
under vacuum and the residue was recrystallized from
-glucopyranosyl]-(1→4)-2-acetamido-3,6-di-O-acetyl-2-
CH OH/Et O to give a first crop of alcohol product (39 mg, deoxy- -glucopyranosyl azide (6). To a solution of azide 18
4%) as white needles. The filtrate was concentrated and the (50 mg, 0.061 mmol, 1 equiv.) in CH OH (1.2 mL) was added
residue was purified by silica gel column chromatography LiOH (920 L, 2 mol/L in H O, 1.8 mmol, 30 equiv.). The
OH 99:1 then 98:2) to afford a second crop of reaction mixture was stirred at room temperature for 22
υ
3
3 3
)
A
A
A
B
B
7
B
A
B
A
β
-D
-
3 3
4 ); 63.5, 63.3 (C-6 and C-6 ); 56.0 (C-2 ); 28.6 (3C, C(CH ) );
+
8
3 3
NAc); 21,3-21.0 (5 COCH OAc). HRMS (ESI ):
+
+
1
4
H
42
F
3
N
5
NaO17 836.2420 [M+Na ]; found
-
1
3
υ (cm ) = 2120, 1747, 1371, 1227, 1155, 1045.
β
-D
3
2
7
β-D
7
3
ꢀ
2
(
CH
product
CH Cl /CH
:1). m.p. 212°C (CH
CDCl : 7.37-7.19 (m, 10H, CH arom.); 4.85 (d, 1H, JHCH 12.2 (580
Hz, CHHPh-1); 4.81 (d, 1H, JHCH 11.2 Hz, CHHPh-3); 4.64 (d, 1H, more and then concentrated. The residue was dissolved in
2
Cl
2
/CH
3
7
(12 mg, 18%) as a white amorphous solid. R
f
0.22 hours, and concentrated to give the deacetylated crude
/CH OH product in quantitative yield. A solution of this residue in
O). H NMR (300 MHz, CD OD + acetic anhydride (580 L, 6.1 mmol, 100 equiv.) and pyridine
L) was then stirred at room temperature for 19 hours
2
D
0
(
2
2
3
OH 96:4). [
α
]
=
−
7.80 (c = 1.00, CHCl
3
3
1
1
3
OH/Et
2
3
ꢀ
2
ε
3
)
δ
ꢀ
2
2
2
J
HCH 11.2 Hz, CHHPh-3); 4.58 (d, 1H, JHCH 12.2 Hz, CHHPh-1); toluene, concentrated three times to remove pyridine traces,
2
.49 (d, 1H, J1,2 8.4 Hz, H-1); 4.38 (dd, 1H, JHCH 15.4, JHCCCH 2.3 and purified by silica gel column chromatography
4
4
2
4
Hz, CHHCCH-4); 4.32 (dd, 1H,
2
CHHCCH-4); 3.93 (dd, 1H, J6a,6b 12.1, J5,6a 2.1 Hz, H-6a); 3.82 as a white amorphous solid. R
J
HCH 15.4,
J
HCCCH 2.3 Hz, (CH
2
Cl
2
/CH
3
OH 98:2 then 97:3) to give product
0.22 (CH Cl /EtOAc 1:1). [
: 5.90 (d,
6 (35 mg, 78%)
20
f
2
2
α]
D
2
1
(
dd, 1H, J2,3 10.2, J1,2 8.4 Hz, H-2); 3.72 (dd, 1H, J6a,6b 11.3, J5,6b
=
−
39.50 (c = 1.00, CHCl
3
). H NMR (300 MHz, CDCl
3
)
δ
A
A
A
B
B
B B
4
1
2
.0 Hz, H-6b); 3.64 (dd, 1H, J2,3 10.2, J3,4 8.7 Hz, H-3); 3.47 (dd, 1H, JNH ,2 8.1 Hz, NH ); 5.22 (dd, 1H, J
B
2
,3 10.3, J
3
,4 9.4 Hz, H-
A
A
A
A
,4 8.5 Hz, H-3 ); 4.99 (dd, 1H,
A
H, J4,5 9.8, J3,4 8.7 Hz, H-4); 3.31 (ddd, 1H, J4,5 9.8, J5,6b 5.3, J5,6a 3 ); 5.09 (dd, 1H, J
2
,3 9.4, J
3
4
.1 Hz, H-5); 2.72 (t, 1H, JHCCCH 2.3 Hz, CHHCCH-4); 1.85 (s, 3H,
B
B
B
B
B
B
B
B
,4 9.4 Hz, H-4 ); 4.89 (d, 1H, JNH ,2 8.7 Hz, NH );
J
4
,5 9.9, J
3
1
3
B
B
,2 8.6 Hz, H-1 ); 4.55 (d, 1H, J
B
A
A
,2 8.8 Hz, H-1 );
A
Ac). C NMR (125 MHz, CD
3
OD +
ε
CDCl
3
)
δ
: 171.9 (COCH
3
); 4.59 (d, 1H, J
1
1
2
A
A
CCH-4); 4.45 (dd, 1H, J6a ,6b 12.2, J
A
A
A
,6a 2.2 Hz, H-6a ); 4.33 (dd, 1H,
1
7
38.3-127.4 (C arom.); 100.0 (C-1); 82.4 (C-3); 79.5 (CH
2
5
2
B
B
B
B
B
2
A
A
7.6 (C-4); 75.5 (C-5); 74.8, 74.7 (CH
2
CCH-4 and CH
2
Ph-3); 70.4
J
6a ,6b 12.6, J
5
,6a 4.5 Hz, H-6a ); 4.28 (dd, 1H, J6a ,6b 12.2,
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DOI: 10.1039/C7OB01201B
ARTICLE
Journal Name
A
A
A
,6b 4.7 Hz, H-6b ); 4.01 (ddd, 1H, J
A
A
A
A
A
A
C
C
J
5
2
,3 9.4, J
1
,2 8.4, JNH ,2
2 2
72.5 (C-5 ); 71.4 (CH Ph-1); 69.5 (C-4 ); 66.0 (CH triaz); 63.1 (C-
A
2
B
B
.1 Hz, H-2 ); 4.01 (dd, 1H, J6a ,6b 12.6, J
B
B
B
B
,6b 2.0 Hz, H-6b ); 6 ); 62.8 (C-6 ); 61.9 (C-6 ); 56.7 (C-2 ); 56.5 (C-2 ); 54.2 (C-2 );
C
A
C
A
B
8
3
8
5
A
A
A
A
A
,4 8.5 Hz, H-4 ); 3.70 (ddd, 1H, J
A
A
+
). HRMS (ESI ): calculated for
+
22 1157.4772 [M+H ]; found 1157.4789. IR:
.80 (dd, 1H, J
4
,5 8.7, J
3
4
,5
28.7 (C(CH
3
)
3
); 23.3-20.8 (7 COCH
3
A
A
A
A
,6a 2.2 Hz, H-5 ); 3.62 (ddd, 1H, J
A
B
B
+
-1
(cm )
.7, J
5
,6b 4.7, J
5
4
,5 9.9,
C
54
H
73
N
6
O
υ
B
B
B
B
B
B
B
B B
J
5
,6a 4.5, J
5
,6b 2.0 Hz, H-5 ); 3.43 (ddd, 1H, J
2
,3 10.3, JNH ,2
= 3341, 2925, 1746, 1684, 1660, 1230, 1111, 1071, 1037, 698.
,2 8.6 Hz, H-2 ); 2.12-1.95 (6 s, 18H, 6 Ac); 1.38 (s, 9H, Benzyl 2-acetamido-3-O-benzyl-2-deoxy-6-O-sodium
); sulfonato-4-O-[{1’-([2-(tert-butyloxycarbonylamino)-3,4,6-tri-
); O-acetyl-2-deoxy-
-glucopyranosyl]-(1→4)-2-acetamido-
-glucopyranosyl)-1H-[1’,2’,3’]-
-glucopyranoside (18). To a solution
(22.0 mg, 0.019 mmol, 1 equiv.) in dry DMF (380
L) was added SO .NEt (17.2 mg, 0.095 mmol, 5 equiv.) under
B
B
B
8
.7, J
1
1
3
Boc). C NMR (125 MHz, CDCl
3
)
δ: 171.3-169.6 (6 COCH
3
B
A
1
7
6
2
55.4 (COC(CH
3
A
)
3
); 101.1 (C-1 ); 88.7 (C-1 ); 80.6 (C(CH
3
)
3
B
β-D
A
A
B
5.2, 75.1 (C-4 and C-5 ); 72.4 (C-3 ); 72.1 (2C, C-3 and C-5 ); 3,6-di-O-acetyl-2-deoxy-
β
-D
B
A
B
B
A
8.6 (C-4 ); 62.2, 62.1 (C-6 and C-6 ); 56.3 (C-2 ); 53.3 (C-2 ); triazol-4’-yl}-methyl]-β-D
8.4 (3C, C(CH ) ); 23,5-20.7 (6 COCH ). HRMS (ESI ): calculated of alcohol 5
3 3 3
+
+
+
-1
for C29
H
44
N
5
O
16 718.2778 [M+H ]; found 718.2784. IR:
υ
(cm )
ꢀ
3
3
=
3356, 2980, 2118, 1746, 1529, 1369, 1230, 1165, 1039.
an argon atmosphere. The reaction mixture was stirred at
room temperature for 15 hours, concentrated and the residue
was purified by silica gel column chromatography
OAc
NHAc
O
N
5'
NHAc
B
3
6
N
4'
2
O
AcO
O
BnO
O
AcO
AcO
N
5
OBn
(CH
2 2 3
Cl /CH OH 95:5 then 90:10). The residue was eluted
1
O
+
through ion exchange Dowex® Na resin to obtain sulfate 18
C
NHBoc
4
A
OAc
OH
(
(
22.0 mg, 87%) as a white amorphous solid. R
f
0.17
/CH OH
δ: 8.15 (s,
2
0
CH
2
Cl
2
/CH
3
OH 9:1). [
α
]
D
=
−
24.60 (c = 0.50, CHCl
CD CN + D2O)
3
3
1
Figure 3 Numbering for the tetrasaccharide analogue
oligosaccharides.
5
and the following 1:1). H NMR (500 MHz, CD OD +
ε
ε
3
3
B B
1
H, H-5’); 7.41-7.28 (m, 10H, CH arom.); 6.11 (d, 1H, J
B
1
,2 9.7
B
Hz, H-1 ); 5.45 (dd, 1H, J
B
B
B
,4 8.8 Hz, H-3 ); 5.20 (dd,
B
Benzyl
butyloxycarbonylamino)-3,4,6-tri-O-acetyl-2-deoxy-
glucopyranosyl]-(1→4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-
-glucopyranosyl)-1H-[1’,2’,3’]-triazol-4’-yl}-methyl]-
glucopyranoside (5). To a mixture of alkyne (40.3 mg, 0.093
mmol, 1.0 equiv.) and azide (66.9 mg, 0.093 mmol, 1.0
(1.1 mL, 1:1) were added CuSO .5H
O, 0.028 mmol, 0.30 equiv.) and
2-acetamido-3-O-benzyl-2-deoxy-4-O-[{1’-([2-(tert-
2
,3 9.1, J
3
C
C
C
C
,4 9.4 Hz, H-3 ); 4.99 (dd, 1H, J
C
C
C
C C
,4
β-D-
1
H, J
2
,3 10.4, J
3
4
,5 10.0, J
3
C
2
.4 Hz, H-4 ); 4.93 (d, 1H, JHCH 11.7 Hz, CHHtriaz); 4.87 (d, 1H,
9
2
2
HCH 11.7 Hz, CHHtriaz); 4.87 (d, 1H, JHCH 12.3 Hz, CHHPh-1);
β
-D
β-D-
J
2
.83 (d, 1H, JHCH 11.4 Hz, CHHPh-3); 4.70 (d, 1H, J
C
C
7
4
1
,2 8.3 Hz,
C
H-1 ); 4.65 (d, 1H, JHCH 11.4 Hz, CHHPh-3); 4.60 (d, 1H, JHCH
2
2
6
t
2
B
B
2.3 Hz, CHHPh-1); 4.54 (brd, 1H, J6a ,6b 12.2 Hz, H-6a ); 4.53
B
equiv.) in BuOH/CH
280 L, 24.6 mg/mL in H
sodium ascorbate (280
2
Cl
2
4
2
O
1
A
A
A
,2 8.1 Hz, H-1 ); 4.45 (dd, 1H, J
B
B
B
B
(
ꢀ
2
(d, 1H, J
1
1
,2 9.7, J
2
,3 9.1 Hz,
B
2
C
C
H-2 ); 4.41 (dd, 1H, J6a ,6b 12.4, J
C
C
C
,6a 3.7 Hz, H-6a ); 4.26
ꢀ
2
L, 39.0 mg/mL in H O, 0.056 mmol,
5
2
A
A
A
2
B
brd, 1H, J6a ,6b 10.8 Hz, H-6a ); 4.21 (dd, 1H, J6a ,6b 12.2,
B
0.60 equiv.) under an argon atmosphere. The reaction mixture
(
B
B
B
2
A
A
A
A
was stirred at room temperature for 2 hours, and then
concentrated under vacuum and the residue was purified by
J
5 5
,6b 4.9 Hz, H-6b ); 4.17 (dd, 1H, J6a ,6b 10.8, J ,6b 3.1 Hz,
A
H-6b ); 4.08 (brd, 1H, J6a ,6b 12.4 Hz, H-6b ); 4.10-4.01 (m,
2
C
C
C
B
B
C
A A
silica gel column chromatography (CH
5:5) to afford product (104 mg, 96%) as a white amorphous
solid. R 0.32 (CH Cl /CH
CHCl /CH
numbering see Figure 3)
0H, CH arom.); 5.90 (d, 1H, J
2 2 3
Cl /CH OH 96:4 then
2
1
5
2
+
H, H-4 and H-5 ); 3.89-3.82 (m, 1H, H-5 ); 3.85 (dd, 1H, J
2 ,3
A
A
A
A
A
9
5
1
0.0, J ,2 8.1 Hz, H-2 ); 3.69-3.57 (m, 3H, H-3 , H-4 and H-
2
D
0
A
C
C
C
C
C
f
2
2
3
OH 94:6). [
OH 1:1). H NMR (500 MHz, CD
: 7.86 (s, 1H, H-5’); 7.36-7.18 (m,
α
]
=
−
35.80 (c = 0.50,
); 3.52 (dd, 1H, J
1H, 7 Ac); 1.42 (s, 9H, Bu). C NMR (125 MHz, CD
CD CN) : 173.9-172.0 (7 COCH ); 146.0 (C-4’); 139.7-128.9
1
,3 10.4, J ,2 8.3 Hz, H-2 ); 2.14-1.68 (6 s,
2
1
t
13
3
3
3
OD + CDCl ) (for the
ε
3
2
OD + εD O
3
δ
ε
3
δ
3
C
B
B
B
A
B
1
1
,2 9.9 Hz, H-1 ); 5.34 (brdd, 1H,
(C arom.); 124.8 (C-5’); 102.6 (C-1 ); 101.7 (C-1 ); 86.3 (C-1 );
A
B
C
C
C
C
C
A
B
); 79.3 (C-4 ); 77.3, 76.7 (C-4 and C-
J 8.6, J 8.1 Hz, H-3 ); 5.23 (dd, 1H, J
C
2
,3 10.0, J
C
,4 9.4 Hz, H-4 ); 4.88 (d, 1H, JHCH
3
,4 9.4 Hz, H-3 );
8
5
5
6
2
C
=
1
2
3.5 (C-3 ); 81.2 (C(CH
3
)
3
C
C
C
2
B
A
B
C
4.97 (dd, 1H, J
4
,5 9.8, J
3
2
); 76.1 (CH Ph-3); 74.5 (C-5 ); 74.1 (C-3 and C-3 ); 72.5 (C-
2
2.1 Hz, CHHtriaz); 4.84 (d, 1H, JHCH 12.2 Hz, CHHPh-1); 4.78
C
C
A
1
2 2
); 72.1 (CH Ph-1); 69.8 (C-4 ); 67.5 (C-6 ); 66.2 (CH triaz);
B
3.5 (C-6 ); 63.2 (C-6 ); 56.8 (C-2 ); 56.4 (C-2 ); 54.8 (C-2 );
2
2
d, 1H, JHCH 11.4 Hz, CHHPh-3); 4.75 (d, 1H, JHCH 12.1 Hz,
C
C
A
B
(
C
2
CHHtriaz); 4.74-4.67 (m, 1H, H-1 ); 4.64 (d, 1H, JHCH 11.4 Hz,
-
8.7 (C(CH
3 3 3
) ); 23.4-20.9 (7 COCH ).HRMS (ESI ): calculated for
2
CHHPh-3); 4.57 (d, 1H, JHCH 12.2 Hz, CHHPh-1); 4.53-4.34 (m,
-
+
-1
54
H
71
N
6
O
25S 1235.4195 [M-H ]; found 1235.4171. IR: υ (cm )
B
A
B
2
C
H, H-6a , H-1 and H-2 ); 4.40 (brd, 1H, J6a ,6b 12.2 Hz, H-
C
3
3800-3200, 2927, 1745, 1685, 1660, 1529, 1370, 1232, 1110,
068, 1045, 699.
C
a ); 4.20 (brd, 1H, J6a ,6b 12.1 Hz, H-6b ); 4.04 (brd, 1H,
B
6a ,6b 12.2 Hz, H-6b ); 4.00-3.91 (m, 2H, H-4 and H-5 ); 3.83
2
B
B
B
6
2
C
C
C
A
J
-Acetamido-2-deoxy-4-O-[{1’-([2-ammoniumyl
A
dd, 1H, J 9.9, J 8.6 Hz, H-2 ); 3.78 (brd, 1H, J6a ,6b 12.3 Hz, H-
2
A
A
(
trifluoroacetate-2-deoxy-
acetamido-2-deoxy- -glucopyranosyl)-1H-[1’,2’,3’]-triazol-
’-yl}-methyl]- -glucopyranose (19). A solution of acetate
37.6 mg, 0.033 mmol, 1.0 equiv.) in CH OH/H O/triethylamine
1.65 mL, 8:1:1) was stirred at 70 °C for 17 hours. The mixture
β-D
-glucopyranosyl]-(1→4)-2-
A
C
a ); 3.80-3.71 (m, 1H, H-5 ); 3.70-3.61 (m, 2H, H-3 and H-
A
6
β
-D
A
b ); 3.57 (dd, 1H, J 9.3, J 8.8 Hz, H-4 ); 3.44 (dd, 1H, J
A
C C
6
2
,3 10.0,
4
D
5
C
C
C
A
,2 7.9 Hz, H-2 ); 3.85-3.26 (m, 1H, H-5 ); 2.10-1.66 (5 s, 21H,
J
1
(
(
3
2
t
13
7
1
1
Ac); 1.40 (s, 9H, Bu). C NMR (125 MHz, CD
72.9-170.9 (7 COCH ); 157.1 (COC(CH ); 145.9 (C-4’); 139.5-
28.3 (C arom.); 122.9 (C-5’); 102.1 (C-1 ); 101.0 (C-1 ); 88.6
3 3
OD + εCDCl ) δ:
3
3 3
)
was then concentrated to give the crude alcohol in
quantitative yield. To a solution of this crude alcohol (48 mg,
C
A
B
A
A
B
(
C-1 ); 83.4 (C-3 ); 80.7 (C(CH
3
)
3
2
); 79.1 (C-4 ); 76.8, 76.7 (C-4
2
0.051 mmol) in EtOAc/H O/EtOH (2.5 mL, 5:3:2) was added
B
A
and C-5 ); 76.5 (C-5 ); 75.8 (CH
B
Ph-3); 73.9 (C-3 ); 73.5 (C-3 );
C
Pd/C (10% wt, 27 mg, 0.025 mmol, 0.5 equiv.). The reaction
6
| J. Name., 2012, 00, 1-3
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Page 7 of 11
Journal Name
Orga Pn l ie ca s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C7OB01201B
ARTICLE
mixture was stirred at room temperature for 15 hours under a 5.5:3:1.5) was added Pd/C (10% wt, 9 mg). The reaction
hydrogen atmosphere. The mixture was then filtered over mixture was stirred at room temperature for 15 hours under
Celite® plug and concentrated to give the crude de-O- hydrogen atmosphere. The mixture was then filtered over a
benzylated derivative in quantitative yield. A solution of this Celite® plug and concentrated. The residue was purified over a
crude derivative (39 mg, 0.051 mmol) in trifluoroacetic C18 cartridge (Phenomenex® 200 mg, H
acid/H O (2  L, 95:5) was stirred at 0°C for 5 min. The reaction alcohol 20 (6.0 mg, mixture, quantitative yield) as a white
mixture was concentrated and the residue was purified over a amorphous solid.
C18 cartridge (Phenomenex® 500 mg, H O 100%) followed by (EtOAc/EtOH/H O 5:3:2). H NMR (500 MHz, D
silica gel column chromatography (EtOAc/EtOH/H O 2:2:1) to 1H, H-5’); 5.93 (d, 1H, J
obtain compound 19 (30.3 mg, mixture, 76%) as a white 2.1 Hz, H-1
mixture 6:4 ( H NMR analysis). 0.21 Hz, CHHtriaz
: 8.31 (brs, 12.0 Hz, CHHtriaz
2
O 100%) to obtain
2
α/β
1
α
/β
mixture 6:4 ( H NMR analysis). R
f
0.25
1
2
2
2
O)
,2 9.8 Hz, H-1 ); 5.23 (d, 0.6H, J
1 ,2
δ: 8.32 (brs,
B
B
B
A A
2
1
A
2
2
α
/
β
α); 5.04, 5.03 (2 d, 1H, JHCH 12.0 Hz and JHCH 12.0
1
2
2
solid.
α
/
β
R
f
α
&
β
); 4.93, 4.92 (2 d, 1H, JHCH 12.0 Hz and JHCH
1
A
A
A
(
EtOAc/EtOH/H
2
O 4:4:2). H NMR (500 MHz, D
2
O)
δ
α
&
β
); 4.75 (d, 0.4H, J
1
,2 8.4 Hz, H-1
β
); 4.61
B
B
B
A
,2
A
C
C
C
B
B
B
B
1
H, H-5’); 5.93 (d, 1H, J
A
1
,2 9.7 Hz, H-1 ); 5.22 (d, 0.6H, J
2
1
(d, 1H, J
B
1
,2 8.2 Hz, H-1 ); 4.34 (dd, 1H, J
A
1
A
,2 9.8, J
2
,3 9.7 Hz,
2
2
A
A
A
2
5
.1 Hz, H-1 α); 5.01, 5.00 (2 d, 1H, JHCH 12.3 Hz and JHCH 12.3 H-2 ); 4.29 (dd, 1H, J6a ,6b 11.2, J ,6a 3.3 Hz, H-6a α&β);
C
C
C
,2 8.4 Hz, H-1 ); 4.89, 4.88 (2 4.26-4.21 (m, 0.4H, H-6b
A
2
A
); 4.23 (dd, 0.6H, J6a ,6b 11.2, J
A
A
A
Hz, CHHtriaz α
2
d, 1H, JHCH 12.3 Hz and JHCH 12.3 Hz, CHHtriaz
&
β
); 4.98 (d, 1H, J
2
1
β
); 4.09 (ddd, 0.6H, J
5
,6b
A
A
A
A
A
A
A
α
&
β
); 4.71 (d, 1.9 Hz, H-6b
A
α
); 4.08-3.88 (m, 7.2H, H-4 , H-3 , H-6a , H-6a , H-2
4
,5 9.9, J
5
,6a 3.3, J
5
C
,6b 1.9
A A
,2 7.7 Hz, H-1
A
B
B
B
B
,2 9.7 Hz, Hz, H-5
B
B
B
A
0
.4H, J
1
β
); 4.37 (dd, 1H, J
B
2
,3 10.3, J
1
α
,4 8.9 Hz, H-4 ); 4.05 (dd, 1H, H-5 , H-6b and H-3
α
,
B
H-2 ); 4.16 (dd, 1H, J
B
B
B
B
B
B
A
2
C
C
); 3.80 (dd, 1H, J6a ,6b 12.5, J
C
C
4
,5 9.9, J
3
α
,4 8.9 Hz, H-3 ); 4.02-3.97 (m, 1H, H-5 ); 3.99 (dd, Hz, H-6b ); 3.80-3.68 (m, 1.2H, H-2
5
,6b 5.7
B
B
B
B
B
B
C
A
A
A
J
2
,3 10.3, J
3
β
, H-3
β, H-5 β); 3.63 (dd,
2
C
C
C
C
C
2
B
B
A
A
A
A
A
1
1
3
H, J6a ,6b 12.3, J
5
,6a 1.7 Hz, H-6a ); 3.97 (dd, 1H, J6a ,6b
B
,6a 1.7 Hz, H-6a ); 3.94-3.91 (m, 1.2H, H-2
0.6H, J ,5 9.9, J ,4 8.7 Hz, H-4 α); 3.62 (dd, 0.4H, J 9.5, J 7.7
4 3
A
B
B
A
C
C
C
C
C
C
2.7, J
5
α
and H- Hz, H-4
B
β
); 3.57 (ddd, 1H, J
C
4
,5 9.4, J
5
,6b 5.7, J
5
,6a 2.2 Hz, H-
A
A
2
); 3.87 (dd, 1H, J6a ,6b 12.7, 5 ); 3.51-3.42 (m, 2H, H-3 , H-4 ); 3.77 (dd, 1H, J
B
C
C
C
C
C C
1 ,2
α
); 3.90-3.86 (m, 0.6H, H-5
B
α
,6b 4.6 Hz, H-6b ); 3.84 (dd, 0.4H, J6a ,6b 12.2, J
2
,3 9.2, J
,6a 1.9 Hz, 8.2 Hz, H-2 ); 2.08, 1.86 (2 s, 6H, 2 Ac). C NMR (125 MHz,
B
B
2
A
A
A
A
C
13
J
5
5
A
2
); 3.82 (dd, 1H, J6a ,6b 12.3, J
C
C
C
C
,6b 5.4 Hz, H-6b ); 3.79
C
H-6a
β
5
2 3
D O) δ: 174.8, 174.5, 174.2 (2 COCH Aα,Aβ,B); 144.0 (C-
2
A
A
A
A
A
C
); 3.75 (dd, 1H, 4’); 124.2 (C-5’); 102.5 (C-1 ); 95.1 (C-1
A
A
(
dd, 0.6H, J6a ,6b 12.3, J
5
,6a 2.1 Hz, H-6a
α
,4 9.1 Hz, H-3 ); 3.75-3.67 (m, 0.8H, H-2
β
); 90.9 (C-1
α); 86.1
); 77.4 (C-4 ); 76.3
C
C
C
C
C
A
B
and H- (C-1 ); 77.9 (C-4
A
B
); 77.7 (C-5 ); 77.6 (C-4
A
B
J
2
,3 10.6, J
A
); 3.72 (dd, 0.6H, J6a ,6b 12.3, J
3
β
α
); 3.68 (C-5 ); 75.5 (C-3 ); 73.9 (C-3
β
β
A
2
A
A
A
A
C
C
A
A
B
3
β
5
,6b 4.8 Hz, H-6b
α
); 3.61 (ddd, 1H, (C-3
β
); 72.7 (C-5
A
); 72.0 (C-3 ); 70.8
2
A
A
A
A
A
A
C
A
A
(
dd, 0.4H, J6a ,6b 12.2, J
5
C
,6b 5.1 Hz, H-6b α
,6a 1.7 Hz, H-5 ); 3.59-3.53 (m, 0.6H, H- 64.9, 64.8 (CH
β
); 69.6 (C-4 ); 68.5 (C-5
triaz ); 60.7 (C-6 ); 60.0 (C-6 ); 56.7 (C-2 β
α); 66.8 (C-6 α); 66.7 (C-6 β);
C
C
C
C
C
C
C
B
A
J
4
,5 9.7, J
5
,6b 5.4, J
5
C
2
α
&
β
,4 9.1 Hz, H-4 ); 3.54 (dd, 0.4H, and C-2 ); 55.2 (C-2 ); 54.1 (C-2
A
α
C
C
C
C
C
B
A
4
); 3.55 (dd, 1H, J
A
,5 9.8, J
4
,5 9.7, J
3
α
); 22.2, 21.9, 21.7 (2 COCH
-
3
A
A
A
,4 8.4 Hz, H-4
A
A
A
A
,6b
A
-
J
4
3
β
); 3.22 (dd, 1H, J
); 3.49 (ddd, 0.4H, J
C
4
,5 9.8, J
5
,A
41 6
Aα β,B). HRMS (ESI ): calculated for C25H N O18S 745.2204
A
A
A
C
C
C
,2 8.4 Hz, [M-TFA-Na ]; found 745.2195. HRMS (ESI ): calculated for
+
+
5.1, J
C
5
,6a 1.9 Hz, H-5
β
H-2 ); 2.08, 1.86 (2 s, 6H, 2 Ac). C NMR (125 MHz, D
2
,3 10.6, J
1
13
+
-
+
+
18S 747.2349 [M-TFA -Na +H ]; found 747.2360. IR:
2
O)
δ:
C
25
H
43
N
-1
6
O
2
,B); 163.6 (q, JC,F 35.4 Hz,
1
75.5, 175.2, 174.7 (2 COCH
3
A
α
); 124.6 (C-5’); 116.9 (q, JC,F 290 1051, 1012, 824.
,A
β
υ (cm ) = 3600-3000, 2934, 1644, 1555, 1375, 1227, 1065,
1
COCF3); 144.7, 144.9 (C-4’α
C
Hz, COCF3); 98.1 (C-1 ); 95.5 (C-1
&
β
A
A
B
); 86.7 (C-1 ); General procedure for acylation with fatty acids. To a solution
β
); 91.3 (C-1
B
α
); 78.0 (C-5 ); 77.1 (C-5 ); 76.5 (C-4 ); of 19 or 20 (1.0 equiv.) in dry DMSO (40-50 mM) were added
A
A
C
B
7
7
7
6
5
8.6 (C-4
α
); 78.3 (C-4
β
A
A
C
); 72.5 (C-3 ); 72.2 (C-3 ); 71.4 (C-3
B
A
5.5 (C-5
1.1 (C-5
β
); 74.6 (C-3
β
α
); triethylamine (1.8 equiv.) and then 21 or 22 (1.2 equiv.) under
A
C
α
); 70.2 (C-4 ); 65.2, 65.1 (CH triaz α&β); 61.1, 61.0, an argon atmosphere. The reaction mixture was stirred at 60°C
2
A
α
B
C
A
C
0.9, 60.8 (C-6
&
β
, C-6 and C-6 ); 57.4 (C-2
β
); 56.4 (C-2 ); overnight in a sealed tube. The mixture was then concentrated
); 22.8, 22.5, 22.3 (2 COCH ,B). and lyophilized. The lyophilized residue was resuspended in
NaO15 689.2600 [M- EtOAc/CH OH (150 mM, 9:1), triturated for 10 min, centrifuged
(cm ) = 3600-3000, 2933, (7000 rpm, 10 min) and the supernatant was removed. This
operation was repeated four times and the residue was
sulfonato-4-O-[{1’-([2- lyophilized to afford final analogues.
-glucopyranosyl]- 2-Acetamido-2-deoxy-4-O-[{1’-([2-palmitamido-2-deoxy-
-glucopyranosyl)-1H-
glucopyranosyl]-(1→4)-2-acetamido-2-deoxy-
-glucopyranose (20). glucopyranosyl)-1H-[1’,2’,3’]-triazol-4’-yl}-methyl]-
solution of acetate 18 (7.3 mg, 0.055 mmol, 1.0 equiv.) in glucopyranose (3). General procedure with 19 (9.3 mg, 0.012
CH OH/H O/triethylamine (300 L, 8:1:1) was stirred at 70°C mmol) and 21 (5.0 mg, 0.014 mmol) gave product (11.0 mg,
for 21 hours. The mixture was then concentrated and the mixture, quantitative yield) as a white lyophilisate.
B
A
α
5.9 (C-2 ); 54.8 (C-2
3
Aα,Aβ
+
+
HRMS (ESI ): calculated for C25
H
42
N
6
-1
3
+
TFA+Na ]; found 689.2596. IR:
υ
1
675, 1646, 1554, 1375, 1203, 1127, 1070, 1039, 838, 801.
2
-Acetamido-2-deoxy-6-O-sodium
ammoniumyl trifluoroacetate-2-deoxy-
1→4)-2-acetamido-2-deoxy-
1’,2’,3’]-triazol-4’-yl}-methyl]-
β
-D
β-D-
(
β
-
D
β-D-
[
D
A
D-
3
2
ꢀ
3
α
/β
α/β
+
1
residue was eluted through ion exchange Dowex® Na resin to ratio 8:2. R
f
0.86 (EtOAc/EtOH/H
2
O 4:4:2). H NMR (600 MHz,
give the crude alcohol in quantitative yield. A solution of this DMSO-d
B
6
+
ε
D
2
O) : 8.09, 8.08 (2 s, 1H, H-5’α&β
δ
); 5.73 (d, 1H,
A
B
,2 10.0 Hz, H-1 ); 4.90 (d, 0.8H, J
B
A
A
,2 2.0 Hz, H-1
alcohol in trifluoroacetic acid/H
2
O (250
ꢀ
L, 85:15) was stirred
J
1
1
α
); 4.87,
);
A A
2
2
at 0°C for 10 min. The reaction mixture was concentrated to 4.85 (2 d, 1H, JHCH 11.9 Hz and JHCH 11.9 Hz, CHHtriaz
α&β
2
obtain the crude ammonium derivative in quantitative yield. 4.63 (d, 1H, JHCH 11.9 Hz, CHHtriaz α&β); 4.43 (d, 0.2H, J
1 ,2
A
β
C
C
,2 8.5 Hz, H-1 ); 4.13 (2 dd, 1H,
C
To a solution of this residue in EtOAc/H
2
O/EtOH (270
ꢀ
L, 8.5 Hz, H-1
); 4.40 (d, 1H, J
1
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C7OB01201B
ARTICLE
Journal Name
B
C
B
B
B
,3 10.0 Hz, H-2
B
2
C
C
B
B
); 3.73 (dd, 1H, J6a ,6b 11.5, 85.8 (C-1 ); 80.2 (C-4 ); 79.1 (C-4
A
A
B
); 77.8 (C-5 );
J
J
1
,2 10.0, J
2
α
&
β
,6a 2.2 Hz, H-6a ); 3.71 (dd, 1H, J
α
); 78.6 (C-4
β
); 74.0 (C-3 ); 72.3 (C-3 );
C
C
B
B
B
B
C
,4 7.8 Hz, H- 77.0 (C-5 ); 75.5 (C-5
B
, H-6a , H-6a
A
A
C
B
5
2
,3 10.0, J
3
β
); 74.4 (C-3
C
β
and C-4 ); 65.1, 64.8 (CH
B
A
A
A
A
A
A
α
3
6
); 3.67-3.37 (m, 9.6H, H-2
B
α
, H-5 , H-2 , H-4 , H-6b , H-3
, H-3
A
α
, H-5
α
α
β
, H- 70.9 (C-5
α
); 3.39 61.0 (C-6 ); 60.7, 60.6 (C-6
); 70.6 (C-3
2
B
triaz
α
&
β
); 59.7 (C-6 ); 57.5 (C-
);
A
B
C
B
A
A
C
A
α
A
a
α
β
, H-6b
C
and H-6b
β
and C-6
β
2
C
C
C
C
A
,3
A
A
2
C
); 55.3 (C-2 ); 54.5 (C-2
A
B
(
dd, 1H, J6a ,6b 11.5, J
5
,6b 7.5 Hz, H-6b ); 3.37 (dd, 0.2H, J
C
2
β
,4 9.2 Hz, 16’’); 29.4-28.6 (C-4’’ to C-7’’ and C-12’’ to C-15’’); 26.8, 26.7
α); 53.9 (C-2 ); 35.8 (C-2’’); 31.4 (C-
A
A
,2 8.5 Hz, H-2
A
C
C C
1
0.5, J
C
1
β
H-3 ); 3.27 (dd, 0.8H, J 9.2, J 8.4 Hz, H-4
); 3.29 (dd, 1H, J
2
,3 9.6, J
3
A
α
); 3.20 (ddd, 1H, J
); 3.21 (dd, 0.2H, (C-8’’ and C-11’’); 25.1 (C-3’’); 23.1, 22.8, 22.7 (2 COCH
C
3
A
A
A
A
,4 8.0 Hz, H-4
A
C
C
C
+
,B); 22.2 (C-17’’); 14.1 (C-18’’). HRMS (ESI ): calculated for
J
4
,5 9.7, J
3
β
,6a 2.2 Hz, H-5 ); 3.10 (ddd, 0.2H, J
4
,5 8.9, J
5
A
,6b
A
α
,A
β
C
C
C
A
A
A
+
+
-
7
.5, J
A
5
A
4
,5 9.7, J
C
,5 8.9 Hz, H-4 );
5
,6a 5.5,
C
1
43 74 6
H N NaO16 953.5054 [M+Na ]; found 953.5073. IR: υ (cm
) = 3600-3000, 2926, 2857, 1649, 1555, 1376, 1322, 1103,
A
β
C
C
C
C
J
5
,6b 1.9 Hz, H-5
); 3.06 (dd, 1H, J
3
,4 9.2, J
4
2
.07 (t, 2H, J2’’,3’’ 7.3 Hz, H-2’’); 1.83, 1.82, 1.62 (3 s, 6H, 2 Ac 1074, 1047.
,A ,B); 1.47 (brtt, 2H, J2’’,3’’ 7.3, J3’’,4’’ 7.0 Hz, H-3’’); 1.29-1.16 2-Acetamido-2-deoxy-6-O-sodium
Aα
β
sulfonato-4-O-[{1’-([2-
-glucopyranosyl]-(1→4)-2-
-glucopyranosyl)-1H-[1’,2’,3’]-triazol-
-glucopyranose (3S). General procedure with
); 85.8 (C- 20 (9.2 mg, 0.010 mmol) and 21 (4.6 mg, 0.013 mmol) gave
1
3
(
m, 24H, H-4’’ to H-15’’); 0.84 (t, 3H, J15’’,16’’ 6.8 Hz, H-16’’).
NMR (150 MHz, DMSO-d O) : 172.5 (C-1’’); 170.0, acetamido-2-deoxy-
,B); 144.7, 144.6 (C-4’ and C-4’ ); 4’-yl}-methyl]-
C
palmitamido-2-deoxy-β-D
6
+
εD
2
δ
β-D
1
69.9, 169.5 (2 COCH
3
A
α
,A
β
α
β
D
C
A
A
1
22.5 (C-5’); 102.0 (C-1 ); 95.3 (C-1
β
); 90.6 (C-1
A
α
B
B
); 80.2 (C-4 ); 79.1 (C-4
A
B
1
C
α
); 78.6 (C-4 β); 77.8 (C-5 ); 77.0 (C- product 3S (7.0 mg, α/β mixture, 67%) as a white lyophilisate.
β
A
A
C
B
1
5
5
6
); 75.5 (C-5
β
); 74.4 (C-3
); 74.0 (C-3 ); 72.3 (C-3 ); 70.8 (C-
α
/
β
ratio 8:2. R
f
0.71 (EtOAc/EtOH/H
2
O 56:26:18). H NMR
);
1 1
and C-6 β); 59.7 (C-6 ); 57.5 (C-2 β); 55.3 5.82 (d, 1H, J ,2 10.0 Hz, H-1 ); 4.89 (d, 0.8H, J ,2 2.7 Hz, H-
A
C
A
C
α
); 70.7 (C-3
α
and C-4 ); 65.1, 64.9 (CH triaz ); 61.1 (C- (600 MHz, DMSO-d
2
B
α
&
β
6
+
εD
2
O) : 8.12, 8.11 (2 s, 1H, H-5’α&β
δ
A
α
A
A
B
B
B
A A
); 60.7, 60.6 (C-6
C
A
α
B
A
1
2
2
(C-2 ); 54.6 (C-2
); 53.9 (C-2 ); 35.8 (C-2’’); 31.4 (C-14’’); 29.4-
α
); 4.85, 4.84 (2 d, 1H,
CHHtriaz ); 4.68, 4.67 (2 d, 1H, JHCH 11.8 Hz and JHCH 11.8
,B); 22.3 (C-15’’); 14.1 (C-16’’). HRMS (ESI ): calculated for Hz, CHHtriaz
JHCH 11.8 Hz and JHCH 11.8 Hz,
2
2
2
A
8.7 (C-4’’ to C-13’’); 25.1 (C-3’’); 23.2, 22.8, 22.7 (2 COCH
3
α&β
+
A
A
A
α
,A
β
1
α&β); 4.43 (d, 0.2H, J ,2 8.2 Hz, H-1 β); 4.40 (d,
+
+
-1
C
C
C
B
B
B B
C
41
H
73
N
6
O
16 905.5078 [M+H ]; found 905.5112. IR: υ (cm ) = 1H, J ,3 9.8
1
,2 8.5 Hz, H-1 ); 3.99, 3.98 (2 dd, 1H, J
A
1
,2 10.0, J
2
B
2
A
A
A
3
1
2
600-3000, 2920, 2851, 1647, 1628, 1550, 1375, 1318, 1105, Hz, H-2 α&β); 3.92 (dd, 0.2H, J6a ,6b 10.9, J ,6a 1.6 Hz, H-
076, 1045.
5
A
A
A
B
A
6a
β); 3.88-3.72 (m, 3.6H, 2 H-6 α, H-3 , H-5 α and H-6b β);
2
C
C
C
C
,6a 2.0 Hz, H-6a ); 3.66-3.57 (m,
B
and H-6a ); 3.51 (dd, 1H, J
C
-Acetamido-2-deoxy-4-O-[{1’-([2-oleamido-2-deoxy-
β
-
D
-
3.73 (dd, 1H, J6a ,6b 11.7, J
5
A
α
A
C
C
C
C
glucopyranosyl]-(1→4)-2-acetamido-2-deoxy-
β
glucopyranosyl)-1H-[1’,2’,3’]-triazol-4’-yl}-methyl]-
C
glucopyranose (4). General procedure with 19 (12.1 mg, 0.016 3.39 (dd, 1H, J6a ,6b 11.7, J
-
D
-
2.6H, H-2 α
C
Hz, H-2 ); 3.50-3.43 (m, 3.2H, H-4 , H-5 , H-6b and H-3
, H-3
,3 9.8, J
,2 8.5
2
1
B
B
B
A
D-
β);
2
C
C
C
,6b 7.2 Hz, H-6b ); 3.37 (dd, 0.2H,
C
5
A
A
A
A
,2 8.2 Hz, H-2
A
β
A
mmol) and 22 (7.1 mg, 0.019 mmol) afforded product
mg, mixture, 95%) as a white lyophilisate. ratio 8:2. R
.86 (EtOAc/EtOH/H
O) : 8.09, 8.08 (2 s, 1H, H-5’
4
(13.7
J
2
,3 10.3, J
1
); 3.32-3.28 (m, 0.2H, H-5 β); 3.28
C
,4 9.1 Hz, H-3 ); 3.24 (dd, 0.8H, J 9.9, J 8.3
C
C
C
C
α
/β
α
/β
f
(dd, 1H, J
A
Hz, H-4
C
2
,3 9.8, J
3
1
C
C
C
C
C
C
0
2
O 4:4:2). H NMR (600 MHz, DMSO-d
6
+
α
); 3.20 (ddd, 1H, J
4
,5 9.4, J
5
,6b 7.0, J
5
,6a 1.8 Hz, H-
B
B
A
β
C
C
C C
εD
2
δ
α
&
β
); 5.73 (brd, 1H, J
1
,2 10.1 5 ); 3.21-3.17 (m, 0.2H, H-4
); 3.06 (dd, 1H, J
4
,5 9.4, J
3
,4 8.9
B
C
Hz, H-1 α&β); 5.35-5.27 (m, 2H, H-9’’ and H-10’’); 4.90 (d, 0.8H, Hz, H-4 ); 2.07 (t, 2H, J2’’,3’’ 7.5 Hz, H-2’’); 1.83, 1.81, 1.62 (3 s,
A
A
A
2
2
J
1
,2 2.3 Hz, H-1
2.1 Hz, CHHtriaz
); 4.43 (d, 0.2H, J
α); 4.87, 4.85 (2 d, 1H, JHCH 12.1 Hz and JHCH 6H, 2 Ac Aα β,B); 1.51-1.44 (m, 2H, H-3’’); 1.28-1.17 (m, 24H,
,A
2
13
1
α&β); 4.63 (brd, 1H, JHCH 12.1 Hz, CHHtriaz H-4’’ to H-15’’); 0.84 (t, 3H, J15’’,16’’ 6.9 Hz, H-16’’). C NMR
A
A
,2 8.4 Hz, H-1
A
C C
α
&
β
1
β
); 4.40 (d, 1H, J
B
1
,2 8.6 (150 MHz, DMSO-d
6
+
ε
D
2
O)
δ
); 144.7 (C-4’); 122.6 (C-5’); 102.0 (C-1 ); 95.2 (C-1
: 172.4 (C-1’’); 169.8, 169.5 (2
C
B
B
B
,3 10.0 Hz, H-2
B
C
A
A
A
Hz, H-1 ); 4.13 (2 dd, 1H, J
C
1
,2 10.1, J
2
α
&
β
); COCH
3
β
β
β
);
);
);
2
C
C
C
C
A
B
B
A
3.73 (dd, 1H, J6a ,6b 11.6, J
5
B
,6a 2.0 Hz, H-6a ); 3.71 (dd, 1H, 90.5 (C-1 α); 85.4 (C-1 ); 80.3 (C-4 ); 79.1 (C-4 α); 78.7 (C-4
B
B
B
B
A
B
C
A
C
J
2 3
,3 10.0, J ,4 7.8 Hz, H-3 ); 3.67-3.57 (m, 2.4H, H-2 α, H- 77.9 (C-5 ); 77.0 (C-5 ); 74.3 (C-3 β); 74.0 (C-3 ); 73.1 (C-5
A
A
2
B
); 3.59 (dd, 1H, J6a ,6b 12.3, J
B
B
B
B
,6a 1.6 Hz, H- 71.9 (C-3 ); 70.7, 70.6 (C-3
A
C
and C-4 ); 68.8 (C-5
A
3
6
3
α
and H-5
α
a ); 3.56 (dd, 0.2H, J6a ,6b 11.9, J
5
α
α
); 65.5 (C-
B
2
A
A
A
A
A
A
C
B
5
,6a 1.7 Hz, H-6a
B
β); 3.54-
, H-6b
6
2
α
&
β
); 65.2 (CH
C
2
triaz
α&β); 61.0 (C-6 ); 59.7 (C-6 ); 57.4 (C-
A
C
B
B
, H-2 , H-4 , H-6b , H-5 , H-3
A
A
A
A
B
.37 (m, 6H, H-6a
α
); 3.39 (dd, 1H, J6a ,6b 11.6, J
β
α
and
β
); 55.2 (C-2 ); 54.4 (C-2 α and C-2 ); 35.8 (C-2’’); 31.4 (C-
A
2
C
C
C
C
C
H-6b
β
dd, 0.2H, J
5
,6b 7.2 Hz, H-6b ); 3.37 14’’); 29.3-28.7 (C-4’’ to C-13’’); 25.1 (C-3’’); 23.1, 22.7, 22.6 (2
A
A
A
A
,2 8.4 Hz, H-2
A
C
C
+
(
2
,3 10.3, J
1
β
); 3.29 (dd, 1H, J
2
,3
COCH
); calculated for
); 3.20 (ddd, 1H, 983.4470. IR: υ
3
Aα,Aβ,B); 22.2 (C-15’’); 14.1 (C-16’’). HRMS (ESI ):
C
C
C
A
-
+
10.0, J
3
,4 8.9 Hz, H-3 ); 3.27 (dd, 0.8H, J 9.7, J 8.7 Hz, H-4
α
C
41
-1
H
71
N
6
O
19
S
983.4500 [M-Na ]; found
(cm ) = 3600-3000, 2922, 2853, 1649, 1554,
1378, 1317, 1226, 1104, 1057.
,5 9.5, 2-Acetamido-2-deoxy-6-O-sodium
-glucopyranosyl]-(1→4)-2-acetamido-2-
-glucopyranosyl)-1H-[1’,2’,3’]-triazol-4’-yl}-methyl]-
-glucopyranose (4S)
to H-17’’); 0.84 (t, 3H, J17’’,18’’ 7.0 Hz, H-18’’). C NMR (150 General procedure with 20 (10.8 mg, 0.012 mmol) and 22 (6.0
MHz, DMSO-d O) : 172.4 (C-1’’); 169.9, 169.8, 169.4 (2 mg, 0.016 mmol) afforded product 4S (8.4 mg, mixture,
COCH ,B); 144.7, 144.6 (C-4’ and C-4’ ); 129.8 (C-9’’ and 67%) as a white lyophilisate. ratio 8:2. R 0.66
C-10’’); 122.4 (C-5’); 102.0 (C-1 ); 95.2 (C-1 O 56:26:18). H NMR (600 MHz, DMSO-d
A
A
A
A
A
3.21 (dd, 0.2H, J
4
,5 9.5, J
3
,4 9.3 Hz, H-4
β
C
C
C
C
C
C
C
A A
J
4
,5 9.5, J
5
,6b 7.2, J
5
A
,6a 2.0 Hz, H-5 ); 3.10 (ddd, 0.2H, J
4
,5
A
A
A
A
C C
9.5, J
5
,6b 5.4, J
5
C
,6a 1.7 Hz, H-5
β
); 3.06 (dd, 1H, J
4
sulfonato-4-O-[{1’-([2-
C
C
J
3
,4 8.9 Hz, H-4 ); 2.09-2.05 (m, 2H, H-2’’); 1.99-1.93 (m, 4H, oleamido-2-deoxy-
H-8’’ and H-11’’); 1.83, 1.82, 1.63 (3 s, 6H, 2 Ac ,A ,B); 1.51- deoxy-
.44 (m, 2H, H-3’’); 1.31-1.18 (m, 20H, H-4’’ to H-7’’ and H-12’’
β-D
Aα
β
β-D
1
D
1
3
6
+
εD
2
δ
α/β
3
Aα
,A
β
α
β
α
/β
f
C
A
β
A
1
); 90.5 (C-1
α
); (EtOAc/EtOH/H
2
6
+
8
| J. Name., 2012, 00, 1-3
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O)
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View Article Online
DOI: 10.1039/C7OB01201B
ARTICLE
B
B
εD
2
δ
: 8.12, 8.11 (2 s, 1H, H-5’
α
&
β
); 5.82 (d, 1H, J
1
,2 10.0 The authors thank Rosemary Beau for comments on the
B
Hz, H-1 ); 5.35-5.27 (m, 2H, H-9’’ and H-10’’); 4.90 (d, 0.8H, manuscript. J.-J. Bono thanks F. Maillet (LIPM) for kindly
2
A
A
,2 2.7 Hz, H-1
A
2
J
1
α); 4.85, 4.84 (2 d, 1H, JHCH 11.7 Hz and JHCH providing the NodRt factors. The authors are grateful to the
2
1
1.7 Hz, CHHtriaz α&β); 4.68, 4.67 (2 d, 1H, JHCH 11.7 Hz and Institut de Chimie des Substances Naturelles (ICSN) – Centre
2
A
A
,2 8.3 Hz, H-1
A
J
HCH 11.7 Hz, CHHtriaz
C
α
,2 8.4 Hz, H-1 ); 3.99, 3.98 (2 dd, 1H, J
&
β
); 4.43 (d, 0.2H, J
C
1
β
,2 10.0, (N.B.). The CHARM AT Labex program (ANR-11-LABX-39) is
); National de la Recherche Scientifique (CNRS) for a PhD grant
C
B B
4
.40 (d, 1H, J
B
1
1
3
B
,3 9.8 Hz, H-2
B
2
A
A
); 3.92 (dd, 0.2H, J6a ,6b 11.3, J
A
A
J
2
α
); 3.88-3.72 (m, 3.6H, 2 H-6
&
β
5
,6a 1.7 acknowledged for its support. The Institut Jean-Pierre Bourgin
and H- benefits from the support of the Labex Saclay Plant Sciences-
A
A
B
, H-3 , H-5
A
Hz, H-6a
A
6
β
); 3.73 (dd, 1H, J6a ,6b 11.8, J
α
,6a 2.0 Hz, H-6a ); 3.66- SPS (ANR-10-LABX-0040-SPS).
α
2
C
C
C
C
C
b
β
5
A
A
B
C C
3
2
.57 (m, 2.6H, H-2 α, H-3 α and H-6a ); 3.51 (dd, 1H, J ,3 9.9,
C
C
C
B
B
B
,2 8.4 Hz, H-2 ); 3.50-3.45 (m, 3H, H-4 , H-5 , H-6b ); 3.45
J
1
A
A
A
A
,4 8.9 Hz, H-3
A
2
C
C
Notes and references
(
dd, 0.2H, J
C
2
,3 10.4, J
3
β
); 3.39 (dd, 1H, J6a ,6b
C
,6b 7.2 Hz, H-6b ); 3.37 (dd, 0.2H, J
C
A
A
A
A
§
e
1
1.8, J
5
2
,3 10.4, J
1
,2 8.3
See Supporting Information.
A
A
C C
Hz, H-2 β); 3.32-3.28 (m, 0.2H, H-5 β); 3.28 (dd, 1H, J
2
,3 9.9, 1. C. Gough and J. Cullimore, Mol. Plant-Microbe Interact., 2011,
C
C
C
,4 8.9 Hz, H-3 ); 3.24 (dd, 0.8H, J 9.7, J 8.2 Hz, H-4
A
J
3
α
,6a 2.0 Hz, H-5 ); 3.21-3.17 (m,
); 3.21
24, 867-878.
C
C
C
C
C
C
C
2
3
4
5
.
.
.
.
K. Akiyama, K. Matsuzaki and H. Hayashi, Nature, 2005, 435,
824-827.
N. K. Peters, J. W. Frost and S. R. Long, Science, 1986, 233, 977-
(
ddd, 1H, J
4
,5 9.6, J
5
,6b 7.2, J
5
A
C
C
C
C
,4 8.9 Hz, H-4 ); 2.07 (t,
C
0
2
1
1
3
.2H, H-4
β
); 3.06 (dd, 1H, J
4
,5 9.6, J
3
H, J2’’,3’’ 7.6 Hz, H-2’’); 1.99-1.93 (m, 4H, H-8’’ and H-11’’);
.83, 1.81, 1.62 (3 s, 6H, 2 Ac ,A ,B); 1.51-1.44 (m, 2H, H-3’’);
.31-1.18 (m, 20H, H-4’’ to H-7’’ and H-12’’ to H-17’’); 0.84 (t,
9
80.
A
α β
X. Perret, C. Staehelin and W. J. Broughton, Microbiol. Mol. Biol.
Rev., 2000, 64, 180-201.
P. Lerouge, P. Roche, C. Faucher, F. Maillet, G. Truchet, J.-C.
1
3
H, J15’’,16’’ 6.9 Hz, H-18’’). C NMR (150 MHz, DMSO-d
O) : 172.4 (C-1’’); 169.8, 169.5 (2 COCH
29.8 (C-9’’ and C-10’’); 122.6 (C-5’); 102.0 (C-1 ); 95.2 (C-1
6
+
εD
2
δ
3
C
); 144.7 (C-4’);
Promé and J. Denarié, Nature, 1990, 344, 781-784.
); 6. F. Maillet, V. Poinsot, O. André, V. Puech-Pages, A. Haouy, M.
A
A
A
1
9
7
7
6
2
1
β
β
β
A
B
B
A
0.5 (C-1
α
7.9 (C-5 ); 77.0 (C-5 ); 74.3 (C-3
); 85.5 (C-1 ); 80.3 (C-4 ); 79.2 (C-4 α
C
); 74.0 (C-3 ); 73.1 (C-5
); 78.7 (C-4
);
);
Gueunier, L. Cromer, D. Giraudet, D. Formey, A. Niebel, E. A.
Martinez, H. Driguez, G. Bécard and J. Denarié, Nature, 2011,
469, 58-63.
B
A
C
β
B
1.9 (C-3 ); 70.7, 70.6 (C-3
A
C
A
α and C-4 ); 68.8 (C-5 α); 65.5 (C-
A
C
B
7
8
.
.
J. Denarié, F. Debellé and J. C. Promé, Annu. Rev. Biochem.,
1996, 65, 503-535.
α
&
β
); 65.2 (CH
2
triaz
); 55.2 (C-2 ); 54.4 (C-2
6’’); 29.4-28.6 (C-4’’ to C-7’’ and C-12’’ to C-15’’); 26.8, 26.7
C-8’’ and C-11’’); 25.1 (C-3’’); 23.1, 22.8, 22.7 (2 COCH
α&β); 61.0 (C-6 ); 59.7 (C-6 ); 57.4 (C-
A
C
A
B
β
α and C-2 ); 35.8 (C-2’’); 31.4 (C-
S. Radutoiu, L. H. Madsen, E. B. Madsen, H. H. Felle, Y.
Umehara, M. Gronlund, S. Sato, Y. Nakamura, S. Tabata, N.
Sandal and J. Stougaard, Nature, 2003, 425, 585-592.
E. B. Madsen, L. H. Madsen, S. Radutoiu, M. Olbryt, M.
Rakwalska, K. Szczyglowski, S. Sato, T. Kaneko, S. Tabata, N.
Sandal and J. Stougaard, Nature, 2003, 425, 637-640.
(
3
+
A
α,Aβ,B); 22.2 (C-17’’); 14.1 (C-18’’). HRMS (ESI ): calculated for
C H N O19S 1009.4657 [M-Na ]; found 1009.4703. IR: υ (cm
43 73 6
9
.
-
+
-
1
)
= 3600-3000, 2925, 2857, 1649, 1559, 1378, 1324, 1228,
1103, 1059.
10. E. Limpens, C. Franken, P. Smit, J. Willemse, T. Bisseling and R.
Geurts, Science, 2003, 302, 630-633.
General procedure for equilibrium binding assays. LYR3 was 11. P. Smit, E. Limpens, R. Geurts, E. Fedorova, E. Dolgikh, C. Gough
and T. Bisseling, Plant Physiol., 2007, 145, 183-191.
produced in Nicotiana benthamiana leaves as a chimeric
protein consisting of the ectodomain of LYR3, the
transmembrane and kinase domain of NFP fused to YFP, and
1
1
2. A. Broghammer, L. Krusell, M. Blaise, J. Sauer, J. T. Sullivan, N.
Maolanon, M. Vinther, A. Lorentzen, E. B. Madsen, K. J. Jensen,
P. Roepstorff, S. Thirup, C. W. Ronson, M. B. Thygesen and J.
Stougaard, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 13859-
1
3
membrane extracts were prepared as previously described.
For equilibrium binding assays, 10 μg of membrane protein
1
3864.
3
were incubated in the presence of 0.5 nM [ S]-NodRt factor
5
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23*), obtained as detailed in Gressent et al., in a total
7
(
volume of 200 μL of binding buffer to determine the amount
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4
represents the specific binding. Competition assays used 2 µM
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Conflicts of interest
There are no conflicts of interest to declare.
17. K. Miyata, T. Kozaki, Y. Kouzai, K. Ozawa, K. Ishii, E. Asamizu, Y.
Okabe, Y. Umehara, A. Miyamoto, Y. Kobae, K. Akiyama, H.
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Acknowledgements
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB01201B
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Page 11 of 11
Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB01201B
Lipo-chitotetrasaccharide analogues have been synthesized from a derivative obtained by controlled chitin
depolymerization and a functionnalized N-acetyl-glucosamine.