Synthesis of a Conjugation-Ready, Phosphorylated, Tetrasaccharide Fragment of the O-PS of Vibrio cholerae O139

Article abstract of DOI:10.1021/acs.joc.5b02105

A new pathway to the tetrasaccharide α-Colp-(1→2)-4,6-P-β-d-Galp-(1→3)-[α-Colp-(1→4)]-β-d-GlcpNAc-1-(OCH₂CH₂)₃NH₂ has been developed. Glycosylation of 8-azido-3,6-dioxaoctyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside with 3,4,6-tri-O-acetyl-2-O-bromoacetyl-α-d-galactopyranosyl bromide afforded the β-linked disaccharide. Debromoacetylation followed by reductive opening of the benzylidene acetal afforded the disaccharide diol acceptor. Halide-assisted glycosylation with 2,4-di-O-benzyl-α-colitosyl bromide gave the 1,2-cis-coupling product. Deacetylation followed by regioselective phosphorylation gave isomeric (R,S)-(P)-4^II,6^II-cyclic phosphates, which were globally deprotected by one-step catalytic (Pd/C) hydrogenation/hydrogenolysis. The target tetrasaccharide, obtained in high overall yield, is amenable for conjugation to proteins.

Full text of DOI:10.1021/acs.joc.5b02105

Brief Communication
pubs.acs.org/joc
Synthesis of a Conjugation-Ready, Phosphorylated, Tetrasaccharide
Fragment of the O‑PS of Vibrio cholerae O139
Sameh E. Soliman†,‡ and Pavol Kovac*,†
́
̌
†NIDDK, LBC, Section on Carbohydrates, National Institutes of Health, Bethesda, Maryland 20892-0815, United States
‡Department of Chemistry, Faculty of Science, Alexandria University, Alexandria 21321, Egypt
S
* Supporting Information
ABSTRACT: A new pathway to the tetrasaccharide α-Colp-(1→2)-4,6-P-β-D-Galp-(1→3)-[α-Colp-(1→4)]-β-D-GlcpNAc-1-
(OCH2CH2)3NH2 has been developed. Glycosylation of 8-azido-3,6-dioxaoctyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-
β-D-glucopyranoside with 3,4,6-tri-O-acetyl-2-O-bromoacetyl-α-D-galactopyranosyl bromide afforded the β-linked disaccharide.
Debromoacetylation followed by reductive opening of the benzylidene acetal afforded the disaccharide diol acceptor. Halide-
assisted glycosylation with 2,4-di-O-benzyl-α-colitosyl bromide gave the 1,2-cis-coupling product. Deacetylation followed by
regioselective phosphorylation gave isomeric (R,S)-(P)-4II,6II-cyclic phosphates, which were globally deprotected by one-step
catalytic (Pd/C) hydrogenation/hydrogenolysis. The target tetrasaccharide, obtained in high overall yield, is amenable for
conjugation to proteins.
holera is a deadly, infectious enteric disease, endemic to
Cmany countries in the Third World. The disease caused
by Vibrio cholerae O1 has been around for centuries, but in
1992, a new type of cholera emerged, which manifested itself by
the same symptoms, i.e., watery diarrhea, but was caused by a
newly discovered pathogen Vibrio cholerae O139. This
prompted the elucidation of the structure1,2 of the O-specific
polysaccharide (O-PS) of this Gram-negative bacterium, which
was the first step toward the rational development of a
conjugate vaccine for the disease. This laboratory has been
involved in development of conjugate vaccines for infectious
diseases from synthetic and bacterial carbohydrates for a
number of years. To that end, among other things, we have
Figure 1. Structure of the O-PS of Vibrio cholerae O139.
synthesized fragments of the protective carbohydrate antigens
fewer steps and in an experimentally more convenient manner
of the disease-causing bacterial pathogens and studied their
than by the approaches described previously.
binding with the homologous protective antibodies in-
Each of the previous pathways to the linker-equipped
volved.3−5 Determination of the minimum structural require-
The disadvantages in the Oscarson approach6 are that the
ments in the antigen which, when transformed into
sequence FD(E)C, shown in Figure 1, has its own merits.
immunogenic conjugates and used as vaccines elicit protective
antibodies, requires assembly of the O-PS fragments in the
spacer-equipped form to make them amenable for conjugation.
In 2006, Oscarson’s6 and our laboratory7 reported independent
syntheses of the linker-equipped upstream tetrasaccharide
[sequence FD(E)C, Figure 1]. Here, we describe a new,
high-yielding approach to the synthesis of the same
carbohydrate sequence. It provides the desired structure in
attachment of a linker requires chemical manipulations with the
fully assembled tetrasaccharide, and the synthetic strategy
requires two steps for the final deprotection. On the other
hand, in the Ruttens approach,7 the isomers-forming
phosphorylation is done in the middle of the whole reaction
Received: September 8, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.5b02105
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The Journal of Organic Chemistry
Brief Communication
Scheme 1. Synthesis of the Disaccharide Diol Acceptor 8
Scheme 2. Synthesis of the Spacer-Equipped, Phosphorylated Tetrasaccharide 14
sequence. Consequently, the two isomeric phosphates must be
resolved to eliminate complications that would arise from
having to continue the sequence with a mixture. Thus, one has
to either complete the sequence with only one isomer or
perform all chemical transformations separately with both
phosphates to increase the overall yield of the final product.
The present tetrasaccharide buildup starts from the down-
stream8 end with the linker-equipped monosaccharide, and the
synthesis is designed in the way allowing the phosphorylation
to be done as a penultimate step. Thus, separation of isomeric
phosphates is not required because the following global
deprotection removes the chirality at the P atom yielding,
thus, the same target product from both phosphates.
The feasible, just outlined strategy toward the tetrasaccharide
fragment 14 (Scheme 2) is an extension of our preparation of
the phosphorylated upstream trisaccharide sequence9 D(E)C,
Figure 1. There, and here as well, to minimize the overall
number of synthetic steps, the choice of protecting groups
allows final, global deprotection involving transformation of
several different functional groups to be achieved in one step
(catalytic hydrogenation/hydrogenolysis with Pd/C).
Accordingly (Scheme 1), 1,3,4,6-tetra-O-acetyl-α-D-galacto-
pyranose (2)10 was bromoacetylated to give 3 (96%), which
was converted into the α-glycosyl bromide 4 with 33% HBr−
HOAc. Silver trifluoromethanesulfonate (triflate) mediated
glycosylation of the linker-equipped glycosyl acceptor 8-azido-
3,6-dioxaoctyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetami-
B
DOI: 10.1021/acs.joc.5b02105
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The Journal of Organic Chemistry
Brief Communication
do-β-D-glucopyranoside11 (5) with the α-glycosyl donor 4 was
performed using our improved protocol11,12 (cf. ref 13) to
afford the desired β-linked disaccharide 6 in 85% yield. In
addition to being a key intermediate here, the foregoing
substance is also a generally useful building block in
oligosaccharide synthesis, as it allows orthogonal deprotection
(debromoacetylation or regioselective opening of the benzyli-
dene acetal). The new methodology using 1,1,3,3-tetramethy-
lurea as an acid scavenger11,12 obviates the use of molecular
sieves, whose use makes it difficult to control the mild acidity of
the reaction medium required to prevent side reactions to
occur. The β-configuration of the interglycosidic linkage in 6
follows from the 1H NMR spectrum (δ 4.74, d, J1,2 = 8.0 Hz, H-
1II). In addition, signals for the two anomeric carbons appeared
in the 13C NMR spectrum at almost identical chemical shifts (δ
99.4 and 99.8 for C-1II and C-1I, respectively). That the acid-
labile benzylidene group was preserved under these conditions
was manifested by the presence of the 13C signal at δ 101.2
(PhCH).
Selective O-debromoacetylation using thiourea in the
presence of a weak, non-nucleophilic organic base to prevent
acyl group migration14 gave the disaccharide acceptor 7 having
O-2II free, in 91% yield. The upfield shift of the signal for H-2II
(δ 3.79 ppm), and the COSY crosspeak between the hydroxyl
group (δ 2.59 ppm) and H-2II confirmed that removal of the
bromoacetyl group occurred without acetyl group migration.
Subsequent regioselective reductive opening of the 4I,6I-O-
benzylidene ring in disaccharide 7 using sodium cyanoborohy-
dride and HCl−Et2O in tetrahydrofuran15 afforded the 6I-O-
benzyl derivative 8 in 88% yield. The significant upfield shift of
the signal for C-4I (by ∼10 ppm, as compared to that in the
spectrum of 7), as well as the COSY crosspeak between H-4I (δ
3.53 ppm) and the newly generated hydroxyl group (δ 4.34
ppm), confirmed that the reductive opening of the benzylidene
acetal led to the 6I-benzyl ether and a free 4I-OH group.
The disaccharide diol glycosyl acceptor 8 (Scheme 2) was
subjected to halide-assisted glycosylation16 with the α-colitosyl
bromide 9 (made by treatment of the corresponding ethyl
thioglycoside17 with bromine) to give the tetrasaccharide 10
(83%). The identification of the coupling product 10 was based
on its 1H and 13C NMR spectra, which included signals
characteristic for both the donor and the acceptor moieties. As
expected, a downfield shift was observed in the spectrum of 10
for the H-4I and H-2II signals, compared to those in the
spectrum of 8, as a result of glycosylation18 at those positions.
The signals for the two anomeric protons of colitose residues
present appeared as doublets at δ 5.22 ppm (J = 3.0 Hz) and
5.07 ppm (J = 3.3 Hz) and confirmed, thus, the exclusive
formation of the α-glycosidic linkages.
The desired transformation of the benzyl ethers, the 2,2,2-
trichloroethyl, N-trichloroacetyl, and the azide groups in 12 was
accomplished in one step by hydrogenation/hydrogenolysis in
the presence of Pd/C catalyst to give the title phosphorylated
tetrasaccharide fragment 14 in 85% yield. Considering the
lability of the α-colitosyl group to acid hydrolysis, the reaction
was conducted in a pH = 7 buffer. In addition to HRMS,
presence of the cyclic phosphate in 14 followed from the 31P
NMR spectrum, showing JP,H = 21.6 Hz.21 As expected,
3
because the deprotection of the phosphate group canceled the
chirality at that site, similar treatment of the phosphate isomer
13 gave the same product 14 (1H NMR, 13C NMR, 31P NMR,
TLC, HRMS) in comparable yield.
CONCLUSIONS
■
We have developed an improved, convenient, and high-yielding
strategy for the stereoselective synthesis of the phosphorylated
tetrasaccharide 14, which is one of the terminal determinants of
the O-specific polysaccharide of Vibrio cholerae O139. The final,
amino-functionalized, linker-equipped fragment 14 was ob-
tained in a form ready for conjugation to proteins. Many
features in the present, successful synthetic approach will be
incorporated in the synthesis of the full O-PS, the
phosphorylated hexasaccharide [sequence FD(E)CBA, Figure
1], which is in progress.
EXPERIMENTAL SECTION
■
General Methods. Optical rotations were measured at ambient
temperature for solution in CHCl3, unless stated otherwise. All
reactions were monitored by thin-layer chromatography (TLC) on
silica gel 60 coated glass slides. Column chromatography was
performed by elution from prepacked columns of silica gel and
monitored with the evaporative light scattering detector. Nuclear
magnetic resonance (NMR) spectra were measured at 600 MHz for
1H, 150 MHz for 13C, and 162 MHz for 31P. Solvent peaks were used
as internal reference relative to TMS for H and 13C chemical shifts
1
(ppm); 31P chemical shifts (ppm) are reported relative to 85% H3PO4
in D2O external reference. Assignments of NMR signals were made by
homonuclear and heteronuclear two-dimensional correlation spectros-
copy, run with the software supplied with the spectrometers. When
reporting assignments of NMR signals, nuclei associated with the
spacer are denoted with a prime; sugar residues are serially numbered,
beginning with the one bearing the aglycon, and are identified by a
Roman numeral superscript in listings of signal assignments, with
nuclei of the colitose residues as III and IV (see Scheme 2, 10). The
density of 2,2,2-trichloroethyl phosphorodichloridate (Aldrich/Sigma,
d ≈ 1.7 g/mL at 20 °C) was determined by weighing of 1 mL of the
reagent. Palladium-on-charcoal catalyst (5%, Escat 103) was purchased
from Engelhard Industries. 1,3,4,6-Tetra-O-acetyl-α-D-galactopyranose
(2) was prepared from the commercially available 1,2,3,4,6-penta-O-
acetyl-β-D-galactopyranose (1) as described,10 except that the
compound was crystallized from i-PrOH. Solutions in organic solvents
were dried with anhydrous Na2SO4, and concentrated at 40 °C/2 kPa.
1,3,4,6-Tetra-O-acetyl-2-O-bromoacetyl-α-D-galactopyranose
(3). To a solution of 1,3,4,6-tetra-O-acetyl-α-D-galactopyranose (2)10
(5.0 g, 14.35 mmol) and 1,1,3,3-tetramethylurea (3.77 mL, 31.57
mmol) in dry CH2Cl2 (45 mL) was added bromoacetyl bromide (2.5
mL, 28.70 mmol) dropwise with stirring under argon at −20 °C. The
cooling was removed, and with continued stirring, the mixture was
allowed to warm to room temperature. After 8 h, analysis by TLC
(12:1 toluene−acetone) indicated complete disappearance of the
starting tetraacetate. The mixture was diluted with CH2Cl2 (100 mL)
and washed successively with ice−water, satd aq NaHCO3, and brine.
The organic layer was dried, concentrated, and coevaporated with
toluene (twice). The residue was crystallized from isopropyl ether to
Subsequent de-O-acetylation of 10 with methanolic sodium
methoxide afforded 8-azido-3,6-dioxaoctyl 2,4-di-O-benzyl-3,6-
dideoxy-α-L-xylo-hexopyranosyl-(1→4)-[2,4-di-O-benzyl-3,6-di-
deoxy-α-L-xylo-hexopyranosyl-(1→2)-β-D-galactopyranosyl-
(1→3)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-gluco-
pyranoside (11) in virtually theoretical yield. Treatment19,20 of
the latter with the phosphorylating reagent 2,2,2-trichloroethyl
phosphorodichloridate gave a mixture of the two isomeric
4II,6II-cyclic 2,2,2-trichloroethyl phosphates 12 and 13 (∼3:1,
31P NMR) in combined 92% yield. For identification and
characterization, the mixture was resolved, but the global
deprotection could be done with a mixture of the isomeric
phosphates.
20
give 3 (6.45 g, 96%). Mp: 86−87 °C (i-Pr2O). [α]D = +92.4 (c 1.0,
CHCl3). 1H NMR (600 MHz, CDCl3): δ = 6.40 (d, 1 H, J1,2 = 3.2 Hz,
C
DOI: 10.1021/acs.joc.5b02105
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The Journal of Organic Chemistry
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H-1), 5.52 (dd, 1 H, J3,4 = 2.9, J4,5 = 1.2 Hz, H-4), 5.40 (dd, 1 H, J2,3
=
(6:1 dichloromethane−acetone) showed that all of the starting
material was consumed and a single product was formed. The mixture
was partitioned between water and CH2Cl2, the organic phase was
dried and concentrated, and the residue was chromatographed (9:1
dichloromethane−acetone) to give 7 (3.8 g, 91%) as a white foam.
10.8 Hz, J3,4 = 3.0 Hz, H-3), 5.37 (dd, 1 H, J1,2 = 3.3 Hz, J2,3 = 10.8 Hz,
H-2), 4.35 (ddd, 1 H, J = 0.8, J = 6.7, J = 7.6 Hz, H-5), 4.14−4.08 (m,
2 H, H-6a,b), 3.80−3.75 (m, 2 H, CH2Br), 2.18, 2.17, 2.05, 2.02 (4 s, 12
H, 4COCH3). 13C NMR (150 MHz, CDCl3): δ = 170.3, 170.1, 170.0,
169.9 (4 OCOCH3), 166.4 (COCH2Br), 89.2 (C-1), 68.8 (C-5), 68.0
(C-2), 67.4 (C-4), 67.1 (C-3), 61.1 (C-6), 24.7 (CH2Br), 20.9, 20.7,
20.6, 20.5 (4OCOCH3). HRMS (ESI-TOF): m/z [M + NH4]+ calcd
for C16H25BrNO11 486.0611, found 486.0608. Anal. Calcd for
C16H21BrO11: C, 40.94; H, 4.51. Found: C, 41.05; H, 4.70.
20
1
[α]D = −9.3 (c 1.2, CHCl3). H NMR (600 MHz, CDCl3): δ =
7.47−7.26 (m, 6 H, Ph, NH), 5.56 (s, 1 H, PhCH), 5.32 (dd, 1 H, J3,4
= 3.4, J4,5 = 0.9 Hz, H-4II), 5.08 (d, 1 H, J1,2 = 8.3 Hz, H-1I), 4.83 (dd,
1 H, J2,3 = 10.3 Hz, J3,4 = 3.5 Hz, H-3II), 4.56 (d, 1 H, J1,2 = 7.9 Hz, H-
1II), 4.48 (t, 1 H, J = 9.9 Hz, H-3I), 4.37 (dd, 1 H, J = 5.0, J = 10.6 Hz,
H-6Ib), 4.11 (dd, 1 H, J = 7.5, J = 11.1 Hz, H-6II ), 3.96−3.92 (m, 2 H,
8-Azido-3,6-dioxaoctyl 4,6-O-Benzylidene-2-deoxy-3-O-(3,4,6-tri-
O-acetyl-2-O-bromoacetyl-β-D-galactopyranosyl)-2-trichloroaceta-
mido-β-D-glucopyranoside (6). To a solution of 3 (6.0 g, 12.80
mmol) in CH2Cl2 (100 mL) at 0 °C (ice−water bath) was added 33%
HBr−HOAc (60 mL) slowly and with stirring (during ∼30 min). After
4 h at 0 °C, the solution was diluted with CH2Cl2 and washed
subsequently with ice−water (twice) and satd aq NaHCO3. The
organic layer was dried, concentrated, and coevaporated with toluene
(twice) to give 3,4,6-tri-O-acetyl-2-O-bromoacetyl-α-D-galactopyrano-
syl bromide (4) as a syrup in almost theoretical yield, which was used
b
H-6II , H-1′b), 3.84−3.80 (m, 2 H, H-6Ia, H-1′a), 3.79 (m, 1 H, H-2II),
a
3.78−3.73 (m, 2 H, H-4I, H-5II), 3.72−3.61 (m, 9 H, H-2I, H-2′, H-3′,
H-4′, H-5′), 3.56−3.52 (m, 1 H, H-5I), 3.41 (t, 2 H, J = 5.0 Hz, H-6′),
2.59 (br s, 1 H, 2II−OH), 2.10, 2.01, 1.98 (3 s, 9 H, 3 COCH3). 13C
NMR (150 MHz, CDCl3): δ = 170.4, 170.3, 170.1 (3 OCOCH3),
162.3 (NCOCCl3), 136.7 (ipso Ph), 129.3−126.0 (Ar), 102.4 (C-1II),
101.5 (PhCH), 100.3 (C-1I), 92.5 (CCl3), 79.5 (C-4I), 76.9 (C-3I),
72.6 (C-3II), 71.0 (C-5II), 70.8, 70.7, 70.5 (C-2′, C-3′, C-4′), 70.0 (C-
5′), 68.9 (C-1′), 68.8 (C-2II), 68.6 (C-6I), 66.9 (C-4II), 66.3 (C-5I),
61.2 (C-6II), 58.2 (C-2I), 50.6 (C-6′), 20.7−20.6 (3 OCOCH3).
HRMS (ESI-TOF): m/z [M + NH4]+ calcd for C33H47Cl3N5O16
874.2083, found 874.2064. Anal. Calcd for C33H43Cl3N4O16: C, 46.19;
H, 5.05; N, 6.53. Found: C, 46.09; H, 5.14; N, 6.45.
1
in the next step without purification. H NMR (600 MHz, CDCl3): δ
= 6.36 (d, 1 H, J1,2 = 3.9 Hz, H-1), 5.53 (br d, 1 H, J = 3.1 Hz, H-4),
5.46 (dd, 1 H, J2,3 = 10.7 Hz, J3,4 = 3.3 Hz, H-3), 5.31 (dd, 1 H, J1,2
=
3.9 Hz, J2,3 = 10.6 Hz, H-2), 4.35 (br t, 1 H, J = 6.7 Hz, H-5), 4.17 (dd,
1 H, J = 6.4, J = 11.5 Hz, H-6a), 4.12 (dd, 1 H, J = 6.7, J = 11.4 Hz, H-
6b), 3.88 (d, 1 H, J = 12.1 Hz, CHHBr), 3.84 (d, 1 H, J = 12.2 Hz,
CHHBr), 2.17, 2.07, 2.02 (3 s, 9 H, 3 COCH3). 13C NMR (150 MHz,
CDCl3): δ = 170.3, 169.9, 169.7 (3 OCOCH3), 166.6 (COCH2Br),
90.5 (C-1), 69.5 (C-2), 69.3 (C-5), 67.2 (C-4), 66.9 (C-3), 60.9 (C-6),
24.7 (CH2Br), 20.7, 20.6, 20.5 (3 OCOCH3).
8-Azido-3,6-dioxaoctyl 6-O-Benzyl-2-deoxy-3-O-(3,4,6-tri-O-ace-
tyl-β-D-galactopyranosyl)-2-trichloroacetamido-β-D-glucopyrano-
side (8). A mixture of the benzylidene acetal 7 (1.12 g, 1.31 mmol) and
freshly activated, powdered molecular sieves (3 Å, 1.7 g) in dry THF
(35 mL) was stirred under nitrogen for 60 min at room temperature.
The solution was cooled to 0 °C, and sodium cyanoborohydride (1.0
g, 15.72 mmol) was added portionwise. After being stirred for 20 min
at 0 °C, 2 M HCl−Et2O was added dropwise at 0 °C until the
effervescence ceased and the pH remained acidic. The mixture was
stirred for an additional 30 min at room temperature, diluted with
CH2Cl2 (50 mL), and filtered through Celite. The filtrate was washed
with cold satd aq NaHCO3 (25 mL) and brine (25 mL), and the
organic extract was dried and concentrated. Chromatography (4:1
A solution of 4 (3.1 g, 6.32 mmol) in anhydrous CH2Cl2 (15 mL)
was added, at −30 °C in one portion, to a mixture of glycosyl
acceptor11 (5, 2.0 g, 3.51 mmol), 1,1,3,3-tetramethylurea (1.0 mL, 8.42
mmol), and powdered AgOTf (1.8 g, 7.02 mmol) in anhydrous
CH2Cl2 (40 mL). The cooling was removed, and with continued
stirring, the mixture was allowed to warm to room temperature. The
stirring was continued until TLC (∼8 h, 3:2 hexane−acetone)
indicated that all of the acceptor was consumed. Et3N (0.5 mL) was
added, and the mixture was diluted with CH2Cl2 (50 mL) and filtered
through a Celite pad. The filtrate was washed successively with 0.5 M
aq HCl, satd aq NaHCO3, and brine, and dried. After concentration,
chromatography (2:1 hexane−acetone) gave the spacer-equipped
disaccharide 6 (2.9 g, 85%).
20
toluene−acetone) afforded 8 (990 mg, 88%). [α]D = +6.7 (c 1.0,
1
acetone). H NMR (600 MHz, acetone-d6): δ = 8.26 (d, 1 H, J2,NH
8.6 Hz, NH), 7.42−7.28 (m, 5 H, Ph), 5.34 (dd, 1 H, J3,4 = 3.5, J4,5
=
=
1.0 Hz, H-4II), 4.93 (dd, 1 H, J2,3 = 10.2 Hz, J3,4 = 3.6 Hz, H-3II), 4.88
(d, 1 H, J1,2 = 8.4 Hz, H-1I), 4.67 (d, 1 H, J1,2 = 7.8 Hz, H-1II), 4.63
(bs, 1 H, PhCH2), 4.34 (br s, 1 H, 4I−OH), 4.33−4.29 (m, 1 H, H-
5II), 4.22 (d, 1 H, J = 3.7 Hz, 2II−OH), 4.18−4.12 (m, 2 H, H-6IIa,b),
4.07−4.04 (m, 1 H, H-3I), 3.95−3.90 (m, 2 H, H-6Ib, H-1′b), 3.81 (m,
1 H, H-2I), 3.78 (m, 1 H, H-2II), 3.76−3.71 (m, 2 H, H-6Ia, H-1′a),
3.70−3.60 (m, 8 H, H-2′, H-3′, H-4′, H-5′), 3.56−3.52 (m, 2 H, H-4I,
H-5I), 3.40 (t, 2 H, J = 5.0 Hz, H-6′), 2.13, 2.00, 1.96 (3 s, 9 H, 3
COCH3). 13C NMR (150 MHz, acetone-d6): δ = 170.8, 170.6, 170.4
(3 OCOCH3), 162.8 (NCOCCl3), 139.9 (ipso Ph), 129.1−128.1 (Ar),
104.7 (C-1II), 101.4 (C-1I), 93.9 (CCl3), 85.6 (C-3I), 76.5 (C-5I), 73.8
(PhCH2), 73.6 (C-3II), 71.8 (C-5II), 71.3, 71.1, 71.0 (C-2′, C-3′, C-4′),
70.7 (C-5′), 70.5 (C-6I), 70.3 (C-4I), 69.5 (C-2II), 69.4 (C-1′), 68.2
(C-4II), 62.5 (C-6II), 57.6 (C-2I), 51.4 (C-6′), 20.7, 20.6, 20,5 (3
OCOCH3). HRMS (ESI-TOF): m/z [M + NH4]+ calcd for
C33H49Cl3N5O16 876.2240, found 876.2241. Anal. Calcd for
C33H45Cl3N4O16: C, 46.08; H, 5.27; N, 6.51. Found: C, 46.15; H,
5.30; N, 6.51.
20
1
[α]D = −11.0 (c 0.7, CHCl3). H NMR (600 MHz, CDCl3): δ =
7.50−7.37 (m, 5 H, Ph), 7.13 (d, 1 H, J2,NH = 7.8 Hz, NH), 5.56 (s, 1
H, PhCH), 5.34 (d, 1 H, J3,4 = 2.9 Hz, H-4II), 5.22 (dd, 1 H, J1,2 = 8.1
Hz, J2,3 = 10.4 Hz, H-2II), 5.06 (d, 1 H, J1,2 = 8.2 Hz, H-1I), 4.96 (dd, 1
H, J2,3 = 10.5 Hz, J3,4 = 3.4 Hz, H-3II), 4.74 (d, 1 H, J1,2 = 8.0 Hz, H-
1II), 4.47 (t, 1 H, J = 9.5 Hz, H-3I), 4.35 (dd, 1 H, J = 4.8, J = 10.6 Hz,
H-6Ib), 4.12 (dd, 1 H, J = 7.3, J = 11.0 Hz, H-6II ), 4.03 (dd, 1 H, J =
b
6.4, J = 11.2 Hz, H-6II ), 3.93−3.89 (m, 1 H, H-1′b), 3.83−3.80 (m, 2
a
H, H-6Ia, H-1′a), 3.77 (t, 1 H, J = 6.9 Hz, H-5II), 3.73−3.68 (m, 3 H,
CH2Br, H-4I), 3.67−3.58 (m, 9 H, H-2′, H-3′, H-4′, H-5′, H-2I),
3.53−3.49 (m, 1 H, H-5I), 3.41 (t, 2 H, J = 5.0 Hz, H-6′), 2.12, 2.01,
1.97 (3 s, 9 H, 3 COCH3). 13C NMR (150 MHz, CDCl3): δ = 170.3,
170.1, 170.0 (3 OCOCH3), 166.2 (COCH2Br), 161.9 (NCOCCl3),
137.0 (ipso Ph), 129.2−126.0 (Ar), 101.2 (PhCH), 99.8 (C-1I), 99.4
(C-1II), 92.5 (CCl3), 78.8 (C-4I), 76.3 (C-3I), 70.8, 70.62, 70.5 (C-2′,
C-3′, C-4′), 70.7 (C-3II), 70.65 (C-5II), 70.2 (C-2II), 70.0 (C-5′), 68.7
(C-1′), 68.5 (C-6I), 66.8 (C-4II), 66.2 (C-5I), 61.0 (C-6II), 58.5 (C-2I),
50.6 (C-6′), 25.3 (CH2Br), 20.59, 20.57, 20.56 (3 OCOCH3). HRMS
(ESI-TOF): m/z [M + NH4]+ calcd for C35H48BrCl3N5O17 994.1294,
found 994.1298. Anal. Calcd for C35H44BrCl3N4O17: C, 42.94; H, 4.53;
N, 5.72. Found: C, 43.01; H, 4.56; N, 5.63.
8-Azido-3,6-dioxaoctyl 2,4-Di-O-benzyl-3,6-dideoxy-α-L-xylo-hex-
opyranosyl-(1→4)-[2,4-di-O-benzyl-3,6-dideoxy-α-L-xylo-hexopyra-
nosyl-(1→2)-3,4,6-tri-O-acetyl-β-D-galactopyranosyl-(1→3)]-6-O-
benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (10).
Br2 (360 μL, 6.96 mmol) was added to a solution of ethyl 2,4-di-O-
benzyl-3,6-dideoxy-1-thio-β-L-xylo-hexopyranoside15 (1.13 g, 3.48
mmol) in CCl4 (20 mL). The mixture was shaken gently, and after
5 min, hex-1-ene (1.75 mL, 13.92 mmol) was added. After
concentration and coevaporation with CCl4 (3 × 10 mL), a solution
in dry CH2Cl2 (10 mL) of the crude α-colitosyl bromide 9 thus
obtained was added to a stirred mixture of 8 (0.50 g, 0.58 mmol),
Bu4NBr (1.13 g, 3.48 mmol), and powdered molecular sieves (4 Å, 4.5
8-Azido-3,6-dioxaoctyl 4,6-O-Benzylidene-2-deoxy-3-O-(3,4,6-tri-
O-acetyl-β-D-galactopyranosyl)-2-trichloroacetamido-β-D-gluco-
pyranoside (7). A solution of thiourea (1.1 g, 14.7 mmol) in methanol
(50 mL) was added at 0 °C to a stirred solution of 6 (4.8 g, 4.90
mmol) and sym-collidine (0.9 mL, 7.35 mmol) in CH2Cl2 (100 mL).
The mixture was stirred overnight at room temperature, when TLC
D
DOI: 10.1021/acs.joc.5b02105
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Brief Communication
g) in 2:1 CH2Cl2−DMF (30 mL). After stirring under argon
atmosphere for 48 h at room temperature, when TLC (6:1
toluene−acetone) indicated the glycosyl acceptor 8 was still present,
more Bu4NBr (0.75 g, 2.32 mmol) and freshly prepared bromide
donor 9 (2.32 mmol/10 mL CH2Cl2) were added into the reaction
mixture, and stirring was continued at room temperature. When TLC
(6:1 toluene−acetone) showed that all glycosyl acceptor 8 had been
consumed (48 h), the mixture was diluted with CH2Cl2 (25 mL) and
filtered through a Celite pad, and the solids were washed with CH2Cl2
(2 × 10 mL). The combined filtrate and washings were successively
washed with satd aq NaHCO3 (25 mL) and water (25 mL), dried, and
concentrated. Chromatography (6:1 chloroform−acetone) gave the
title tetrasaccharide 10 (0.71 g, 83%). [α]D20 = −23.6 (c 0.6, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 7.34−7.22 (m, 25 H, Ph), 7.09 (d, 1
H, J2,NH = 8.0 Hz, NH), 5.27 (d, 1 H, J3,4 = 2.9 Hz, H-4II), 5.22 (d, 1
H, J1,2 = 3.0 Hz, H-1IV), 5.07 (d, 1 H, J1,2 = 3.3 Hz, H-1III), 4.93 (d,
partial overlap, 1 H, J1,2 = 7.0 Hz, H-1I), 4.90 (dd, partial overlap, 1 H,
J2,3 = 10.1, J3,4 = 3.5 Hz, H-3II), 4.76 (d, 1 H, J1,2 = 7.9 Hz, H-1II),
4.66−4.62 (m, 1 H, H-5III), 4.57 (d, 1 H, 2J = 12.1 Hz, PhCHH), 4.56
(d, 1 H, 2J = 12.3 Hz, PhCHH), 4.55 (d, 1 H, 2J = 12.2 Hz, PhCHH),
and chromatography (3:1 toluene−acetone) gave 12 (260 mg, 69%)
and 13 (87 mg, 23%), in that order.
20
1
Data for 12. [α]D = −16.5 (c 0.5, CHCl3). H NMR (600 MHz,
CDCl3): δ = 7.38−7.23 (m, 25 H, Ph), 7.06 (d, 1 H, J2,NH = 7.1 Hz,
NH), 5.26 (d, 1 H, J1,2 = 3.0 Hz, H-1III), 5.07 (d, 1 H, J1,2 = 8.1 Hz, H-
1I), 5.04 (d, 1 H, J1,2 = 3.4 Hz, H-1IV), 4.76 (d, 1 H, J3,4 = 3.1 Hz, H-
4II), 4.72 (q, 1 H, J5,6 = 7.0 Hz, H-5IV), 4.68−4.61 (m, 2 H, CH2CCl3),
4.64 (d, 1 H, J1,2 = 7.9 Hz, H-1II), 4.60 (dd, partial overlap, 1 H, J2,3
=
8.2, J3,4 = 2.7 Hz, H-3I), 4.58−4.52 (m, 7 H, 5 x PhCHH, H-6IIa,b), 4.51
(d, 1 H, 2J = 11.6 Hz, PhCHH), 4.48 (d, 1 H, 2J = 11.7 Hz, PhCHH),
2
2
4.45 (d, 1 H, J = 12.2 Hz, PhCHH), 4.41 (d, 1 H, J = 12.1 Hz,
2
PhCHH), 4.36 (d, 1 H, J = 12.2 Hz, PhCHH), 4.28 (d, 1 H, J = 3.3
Hz, 3II−OH), 4.13 (q, 1 H, J5,6 = 7.3 Hz, H-5III), 3.98−3.95 (m, 2 H,
H-4I, H-2III), 3.93−3.87 (m, 4 H, H-6Ib, H-4IV, H-2IV, H-1′b), 3.83 (dd,
1 H, J1,2 = 8.0, J2,3 = 9.9 Hz, H-2II), 3.73−3.71 (m, 2 H, H-6Ia, H-1′a),
3.70−3.66 (m, 1 H, H-3II), 3.63−3.57 (m, 8 H, H-2′, H-3′, H-4′, H-
5′), 3.56−3.50 (m, 2 H, H-5I, H-2I), 3.48 (br s, 2 H, H-4III, H-5II), 3.34
(t, 2 H, J = 5.1 Hz, H-6′), 2.17 (dt, 1 H, J2,3 = J3,4 = 3.7, 2J = 13.1 Hz, 1
H, H-3IIIeq), 2.06 (dt, 1 H, J2,3 = J3,4 = 3.7, 2J = 12.6 Hz, 1 H, H-3IVeq),
1.85 (dt, 1 H, J3,4 = 2.2, J2,3 = 2J = 12.7 Hz, 1 H, H-3IVax), 1.82 (dt, 1 H,
J3,4 = 2.3, J2,3 = 2J = 13.0 Hz, 1 H, H-3IIIax), 1.28 (d, 3 H, J5,6 = 6.5 Hz,
H-6IV), 1.22 (d, 3 H, J5,6 = 6.5 Hz, H-6III). 13C NMR (150 MHz,
CDCl3): δ = 161.3 (NCOCCl3), 139.5, 138.3, 138.2, 138.1, 136.8 (5
ipso Ph), 128.7−127.1 (Ar), 101.9 (C-1II), 98.9 (C-1III), 98.1 (C-1I),
96.7 (C-1IV), 94.9 (d, JC,P = 9.9 Hz, CH2CCl3), 92.6 (COCCl3), 78.3
(d, JC,P = 6.9 Hz, C-4II), 77.7 (C-2II), 76.9 (C-4IV), 76.8 (CH2CCl3),
75.8 (C-3I), 75.8 (C-5I), 75.2 (C-4III), 73.2 (PhCH2), 72.4 (PhCH2),
72.3 (C-4I), 72.1 (C-2III), 71.6 (d, JC,P = 7.1 Hz, C-3II), 71.4 (2
PhCH2), 71.1 (C-2IV), 70.9 (PhCH2), 70.8 (d, JC,P = 7.7 Hz, C-6II),
70.7, 70.6, 70.5, 70.0 (C-2′, C-3′, C-4′, C-5′), 68.6 (C-1′), 68.4 (C-
5III), 67.6 (C-6I), 66.5 (C-5IV), 66.2 (d, JC,P = 6.3 Hz, C-5II), 60.6 (C-
2I), 50.6 (C-6′), 27.5 (C-3IV), 26.9 (C-3III), 16.6 (C-6III), 16.4 (C-6IV).
31P NMR (162 MHz, CDCl3): δ = −10.62. HRMS (ESI-TOF): m/z
[M + Na]+ calcd for C69H83Cl6N4O21PNa 1567.3316, found
1567.3302.
2
2
4.52 (d, 1 H, J = 12.2 Hz, PhCHH), 4.46 (d, 1 H, J = 12.1 Hz,
PhCHH), 4.43 (d, 1 H, 2J = 12.2 Hz, PhCHH), 4.41−4.38 (m, 4 H, 3
x PhCHH, H-3I), 4.28 (d, 1 H, 2J = 12.4 Hz, PhCHH), 4.29−4.25 (m,
1 H, H-5IV), 4.02−3.95 (m, 4 H, H-4I, H-1′a, H-6IIa,b), 3.90 (dd, 1 H,
J1,2 = 8.1, J2,3 = 10.1 Hz, H-2II), 3.88−3.84 (m, 3 H, H-2III, H-2IV, H-
6Ia), 3.76 (t, 1 H, J = 6.9 Hz, H-5II), 3.74−3.68 (m, 2 H, H-1′b, H-6Ib),
3.65−3.55 (m, 10 H, H-2I, H-2′, H-3′, H-4′, H-5′, H-5I), 3.51 (br s, 1
H, H-4IV), 3.36 (t, 2 H, J = 5.1 Hz, H-6′), 3.33 (br s, 1 H, H-4III),
2.08−2.16 (m, 2 H, H-3IIIeq, H-3IVeq), 2.01, 1.81, 1.68 (3 s, 9 H, 3 x
COCH3), 1.73−1.81 (m, 2 H, H-3IIIax, H-3IVax), 1.24 (d, 3 H, J5,6 = 6.2
Hz, H-6IV), 1.20 (d, 3 H, J5,6 = 6.2 Hz, H-6III). 13C NMR (150 MHz,
CDCl3): δ = 170.4, 169.8, 169.7 (3 OCOCH3), 161.5 (NCOCCl3),
138.5, 138.4, 138.3, 138.1, 138.0 (5 ipso Ph), 128.4−127.3 (Ar), 101.7
(JC,H = 161.7 Hz, C-1II), 99.0 (JC,H = 165.0 Hz, C-1I), 97.3 (JC,H
=
171.3 Hz, C-1IV), 96.6 (JC,H = 172.1 Hz, C-1III), 92.4 (CCl3), 75.7 (C-
3I, C-4IV), 75.4 (C-4III), 75.2 (C-5I), 73.2 (PhCH2), 73.1 (C-3II), 72.8
(C-4I), 72.5 (C-2II), 71.4 (C-2III), 71.3 (PhCH2), 71.2 (PhCH2), 70.9
(C-2IV), 70.7, 70.6, 70.5 (C-2′, C-3′, C-4′), 70.4 (C-5II), 70.3
(PhCH2), 70.0 (C-5′), 68.4 (C-6I), 67.6 (C-1′), 67.4 (C-4II), 67.3 (C-
5IV), 66.0 (C-5III), 60.6 (C-6II), 59.3 (C-2I), 50.5 (C-6′), 26.7 (C-3IV),
26.4 (C-3III), 20.7, 20.6, 20.4 (3 x OCOCH3), 16.6 (C-6III), 16.3 (C-
6IV). HRMS (ESI-TOF): m/z [M + NH4]+ calcd for C73H93Cl3N5O22
1496.5372, found 1496.5388. Anal. Calcd for C73H89Cl3N4O22: C,
59.21; H, 6.06; N, 3.78. Found: C, 59.44; H, 6.17; N, 3.69.
20
1
Data for 13. [α]D = −8.7 (c 0.5, CHCl3). H NMR (600 MHz,
CDCl3): δ = 7.35−7.24 (m, 25 H, Ph), 7.16 (d, 1 H, J2,NH = 7.1 Hz,
NH), 5.24 (d, 1 H, J1,2 = 2.8 Hz, H-1III), 5.08 (d, 1 H, J1,2 = 8.0 Hz, H-
1I), 5.06 (d, 1 H, J1,2 = 3.4 Hz, H-1IV), 4.90 (d, 1 H, J3,4 = 3.2 Hz, H-
4II), 4.71−4.66 (m, 2 H, H-5IV, H-6II ), 4.65−4.56 (m, 2 H, CH2CCl3),
b
4.63 (d, 1 H, J1,2 = 7.8 Hz, H-1II), 4.58 (d, 1 H, 2J = 12.2 Hz, PhCHH),
4.54−4.39 (m, 9 H, 7 x PhCHH, H-3I, H-6II ), 4.38 (d, 1 H, 2J = 11.8
a
Hz, PhCHH), 4.36 (d, 1 H, 2J = 11.8 Hz, PhCHH), 4.27 (d, 1 H, J =
3.9 Hz, 3II−OH), 4.13 (q, 1 H, J5,6 = 7.4 Hz, H-5III), 4.00 (t, 1 H, J3,4
=
J4,5 = 8.8 Hz, H-4I), 3.95−3.92 (m, 1 H, H-2III), 3.91−3.86 (m, 2 H, H-
8-Azido-3,6-dioxaoctyl 2,4-Di-O-benzyl-3,6-dideoxy-α-L-xylo-hex-
opyranosyl-(1→4)-[2,4-di-O-benzyl-3,6-dideoxy-α-L-xylo-hexopyra-
nosyl-(1→2)-β-D-galactopyranosyl-(S)-(P)-4,6-cyclic 2,2,2-trichlor-
oethyl phosphate-(1→3)]-6-O-benzyl-2-deoxy-2-trichloroacetami-
do-β-D-glucopyranoside (12) and 8-Azido-3,6-dioxaoctyl 2,4-Di-O-
benzyl-3,6-dideoxy-α-L-xylo-hexopyranosyl-(1→4)-[2,4-di-O-benzyl-
3,6-dideoxy-α-L-xylo-hexopyranosyl-(1→2)-β-D-galactopyranosyl-
(R)-(P)-4,6-cyclic 2,2,2-trichloroethyl phosphate-(1→3)]-6-O-benzyl-
2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (13). A solution
of sodium methoxide in methanol (1 M, ∼ 1.2 mL) was added under
argon to a solution of 10 (400 mg, 0.27 mmol) in 1:5 CH2Cl2−MeOH
(30 mL), and the mixture was stirred at room temperature for 2 h. The
mixture was neutralized with Amberlite IR-120 (H+) resin and filtered,
and the solids were washed with MeOH (2 × 10 mL). The filtrate was
concentrated and coevaporated with toluene (twice) to give
compound 11 as an amorphous solid in almost theoretical yield.
HRMS (ESI-TOF): m/z [M + NH4]+ calcd for C67H87Cl3N5O19
1370.5055, found 1370.5061.
To a solution of triol 11 (330 mg, 0.24 mmol) and pyridine (200
μL, 2.44 mmol) in CH2Cl2 (5 mL) was added 2,2,2-trichloroethyl
phosphorodichloridate (57 μL, 0.36 mmol) dropwise at −20 °C.
When TLC (∼20 min, 4:1 toluene−acetone) indicated complete
conversion of 11, excess of reagent was destroyed by addition of
MeOH (400 μL). The mixture was concentrated, and EtOAc (3 mL)
was added to the residue. The precipitate was filtered off and washed
with EtOAc (2 × 2 mL). The combined filtrates were concentrated,
6Ib, H-1′b), 3.85−3.83 (m, 1 H, H-2IV), 3.79 (dd, 1 H, J1,2 = 7.8, J2,3
=
9.9 Hz, H-2II), 3.75−3.72 (m, 1 H, H-3II), 3.71−3.66 (m, 2 H, H-6Ia,
H-1′a), 3.62−3.57 (m, 11 H, H-4IV, H-2′, H-3′, H-4′, H-5′, H-5I, H-
2I), 3.52 (br s, 1 H, H-5II), 3.46 (br s, 1 H, H-4III), 3.34 (t, 2 H, J = 5.0
2
Hz, H-6′), 2.16 (dt, 1 H, J2,3 = J3,4 = 3.1, J = 12.2 Hz, 1 H, H-3IVeq),
2.12 (dt, 1 H, J2,3 = J3,4 = 3.6, 2J = 12.7 Hz, 1 H, H-3IIIeq), 1.85 (dt, 1 H,
J3,4 = 2.2, J2,3 = 2J = 12.8 Hz, 1 H, H-3IIIax), 1.82 (dt, 1 H, J3,4 = 2.2, J2,3
= 2J = 12.7 Hz, 1 H, H-3IVax), 1.26 (d, 3 H, J5,6 = 6.3 Hz, H-6IV), 1.20
(d, 3 H, J5,6 = 6.5 Hz, H-6III). 13C NMR (150 MHz, CDCl3): δ = 161.4
(NCOCCl3), 138.9, 138.2, 138.1, 138.0, 137.1 (5 ipso Ph), 130.9−
127.4 (Ar), 102.1 (C-1II), 98.7 (C-1III), 98.2 (C-1I), 96.2 (C-1IV), 94.5
(d, JC,P = 8.2 Hz, CH2CCl3), 92.6 (COCCl3), 77.6 (d, JC,P = 2.2 Hz,
CH2CCl3), 78.3 (C-4II, C-2II, C-3I), 76.5 (C-4IV), 75.3 (C-5I), 75.2 (C-
4III), 73.3 (PhCH2), 72.3 (C-4I), 71.8 (d, partial overlap JC,P = 7.5 Hz,
C-3II), 71.7 (C-2III), 71.3 (PhCH2), 71.2 (PhCH2), 71.1 (PhCH2), 71.0
(PhCH2), 70.8 (C-2IV), 70.7, 70.6, 70.5, 70.0 (C-2′, C-3′, C-4′, C-5′),
70.2 (d, JC,P = 4.4 Hz, C-6II), 68.6 (C-1′), 68.4 (C-5III), 67.9 (C-6I),
67.1 (d, JC,P = 5.6 Hz, C-5II), 66.4 (C-5IV), 60.1 (C-2I), 50.6 (C-6′),
27.0 (C-3III, C-3IV), 16.5 (C-6III), 16.4 (C-6IV). 31P NMR (162 MHz,
CDCl3): δ = −1.88. HRMS (ESI-TOF): m/z [M + NH4]+ calcd for
C69H87Cl6N5O21P 1562.3756, found 1562.3749.
8-Amino-3,6-dioxaoctyl 3,6-Dideoxy-α-L-xylo-hexopyranosyl-
(1→4)-[3,6-dideoxy-α-L-xylo-hexopyranosyl-(1→2)-β-D-galactopyr-
anosyl-4,6-cyclic phosphate-(1→3)]-2-deoxy-2-acetamido-β-D-glu-
copyranoside (14). (a) A mixture of 12 (150 mg, 0.11 mmol) and Pd/
E
DOI: 10.1021/acs.joc.5b02105
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Brief Communication
C (150 mg) in a mixture of MeOH (6 mL) and 0.1 M potassium
phosphate buffer (6 mL; pH = 7) was stirred under H2 (1 atm) at
room temperature. After 5 days, when TLC (2:1 i-PrOH−30%
NH4OH) showed complete conversion of the starting material into a
more polar product, the mixture was filtered through a Celite pad, the
catalyst was washed with H2O (2 × 5 mL), and the filtrate was
concentrated. Chromatography (2:1:0.1 i-PrOH−H2O−30%
NH4OH) followed by lyophilization afforded the desired tetrasacchar-
ide 14 (69 mg, 85%). 1H NMR (600 MHz, D2O): δ = 4.94 (d, 1 H, J1,2
B.; Kovac
273, 2777−2783.
(5) Liao, X.; Poirot, E.; Chang, A. H. C.; Zhang, X.; Zhang, J.; Nato,
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,
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P.; Fournier, J. M.; Glaudemans, C. P. J. J. Biol. Chem. 1998,
F.; Fournier, J.-M.; Kovac
2002, 337, 2437−2442.
́ ̌
, P.; Glaudemans, C. P. J. Carbohydr. Res.
(6) Turek, D.; Sundgren, A.; Lahmann, M.; Oscarson, S. Org. Biomol.
Chem. 2006, 4, 1236−1241.
́ ̌
(7) Ruttens, B.; Kovac, P. Carbohydr. Res. 2006, 341, 1077−1080.
(8) McNaught, A. D. Carbohydr. Res. 1997, 297, 1−92.
(9) Soliman, S. E.; Kovac P. J. Org. Chem. 2015, 80, 4851−4860.
(10) Chittenden, G. J. F. Carbohydr. Res. 1988, 183, 140−143.
(11) Soliman, S. E.; Kovac P. Synthesis 2014, 46, 748−751.
(12) Soliman, S. E.; Zoeisha, C.; Kovac P. In Carbohydrate Chemistry:
Proven Synthetic Methods; Kovac P., Ed.; CRC/Taylor and Francis:
Boca Raton, 2014.
= 3.7 Hz, H-1III), 4.83 (d, 1 H, J1,2 = 3.5 Hz, H-1IV), 4.73 (q, 1 H, J5,6
6.9 Hz, H-5III), 4.64 (d, 1 H, J1,2 = 7.8 Hz, H-1II), 4.50 (d, 1 H, J3,4
=
=
́ ̌
,
3.7 Hz, H-4II), 4.35 (d, partial overlap, 1 H, J1,2 = 8.8 Hz, H-1I), 4.33−
4.27 (m, 2 H, H-6II a,b), 4.25−4.22 (m, 1 H, H-5III), 4.13 (br t, 1 H, J =
3.1 Hz, H-4IV), 3.84 (t, 1 H, J2,3 = J3,4 = 9.8 Hz, H-3I), 3.95−3.89 (m, 4
H, H-2IV, H-2III, H-6Ib, H-1′b), 3.84−3.79 (m, 2 H, H-3II, H-6Ia), 3.77
(dd, 1 H, J1,2 = 8.8, J 2,3 = 10.4 Hz, H-2I), 3.72 (br t, 1 H, J = 3.3 Hz,
H-4III), 3.66−3.64 (m, 4 H, H-4I, H-1′b, H-5′), 3.62−3.58 (m, 6 H, H-
2′, H-3′, H-4′), 3.58−3.56 (m, 1 H, H-5I), 3.55 (dd, partial overlap, 1
H, J1,2 = 7.9, J2,3 = 9.5 Hz, H-2II), 3.44 (dt, 1 H, J = 3.1, J = 9.9 Hz, H-
́ ̌
,
́ ̌
,
́ ̌
,
(13) Sherman, A. A.; Yudina, O. N.; Mironov, Y. V.; Sukhova, E. V.;
Shashkov, A. S.; Menshov, V. M.; Nifantiev, N. E. Carbohydr. Res.
2001, 336, 13−46.
(14) Kovac
298.
́ ̌
, P.; Glaudemans, C. P. J. Carbohydr. Res. 1985, 140, 289−
5I), 3.08 (t, 2 H, J = 5.1 Hz, H-6′), 2.01−1.98 (dt, partial overlap, 1 H,
2
J3,4 = 2.9, J = J = 12.9 Hz, H-3IVax), 1.97 (s, 3 H, COCH3), 1.84−
2,3
(15) Garegg, P. J.; Hultberg, H.; Wallin, S. Carbohydr. Res. 1982, 108,
97−101.
1.80 (m, 2 H, H-3III), 1.81−1.78 (dt, partial overlap, 1 H, J3,4 = 3.4, J2,3
= 2J = 12.9 Hz, H-3IVeq), 1.14 (d, 6 H, J 5,6 = 6.6 Hz, H-6III, H-6IV). 13
C
(16) Lemieux, R. U.; Hendriks, K. B.; Stick, R. V.; James, K. J. Am.
Chem. Soc. 1975, 97, 4056−4062.
NMR (150 MHz, D2O): δ = 173.6 (COCH3), 101.7 (C-1I), 100.9 (C-
1II), 99.4 (C-1III), 97.7 (C-1IV), 76.3 (d, JC,P = 4.6 Hz, C-4II), 76.1 (C-
2II), 75.5 (C-5I), 75.3 (C-3I), 72.5 (C-4I), 72.3 (d, JC,P = 7.4 Hz, C-3II),
69.8, 69.7, 69.5 (C-2′, C-3′, C-4′), 69.1 (C-1′), 68.7 (C-4III), 68.6 (d,
JC,P = 5.0 Hz, C-6II), 68.3 (C-4IV), 67.3 (d, JC,P = 4.6 Hz, C-5II), 67.1
(C-5′), 66.6 (C-5IV), 66.0 (C-5III), 63.5 (C-2III), 63.3 (C-2IV), 59.5 (C-
6I), 55.6 (C-2I), 39.2 (C-6′), 32.7 (C-3III), 32.5 (C-3IV), 22.1
(COCH3), 15.5, 15.3 (C-6III, C-6IV). 31P NMR (162 MHz, D2O): δ
= −3.74 (3JP,H 21.6 Hz). HRMS (ESI-TOF): m/z [M − H]− calcd for
C32H56N2O21P 835.3113, found 835.3109.
́ ̌
(17) Ruttens, B.; Kovac, P. Synthesis 2004, 2004, 2505−2508.
(18) Shashkov, A. S.; Chizhov, O. S. Bioorg. Khim. 1976, 2, 437−496.
(19) Ruttens, B.; Kovac P. Helv. Chim. Acta 2006, 89, 320−332.
(20) Ruttens, B.; Saksena, R.; Kovac P. Eur. J. Org. Chem. 2007,
́ ̌
,
́ ̌
,
2007, 4366−4375.
(21) Knirel, Y. A.; Paredes, L.; Jansson, P.-E.; Weintraub, A.;
Widmalm, G.; Albert, M. J. Eur. J. Biochem. 1995, 232, 391−396.
(b) Compound 13 (120 mg, 0.08 mmol) when treated with Pd/C
(120 mg) and worked up, as described for 12, afforded 14 (53 mg,
81%), which was in all aspects identical with the compound described
above.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.5b02105.
1H and 13C NMR spectra for all new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: kpn@helix.nih.gov.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Intramural Research
Program of the National Institutes of Health (NIH) and the
National Institute of Diabetes and Digestive and Kidney
Diseases (NIDDK).
REFERENCES
■
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DOI: 10.1021/acs.joc.5b02105
J. Org. Chem. XXXX, XXX, XXX−XXX