Synthesis of oligo(α- d -glycosyl phosphate) derivatives by a phosphoramidite method via boranophosphate intermediates

Article abstract of DOI:10.1021/jo102584g

An efficient method for the synthesis of short oligo(α-d-glycosyl boranophosphate) derivatives by using an α-d-glycosyl phosphoramidite as a monomer unit was developed. The synthesis of oligomers was carried out by repeating a cycle consisting of the cond

Full text of DOI:10.1021/jo102584g

ARTICLE
pubs.acs.org/joc
Synthesis of Oligo(r-D-glycosyl phosphate) Derivatives by a
Phosphoramidite Method via Boranophosphate Intermediates
Shoichi Fujita, Natsuhisa Oka, Fumiko Matsumura, and Takeshi Wada*
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bioscience Building 702,
5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
S Supporting Information
b
ABSTRACT: An efficient method for the synthesis of short
oligo(R-D-glycosyl boranophosphate) derivatives by using an R-
D-glycosyl phosphoramidite as a monomer unit was developed.
The synthesis of oligomers was carried out by repeating a cycle
consisting of the condensation of the monomer unit with a
terminal hydroxy group of carbohydrates, boronation of the
resultant phosphite intermediates, and terminal deprotection.
The phosphoramidite monomer unit was synthesized from the
corresponding glycosyl iodide and methyl N,N-diisopropylpho-
sphonamidate in a highly R-selective manner. Di- and tri(R-D-
glycosyl boranophosphate) derivatives obtained by the synthetic cycle were converted into the corresponding H-phosphonate
diester derivatives, which were then used to synthesize di- and tri(R-D-glycosyl phosphate) derivatives including a fragment of
Leishmania glycocalyx lipophosphoglycans.
’ INTRODUCTION
on the synthesis of disaccharide phosphates have been reported
to date,4a,8 and the synthesis of more complex molecules has not
been achieved. It is attributed to the following reasons: (1) a
versatile method to synthesize diastereopure R- or β-glycosyl
phosphoramidites has not been developed, and (2) glycosyl
phosphite and phosphate triester intermediates, which are gener-
ated by condensation of the glycosyl phosphoramidites with other
saccharides and subsequent oxidation, are prone to decomposition
by acid activators (e.g., 1H-tetrazole), neighboring group partici-
pation, and/or during chromatographic purification.3,11
Very recently, we have developed a method to synthesize
glycosyl phosphites and phosphoramidites in a highly R-selective
manner.7a This method would solve the first problem in applying
the phosphoramidite method to the synthesis of phosphogly-
cans. It should be noted that most of the phosphoglycans in
nature consist of R-glycosyl phosphates.3 We have also found
that glycosyl phosphite triester intermediates can be temporarily
protected by boronation of the phosphorus atom instead of
oxidation and that the resultant glycosyl boranophosphate triesters
can be converted into the corresponding glycosyl phosphate diesters
as well as some other P-modified analogues, such as phosphorothi
oates.7b,c,12 The boranophosphate intermediates are chemically
stable under various reaction conditions for the synthesis of
phosphoglycans, and the new strategy utilizing boranophosphates
would solve the second problem. In this paper, we report the
development of an efficient method to synthesize phosphoglycans
by using an R-glycosyl phosphoramidite monomers and glycosyl
Natural phosphoglycans containing glycosyl phosphate units
are found in the cell wall or capsule of bacteria and the glycocalyx
of protozoan parasites, such as Neisseria meningitidis, Streptococcus
pneumoniae, and Leishmania, and work asantigenicdeterminants.1,2
Therefore, structural and functional studies of these biomole-
cules are important to develop therapeutics against these patho-
genic organisms.1,2 Since chemically synthesized phosphoglycans
and their analogues are quite useful for these studies, a variety of
studies have been conducted to develop an efficient method to
synthesize glycosyl phosphate derivatives and phosphoglycans.3
Currently, these molecules, particularly those containing multi-
ple glycosyl phosphate units, are usually synthesized by the H-
phosphonate method.2a,3ꢀ6 For example, phosphoglycans con-
taining two to four glycosyl phosphate repeating units have been
synthesized by this method.2a,3ꢀ5 However, it has been reported
that the synthesis of phosphoglycans containing more glycosyl
phosphate units is still difficult.5 It may be due to the instability of
glycosyl H-phosphonodiester intermediates and/or the existence
of polar and nucleophilic PꢀOꢀ groups of glycosyl phospho-
diester intermediates, which may cause some side reactions or
difficulty in chromatographic purification.
With this background, we have sought to develop an efficient
method to synthesize phosphoglycans by a different approach
and focused on glycosyl phosphoramidites4a,7a,8 and borano
phosphates.4d,7b,7c,9 Phosphoramidites are by far the most fre-
quently used compounds to synthesize phosphate-containing
biomolecules, especially oligonucleotides (phosphoramidite
method).10 In contrast, the phosphoramidite method has been
scarcely used to synthesize phosphoglycans. Only a few studies
Received: December 29, 2010
Published: March 08, 2011
r
2011 American Chemical Society
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Scheme 1. Stereoselective Synthesis of R-D-Glucosyl Bora-
nophosphate Derivatives 4ꢀ6 and an Attempt To Synthesize
Boranophosphotriester-Linked Disaccharide 7 via Conden-
sation Reaction
Scheme 2. Plausible Mechanism for Generation of 8 and 9
Scheme 3. Stereoselective Synthesis of R-D-Glucosyl Phos-
phoramidite 10
phosphate diesters. We have also reported that the reaction of
glycosyl iodides with an H-phosphonate diester afforded glycosyl
phosphites in a highly R-selective manner.7a The glycosyl
phosphites can be converted into the boranophosphate deriva-
tives by P-boronation. By combining these methods, we tried to
synthesize an R-boranophosphotriester-linked disaccharide.
As shown in Scheme 1, dimethyl 2,3,6-tri-O-benzyl-4-O-(tert-
butyldimethylsilyl)-R-D-glucopyranosyl boranophosphate 4 was
synthesized by using a glycosyl iodide 2, dimethyl H-phospho-
nate, and 1,8-bis(dimethylamino)naphthalene (DMAN) accord-
ing to our recent procedure.7a The R-isomer was obtained R-
selectively (R:β = 92:8). One of the two methyl groups of 4 was
then removed by treatment with PhSH and triethylamine to give
methyl 2,3,6-tri-O-Bn-4-O-TBDMS-R-D-glucopyranosyl bora-
nophosphate diester 5. The TBDMS group of 4 was also removed
by treatment with tetrabutylammonium fluoride (TBAF) to give
dimethyl 2,3,6-tri-O-Bn-R-D-glucopyranosyl boranophosphate 6.
Then, we attempted to condense these two building blocks (5, 6)
under the same conditions as we used to synthesize β-D-glucopyr-
anosyl boranophosphate derivatives with bis(2-oxo-3-oxazolidi-
nyl)phosphinic chloride (Bop-Cl) as a condensing reagent,
3-nitro-1,2,4-triazole (NT) as a nucleophilic catalyst, and i-Pr2NEt
(boranophosphotriester method).7b However, the desired bora-
nophosphotriester-linked disaccharide 7 was not obtained, but the
formation of a glucosyl chloride 8 and a glucosyl nitrotriazolide 9
boranophosphate intermediates. An attempt to use glycosyl
boranophosphates as monomers is also described.
1
was observed by H NMR and MALDI-TOF MS. The use of
’ RESULTS AND DISCUSSION
3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium hexa-
fluorophosphate (PyNTP) as a condensing reagent7c gave a
similar result. All the attempts to improve the result by changing
the reaction conditions resulted in failure. The results were
completely opposite from those previously obtained with β-D-
glucopyranosyl boranophosphate derivatives,7b,c indicating that
the anomeric configuration and/or the protecting groups of
substrates were important factors in the boranophosphotriester
method. Althoughthedetailed reaction mechanismisnot clear yet,
the formation of the byproducts 8 and 9 indicates that an
First, we describe our attempt to synthesize disaccharide
phosphate derivatives by using an R-glycosyl boranophosphate
derivative as a monomer unit (Scheme 1). We have recently
reported that a β-D-glucopyranosyl boranophosphate diester and
an R-D-mannopyranosyl boranophosphate diester underwent
smooth condensation reactions to give the corresponding dis-
accharide boranophosphates and some other glycosyl borano-
phosphate derivatives in excellent yields.7b,c The resultant
boranophosphates can be converted into the corresponding
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Scheme 4. Synthesis of Oligo(R-D-glucosyl boranophosphate) Derivatives by the Phosphoramidite Method
Scheme 5. Synthesis of Oligo(R-D-glucosyl phosphate) Derivatives from Boranophosphate Counterparts
oxocarbenium ion may be generated13 by the elimination of the
activated boranophosphate moiety (Scheme 2). The active inter-
mediates generated from 5 must be less reactive to the hydroxy
group of 6 than the β-isomeric counterparts due to the steric
hindrance of the glucopyranose moiety. The 4-O-TBDMS group
may also increase the steric hindrance. Furthermore, the oxocarbe-
nium ion is generated more easily in the case of per-O-Bn glycosyl
phosphate derivatives compared to their per-O-acyl counterparts,13
which we successfully used in the boranophosphotriester method
before.7b,c We speculate that the results shown in Scheme 1 reflect
the combined effect of these factors.
Given these results, we turned our attention to the phosphor-
amidite method to synthesize R-glycosyl boranophosphotriester-
linked phosphoglycans. The requisite R-glycosyl phosphorami-
dite monomer 10 can be synthesized by the same R-selective
phosphitylation of the glucosyl iodide 2 (Scheme 3).7a The
reaction was also almost completely R-selective (R:β = 98:2).
Condensation of 10 with a glucose derivative 11 was performed
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Scheme 6. Synthesis of R-D-Glc-(1-P-6)-D-Man 35 by the Phosphoramidite Method
in the presence of 1H-tetrazole as an activating agent (Scheme 4).
The reaction was completed within 25 min at rt. Subsequently,
the phosphite intermediate 12 was in situ boronated by treatment
MS (calcd for C83H91NO21P2Na [M ꢀ 2H þ Na]ꢀ 1522.5462,
found 1522.5399)], though the aminoglycoside was easily hydro-
lyzed by treatment with ammonium acetate buffer (pH 4.6) to give
the desired product 26.14 All the Bn groups were then removed by
catalytic hydrogenolysis to give the desired phosphodiester-linked
fully deprotected disaccharide 27 and trisaccharide 28.
Finally, we applied the method to the synthesis of R-D-Glc-
(1-P-6)-D-Man 35, a fragment of Leishmania glycocalyx lipopho-
sphoglycans (Scheme 6).2b,3 The synthesis of the boranopho-
sphotriester-linked disaccharide 30 by condensation of the
phosphoramidite 10, subsequent conversion to the phosphodie-
ster-linked disaccharide 33 via the H-phosphonate diester inter-
mediate 32, and deprotection was performed according to the
same procedure without significant side reactions to give 35 in
good total yield.
with BH3 THF. A 31P NMR analysis showed that these reactions
3
proceeded without any observable side reactions. The desired
disaccharide boranophosphate 13 was isolated in good yield.
For a stepwise synthesis of a phosphoglycan chain, a terminal
protecting group must be selectively removed. In the case of 13,
the 4-O-TBDMS group was selectively removed to give 14, which
was then coupled with the phosphoramidite monomer 10 again.
Subsequent P-boronation of the phosphite intermediate gave the
boranophosphotriester-linked trisaccharide 15. Thus, we demon-
strated that the phosphoramidite method was applicable to a
stepwise chain elongation of oligo(R-glycosyl boranophosphate)
derivatives.
Next, we attempted to convert the boranophosphotriester-
linked disaccharide 13 and trisaccharide 15 into the phosphodie-
ster-linked counterparts according to the method we recently
developed (Scheme 5).7b,c First, the methyl group on the bor-
anophosphotriester of 13 and 15 was removed by treatment with
PhSH and Et3N to give the boranophosphodiesters 16 and 17.
The compounds 16 and 17 were then treated with 4,40-dimethox-
ytrityl (DMTr) cation generated in situ from DMTrOMe and
dichloroacetic acid (DCA) to convert the boranophosphodiester-
linked disaccharides into the H-phosphonodiester-linked 18 and
19. A 31P NMR analysis showed that the conversion was virtually
quantitative. The use of trityl (Tr) cation generated from TrOMe
gave the same results. The resultant H-phosphonate diesters were
oxidized in situ with I2ꢀH2Oꢀpyridine (Py) to give the desired
phosphodiester-linked disaccharide 20 and trisaccharide 21. In
addition, thiophosphodiester-linked disaccharide 22 was synthe-
sized by the sulfurization of 18. The TBDMS groups of 20 and 21
’ CONCLUSION
In conclusion, the present study showed that the phosphor-
amidite method can be applicable to the synthesis of short
oligo(R-glycosyl phosphate) derivatives via the boranopho-
sphate intermediates. To the best of our knowledge, this is the
first report on the synthesis of oligo(glycosyl phosphate) deri-
vatives longer than disaccharide phosphates by using the phos-
phoramidite method. The high efficiency of the chain elongation
by this method owing to the reactivity of the phosphoramidite
monomers would be advantageous for the synthesis of biomo-
lecules consisting of multiple glycosyl phosphate units.
’ EXPERIMENTAL SECTION
2,3,6-Tri-O-Bn-4-O-TBDMS-D-glucopyranosyl Acetate (1).
2,3,6-Tri-O-Bn-4-O-TBDMS-R-D-glucopyranose15 (8.20 g, 14.5 mmol)
was dried by repeated coevaporation with dry Py and dissolved in dry Py
(29.5 mL) under Ar. Acetic anhydride (2.80 mL, 29.6 mmol) was added
to the solution at 0 °C and the mixture was allowed to stir for 19 h at rt.
A saturated NaHCO3 aqueous solution (30 mL) and AcOEt (30 mL)
were then added successively to the mixture. The organic layer was sepa-
rated, washed with saturated NaHCO3 aqueous solutions (2 ꢁ 50 mL),
were removed by treatment with Et3N 3HF at 40 °C to give 23
3
and 24. The anomeric acetyl group of the disaccharide 23 was
successfully removed by treatment with Me2NH to give 25 in
excellent yield. In contrast, the treatment of 24 with Me2NH
afforded a mixture of the desired 26 and an N,N-dimethylamino-
1
glycoside [analyzed by H NMR, 13C NMR, and MALDI-TOF
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dried over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography using
hexaneꢀAcOEt (5:2, v/v) as an eluent to give 1 as a colorless oil (8.00 g,
13.2 mmol, 91%). 1H NMR (CDCl3, 300 MHz): δ 7.37ꢀ7.18 (m, 15H,
CH2Ph), 6.43 (d, J = 3.0 Hz, R-H-1), 5.71 (d, J = 8.0 Hz, β-H-1),
5.11ꢀ4.49 (m, 6H, CH2Ph), 3.87ꢀ3.51 (m, 6H, H-2, H-3, H-4, H-5,
H-6), 2.17 (s, R-OCOCH3), 2.04 (s, β-OCOCH3), 0.87ꢀ0.82 (m, 9H,
SiC(CH3)3), 0.05ꢀ0.02 (m, 6H, Si(CH3)2). 13C NMR (CDCl3, 75.5
MHz): δ 169.4, 169.1 (OCOCH3), 138.9, 138.6, 138.1, 138.0, 137.9,
137.3 (C, Ar), 128.3, 128.2, 128.1, 128.0, 127.8, 127.6, 127.5, 127.4,
127.3, 127.1, 127.0, 126.8 (CH, Ar), 93.9 (β-C-1), 89.6 (R-C-1), 84.6
(CH), 81.5 (CH), 81.0 (CH), 79.4 (CH), 77.2 (CH), 76.9 (CH), 74.7
(CH2Ph), 74.7 (CH2Ph), 74.6 (CH2Ph), 74.2 (CH), 73.2 (CH2Ph),
73.1 (CH2Ph), 72.9 (CH2Ph), 70.2 (CH), 69.9 (CH), 68.5 (C-6), 25.9,
25.8 (SiC(CH3)3), 21.1, 21.0 (OCOCH3), 18.0, 17.9 (SiC(CH3)3),
ꢀ3.86, ꢀ3.90, ꢀ5.00, ꢀ5.10 (Si(CH3)2). HRMS (ESI): calcd for
C35H46O7SiK [M þ K]þ 645.2644, found 645.2647.
successively to the mixture. The organic layer was separated and washed
with 1 M TEAB aqueous solutions (2 ꢁ 5.0 mL). The aqueous layers
were combined and back-extracted with CHCl3 (5.0 mL). The organic
layers were combined, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography using CH2Cl2ꢀEt3NꢀMeOH (98:1:1ꢀ97:1:2, v/v/
v) as an eluent. The fractions containing 5 were collected and concen-
trated under reduced pressure. The residue was dissolved in CHCl3
(5.0 mL) and washed with a 1 M TEAB aqueous solution (5.0 mL). The
aqueous layer was back-extracted with CHCl3 (10 mL). The organic
layers were combined, dried over Na2SO4, filtered, and concentrated
under reduced pressure to give 5 as a yellow oil (146 mg, 0.19 mmol,
95%, R:β = 97:3). 1H NMR (CDCl3, 300 MHz): δ 7.35ꢀ7.18 (m, 15H,
CH2Ph), 5.93 (td, JHH = 3.3 Hz, JPH = 8.5 Hz, H-1), 5.09ꢀ4.47 (m, 6H,
CH2Ph), 4.07ꢀ3.55 (m, 9H, H-2, H-3, H-4, H-5, H-6, POCH3),
2.96ꢀ2.87 (q, 6H, NCH2CH3), 1.20 (t, 9H, NCH2CH3) 0.87ꢀ0.78
(m, 9H, SiC(CH3)3), 0.01 to ꢀ0.03 (m, 6H, Si(CH3)2). 13C NMR
(CDCl3, 75.5 MHz): δ 139.3, 139.2, 138.4, 138.3, 138.2 (C, Ar), 128.2,
128.1, 128.0, 127.9, 127.8, 127.5, 127.4, 127.3, 127.2, 127.1, 127.0, 126.9,
126.8 (CH, Ar), 91.1, 91.0, 90.7, 90.6 (C-1), 80.9 (CH), 80.7 (CH), 80.6
(CH), 80.5 (CH), 80.4 (CH), 77.2 (CH), 74.5 (CH2Ph), 74.4 (CH2Ph),
73.1 (CH2Ph), 73.0 (CH2Ph), 72.4 (CH), 72.3 (CH), 72.2 (CH2Ph),
71.7 (CH2Ph), 70.5 (CH), 70.4 (CH), 69.1 (C-6), 68.9 (C-6), 51.1,
51.0, 50.7, 50.6 (POCH3), 45.1 (NCH2CH3), 25.9 (SiC(CH3)3), 18.1,
18.0 (SiC(CH3)3), 8.36 (NCH2CH3), ꢀ3.75, ꢀ3.79, ꢀ5.02, ꢀ5.03
(Si(CH3)2). 31P NMR (CDCl3, 121.5 MHz): δ 100.2ꢀ94.6 (m).
HRMS (ESI): calcd for C34H50BO8PSiNa [M þ Na]þ 679.2998, found
679.3022.
Dimethyl 2,3,6-Tri-O-Bn-r-D-glucopyranosyl Boranopho-
sphate (6). Dimethyl 2,3,6-tri-O-Bn-4-O-TBDMS-R-D-glucopyrano-
syl boranophosphate 4 (0.133 g, 0.20 mmol), which was dried by
repeated coevaporation with dry toluene, was added to dry THF
(1.6 mL). A 1 M solution of TBAF in THF (0.40 mL, 0.40 mmol),
which was dried over MS 3A prior to use, was added to the solution at rt.
The mixture was allowed to stir for 30 min at rt. Then a saturated
NaHCO3 aqueous solution (3.0 mL) and CHCl3 (5.0 mL) were added
successively to the mixture. The organic layer was separated and washed
with saturated NaHCO3 aqueous solutions (2 ꢁ 5.0 mL). The aqueous
layers were combined and back-extracted with CHCl3 (2 ꢁ 5.0 mL).
The organic layers were combined and dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by silica
gel column chromatography using hexaneꢀAcOEt (2:1, v/v) as an
eluent to give 6 as a colorless oil (0.102 g, 0.182 mmol, 91%, R:β = 96:4).
1H NMR (CDCl3, 300 MHz): δ 7.38ꢀ7.24 (m, 15H, CH2Ph), 5.82 (dd,
JHH = 3.3 Hz, JPH = 7.7 Hz, R-H-1), 5.00ꢀ4.50 (m, 6H, CH2Ph),
3.95ꢀ3.57 (m, 12H, H-2, H-3, H-4, H-5, H-6, POCH3). 13C NMR
(CDCl3, 75.5 MHz): δ 138.4, 137.7, 137.4 (C, Ar), 128.5, 128.4, 128.3,
128.1, 127.9, 127.8, 127.7, 127.6 (CH, Ar), 94.5 (C-1), 80.3 (CH), 78.7
(CH), 78.6 (CH), 75.3 (CH2Ph), 73.5 (CH2Ph), 72.9 (CH2Ph), 72.1
(CH), 70.1 (CH), 69.1 (C-6), 53.5, 53.4, 53.3 (POCH3). 31P NMR
(CDCl3, 121.5 MHz): δ 121.4ꢀ117.6 (m). HRMS (ESI): calcd for
C29H38BO8PK [M þ K]þ 595.2029, found 595.2041.
Dimethyl 2,3,6-Tri-O-Bn-4-O-TBDMS-r-D-glucopyranosyl
Boranophosphate (4). 2,3,6-Tri-O-Bn-4-O-TBDMS-D-glucopyra-
nosyl acetate 1 (0.975 g, 1.6 mmol) was dried by repeated coevaporation
with dry Py and dry toluene and dissolved in dry CH2Cl2 (32.5 mL). MS
4A (4A molecular sieves) were added to the mixture. Trimethylsilyl iodide
(0.260 mL, 1.8 mmol) was added to the solution at ꢀ40 °C with stirring.
Additional trimethylsilyl iodide (92.5 μL, 0.65 mmol) was added after 3 h
and again added (70 μL, 0.37 mmol) after 1 h. The mixture was then
allowed to stir for 1 h at the same temperature. The mixture was then
coevaporated with dry toluene (8 ꢁ 16 mL) at 0 °C to remove any
volatiles. A solution of dimethyl H-phosphonate (1.50 mL, 16.3 mmol)
and DMAN (0.691 g, 4.0 mmol) in dry MeCN (16.1 mL), which was
dried over MS 3A (3A molecular sieves) prior to use, was added to the
residue at rt, and the mixture was stirred for 1 d. A saturated NaHCO3
aqueous solution (20 mL) and CHCl3 (20 mL) were then added
successively to the mixture. The organic layer was separated and washed
with saturated NaHCO3 aqueous solutions (2 ꢁ 20 mL), dried over
Na2SO4, filtered, and concentrated under reduced pressure. The residue
was then dissolved in dry MeCN (16.5 mL) and a 1 M solution of
BH3 THF in THF (16.5 mL, 16.5 mmol) was added to the solution at rt.
3
After stirring for 1 h, a saturated NaHCO3 aqueous solution (20 mL) and
CHCl3 (20mL) were added successively. The organiclayerwasseparated,
washed with saturated NaHCO3 aqueous solutions (2 ꢁ 20 mL), dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography using hexa-
neꢀAcOEt (5:2, v/v) as an eluent to give 4 as a brown oil (0.801 g, 1.2
mmol, 74%, R:β = 92:8). 1H NMR (CDCl3, 300 MHz): δ 7.33ꢀ7.22 (m,
15H, CH2Ph), 5.85 (dd, JHH = 3.0 Hz, JPH = 7.7 Hz, R-H-1), 5.06ꢀ4.47
(m, 6H, CH2Ph), 3.92ꢀ3.58 (m, 12H, H-2, H-3, H-4, H-5, H-6, POCH3),
0.84ꢀ0.81 (m, 9H, SiC(CH3)3), 0.02 to ꢀ0.03 (m, 6H, Si(CH3)2). 13
C
NMR (CDCl3, 75.5 MHz): δ 138.8, 138.0, 137.3 (C, Ar), 128.3, 128.2,
128.1, 128.0, 127.9, 127.5, 127.4, 127.1, 127.0 (CH, Ar), 94.3 (C-1), 80.6
(CH), 79.8 (CH), 79.7 (CH), 74.8 (CH2Ph), 73.8 (CH), 73.3 (CH2Ph),
73.0 (CH2Ph), 70.1 (CH), 68.6 (C-6), 53.5, 53.4, (POCH3), 25.9
(SiC(CH3)3), 18.1 (SiC(CH3)3), ꢀ3.78, ꢀ4.98 (Si(CH3)2). 31P NMR
(CDCl3, 121.5 MHz): δ 119.0ꢀ115.6 (m). HRMS (ESI): calcd for
C35H52BO8PSiNa [M þ Na]þ 693.3154, found 693.3155.
Methyl 2,3,6-Tri-O-Bn-4-O-TBDMS-r-D-glucopyranosyl N,
N-diisopropylphosphoramidite (10). 2,3,6-Tri-O-Bn-4-O-TBD
MS-D-glucopyranosyl acetate 1 (2.33 g, 3.8 mmol) was dried by repeated
coevaporation with dry Py and dry toluene and dissolved in dry CH2Cl2
(32.5 mL). MS 4A were added to the solution. The solution was cooled
to ꢀ15 °C and trimethylsilyl iodide (0.650 mL, 4.6 mmol) was added.
The mixture was then allowed to stir for 2 h at 0 °C and additional
trimethylsilyl iodide (54.0 μL, 0.365 mmol) was added. After 20 min, the
mixture was coevaporated with dry toluene (5 ꢁ 30 mL) at 0 °C.
A solution of methyl N,N-diisopropylphosphonamidate (6.94 g, 38.8
mmol) and diisopropylethylamine (1.35 mL, 9.5 mmol) in dry toluene
(38 mL), which was dried over MS 4A prior to use, was added to the
Methyl 2,3,6-Tri-O-Bn-4-O-TBDMS-r-D-glucopyranosyl Bo
ranophosphate Triethylammonium Salt (5). Triethylamine
(1.15 mL, 8.3 mmol) and benzenethiol (0.850 mL, 8.2 mmol) were
added successively to a solution of dimethyl 2,3,6-tri-O-Bn-4-O-
TBDMS-R-D-glucopyranosyl boranophosphate 4 (137 mg, 0.20 mmol)
in dry THF (2.0 mL). Additional triethylamine (0.55 mL, 4.0 mmol) and
benzenethiol (0.42 mL, 4.1 mmol) were added after 14 h, and after 5 h
more triethylamine (0.55 mL, 4.0 mmol) and benzenethiol (0.42 mL, 4.1
mmol) were added. The mixture was then allowed to stir for 6 h at rt. A 1
M TEAB aqueous solution (5.0 mL) and CHCl3 (5.0 mL) were added
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The Journal of Organic Chemistry
ARTICLE
residue at rt. After 1 d, the mixture was filtered, and a saturated NaHCO3
aqueous solution (40 mL) and AcOEt (40 mL) were added successively
to the filtrate. The organic layer was separated, washed with saturated
NaHCO3 aqueous solutions (2 ꢁ 30 mL), dried over Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified by
NH silica gel (Fuji Silysia Chemical, Inc. DM1020 100ꢀ200 mesh)
column chromatography using hexaneꢀAcOEt (10:1, v/v) as an eluent
to give 10 as a colorless oil (2.24 g, 3.1 mmol, 81%). 1H NMR (CDCl3,
300 MHz): δ 7.35ꢀ7.18 (m, 15H, CH2Ph), 5.49ꢀ5.45 (m, 1H, R-H-1),
5.08ꢀ4.47 (m, 6H, CH2Ph), 3.98ꢀ3.55 (m, 8H, H-2, H-3, H-4, H-5,
H-6, NCH(CH3)2), 3.47, 3.43, 3.38 (s, 3H, POCH3), 1.26ꢀ1.12
(m, 12H, NCH(CH3)2), 0.87ꢀ0.81 (m, 9H, SiC(CH3)3), 0.03 to
ꢀ0.04 (m, 6H, Si(CH3)2). 13C NMR (CDCl3, 75.5 MHz): δ 139.2,
139.1, 138.3, 138.2, 138.1, 138.0 (C, Ar), 128.1, 128.0, 127.9, 127.7,
127.5, 127.4, 127.3, 127.2, 127.1, 127.0, 126.9 (CH, Ar), 92.7, 92.4, 91.9,
91.6 (C-1), 81.3 (CH), 81.1 (CH), 81.0 (CH), 80.9 (CH), 80.6 (CH),
80.5 (CH), 77.2 (CH), 74.6 (CH2Ph), 73.1 (CH2Ph), 73.0 (CH2Ph),
72.4 (CH), 72.2 (CH, CH2Ph), 70.6 (CH), 70.2 (CH), 68.9 (C-6), 68.5
(C-6), 51.1, 50.9, 50.7, 50.5 (POCH3), 43.5, 43.3, 43.2, 43.0
(NCH(CH3)2), 25.9 (SiC(CH3)3), 24.8, 24.7, 24.6, 24.5, 24.4, 24.3
(NCH(CH3)2), 18.0 (SiC(CH3)3), ꢀ3.78, ꢀ4.98, ꢀ5.06 (Si(CH3)2).
31P NMR (CDCl3, 121.5 MHz): δ 152.6, 149.8 (s). HRMS (ESI): calcd
for C40H60NO7PSiK [M þ K]þ 764.3508, found 764.3539.
R-H-1:CHOP), 5.58, 5.56 (d, J = 8.3 Hz, β-H-1), 4.98ꢀ4.77 (m, 3H,
CH2Ph), 4.70ꢀ3.45 (m, 24H, CH2Ph, H-2, H-3, H-4, H-5, H-6,
POCH3), 2.14, 2.10, 2.03, 2.02 (s, 3H, OCOCH3), 0.86ꢀ0.79 (m, 9H,
SiC(CH3)3), 0.01 to ꢀ0.06 (m, 6H, Si(CH3)2). 13C NMR (CDCl3, 75.5
MHz): δ 169.1 (OCOCH3), 138.7, 138.6, 138.4, 138.2, 138.1, 138.0,
137.8, 137.3 (C, Ar), 128.3, 128.1, 128.0, 127.9, 127.7, 127.6, 127.4,
127.3, 127.2, 127.1, 127.0, 126.8 (CH, Ar), 94.9, 94.5 (C-1), 93.6
(β-COCOCH3), 89.5 (R-COCOCH3), 82.7 (CH), 80.8 (CH), 80.6
(CH), 79.8 (CH), 79.3 (CH), 78.8 (CH), 75.4, 75.2, 75.1, 74.9, 74.7,
74.6, 73.8, 73.5, 73.4, 73.3, 73.2, 73.1, 73.0, 72.8 (CH, CH2), 70.1 (CH),
69.8 (CH), 68.5 (CH2), 68.3 (CH2), 68.1 (CH2), 68.0 (CH2), 54.1
(POCH3), 25.9 (SiC(CH3)3), 21.0 (OCOCH3), 18.0 (SiC(CH3)3),
ꢀ3.85, ꢀ5.08 (Si(CH3)2). 31P NMR (CDCl3, 121.5 MHz): δ 123.3ꢀ
116.2 (m). HRMS (ESI): calcd for C63H80BO14PSiNa [M þ Na]þ
1153.5040, found 1153.5065.
Boranophosphotriester-Linked r-D-Glc-(1-P-4)-D-Glc-OAc
Derivative (14). Compound 13 (1.12 g, 0.99 mmol) was dried by
repeated coevaporation with dry toluene and dissolved in dry THF
(8.4 mL). A 1 M solution of TBAF in THF (2.0 mL, 2.0 mmol), which
was dried over MS 3A prior to use, was then added at rt. The mixture was
allowed to stir for 1.5 h at rt. A saturated NaHCO3 aqueous solution
(10 mL) and AcOEt (10 mL) were added successively to the mixture.
The organic layer was separated and washed with saturated NaHCO3
aqueous solutions (2 ꢁ 20 mL). The aqueous layers were combined and
back-extracted with AcOEt (2 ꢁ 20 mL). The organic layers were
combined, dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
using hexaneꢀAcOEt (3:1, v/v) as an eluent to give 14 as a colorless
2,3,6-Tri-O-Bn-D-glucopyranosyl Acetate (11). 2,3,6-Tri-O-
Bn-4-O-TBDMS-D-glucopyranosyl acetate 1 (1.21 g, 2.0 mmol) was
dried by repeated coevaporation with dry Py and dry toluene and
dissolved in dry THF (20 mL) under Ar. A 1 M solution of TBAF in
THF (3.00 mL, 3.0 mmol), which was dried over MS 3A prior to use, was
added to the solution at rt, and the mixture was allowed to stir for 15 min.
A saturated NaHCO3 aqueous solution (20 mL) and AcOEt (20 mL)
were then added successively to the mixture. The organic layer was
separated and washed with saturated NaHCO3 aqueous solutions (2 ꢁ
50 mL). The aqueous layers were combined and back-extracted with
AcOEt (2 ꢁ 50 mL). The organic layers were combined, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography using hexaneꢀAcOEt
(5:2, v/v) as an eluent to give 11 as a colorless oil (0.804 g, 1.6 mmol,
82%). 1H NMR (CDCl3, 300 MHz): δ 7.33ꢀ7.16 (m, 15H, CH2Ph),
6.36 (d, J = 3.6 Hz, R-H-1), 5.61 (d, J = 7.7 Hz, β-H-1), 4.97ꢀ4.45 (m,
6H, CH2Ph), 3.86ꢀ3.48 (m, 6H, H-2, H-3, H-4, H-5, H-6), 2.10 (s, R-
OCOCH3), 1.99 (s, β-OCOCH3). 13C NMR (CDCl3, 75.5 MHz): δ
169.0 (OCOCH3), 138.4, 138.2, 137.9, 137.6, 137.5, 137.3 (C, Ar),
128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4 (CH, Ar),
93.8 (β-C-1), 89.7 (R-C-1), 83.9 (CH), 80.7 (CH), 80.4 (CH), 78.2
(CH), 77.2 (CH), 75.1 (CH2Ph), 74.7 (CH2Ph), 74.6 (CH), 73.4
(CH2Ph), 73.3 (CH2Ph), 72.8 (CH2Ph), 72.4 (CH), 70.7 (CH), 70.0
(CH), 69.1 (C-6), 69.0 (C-6), 20.9, 20.8 (OCOCH3). HRMS (ESI):
calcd for C29H32O7K [M þ K]þ 531.1780, found 531.1760.
1
foam (0.738 g, 0.73 mmol, 73%). H NMR (CDCl3, 300 MHz): δ
7.35ꢀ7.21 (m, 30H, CH2Ph), 6.32 (m, R-H-1), 5.83ꢀ5.80 (m, 1H, R-
H-1:CHOP), 5.59ꢀ5.57 (m, β-H-1), 4.93ꢀ3.44 (m, 27H, H-2, H-3,
H-4, H-5, H-6, CH2Ph, POCH3), 2.16, 2.13, 2.04, 2.03 (s, 3H,
OCOCH3), 1.15ꢀ0.11 (br, 3H, BH3). 13C NMR (CDCl3, 75.5 MHz):
δ 169.1 (OCOCH3), 138.6, 138.4, 138.2, 137.7, 137.4, 137.3, 137.2 (C,
Ar), 128.5, 128.4, 128.2, 128.1, 128.0, 127.8, 127.6, 127.5, 127.4, 127.3,
127.2, 127.0, 126.9 (CH, Ar), 95.1, 94.8 (C-1), 93.6 (β-COCOCH3),
89.4 (R-COCOCH3), 82.7 (CH), 80.8 (CH), 80.3 (CH), 79.3 (CH),
78.6 (CH), 77.2 (CH), 75.5, 75.4, 75.3, 75.2, 75.0, 74.9, 74.7, 74.5 (CH,
CH2), 73.5 (CH2), 73.4 (CH2), 73.2 (CH2), 73.0 (CH2), 72.8 (CH),
72.4 (CH), 72.2 (CH), 71.9 (CH), 70.3 (CH), 70.2 (CH), 70.0 (CH),
68.9 (CH2), 68.8 (CH2), 68.3 (CH2), 68.2 (CH2), 68.0 (CH2), 54.1
(POCH3), 21.0 (OCOCH3). 31P NMR (CDCl3, 121.5 MHz): δ 119.8ꢀ
114.2 (m). HRMS (ESI): calcd for C57H66BO14PNa [M þ Na]þ
1039.4175, found 1039.4202.
Boranophosphotriester-Linked 4-O-TBDMS-r-D-Glc-(1-P-
4)-r-D-Glc-(1-P-4)-D-Glc-OAc Derivative (15). Compounds 10
(0.625 g, 0.86 mmol) and 14 (0.738 g, 0.73 mmol) were dried by
repeated coevaporation with dry toluene and in vacuo overnight, and a
solution of 1H-tetrazole (112 mg, 1.6 mmol) in dry MeCN (8.0 mL),
which was dried over MS 3A prior to use, was added. The mixture was
Boranophosphotriester-Linked 4-O-TBDMS-r-D-Glc-(1-P-
4)-D-Glc-OAc Derivative (13). Compounds 10 (0.192 g, 0.26 mmol)
and 11 (0.123 g, 0.25 mmol) were dried by repeated coevaporation with
dry toluene and in vacuo overnight. A solution of 1H-tetrazole (35 mg,
0.50 mmol) in dry MeCN (2.5 mL), which was dried over MS 3A prior
to use, was added to the mixture and the mixture was allowed to stir for
allowed to stir for 15 min at rt. A 1 M solution of BH3 THF in THF
3
(4.0 mL, 4.0 mmol) was then added to the mixture at rt. The mixture was
allowed to stir for 20 min. A saturated NaHCO3 aqueous solution
(5.0 mL) and AcOEt (10 mL) were added successively to the mixture.
The organic layer was separated and washed with saturated NaHCO3
aqueous solutions (2 ꢁ 10 mL). The aqueous layers were combined and
back-extracted with AcOEt (2 ꢁ 10 mL). The organic layers were
combined, dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
using hexaneꢀAcOEt (2:1, v/v) as an eluent to give 15 as a colorless
20 min at rt. A 1 M solution of BH3 THF in THF (1.25 mL, 1.25 mmol)
3
was then added, and the mixture was allowed to stir for 20 min at rt.
A saturated NaHCO3 aqueous solution (10 mL) and CHCl3 (10 mL)
were added successively to the mixture. The organic layer was separated,
washed with saturated NaHCO3 aqueous solutions (2 ꢁ 10 mL), dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography using hex-
aneꢀAcOEt (5:2, v/v) as an eluent to give 13 as a colorless foam (0.230
1
foam (0.871 g, 0.52 mmol, 72%). H NMR (CDCl3, 300 MHz): δ
7.35ꢀ7.18 (m, 45H, CH2Ph), 6.32ꢀ6.29 (m, R-H-1), 5.86ꢀ5.72
(m, 2H, R-H-1:CHOP), 5.58ꢀ5.47 (m, β-H-1), 4.98ꢀ3.38 (m, 42H,
CH2Ph, H-2, H-3, H-4, H-5, H-6, POCH3), 2.14, 2.07, 2.06, 2.03, 2.02
1
g, 0.20 mmol, 82%). H NMR (CDCl3, 300 MHz): δ 7.41ꢀ7.21 (m,
30H, CH2Ph), 6.33, 6.30 (d, J = 3.6 Hz, R-H-1), 5.91ꢀ5.83 (m, 1H,
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The Journal of Organic Chemistry
ARTICLE
3
3
(s, 3H, OCOCH3), 0.86ꢀ0.79 (m, 9H, SiC(CH3)3), 0.00 to ꢀ0.07 (m,
6H, Si(CH3)2). 13C NMR (CDCl3, 75.5 MHz): δ 169.0 (OCOCH3),
138.7, 138.6, 138.4, 138.3, 138.2, 138.0, 137.9, 137.7, 137.2 (C, Ar),
128.3, 128.2, 128.0, 127.9, 127.7, 127.6, 127.4, 127.3, 127.1, 126.8, 126.7
(CH, Ar), 94.7, 94.2, 94.0 (C-1), 93.5 (β-COCOCH3), 89.4 (R-
COCOCH3), 82.5 (CH), 80.7 (CH), 80.5 (CH), 79.8 (CH), 79.5
(CH), 79.2 (CH), 79.1 (CH), 78.7 (CH), 78.6 (CH), 77.2 (CH), 74.9,
74.6, 73.8, 73.7, 73.5, 73.4, 73.3, 73.2, 73.0, 72.7 (CH, CH2), 72.2 (CH),
71.7 (CH), 70.0 (CH), 69.8 (CH), 68.5 (CH2), 68.1 (CH2), 67.8
(CH2), 54.1 (POCH3), 25.9 (SiC(CH3)3), 21.0, 20.9 (OCOCH3), 18.0
(SiC(CH3)3), ꢀ3.89, ꢀ5.14 (Si(CH3)2). 31P NMR (CDCl3, 121.5
MHz): δ 122.7ꢀ117.3 (m). HRMS (ESI): calcd for C91H114B2O21P2-
SiNa [M þ Na]þ 1677.7175, found 1677.7155.
0.90 (t, 18H, NCH2CH3), 0.84ꢀ0.75 (m, 9H, SiC(CH ) ), 0.00 to
ꢀ0.08 (m, 6H, Si(CH3)2). 13C NMR (CDCl3, 75.5 MHz): δ 169.4,
169.0 (OCOCH3), 140.4, 140.1, 139.9, 139.5, 139.2, 139.0, 138.5, 138.1,
137.8 (C, Ar), 128.4, 128.0, 127.8, 127.3, 127.0, 126.6, 126.3 (CH, Ar),
93.6 (β-COCOCH3), 91.9, 91.6, 91.0 (C-1), 89.9, 89.6 (R-
COCOCH3), 84.0 (CH), 81.8 (CH), 81.1 (CH), 80.6 (CH), 79.7
(CH), 78.6 (CH), 78.3 (CH), 77.2 (CH), 75.9, 75.6, 75.2, 74.8, 74.2,
73.8, 73.2, 73.0, 72.5, 72.0 (CH, CH2), 71.2 (CH), 70.3 (CH), 70.0
(CH), 69.3 (CH2), 68.6 (CH2), 44.7 NCH2CH3, 25.9 (SiC(CH3)3),
20.9 (OCOCH3), 18.0 (SiC(CH3)3), 8.15 NCH2CH3, ꢀ3.93, ꢀ5.22
(Si(CH3)2). 31P NMR (CDCl3, 121.5 MHz): δ 100.2ꢀ92.2 (m).
HRMS (ESI): calcd for C95H126B2NO21P2Si [M þ H]þ 1728.8248,
found 1728.8240.
Boranophosphodiester-Linked 4-O-TBDMS-r-D-Glc-(1-P-
4)-D-Glc-OAc Derivative (16). Triethylamine (1.13 mL, 8.1 mmol)
and benzenethiol (0.850 mL, 8.2 mmol) were added successively to a
solution of 13 (0.227 g, 0.20 mmol) in dry THF (2.0 mL), and the
mixture was allowed to stir for 16 h at rt. A 1 M TEAB aqueous solution
(5.0 mL) and AcOEt (5.0 mL) were added successively to the mixture.
The organic layer was separated and washed with 1 M TEAB aqueous
solutions (2 ꢁ 5.0 mL). The aqueous layers were combined and back-
extracted with AcOEt (5.0 mL). The organic layers were combined,
filtered through cotton, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography using
CH2Cl2ꢀEt3NꢀMeOH (98:1:1ꢀ97:1:2, v/v/v) as an eluent. The
fractions containing 16 were collected and concentrated under reduced
pressure. The residue was dissolved in CHCl3 (5.0 mL) and washed with
a 1 M TEAB aqueous solution (5.0 mL). The aqueous layer was back-
extracted with CHCl3 (10 mL). The organic layers were combined,
filtered through cotton, and concentrated under reduced pressure to
give 16 as a colorless foam (0.226 g, 0.19 mmol, 93%). 1H NMR
(CDCl3, 300 MHz): δ 7.52ꢀ7.13 (m, 30H, CH2Ph), 6.37, 6.31 (d, J =
3.6 Hz, R-H-1), 6.06ꢀ5.91 (m, 1H, R-H-1:CHOP), 5.59, 5.53 (d, J = 8.3
Hz, β-H-1), 5.31ꢀ3.45 (m, 24H, CH2Ph, H-2, H-3, H-4, H-5, H-6), 2.61
(q, 6H, NCH2CH3), 2.09, 2.07, 2.03, 2.01 (s, 3H, OCOCH3), 0.96 (t,
9H, NCH2CH3), 0.83ꢀ0.79 (m, 9H, SiC(CH3)3), 0.02 to ꢀ0.07 (m,
6H, Si(CH3)2). 13C NMR (CDCl3, 75.5 MHz): δ 169.5, 169.1
(OCOCH3) 140.2, 140.0, 139.6, 139.3, 139.0, 138.8, 138.4, 138.2,
137.8 (C, Ar), 128.2, 128.0, 127.8, 127.5, 127.3, 127.1, 126.8, 126.6
(CH, Ar), 93.7 (β-COCOCH3), 91.8, 91.2 (C-1), 89.9, 89.6 (R-
COCOCH3), 84.0 (CH), 81.1, 81.0, 80.8, 80.5, 80.3, 78.8, 78.5, 77.2,
76.4, 75.9, 75.2, 74.9, 74.4, 74.3, 73.5, 73.2, 73.1, 72.5, 72.4, 72.2, 71.8,
70.5, 70.3, 69.3, 69.1 (CH, CH2), 68.7 (CH2), 44.9 (NCH2CH3), 26.0
(SiC(CH3)3), 21.1 (OCOCH3), 18.1 (SiC(CH3)3), 8.25 (NCH2CH3),
ꢀ3.79, ꢀ5.09 (Si(CH3)2). 31P NMR (CDCl3, 121.5 MHz): δ
103.0ꢀ93.0 (m). HRMS (ESI): calcd for C62H77BO14PSi [M ꢀ H]ꢀ
1115.4919, found 1115.4896.
Phosphodiester-Linked
4-O-TBDMS-r-D-Glc-(1-P-4)-D-
Glc-OAc Derivative (20). Dichloroacetic acid (56 μL) was added
to a solution of DMTrOMe (95.8 mg, 0.28 mmol) and compound 16
(0.114 g, 94 μmol) in dry CH2Cl2 (1.9 mL) at 0 °C. The mixture was
allowed to stir for 1 h at 0 °C. Et3SiH (0.2 mL) and CHCl3 (5.0 mL)
were added successively to the mixture, and the mixture was washed with
saturated NaHCO3 aqueous solutions (2 ꢁ 7.0 mL). The aqueous layers
were combined and back-extracted with CHCl3 (7.0 mL). The organic
layers were combined, dried over Na2SO4, filtered, and concentrated
under reduced pressure to give the H-phosphonate intermediate 18. A
solution of I2 (47.6 mg, 0.19 mmol) in dry Py (1.78 mL) and H2O (94
μL) was then added to crude 18, and the mixture was allowed to stir for 2
h at rt. The mixture was then diluted with CHCl3 (5.0 mL) and washed
with a 0.5 M Na2S2O3 aqueous solution (5.0 mL) and 1 M TEAB
aqueous solutions (2 ꢁ 5.0 mL). The aqueous layers were combined and
back-extracted with CHCl3 (2 ꢁ 15 mL). The organic layers were
combined, filtered through cotton, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
using CH2Cl2ꢀEt3N (99:1, v/v) as an eluent. The fractions containing
20 were collected and concentrated under reduced pressure. The residue
was dissolved in CHCl3 (5.0 mL) and washed with a 1 M TEAB aqueous
solution (5.0 mL). The aqueous layer was back-extracted with CHCl3
(10 mL). The organic layers were combined, filtered through cotton,
and concentrated under reduced pressure to give 20 as a yellow oil
(0.107 g, 88 μmol, 94%). 1H NMR (CDCl3, 300 MHz): δ 7.53ꢀ7.16
(m, 30H, CH2Ph), 6.34 (d, J = 3.6 Hz, R-H-1), 5.94 (td, 1H, JHH = 3.3
Hz, JPH = 7.7 Hz, R-H-1:CHOP), 5.54 (d, J = 8.3 Hz, β-H-1), 5.30ꢀ4.99
(m, 2H, CH2Ph), 4.86ꢀ3.45 (m, 22H, CH2Ph, H-2, H-3, H-4, H-5,
H-6), 2.64 (q, 6H, NCH2CH3), 2.07, 2.03 (s, 3H, OCOCH3), 1.00 (t,
9H, NCH2CH3), 0.81ꢀ0.79 (m, 9H, SiC(CH3)3), 0.01 to ꢀ0.07 (m,
6H, Si(CH3)2). 13C NMR (CDCl3, 75.5 MHz): δ 169.4, 169.0
(OCOCH3), 139.8, 139.6, 139.1, 139.0, 138.9, 138.2, 138.1, 137.7 (C,
Ar), 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5, 127.3, 127.2, 127.0,
126.9, 126.8 (CH, Ar), 93.7 (β-COCOCH3), 92.7 (C-1), 89.8 (R-
COCOCH3), 83.9 (CH), 81.0 (CH), 80.6 (CH), 80.5 (CH), 78.2
(CH), 77.2 (CH), 76.0 (CH), 74.8 (CH2Ph), 74.4 (CH2Ph), 74.0 (CH),
73.9 (CH), 73.7 (CH), 73.6 (CH), 73.3, 73.2, 73.1 (CH, CH2), 72.3
(CH), 71.9 (CH2Ph), 70.5 (CH), 70.4 (CH), 69.3 (C-6), 69.2 (C-6),
69.0 (C-6), 44.9 NCH2CH3, 25.9, (SiC(CH3)3), 21.1, 21.0
(OCOCH3), 18.0 (SiC(CH3)3), 8.22 NCH2CH3, ꢀ3.83, ꢀ5.14
(Si(CH3)2). 31P NMR (CDCl3, 121.5 MHz): δ ꢀ1.54, ꢀ1.66 (s).
HRMS (ESI): calcd for C68H91NO15PSi [M þ H]þ 1220.5890, found
1220.5885.
Boranophosphodiester-Linked 4-O-TBDMS-r-D-Glc-(1-P-
4)-r-D-Glc-(1-P-4)-D-Glc-OAc Derivative (17). Triethylamine
(1.33 mL, 9.5 mmol) and benzenethiol (0.983 mL, 9.5 mmol) were
added successively to a solution of 15 (0.197 g, 0.12 mmol) in dry THF
(1.2 mL), and the mixture was allowed to stir for 6 h at rt. A 1 M TEAB
aqueous solution (5.0 mL) and AcOEt (5.0 mL) were added successively
to the mixture. The organic layer was separated and washed with 1 M
TEAB aqueous solutions (2 ꢁ 5.0 mL). The aqueous layers were
combined and back-extracted with AcOEt (5.0 mL). The organic layers
were combined, filtered through cotton, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography using CH2Cl2ꢀEt3NꢀMeOH (98:1:1, v/v/v) as an
Phosphodiester-Linked 4-O-TBDMS-r-D-Glc-(1-P-4)-r-D-
Glc-(1-P-4)-D-Glc-OAc Derivative (21). Dichloroacetic acid (62
μL) was added to a solution of DMTrOMe (210 mg, 0.62 mmol) and
the compound 17 (0.189 g, 0.10 mmol) in dry CH2Cl2 (2.0 mL) at 0 °C.
Additional DCA (10 μL) and DMTrOMe (30.7 mg, 90 μmol) were
added after 1 h. The mixture was then allowed to stir for 30 min at 0 °C.
Et3SiH (0.9 mL) and CHCl3 (5.0 mL) were added successively to the
mixture, and the mixture was washed with saturated NaHCO3 aqueous
1
eluent to give 17 as a colorless foam (0.212 g, 0.11 mmol, 97%). H
NMR (CDCl3, 300 MHz): δ 7.51ꢀ7.14 (m, 45H, CH2Ph), 6.34ꢀ6.27
(m, R-H-1), 6.04ꢀ5.85 (m, 2H, R-H-1:CHOP), 5.51ꢀ5.42 (m, β-H-1),
5.32ꢀ3.41 (m, 36H, CH2Ph, H-2, H-3, H-4, H-5, H-6), 2.55 (q, 12H,
NCH2CH3), 2.03, 2.02, 2.00, 1.99, 1.97, 1.96, 1.89 (s, 3H, OCOCH3),
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solutions (3 ꢁ 7.0 mL). The aqueous layers were combined and back-
extracted with CHCl3 (7.0 mL). The organic layers were combined,
dried over Na2SO4, filtered, and concentrated under reduced pressure to
give the H-phosphonate intermediate 19. A solution of I2 (103 mg, 0.41
mmol) in dry PyꢀH2O (2.05 mL, 95:5, v/v) was then added, and the
mixture was allowed to stir for 3 h at rt. The mixture was diluted with
CHCl3 (5.0 mL) and washed with a 0.5 M Na2S2O3 aqueous solution
(5.0 mL) and 1 M TEAB aqueous solutions (2 ꢁ 5.0 mL). The aqueous
layers were combined and back-extracted with CHCl3 (2 ꢁ 15 mL). The
organic layers were combined, filtered through cotton, and concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography using CH2Cl2ꢀEt3NꢀMeOH (99:1:0ꢀ96:2:2, v/v/v)
as an eluent. The fractions containing 21 were collected and concentrated
under reduced pressure. The residue was dissolved in CHCl3 (5.0 mL)
and washed with a 1 M TEAB aqueous solution (5.0 mL). The aqueous
layer was back-extracted with CHCl3 (10 mL). The organic layers were
combined, filtered through cotton, and concentrated under reduced
(m, 9H, SiC(CH3)3), 0.01 to ꢀ0.07 (m, 6H, Si(CH3)2). 13C NMR
(CDCl3, 75.5 MHz): δ 169.4, 169.1 (OCOCH3), 140.3, 140.1, 139.3,
139.2, 139.0, 138.8, 138.4, 138.3, 138.1, 137.7 (C, Ar), 128.5, 128.3,
128.1, 127.8, 127.6, 127.5, 127.3, 127.0, 126.7, 126.6 (CH, Ar), 93.7 (β-
COCOCH3), 93.3 (C-1), 89.8 (R-COCOCH3), 83.7 (CH), 81.2 (CH),
80.9 (CH), 80.6 (CH), 80.1 (CH), 78.6 (CH), 77.2 (CH), 76.2, 74.9,
74.8, 74.6, 74.4, 74.2, 73.3, 73.0, 72.6, 72.0, 71.5, 70.4, 70.1, 69.4, 69.2,
68.7 (CH, CH2), 68.6 (CH2), 45.0 NCH2CH3, 26.0, 25.8 (SiC(CH3)3),
21.0 (OCOCH3), 18.0 (SiC(CH3)3), 8.25 NCH2CH3, ꢀ3.86, ꢀ5.12
(Si(CH3)2). 31P NMR (CDCl3, 121.5 MHz): δ 59.3, 59.2, 56.8, 56.6 (s).
HRMS (ESI): calcd for C68H91NO14PSSi [M þ H]þ 1236.5662, found
1236.5668.
Phosphodiester-Linked r-D-Glc-(1-P-4)-D-Glc-OAc Deriva-
tive (23). Et3N 3HF (0.157 mL, 0.97 mmol) was added to a solution of
3
the compound 20 (78.5 mg, 64 μmol) in dry THF (1.2 mL) at 40 °C.
The mixture was allowed to stir for 32 h at 40 °C. A 1 M TEAB aqueous
solution (5.0 mL) and CH2Cl2 (2.0 mL) were added successively to the
mixture and the mixture was washed with 1 M TEAB aqueous solutions
(2 ꢁ 5.0 mL). The aqueous layers were combined and back-extracted
with CH2Cl2 (5.0 mL). The organic layers were combined, filtered
through cotton, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography using CH2Cl2ꢀ
Et3NꢀMeOH (99:1:0ꢀ98:1:1, v/v/v) as an eluent. The fractions
containing 23 were collected and concentrated under reduced pressure.
The residue was dissolved in CHCl3 (5.0 mL) and washed with a 1 M
TEAB aqueous solution (5.0 mL). The aqueous layer was back-extracted
with CHCl3 (10 mL). The organic layers were combined, filtered
through cotton, and concentrated under reduced pressure to give 23
as a yellow oil (63.4 mg, 57 μmol, 89%). 1H NMR (CDCl3, 300 MHz): δ
7.53ꢀ7.17 (m, 30H, CH2Ph), 6.32 (d, J = 3.6 Hz, R-H-1), 5.95ꢀ5.90
(m, 1H, R-H-1:CHOP), 5.56 (d, J = 8.3 Hz, β-H-1), 5.27ꢀ4.78 (m, 4H,
CH2Ph), 4.73ꢀ3.45 (m, 20H, CH2Ph, H-2, H-3, H-4, H-5, H-6), 2.60 (q,
6H, NCH2CH3), 2.08, 2.03 (s, 3H, OCOCH3), 1.02 (t, 9H, NCH2CH3).
13C NMR (CDCl3, 75.5 MHz): δ 169.4, 169.1 (OCOCH3), 139.9, 139.6,
139.1, 139.0, 138.9, 138.3, 138.2, 137.9, 137.7 (C, Ar), 128.3, 128.2, 128.1,
128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.0, 126.9 (CH, Ar), 93.7
(β-COCOCH3), 93.1, 93.0 (C-1), 89.8 (R-COCOCH3), 84.0 (CH), 81.1
(CH), 80.8 (CH), 80.6 (CH), 79.8 (CH), 79.7 (CH), 78.2 (CH), 77.2
(CH), 76.2 (CH), 76.1 (CH), 75.1 (CH2), 74.9 (CH2), 73.9 (CH2), 73.8,
73.7, 73.6, 73.5, 73.2 (CH, CH2), 73.1 (CH2), 72.0 (CH2), 71.2 (CH), 70.8
(CH), 70.4 (CH), 70.0 (CH2), 69.4 (CH2), 45.5 (NCH2CH3), 21.1, 21.0
(OCOCH3), 9.79 (NCH2CH3). 31PNMR(CDCl3, 121.5 MHz): δꢀ1.85,
ꢀ1.97 (s). HRMS (ESI): calcd for C56H60O15P [M ꢀ H]ꢀ 1003.3675,
found 1003.3680.
1
pressure to give 21 as a yellow oil (0.114 g, 62 μmol, 61%). H NMR
(CDCl3, 300 MHz): δ 7.53ꢀ7.12 (m, 45H, CH2Ph), 6.30 (d, J = 3.6 Hz,
R-H-1), 5.96ꢀ5.92 (m, 2H, R-H-1:CHOP), 5.45 (d, J = 8.0 Hz, β-H-1),
5.32ꢀ4.98, (m, 3H, CH2Ph), 4.89ꢀ3.42 (m, 33H, H-2, H-3, H-4, H-5,
H-6, CH2Ph), 2.56 (q, 12H, NCH2CH3), 1.99 (s, 3H, OOCCH3) 0.93 (t,
18H, NCH2CH3) 0.78 (s, 9H, SiC(CH3)3), ꢀ0.02, ꢀ0.09 (s, 6H,
Si(CH3)2). 13C NMR (CDCl3, 75.5 MHz): δ 169.4, 169.0 (OCOCH3)
140.1, 140.0, 139.7, 139.2, 139.0, 138.7, 138.5, 138.4, 138.2, 137.8 (C,Ar),
128.1, 127.9, 127.7, 127.5, 127.3, 127.2, 127.0, 126.7, 126.6 (CH, Ar), 93.6
(β-COCOCH3), 92.7, 92.4 (C-1), 89.9 (R-COCOCH3), 83.9 (CH), 81.0
(CH), 80.7 (CH), 80.5 (CH), 80.3 (CH), 79.7 (CH), 78.0 (CH), 77.2
(CH), 75.6 (CH), 74.6, 74.5, 74.2, 73.9, 73.6, 73.0, 72.8, 72.2, 71.9, 71.5,
71.4, 70.4, 70.1 (CH, CH2), 69.1 (CH2), 68.8 (CH2), 44.7 NCH2CH3,
25.9 (SiC(CH3)3), 21.0 (OCOCH3), 18.0 (SiC(CH3)3), 8.21 NCH2CH3,
ꢀ3.92, ꢀ5.23 (Si(CH3)2). 31P NMR (CDCl3, 121.5 MHz): δ ꢀ1.55,
ꢀ1.58, ꢀ1.65, ꢀ1.69 (s). HRMS (ESI): calcd for C95H121NO23P2Si
[M þ 2H]2þ 1733.7563, found 1733.7544.
Thiophosphodiester-Linked 4-O-TBDMS-r-D-Glc-(1-P-4)-
D-Glc-OAc Derivative (22). Trifluoroacetic acid (27 μL) was added
to a solution of TrOMe (62.9 mg, 0.23 mmol) and the compound 16
(89.3 mg, 73 μmol) in dry CH2Cl2 (1.5 mL) at 0 °C. The mixture was
allowed to stir for 1 h at 0 °C. Et3SiH (0.2 mL) and CH2Cl2 (5.0 mL)
were successively added, and the mixture was washed with saturated
NaHCO3 aqueous solutions (2 ꢁ 7.0 mL). The aqueous layers were
combined and back-extracted with CH2Cl2 (7.0 mL). The organic layers
were combined, dried over Na2SO4, filtered, and concentrated under
reduced pressure to give the H-phosphonate intermediate 18. The crude
18 was dissolved in dry Py (1.5 mL) and S8 (12.3 mg, 0.38 mmol) was
added at rt. The mixture was allowed to stir for 2.5 h at rt. A 1 M TEAB
aqueous solution (5.0 mL) and AcOEt (3.0 mL) were added successively
to the mixture. The organic layer was separated and washed with 1 M
TEAB aqueous solutions (2 ꢁ 5.0 mL). The aqueous layers were
combined and back-extracted with AcOEt (5.0 mL). The organic layers
were combined, filtered through cotton, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography using CH2Cl2ꢀEt3NꢀMeOH (96:1:3, v/v/v) as an
eluent. The fractions containing 22 were collected and concentrated
under reduced pressure. The residue was dissolved in CHCl3 (5.0 mL)
and washed with 1 M TEAB aqueous solution (5.0 mL). The aqueous
layer was back-extracted with CHCl3 (10 mL). The organic layers were
combined, filtered through cotton, and concentrated under reduced
pressure to give 22 as a yellow oil (76.9 mg, 62 μmol, 85%). 1H NMR
(CDCl3, 300 MHz): δ 7.56ꢀ7.14 (m, 30H, CH2Ph), 6.37, 6.33 (d, J =
3.3 Hz, R-H-1), 6.31ꢀ6.08 (m, 1H, H-1:CHOP), 5.59, 5.55 (d, J = 8.0
Hz, β-H-1), 5.47ꢀ5.24 (m, 1H, CH2Ph), 5.07ꢀ3.41 (m, 23H, CH2Ph,
H-2, H-3, H-4, H-5, H-6), 2.68 (q, 6H, NCH2CH3), 2.08, 2.07, 2.06,
2.05, 2.03, 2.00 (s, 3H, OCOCH3), 0.98 (t, 9H, NCH2CH3), 0.90ꢀ0.78
Phosphodiester-Linked r-D-Glc-(1-P-4)-r-D-Glc-(1-P-4)-D-
Glc-OAc Derivative (24). Et3N 3HF (0.16 mL, 0.95 mmol) was
3
added to a solution of the compound 21 (114 mg, 62 μmol) in dry THF
(1.2 mL) at 40 °C. The mixture was allowed to stir for 30 h at 40 °C. A 1
M TEAB aqueous solution (5.0 mL) and CH2Cl2 (5.0 mL) were added
successively to the mixture, and the mixture was washed with 1 M TEAB
aqueous solutions (2 ꢁ 5.0 mL). The aqueous layers were combined and
back-extracted with CH2Cl2 (10 mL). The organic layers were com-
bined, filtered through cotton, and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
using CH2Cl2ꢀEt3NꢀMeOH (98:2:0ꢀ96:2:2, v/v/v) as an eluent.
The fractions containing 24 were collected and concentrated under
reduced pressure. The residue was dissolved in CHCl3 (5.0 mL) and
washed with a 1 M TEAB aqueous solution (5.0 mL). The aqueous layer
was back-extracted with CHCl3 (10 mL). The organic layers were
combined, filtered through cotton, and concentrated under reduced
1
pressure to give 24 as a colorless foam (87.8 mg, 51 μmol, 82%). H
NMR (CDCl3, 300 MHz): δ 7.53ꢀ7.12 (m, 45H, CH2Ph), 6.30 (d, J =
3.6 Hz, R-H-1), 5.94ꢀ5.89 (m, 2H, R-H-1:CHOP), 5.44 (d, J = 8.0 Hz,
β-H-1), 5.31ꢀ5.07 (m, 2H, CH2Ph), 4.87ꢀ3.36 (m, 34H, CH2Ph, H-2,
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H-3, H-4, H-5, H-6), 2.58 (q, 12H, NCH2CH3), 2.08, 1.99, 1.95 (s, 3H,
OCOCH3), 0.94 (t, 18H, NCH2CH3). 13C NMR (CDCl3, 75.5 MHz): δ
169.5, 169.0 (OCOCH3), 140.1, 139.9, 139.7, 139.1, 139.0, 138.9, 138.6,
138.5, 138.3, 138.0, 137.8 (C, Ar), 128.2, 128.0, 127.9, 127.8, 127.5,
127.4, 127.3, 127.1, 126.9, 126.7 (CH, Ar), 93.6 (β-COCOCH3), 92.9,
92.7 (C-1), 89.9 (R-COCOCH3), 83.9 (CH), 81.1 (CH), 80.6 (CH),
79.7 (CH), 79.5 (CH), 79.4 (CH), 78.1 (CH), 77.2 (CH), 75.0 (CH2),
74.7 (CH2), 74.3, 73.9, 73.7, 73.6, 73.5, 73.2, 73.0, 72.9, 72.0, 71.7 (CH,
CH2), 71.1 (CH), 70.7 (CH), 70.5 (CH2), 70.1 (CH2), 69.9 (CH2),
69.1 (CH2), 44.8 (NCH2CH3), 21.0 (OCOCH3), 8.21 (NCH2CH3).
31P NMR (CDCl3, 121.5 MHz): δ ꢀ1.73, ꢀ1.77, ꢀ1.87 (s). HRMS
(ESI): calcd for C83H89O23P2 [M ꢀ H]ꢀ 1515.5275, found 1515.5271.
Phosphodiester-Linked r-D-Glc-(1-P-4)-D-Glc Derivative
(25). A 2 M solution of Me2NH in THF (0.210 mL, 0.42 mmol) was
added to a solution of the compound 23 (30.8 mg, 28 μmol) in dry
MeCN (0.31 mL) at rt. The mixture was allowed to stir for 4 h at rt. A 1
M TEAB aqueous solution (5.0 mL) and AcOEt (5.0 mL) were added
successively to the mixture, and the mixture was washed with 1 M
TEAB aqueous solutions (2 ꢁ 5.0 mL). The aqueous layers were
combined and back-extracted with AcOEt (5.0 mL). The organic layers
were combined, filtered through cotton, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography using CH2Cl2ꢀEt3N (99:1, v/v) as an eluent. The
fractions containing 25 were collected and concentrated under re-
duced pressure. The residue was dissolved in CHCl3 (5.0 mL) and
washed with a 1 M TEAB aqueous solution (5.0 mL). The aqueous
layer was back-extracted with CHCl3 (10 mL). The organic layers were
combined, filtered through cotton, and concentrated under reduced
pressure to give 25 as a colorless foam (27.8 mg, 26 μmol, 94%). 1H
NMR (CDCl3, 300 MHz): δ 7.52ꢀ7.14 (m, 30H, CH2Ph), 5.90 (td,
1H, JHH = 3.0 Hz, JPH = 7.2 Hz, R-H-1:CHOP), 5.24ꢀ4.47 (m, 13H,
R,β-H-1, CH2Ph), 4.39ꢀ3.36 (m, 12H, H-2, H-3, H-4, H-5, H-6), 2.65
(q, 6H, NCH2CH3), 1.00 (t, 9H, NCH2CH3). 13C NMR (CDCl3, 75.5
MHz): δ 140.0, 139.9, 138.9, 138.8, 138.6, 138.4, 138.3, 138.1 (C, Ar),
128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 127.6, 127.5, 127.4, 127.2,
127.1, 126.9, 126.8 (CH, Ar), 97.3 (β-C-1), 93.1 (C-1:COP), 91.0 (R-
C-1), 83.9 (CH), 82.9 (CH), 81.2 (CH), 81.1 (CH), 80.8 (CH), 79.7
(CH), 79.3 (CH), 77.2 (CH), 75.2, 75.1, 74.9, 74.8, 74.7, 74.6, 74.5,
74.4 (CH, CH2), 73.5 (CH2), 73.3 (CH2), 73.2 (CH2), 72.0 (CH2),
71.9 (CH2), 71.3 (CH), 71.2 (CH), 70.8 (CH), 70.7 (CH), 70.5
(CH2), 70.1 (CH2), 45.0 (NCH2CH3), 8.52 (NCH2CH3). 31P NMR
(CDCl3, 121.5 MHz): δ ꢀ1.75, ꢀ1.81 (s). HRMS (ESI): calcd for
C54H58O14P [M ꢀ H]ꢀ 961.3570, found 961.3591.
10 h at rt. The solution was concentrated under reduced pressure. A 1 M
TEAB aqueous solution (5.0 mL) and CHCl3 (5.0 mL) were added
successively to the residue, and the organic layer was separated. The
organic layer was then washed with 1 M TEAB aqueous solutions (2 ꢁ
5.0 mL). The aqueous layers were combined and back-extracted with
CHCl3 (10 mL). The organic layers were combined, filtered through
cotton, and concentrated under reduced pressure. The residue was
purified by Sephadex LH-20 (CH2Cl2) to give 26 as colorless foam (48.0
mg, 29 μmol, 67%). 1H NMR (CDCl3, 300 MHz): δ 7.52ꢀ7.13
(m, 45H, CH2Ph), 5.92ꢀ5.89 (m, 2H, R-H-1:CHOP), 5.30ꢀ3.42
(m, 37H, CH2Ph, R,β-H-1, H-2, H-3, H-4, H-5, H-6), 2.56 (q, 12H,
NCH2CH3), 0.92 (t, 18H, NCH2CH3). 13C NMR (CDCl3, 75.5 MHz):
δ 140.2, 140.0, 139.2, 139.0, 138.6, 138.4, 138.2, 138.1 (C, Ar), 128.3,
128.2, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3,
127.1, 127.0 (CH, Ar), 97.0 (β-C-1), 93.0, 92.7 (C-1:COP), 91.1 (R-C-
1), 81.1 (CH), 80.6 (CH), 79.7 (CH), 79.5 (CH), 79.1 (CH), 77.2
(CH), 75.1 (CH2), 74.6 (CH2), 73.5 (CH2), 73.1 (CH2), 73.0 (CH2),
72.0 (CH2), 71.6 (CH2), 71.1 (CH), 70.6 (CH), 70.5 (CH2), 70.3
(CH), 69.9 (CH2), 44.7 (NCH2CH3), 8.16 (NCH2CH3). 31P NMR
(CDCl3, 121.5 MHz): δ ꢀ4.11, ꢀ4.37 (s). HRMS (ESI): calcd for
C81H88O22P2Na [M þ Na]þ 1497.5135, found 1497.5124.
r-D-Glc-(1-P-4)-D-Glc (27). Compound 25 (27.2 mg, 26 μmol)
and palladium on activated carbon (10%, 31 mg) in MeOH (2.6 mL)
were allowed to stir under atmospheric pressure of H2 for 11 h. The
residue was then filtered through a filter paper and a membrane filter
(DISMIC-25JP, 0.50 μm). MeOHꢀEt3N (10 mL, 9:1, v/v) was added
to the solution and the solvent was removed under reduced pressure.
H2O was added to the residue. The insoluble materials were removed by
membrane filtration (DISMIC-13HP, 0.45 μm) to give 27 as a colorless
foam (12.0 mg, 23 μmol, 90%). 1H NMR (D2O, 300 MHz): δ
5.59ꢀ5.56 (m, 1H, R-H-1:CHOP), 5.22 (d, J = 3.9 Hz, R-H-1), 4.65
(d, J = 8.0 Hz, β-H-1), 3.98ꢀ3.26 (m, 12H, H-2, H-3, H-4, H-5, H-6),
3.19 (q, 6H, NCH2CH3), 1.27 (t, 9H, NCH2CH3). 13C NMR (D2O,
75.5 MHz): δ 98.9 (β-C-1), 98.7, 98.6 (C-1:COP), 94.8 (R-C-1), 77.8,
77.7, 77.6, 76.9, 76.6, 75.6, 74.7, 74.3, 74.2, 73.3, 71.9, 63.3, 63.2, 63.0,
48.8 (NCH2CH3), 11.8 (NCH2CH3). 31P NMR (D2O, 121.5 MHz):
δ ꢀ1.10, ꢀ1.13 (s). HRMS (ESI): calcd for C12H23O14PNa [M þ Na]þ
445.0718, found 445.0742.
r-D-Glc-(1-P-4)-r-D-Glc-(1-P-4)-D-Glc (28). The compound 26
(33.6 mg, 20 μmol) and palladium on activated carbon (10%, 30 mg) in
MeOH (2.0 mL) were allowed to stir under atmospheric pressure of H2
for 2 h. The residue was then filtered through a filter paper and a
membrane filter (DISMIC-25JP, 0.50 μm). MeOHꢀEt3N (10 mL, 9:1,
v/v) was added to the solution and the solvent was removed under
reduced pressure. H2O was added to the residue. The insoluble materials
were removed by membrane filtration (DISMIC-13HP, 0.45 μm) to
give 28 as a colorless foam (14.6 mg, 17 μmol, 84%). 1H NMR (D2O,
300 MHz): δ 5.60ꢀ5.56 (m, 2H, R-H-1:CHOP), 5.22 (d, J = 3.9 Hz, R-
H-1), 4.65 (d, J = 8.0 Hz, β-H-1), 4.05ꢀ3.27 (m, 18H, H-2, H-3, H-4,
H-5, H-6), 3.20 (q, 12H, NCH2CH3), 1.27 (t, 18H, NCH2CH3). 13C
NMR (D2O, 75.5 MHz): δ 98.7, 98.6 (β-C-1), 98.3, 98.2 (C-1:COP),
94.7 (R-C-1), 77.8, 77.6, 76.8, 76.7, 76.2, 75.6, 74.8, 74.4, 74.3, 74.1, 71.9,
63.3, 63.2, 63.0, 62.9, 61.6, 49.4 (NCH2CH3), 11.0 (NCH2CH3). 31P
NMR (D2O, 121.5 MHz): δ ꢀ1.12 (s). HRMS (ESI): calcd for
C18H34O22P2Na [M þ Na]þ 687.0909, found 687.0922.
Phosphodiester-Linked r-D-Glc-(1-P-4)-r-D-Glc-(1-P-4)-D-
Glc Derivative (26). A 2 M solution of Me2NH in THF (0.32 mL,
0.64 mmol) was added to a solution of the compound 24 (73.6 mg, 43
μmol) in dry MeCN (0.48 mL) at rt. The mixture was allowed to stir at
rt. Additional 2 M solutions of Me2NH in THF were added after 6 h
(0.53 mL, 1.1 mmol) and 36 h (0.43 mL, 0.86 mmol), and the mixture
was stirred for another 27 h. A 1 M TEAB aqueous solution (5.0 mL) and
CHCl3 (5.0 mL) were then added successively to the mixture, and the
mixture was washed with 1 M TEAB aqueous solutions (2 ꢁ 5.0 mL).
The aqueous layers were combined and back-extracted with CHCl3
(5.0 mL). The organic layers were combined, filtered through cotton,
and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography using CH2Cl2ꢀEt3NꢀMeOH
(97:2:1ꢀ96:2:2, v/v/v) as an eluent. The fractions containing 26 and
the N,N-dimethylaminoglycoside derivative were collected and concen-
trated under reduced pressure. The residue was dissolved in CHCl3
(5.0 mL) and washed with a 1 M TEAB aqueous solution (5.0 mL). The
aqueous layer was back-extracted with CHCl3 (10 mL). The organic
layers were combined, filtered through cotton, and concentrated under
reduced pressure. The residue was dissolved in an ammonium acetate
buffer solution (0.5 mL, pH 4.6) and the solution was allowed to stir for
2,3,4-Tri-O-Bn-r-D-mannopyranosyl Acetate (29). 2,3,4-Tri-
O-Bn-6-O-(4,40-dimethoxytrityl)-D-mannopyranosyl acetate S6 (0.182
g, 0.23 mmol) was dissolved in dry CH2Cl2 (2.3 mL). Dichloroacetic
acid (0.115 mL) was added to the solution at 0 °C. The mixture was
allowed to stir for 1 h. Then dry MeOH (5.0 mL) and a saturated
NaHCO3 aqueous solution (5.0 mL) were added successively to the
mixture. The organic layer was separated and washed with saturated
NaHCO3 aqueous solutions (2 ꢁ 5.0 mL). The aqueous layers were
combined and back-extracted with CH2Cl2 (5.0 mL). The organic layers
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ARTICLE
were combined, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column chroma-
tography using hexaneꢀAcOEt (2:1, v/v) as an eluent to give 29 as a
colorless oil (99.0 mg, 0.20 mmol, 87%). 1H NMR (CDCl3, 300 MHz): δ
7.40ꢀ7.16 (m, 15H, CH2Ph), 6.14 (d, J = 1.9 Hz, R-H-1), 5.60 (s, β-H-1),
4.97ꢀ4.57 (m, 6H, CH2Ph), 4.09ꢀ3.69 (m, 6H, H-2, H-3, H-4, H-5,
H-6), 2.07 (s, β-OCOCH3), 2.02 (s, R-OCOCH3). 13C NMR (CDCl3,
75.5 MHz): δ 169.0 (OCOCH3), 138.1, 138.0, 137.7 (C, Ar), 128.4,
128.3, 128.1, 127.9, 127.8, 127.7, 127.6 (CH, Ar), 92.9 (β-C-1), 91.6 (R-
C-1), 79.1 (C-3), 75.3 (CH2Ph), 74.5 (C-2), 73.9 (C-4), 73.5 (C-5), 72.6
(CH2Ph), 72.2 (CH2Ph), 61.9 (C-6), 20.9 (OCOCH3). HRMS (ESI):
calcd for C29H32O7K [M þ K]þ 531.1780, found 531.1805.
74.2, 74.1, 73.9, 73.7, 73.4, 73.0, 72.6, 72.4, 72.3, 71.8, 71.4 (CH, CH2), 70.1
(CH), 69.9 (CH), 68.6 (CH2Ph), 68.5 (CH2Ph), 62.8 (CH2Ph), 62.6
(CH2Ph), 45.0 (NCH2CH3), 25.9 (SiC(CH3)3), 21.0 (OOCCH3), 18.0
(SiC(CH3)3), 8.47 (NCH2CH3), ꢀ3.86, ꢀ3.97, ꢀ5.16 (Si(CH3)2). 31
P
NMR (CDCl3, 121.5 MHz): δ 101.2ꢀ95.0 (m). HRMS (ESI): calcd for
C62H77BO14PSi [M ꢀ H]ꢀ 1115.4919, found 1115.4892.
Phosphodiester-Linked 4-O-TBDMS-D-Glc-(1-P-6)-r-D-
Man-OAc Derivative (33). Dichloroacetic acid (0.18 mL) was added
to a solution of DMTrOMe (0.307 g, 0.90 mmol) and the compound 31
(0.362 g, 0.30 mmol) in dry CH2Cl2 (6.0 mL) at 0 °C. The mixture was
allowed to stir for 1 h at 0 °C. Et3SiH (0.65 mL) and CHCl3 (5.0 mL)
were added successively to the mixture and the mixture was washed with
saturated NaHCO3 aqueous solutions (2 ꢁ 10 mL). The aqueous layers
were combined and back-extracted with CHCl3 (10 mL). The organic
layers were combined, dried over Na2SO4, filtered, and concentrated
under reduced pressure to give the H-phosphonate intermediate 32.
A solution of I2 (0.152 g, 0.60 mmol) in dry PyꢀH2O (6.0 mL, 95:5,
v/v) was then added to the crude 32 and the mixture was allowed to stir
for 1 h at rt. The mixture was diluted with CHCl3 (5.0 mL) and washed
with a 0.5 M Na2S2O3 aqueous solution (10 mL) and 1 M TEAB
aqueous solutions (2 ꢁ 10 mL). The aqueous layers were combined and
back-extracted with CHCl3 (10 mL). The organic layers were combined,
filtered through cotton, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography using
CH2Cl2ꢀEt3NꢀMeOH (98:1:1, v/v/v) as an eluent. The fractions
containing 33 were collected and concentrated under reduced pressure.
The residue was dissolved in CHCl3 (5.0 mL) and washed with a 1 M
TEAB aqueous solution (5.0 mL). The aqueous layer was back-extracted
with CHCl3 (10 mL). The organic layers were combined, filtered
through cotton, and concentrated under reduced pressure to give 33
containing ca. 8 wt % of triethylammonium dichloroacetate as a yellow
Boranophosphotriester-Linked 4-O-TBDMS-r-D-Glc-(1-P-
6)-D-Man-OAc Derivative (30). Compounds 10 (0.327 g, 0.45
mmol) and 29 (0.184 g, 0.37 mmol) were dried by repeated coevapora-
tion with dry toluene and dry MeCN and in vacuo overnight. A solution
of1H-tetrazole (62.8 mg, 0.90 mmol) indry MeCN (3.75 mL), which was
dried over MS 3A prior to use, was added, and the mixture was allowed to
stir for 25 min at rt. A 1 M BH3 THF solution in THF (1.87 mL, 1.87
3
mmol) was added and the mixture was allowed to stir for 15 min at rt.
A saturated NaHCO3 aqueous solution (5.0 mL) and CHCl3 (5.0 mL)
were added successively to the mixture. The organic layer was separated,
washed with saturated NaHCO3 aqueous solutions (2 ꢁ 10 mL), dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography using hexa-
neꢀAcOEt (5:2, v/v) as an eluent to give 30 as a colorless foam (0.354 g,
0.31 mmol, 84%). 1H NMR (CDCl3, 300 MHz): δ 7.37ꢀ7.14 (m, 30H,
CH2Ph), 6.20, 6.16 (d, J = 1.9, 1.7 Hz, R-H-1), 5.91ꢀ5.85 (m, 1H, R-H-1:
CHOP), 5.59 (s, β-H-1), 5.03ꢀ4.20 (m, 12H, CH2Ph), 4.01ꢀ3.50
(m, 15H, H-2, H-3, H-4, H-5, H-6, POCH3), 1.99, 1.96 (s, 3H,
OCOCH3), 0.84ꢀ0.81 (m, 9H, SiC(CH3)3), 0.02 to ꢀ0.03 (m, 6H,
Si(CH3)2). 13C NMR (CDCl3, 75.5 MHz): δ 168.7, 168.6 (OCOCH3),
138.9, 138.1, 137.9, 137.6, 137.5, 137.4 (C, Ar), 128.3, 128.2, 128.1, 128.0,
127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 126.9(CH, Ar), 94.4(C-1),
91.5 (R-COCOCH3), 80.5 (CH), 79.7 (CH), 79.6 (CH), 79.5 (CH),
79.0 (CH), 78.9 (CH), 75.2 (CH2), 74.6 (CH2), 73.7, 73.5, 73.4, 73.3,
73.1, 73.0, 72.6, 72.5, 72.0, 71.9 (CH, CH2), 69.8 (CH), 69.7 (CH), 68.4
(CH2), 68.3 (CH2), 65.7 (CH2), 65.6 (CH2), 53.3, 53.2, 53.0, 52.9
(POCH3), 25.9 (SiC(CH3)3), 20.9 (OCOCH3), 18.0 (SiC(CH3)3),
ꢀ3.87, ꢀ3.92, ꢀ5.10 (Si(CH3)2). 31P NMR (CDCl3, 121.5 MHz): δ
119.7ꢀ115.9 (m). HRMS (ESI): calcd for C63H80BO14PSiNa [M þ
Na]þ 1153.5040, found 1153.5028.
1
oil (0.302 g, 0.25 mmol, 83%, estimated by H NMR analysis). The
mixture was purified by Sephadex LH-20 (CH2Cl2) to give 33 in 14%
1
isolated yield (49.0 mg, 40 μmol). H NMR (CDCl3, 300 MHz): δ
7.37ꢀ7.04 (m, 30H, CH2Ph), 6.13 (d, J = 1.9 Hz, R-H-1), 5.91 (dd, 1H,
JHH = 3.0 Hz, JPH = 8.0 Hz, R-H-1:CHOP), 5.59 (s, β-H-1), 5.00ꢀ4.44
(m, 12H, CH2Ph), 4.40ꢀ3.44 (m, 12H, H-2, H-3, H-4, H-5, H-6), 2.82
(q, 6H, NCH2CH3), 2.02, 1.99, 1.96 (s, 3H, OCOCH3), 1.14 (t, 9H,
NCH2CH3), 0.82ꢀ0.78 (m, 9H, SiC(CH3)3), ꢀ0.01 to ꢀ0.09 (m, 6H,
Si(CH3)2). 13C NMR (CDCl3, 75.5 MHz): δ 169.0 (OCOCH3), 139.4,
138.6, 138.4, 138.3, 138.2, 137.9 (C, Ar), 128.3, 128.2, 128.1, 128.0,
127.9, 127.8, 127.6, 127.5, 127.4, 127.2, 127.1, 127.0, 126.7 (CH, Ar),
92.5, 92.4 (C-1), 91.9 (R-COCOCH3), 80.8 (CH), 80.3 (CH), 80.2
(CH), 79.2 (CH), 77.2 (CH), 75.1 (CH2Ph), 74.3, 74.0, 73.9 (CH,
CH2), 72.9 (CH2Ph), 72.4 (CH), 72.3 (CH2Ph), 71.5 (CH2Ph), 70.0
(CH), 68.7 (CH2Ph), 64.3 (CH2Ph), 45.1 NCH2CH3, 26.0 (SiC
(CH3)3), 21.0 (OCOCH3), 18.0 (SiC(CH3)3), 8.35 NCH2CH3,
ꢀ3.89, ꢀ5.11 (Si(CH3)2). 31P NMR (CDCl3, 121.5 MHz): δ ꢀ3.07 (s).
HRMS (ESI): calcd for C62H74O15PSi [M ꢀ H]ꢀ 1117.4540, found
1117.4530.
Boranophosphodiester-Linked 4-O-TBDMS-r-D-Glc-(1-P-
6)-D-Man-OAc Derivative (31). Triethylamine (1.75 mL, 12.6
mmol) and benzenethiol (1.30 mL, 12.6 mmol) were added successively
to a solution of 30 (0.354 g, 0.31 mmol) in dry THF (3.1 mL), and the
mixture was allowed to stir for 17 h at rt. A 1 M TEAB aqueous solution
(5.0 mL) and AcOEt (5.0 mL) were added successively to the mixture.
The organic layer was separated and washed with 1 M TEAB aqueous
solutions (2 ꢁ 5.0 mL). The aqueous layers were combined and back-
extracted with AcOEt (5.0 mL). The organic layers were combined,
filtered through cotton, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography using
CH2Cl2ꢀEt3NꢀMeOH (99:1:0ꢀ98:1:1, v/v/v) as an eluent to give 31
as colorless foam (0.369 g, 0.30 mmol, 97%). 1HNMR(CDCl3, 300 MHz):
δ 7.46ꢀ7.16 (m, 30H, CH2Ph), 6.16ꢀ6.14 (m, R-H-1), 6.00ꢀ5.95 (m,
1H, R-H-1:CHOP), 5.62 (s, β-H-1), 5.09ꢀ4.45 (m, 12H, CH2Ph),
Phosphodiester-Linked r-D-Glc-(1-P-6)-D-Man Derivative
(34). Et3N 3HF (0.44 mL, 2.7 mmol) was added to a solution of
3
the compound 33 (0.219 g) containing ca. 4 wt % triethylammonium
dichloroacetate in dry THF (3.3 mL) at 40 °C. The mixture was allowed
to stir for 44 h at 40 °C. A 1 M TEAB aqueous solution (5.0 mL)
and CH2Cl2 (2.0 mL) were added successively to the mixture, and the
mixture waswashedwith 1 M TEAB aqueoussolutions (2ꢁ 5.0 mL). The
aqueous layers were combined and back-extracted with CH2Cl2 (5.0 mL).
The organic layers were combined, filtered through cotton, and concen-
trated under reduced pressure. The residue was purified by silica gel
column chromatography using CH2Cl2ꢀEt3NꢀMeOH (99:1:0ꢀ98:1:1,
v/v/v) as an eluent. The fractions containing the desired product were
collected and concentrated under reduced pressure. The residue was
dissolved in CHCl3 (5.0 mL) and washed with a 1 M TEAB aqueous
3
4.31ꢀ3.58 (m, 12H, H-2, H-3, H-4, H-5, H-6), 2.79 (q, 6H, NCH2CH ),
2.08, 2.02, 2.00 (s, 3H, OCOCH3), 1.13 (t, 9H, NCH2CH3), 0.92ꢀ0.81
(m, 9H, SiC(CH3)3), 0.02 to ꢀ0.04 (m, 6H, Si(CH3)2). 13CNMR(CDCl3,
75.5 MHz): δ 169.0 (OCOCH3) 139.3, 138.5, 138.2, 137.8, (C, Ar), 128.3,
128.0, 127.8, 127.7, 127.5, 127.4, 127.3, 127.2, 127.1, 127.0, 126.8, 126.7
(CH, Ar), 92.1, 91.7 (R-COCOCH3), 91.2, 90.9 (C-1), 81.0 (CH), 80.8
(CH), 80.3 (CH), 80.2 (CH), 79.3 (CH), 79.0 (CH), 75.1, 75.0, 74.7, 74.3,
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The Journal of Organic Chemistry
ARTICLE
solution (5.0 mL). The aqueous layer was back-extracted with CHCl3
(10 mL). The organic layers were combined, filtered through cotton, and
concentrated under reduced pressure to give the desired 4-O-desilylated
R-D-Glc-(1-P-6)-D-Man-OAc derivative as a yellow oil (0.136 g, 0.12
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details and char-
b
acterizing data, including 1H, 13C, and 31P spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
1
mmol, 41% isolated yield from 31). H NMR (CDCl3, 300 MHz): δ
7.40ꢀ7.20 (m, 30H, CH2Ph), 6.13 (d, J = 1.9 Hz, R-H-1), 5.91 (dd, 1H,
JHH = 3.0 Hz, JPH = 8.0 Hz, R-H-1:CHOP), 5.58 (s, β-H-1), 4.94ꢀ4.40
(m, 12H, CH2Ph), 4.29ꢀ3.53 (m, 12H, H-2, H-3, H-4, H-5, H-6), 2.80
(q, 6H, NCH2CH3), 2.01, 1.99 (s, 3H, OCOCH3), 1.13 (t, 9H,
NCH2CH3). 13C NMR (CDCl3, 75.5 MHz): δ 169.0 (OCOCH3),
139.0, 138.5, 138.3, 138.2, 137.8 (C, Ar), 128.3, 128.2, 128.1, 128.0,
127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 127.2 (CH, Ar), 92.9, 92.8
(C-1), 91.8 (R-COCOCH3), 80.8 (CH), 79.4 (CH), 79.3 (CH), 79.1
(CH), 77.2 (CH), 75.0 (CH2), 74.9 (CH2), 74.3 (CH), 74.2 (CH), 74.0
(CH), 73.9 (CH), 73.4 (CH2), 72.9 (CH2), 72.3 (CH2), 71.5 (CH2),
70.7 (CH), 69.5 (CH2), 64.2 (CH2), 64.1 (CH2), 45.1 NCH2CH3,
21.0 (OCOCH3), 8.35 NCH2CH3. 31P NMR (CDCl3, 121.5 MHz):
δ ꢀ2.78, ꢀ3.19 (s). HRMS (ESI): calcd for C56H61O15PNa [M þ Na]þ
1027.3640, found 1027.3626. Next, a 2 M solution of Me2NH in THF
(0.65 mL, 1.30 mmol) was added to a solution of the 4-O-desilylated R-D-
Glc-(1-P-6)-D-Man-OAc derivative thus obtained (96.5 mg, 87 μmol)
in dry MeCN (0.87 mL) at rt. The mixture was allowed to stir for 3 h at rt.
A 1 M TEAB aqueous solution(5.0 mL) and CH3Cl(5.0 mL) wereadded
successively to the mixture and the mixture was washed with 1 M TEAB
aqueous solutions (2 ꢁ 5.0 mL). The aqueous layers were combined and
back-extracted with CH3Cl (2 ꢁ 5.0 mL). The organic layers were
combined, filtered through cotton, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
using CH2Cl2ꢀEt3NꢀMeOH (99:1:0ꢀ98:1:1, v/v/v) as an eluent. The
fractions containing 34 were collected and concentrated under reduced
pressure. The residuewas dissolved inCHCl3 (5.0 mL) and washedwitha
1 M TEAB aqueous solution (5.0 mL). The aqueous layer was back-
extracted with CHCl3 (10 mL). The organic layers were combined,
filtered through cotton, and concentrated under reduced pressure to give
34 as a colorless foam (80.3 mg, 76 μmol, 86%). 1H NMR (CDCl3, 300
’ AUTHOR INFORMATION
Corresponding Author
wada@k.u-tokyo.ac.jp
’ ACKNOWLEDGMENT
We thank Prof. Kazuhiko Saigo (Kochi University of Tech-
nology) for helpful suggestions. This work was financially
supported by MEXT Japan.
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MHz): δ 7.37ꢀ7.16 (m, 30H, CH2Ph), 5.84 (dd, 1H, JHH = 3.3 Hz, JPH
=
7.4 Hz, R-H-1:CHOP), 4.88ꢀ4.43 (m, 12H, CH2Ph), 4.39ꢀ3.49 (m,
13H, R,β-H-1, H-2, H-3, H-4, H-5, H-6), 2.69 (q, 6H, NCH2CH3), 1.04
(t, 9H, NCH2CH3). 13C NMR (CDCl3, 75.5 MHz): δ 139.1, 138.8, 138.7,
138.6, 138.4, 138.2 (C, Ar), 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
127.5, 127.4, 127.3, 127.2 (CH, Ar), 93.0, 92.9 (C-1:COP), 92.1 (C-1),
81.1 (CH), 80.0 (CH), 79.3 (CH), 79.2 (CH), 77.2 (CH), 76.2 (CH),
75.4 (CH), 74.9 (CH2), 74.7 (CH2), 73.3 (CH2), 72.7 (CH2), 71.8
(CH2), 70.9 (CH), 70.8 (CH), 70.7 (CH), 69.5 (CH2), 65.4 (CH2), 45.2
NCH2CH3, 8.34 NCH2CH3. 31P NMR (CDCl3, 121.5 MHz): δ ꢀ3.30,
ꢀ3.50 (s). HRMS (ESI): calcd for C54H59O14PNa [M þ Na]þ 985.3535,
found 985.3516.
r-D-Glc-(1-P-6)-D-Man (35). The compound 34 (28.4 mg, 27
μmol) and palladium on activated carbon (10%, 30 mg) in MeOH
(2.7 mL) were allowed to stir under atmospheric pressure of H2 for 3 h.
The residue was filtered through a filter paper and a membrane filter
(DISMIC-25JP, 0.50 μm). MeOHꢀEt3N (10 mL, 9:1, v/v) was added
to the solution. The solvent was removed under reduced pressure. H2O
was added to the residue. The insoluble materials were removed by
membrane filtration (DISMIC-13HP, 0.45 μm) to give 35 as a colorless
foam (10.3 mg, 20 μmol, 74%). 1H NMR (D2O, 300 MHz): δ
5.53ꢀ5.50 (m, 1H, R-H-1:CHOP), 5.17 (d, J = 1.4 Hz, R-H-1), 4.90
(s, β-H-1), 4.22ꢀ3.44 (m, 12H, H-2, H-3, H-4, H-5, H-6), 3.19 (q, 6H,
NCH2CH3), 1.27 (t, 9H, NCH2CH3). 13C NMR (D2O, 75.5 MHz): δ
98.2, 98.1 (C-1:COP), 96.9 (R-C-1), 96.6 (β-C-1), 75.5 (CH), 75.3
(CH), 74.1 (CH), 74.0 (CH), 73.4 (CH), 72.9 (CH), 71.9 (CH), 69.2
(CH), 67.6 (C-6), 63.0 (C-6), 49.4 (NCH2CH3), 11.0 (NCH2CH3).
31P NMR (D2O, 121.5 MHz): δ ꢀ0.40, ꢀ0.46 (s). HRMS (ESI): calcd
for C12H23O14PNa [M þ Na]þ 445.0718, found 445.0725.
(10) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223–2311.
2658
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(15) See Supporting Information.
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