Synthesis and properties of 2-deoxy-2-fluoromannosyl phosphate derivatives

Article abstract of DOI:10.1016/j.tet.2017.06.015

2-Deoxy-2-fluoromannosyl phosphotriester and phosphodiester derivatives were synthesized and they were demonstrated to be more stable than the 2-OH compounds under acidic conditions. 2-Fluoro substituted analog of α-mannopyranosyl 1-phosphodiester 20 was

Full text of DOI:10.1016/j.tet.2017.06.015

Tetrahedron 73 (2017) 4560e4565
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and properties of 2-deoxy-2-fluoromannosyl phosphate
derivatives
,
*
Rintaro Iwata Hara a, Satoshi Kobayashi b, Mihoko Noro b, Kazuki Sato b, Takeshi Wada a
a Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
b 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
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Deoxy-2-fluoromannosyl phosphotriester and phosphodiester derivatives were synthesized and they
were demonstrated to be more stable than the 2-OH compounds under acidic conditions. 2-Fluoro
Received 16 March 2017
Received in revised form
5 June 2017
Accepted 8 June 2017
Available online 13 June 2017
substituted analog of
a-mannopyranosyl 1-phosphodiester 20 was found to be significantly stable at
pH 1, where 2-OH compound 21 was gradually hydrolyzed. In addition, 2-F analogs of
a
-mannopyranosyl
1-phosphotriesters also was found to be stable under both Brønsted and Lewis acidic conditions. These
results strongly suggested that the 2-F substitution is useful for both the synthesis of mannosyl phos-
phodiester derivatives and improvement of their chemical stability.
© 2017 Elsevier Ltd. All rights reserved.
1. Introduction
phosphate structure.6 In this study, we focus on 2-deoxy-2-
fluoromannopyranosyl 1-phosphate derivatives as glycosyl phos-
The
a
-glycosyl phosphate unit, which has a phosphodiester
phate analogs.
Derivatives of a-mannopyranosyl 1-phosphate can be seen in
linkage between the anomeric carbon in one sugar and a carbon
atom in another sugar, is known as a constituent of capsular
polysaccharides (CPS) in pathogenic bacteria such as Neisseria
meningitidis and Streptococcus pneumoniae,1 and the glycocalyx of
parasitic protozoans such as Leishmania and Trypanosoma.2 These
units are considered to be important components in biological
phenomena including immunological responses and infection.3
Furthermore, there have been some reports in which some phos-
phoglycans have shown vaccine activity, and vaccines based on
them have been developed.4
Therefore, stable provision of pure compounds containing the
glycosyl phosphate structure is useful for both the elucidation of
their functions and the development of -glycosyl phosphate-
based vaccines and drugs. However, this remains a challenge for
both biologists and chemists, because sugar chains in living sys-
tems have structural heterogeneity and are present in very small
quantities; furthermore, their structures and their synthetic in-
termediates are chemically unstable in some cases.5 Therefore,
chemical modification has been spotlighted as a method for the
the glycocalyx of Leishmania1a and Hansenula capsulata Y-1842
exophosphomannan,7 and there are some derivatives containing
2-N-acetylmannosamine 1-phosphate units in CPS in Neisseria
meningitidis (Fig. 1).1a In this study, we chose the Man-
-(1 / 6)-P-
a-
a
Man structure as a synthetic target in the model compounds, poly-
(mannopyranosyl 1-phosphates), in Hansenula capsulata Y-1842
exophosphomannan.
A fluorine atom is electronically equivalent to a hydroxyl group.
Indeed, 2-deoxy-2-(18F)fluoro-
D-glucose is widely used as a glucose
a
-
analog for positron emission tomography (PET),8 and fluorinated
sugars have been reported as sugar analogs that serve as substrates
or inactivators for glycosidases, glycosyltransferases, and other
enzymes.9 Furthermore, a fluorine atom shows strong electron
withdrawing properties, and it can be expected to be advantageous
a
for the stabilization of a-glycosyl phosphate structures and their
synthetic intermediates. Some glycosyl 1-P structures and their
synthetic intermediates are degraded mainly through elimination
of the phosphorous containing groups at the anomeric position and
generation of the glycosyl oxocarbenium ion.5,10 A fluorine atom at
the 2-position of a pyranose can destabilize such a cation and make
stable supply of pure compounds containing the
a-glycosyl
a-glycosyl phosphate structures and their intermediates harder to
chemically degrade.
* Corresponding author.
E-mail address: twada@rs.tus.ac.jp (T. Wada).
Therefore, we firstly aimed to synthesize mannopyranosyl 1-
http://dx.doi.org/10.1016/j.tet.2017.06.015
0040-4020/© 2017 Elsevier Ltd. All rights reserved.

R.I. Hara et al. / Tetrahedron 73 (2017) 4560e4565
4561
them were recovered. On the other hand, 2-OBn compound 12 was
slowly degraded (almost completely disappeared in 24 h by TLC
monitoring, see SI) and nothing was recovered after the reaction.
The major degraded products were hydrolyzed product 4 and
anomeric dichloro acetate. By comparing the results for 9e11 and
12, it is clearly demonstrated that electron withdrawing 2-F and 2-
OAc substituents stabilized the mannosyl phosphotriesters in the
presence of dichloroacetic acid.
Fig. 1.
a-mannopyranosyl 1-phosphate repeating units seen in (a) Hansenula capsulata
Y-1842 exophosphomannan (b) CPS from Neisseria meningitidis (c) Leishmania
glycocalyx.
Next, the stability of these compounds under Lewis acidic con-
ditions was also investigated (Table 1, right column). In these ex-
periments, dichloromethane containing 0.1 M of BF3‧Et2O was used
to achieve Lewis acidic conditions. Under the conditions, the 2-O-
benzyl mannosyl phosphotriester 12 was completely degraded in
1 h (see SI). Furthermore, 2-O-acetyl one 10 was slowly degraded,
and nothing was recovered after 24 h of exposure to the acidic
solution. On the other hand, 2-fluoro mannosyl phosphotriesters 9
and 11 were observed to be intact even after 24 h, and 83% and 71%
of 9 and 11, respectively, were recovered. From these results, it was
shown that introducing a fluoro group instead of an OAc or OBn
groups at the 2-position of mannopyranose is much effective for
stabilization of mannosyl phosphotriesters under both Brønsted
and Lewis acidic conditions.
diethylphosphate and 2-deoxy-2-fluoromannopyranosyl 1-
diethylphosphate derivatives as analogs of the synthetic in-
termediates using the phosphoramidite method, which is one of
the most commonly used synthetic methods of synthesizing
a
a
-
-
glycosyl phosphate derivatives.1a Secondly, we synthesized
glycosyl phosphate derivatives containing an
a
-Man-(1-POꢀ4 -6)-
Man structure. Furthermore, the chemical stabilities of man-
nopyranosyl and 2-deoxy-2-fluoromannopyranosyl derivatives
were compared.
2. Results and discussion
2.1. Synthesis of phosphotriester analogs
2.3. Synthesis of mannosyl phosphates
Compounds 1e4 were known compounds and their synthetic
procedures are described in the literature.11 The anomeric hydroxyl
group of each compound was phosphitylated using diethyl chlor-
ophosphite to afford compounds 5e8. All compounds were sub-
sequently oxidized with (þ)-(8,8-dichlorocamphorylsulfonyl)
Next, we synthesized compounds containing the Man-a-
(1 / 6)-P-Man structure as analogs of Hansenula capsulata Y-1842
exophosphomannan. Firstly, mannose derivatives containing a hy-
droxyl group on their anomeric carbon atom, 13 and 14, were
prepared. Compound 13 was synthesized from a known acetyl-
protected compound (see SI),11a and 14 was prepared by a pro-
cedure described in the literature.14 Compounds 13 and 14 were
phosphitylated using 2-cyanoethyl-N,N-diisopropylchlorophosp-
horamidite to afford 15 and 16 in 86% and 73% yields, respectively.
The 6-hydroxy compound of thiomannoside 17 was prepared by
slight modification of a procedure described in the literature.15 In
the condensation reaction of phosphoramidite monomer 16 with
alcohol 17, the reaction did not proceed efficiently and over 80% of
unreacted 17 was recovered when 1-H-tetrazole was used as an
acidic activator. These condensation reactions proceeded well
when 5-ethylthio-1-H-tetrazole (ETT) was used as a more acidic
activator,16 and subsequently oxidation using tert-butyl hydroper-
oxaziridine (DCSO) and then mannosyl a-1-diethylphosphate de-
rivatives 9e12 were obtained in good yields (82%, 61%, 69%, 77%
respectively).12 Incidentally, the
anomer was not observed in
these reactions except in the synthesis of compound 12, in which
the anomer was isolated in 21% yield (Scheme 1).
b
b
2.2. Stability of mannosyl phosphotriesters under acidic conditions
A glycosyl phosphotriester structure is an intermediate in the
synthesis of glycosyl phosphate derivatives in the phosphoramidite
method. One of the most serious concerns regarding these re-
actions is the instability of the phosphotriester intermediates under
acidic conditions, such as in deprotection. Therefore, the stability of
mannosyl phosphotriesters 9e12 in dichloromethane containing
3% (v/v) dichloroacetic acid (DCA), which is prescribed for the
removal of trityl type protecting groups,13 was evaluated (Table 1,
left column). In these experiments, the reaction time was 24 h, and
the unreacted phosphotriester was recovered by silica gel column
chromatography. Most large quantities of 2-F and 2-OAc com-
pounds 9e11 remained intact under the conditions and 69e79% of
oxide17 afforded Man-
a-(1 / 6)-P-Man precursors 18 and 19 in
good yield as shown in Scheme 2. The cyanoethyl groups on com-
pounds 18 and 19 were removed with triethylamine and the ben-
zoyl groups were subsequently removed in the presence of
ammonia to afford the ammonium salts of the Man-a-(1 / 6)-P-
Man derivatives. These compounds were purified by reversed
phase HPLC (RP-HPLC) and were finally transformed to the sodium
salts on ion-exchange resin to afford compounds 20 and 21 (41%
and 26% yields, respectively) (see Scheme 3).
2.4. Stability of Man-
conditions
a-(1 / 6)-P-Man structures under acidic
To evaluate the properties of
a-2-deoxy-2-fluoromannosyl 1-
phosphate 20 and -mannosyl 1-phosphate 21, these compounds
a
were exposed to aqueous solutions at pH 7.0, 5.0, and 1.0 and the
degradation was monitored by RP-HPLC. At pH 7.0 and 5.0, no
degradation was observed with compounds 20 and 21. On the other
hand, degradation of 2-OH compound 21 was observed at pH 1.0 at
37 ꢁC and it was almost completely decomposed after 48 h (Fig. 2).
In contrast, 2-F derivative 20 was found to be completely
stable under the same acidic conditions. These results clearly
indicated that the 2-F substituent stabilized not only mannosyl
Scheme 1. Synthesis of mannopyranosyl 1-diethylphosphate and 2-deoxy-2-
fluoromannopyranosyl 1-diethylphosphate derivatives.

4562
R.I. Hara et al. / Tetrahedron 73 (2017) 4560e4565
Table 1
400 spectrometer with trifluoroacetic acid as an external standard
-76.55). Mass spectra were recorded on a Voyager System 4327
(Applied Biosystem) or a 910-MS FTMS system (Varian). Analytical
TLC was performed on Merck Kieselgel 60-F254 plates. Silica gel
column chromatography was carried out using Silica gel 60N
Recovery ratio of mannosyl phosphotriesters after treatment with DCA or BF3‧Et2O
over 24 h.
(d
Compound
3% (v/v)
DCA /CH2Cl2 at rt
0.1 M BF3‧Et2O/CH2Cl2-Et3SiH
(9:1, v/v) at 0ꢁꢁ
C
9
76%
79%
69%
0%
83%
0%
71%
0%
(63e210
m
m or 40e50
m
m) unless otherwise noted. Reversed
10
11
12
phase HPLC was carried out using a
m
Bondasphere 5 m C18,
m
100 Å, 3.9 mm ꢂ 150 mm (Waters) with a gradient of 0e20%
acetonitrile in 0.1 M triethylammonium acetate buffer at 25 ꢁC at a
phosphotriesters under both Brønsted and Lewis acidic conditions.
rate of 0.5 mL/min, or SunFire 5
mm C18, 100 Å, 19 mm ꢂ 150 mm
(Waters) with a gradient of 0e40% acetonitrile in 0.1 M triethy-
lammonium acetate buffer at 25 ꢁC at a rate of 2.5 mL/min.
Organic solvents were purified and dried by the appropriate pro-
cedure. all the new compounds were not crystalized and obtained
as oil or amorphous solid, and so melting temperatures were not
measured.
phosphotriesters under acidic conditions but also mannosyl phos-
phodiesters even at a pH of 1.0, which represents the most acidic
conditions in living systems such as in the stomach.
3. Conclusion
4.2. Elucidation of stability of Man-
under acidic conditions
a-(1 / 6)-P-Man structures
In this study, 2-deoxy-2-fluoromannosyl phosphotriester and
phosphodiester derivatives were synthesized and their stability
under acidic conditions was investigated. Phosphotriester de-
rivatives 9 and 11 and phosphodiester derivative 20 were demon-
strated to be more stable under acidic conditions than the
corresponding mannosyl phosphate derivatives. These results
strongly suggested that the 2-F substitution is useful for stabilizing
both mannosyl phosphotriester derivatives and their synthetic in-
To evaluate the stability of 20 or 21 pH 1.0, to a 2.0 mM aqueous
solution of compound 20 or 21 (50 L), an acidic aqueous solution
containing 0.2 M hydrochloride and 0.2 M potassium chloride
(50
L) was added. The solution was maintained at 37 ꢁC on a
thermal cycler. After 1, 4.45, 8, 19, 30, and 48 h, 5 L was transferred
out of the solution and diluted with 100 L of 0.1 M triethy-
m
m
m
m
termediates. The properties of these analog molecules of Man-a-
lammonium acetate buffer containing 30 nmol of benzamide. The
mixture was analyzed by RP-HPLC, and the ratio of intact mannosyl
phosphodiester 20 or 21 was calculated based on area ratio be-
tween benzamide and 20 or 21.
To evaluate the stability of 20 or 21 pH 7.0 and 5.0, the same
procedure was used in the following solutions; pH 7.0: 0.1 M
phosphate buffer (NaH2PO4-Na2HPO4); pH 5.0: citric-phosphate
buffer (citric acid- Na2HPO4).
(1 / 6)-P-Man, including their biological activities are currently
being investigated.
4. Experimental section
4.1. General information
All reactions were carried out under an argon atmosphere. 1
H
NMR spectra were obtained at 300 MHz on a Varian MERCURY
300 spectrometer or at 400 MHz on a JEOL Lambda 400 spec-
trometer with tetramethylsilane (TMS) as an internal standard (
0.0) in CDCl3 and with trimethylsilyl propanoic acid (TSP) as an
external standard (
0.0) in D2O. 13C NMR spectra were obtained at
75.5 MHz on a Varian MERCURY 300 spectrometer with CDCl3 as
an internal standard ( 77.0) in CDCl3, or at 100.8 MHz on a JEOL
d
d
d
Lambda 400 spectrometer with TPS as an external standard (d 0.0)
in D2O. 31P NMR spectra were obtained at 121.5 MHz on a Varian
MERCURY 300 spectrometer or at 162.0 MHz on a JEOL Lambda
400 spectrometer with 85% H3PO4
(d 0.0) as an external standard.
19F NMR spectra were obtained at 376.3 MHz on a JEOL Lambda
Scheme 3. Synthesis of Man-a-(1 / 6)-P-Man derivatives 20 and 21.
Fig. 2. Stability of 2-deoxy-2-fluoro mannosyl phosphate 20 and mannosyl phosphate
21 in acidic aqueous solution (pH 1.0, 37 ꢁC).
Scheme 2. Synthesis of Man-a-(1 / 6)-P-Man precursors 18 and 19.

R.I. Hara et al. / Tetrahedron 73 (2017) 4560e4565
4563
4.3. Elucidation of stability of mannosyl phosphotriesters under
Brønsted acidic conditions
5.40e5.30 (3H, m, H-2, H-3, H-4), 4.31 (1H, dd, J ¼ 12.3, 4.8 Hz, H-
6a), 4.24e4.09 (6H, m, H-5, H-6b, POCH2CH3), 2.18 (3H, s, OAc), 2.10
(3H, s, OAc), 2.06 (3H, s, OAc), 2.01 (3H, s, OAc), 1.38 (6H, tt, J ¼ 6.9,
Compound 9, 10, 11, and 12 (0.12 mmol) were dissolved in
dichloromethane (12 mL) respectively. To each solution, dichloro-
acetic acid (36 mL), and the reactions were monitored by TLC. After
1.2 Hz, POCH2CH3) 31P NMR (CDCl3, 121.5 MHz)
4.7. Compound 11
d
ꢀ3.0.
24 h, each reaction was quenched by addition of a saturated
aqueous solution of NaHCO3. The organic layer was dried over
Na2SO4, filtered, and concentrated, and compound 9, 10, 11, and 12
were recovered by silica gel column chromatography.
Compound 3 (1.2 g, 2.7 mmol) was dissolved in dichloro-
methane (27 mL), and the solution was stirred and cooled to 0 ꢁC.
Triethylamine (1.35 mL, 10.8 mmol) and diethyl chlorophosphite
(1.08 mL, 5.4 mmol) were added to the solution, and the mixture
was warmed to room temperature. After 20.5 h, the solution con-
taining compound 7 was cooled to 0 ꢁC again, and then (þ)-(8,8-
dichlorocamphorylsulfonyl)oxaziridine (2.4 g, 8.1 mmol) was
added. The solution was warmed to room temperature, stirred for
1 h, and then diluted with chloroform (300 mL). The solution was
washed with a saturated aqueous solution of NaHCO3 (50 mL ꢂ 2).
The organic layer was dried over Na2SO4, filtered, and concentrated.
The crude product was purified by silica gel column chromatog-
raphy (hexane-ethyl acetate (17:3, v/v)) to afford 11 as a pale-
yellow oil (1.11 g, 1.89 mmol, 69%).
4.4. Elucidation of stability of mannosyl phosphotriesters under
Lewis acidic conditions
Compound 9, 10, 11, and 12 (0.1 mmol) were dissolved in a
mixed solvent of dichloromethane and triethylsilane (10:1, v/v,
1.0 mL) respectively. Each solution was cooled to 0 ꢁC, BF3-Et2O
(17 mL) was added, and the reactions were monitored by TLC. After
24 h, each reaction was quenched by addition of a saturated
aqueous solution of NaHCO3. The organic layer was dried over
Na2SO4, filtered, and concentrated, and compound 9, 10, 11, and 12
were recovered by silica gel column chromatography.
1H NMR (CDCl3, 300 MHz)
J ¼ 1.8, 7.5 Hz, H-1), 4.89e4.47 (7H, m, H-2, PhCH2), 4.12e3.67 (9H,
m, H-3, H-4, H-5, H-6, POCH2CH3), 1.30e1.24 (6H, m, POCH2CH3) 31
d 7.40e7.17 (m, 15H, Ph) 5.79 (1H, dt,
4.5. Compound 9
P
NMR (CDCl3, 121.5 MHz)
d
ꢀ2.8.
Compound 1 (0.266 g, 0.86 mmol) was dissolved in dichloro-
methane (8.6 mL), and the solution was stirred and cooled to 0 ꢁC.
Triethylamine (0.48 mL, 3.84 mmol) and diethyl chlorophosphite
(0.38 mL, 1.92 mmol) were added to the solution, and the mixture
was warmed to room temperature. After 2.5 h, the solution con-
taining compound 5 was cooled to 0 ꢁC again, and then (þ)-(8,8-
dichlorocamphorylsulfonyl)oxaziridine (0.769 g, 2.58 mmol) was
added. The solution was warmed to room temperature, stirred for
4 h, and then diluted with chloroform (10 mL). The solution was
washed with a saturated aqueous solution of NaHCO3 (20 mL ꢂ 2).
The organic layer was dried over Na2SO4, filtered, and concentrated.
The crude product was purified by silica gel column chromatog-
raphy (hexane-ethyl acetate (1:1, v/v)) to afford 9 as a colorless
solid (0.331 g, 0.74 mmol, 87%).
MALDI-TOF MS: m/z calcd for C31H38FNaO8P [MþNa]þ 611.22;
found 611.30.
4.8. Compound 12
Compound 4 (1.4 g, 2.6 mmol) was dissolved in dichloro-
methane (26 mL), and the solution was stirred and cooled to 0 ꢁC.
Triethylamine (1.3 mL, 10.4 mmol) and diethyl chlorophosphite
(1.04 mL, 5.2 mmol) were added to the solution, and the mixture
was warmed to room temperature. After 33 h, more diethyl chlor-
ophosphite (0.52 mL, 2.6 mmol) was added and the mixture was
stirred for 3 h. The solution containing compound 8 was cooled to
0
ꢁC again, and then (þ)-(8,8-dichlorocamphorylsulfonyl)oxazir-
idine (2.30 g, 7.8 mmol) was added. The solution was warmed to
room temperature, stirred for 12 h, and then diluted with chloro-
form. The solution was washed twice with a saturated aqueous
solution of NaHCO3 and twice with phosphate buffer (pH 7). The
organic layer was dried over Na2SO4, filtered, and concentrated. The
crude product was purified by silica gel column chromatography
(hexane-ethyl acetate (3:7, v/v)) to afford 12 as a colorless solid
(1.2818 g, 1.90 mmol, 73%).
1H NMR (CDCl3, 300 MHz)
d
5.79 (1H, dt, J ¼ 2.4, 7.5 Hz, H-1),
5.39 (1H, t, J ¼ 10.1 Hz, H-3), 5.27 (1H, m, H-4), 4.82 (td, J ¼ 2.1,
48.9 Hz, H-2), 4.30e4.08 (7H, m, H-5, H-6, POCH2CH3), 2.09 (3H, s,
OAc), 2.07 (3H, s, OAc), 2.04 (3H, s, OAc), 1.36 (6H, tt, J ¼ 6.9, 1.5 Hz,
POCH2CH3) 31P NMR (CDCl3, 121.5 MHz)
d
ꢀ2.9.
MALDI-TOF MS: m/z calcd for C16H26FNaO11P [MþNa]þ 467.11;
found 466.98.
1H NMR (CDCl3, 300 MHz)
d 7.40e7.18 (m, 20H, Ph), 5.73 (1H, dd,
4.6. Compound 10
J ¼ 6.3, 2.1 Hz, H-1), 4.90 (1H, d, J ¼ 10.8, PhCH2) 4.74 (2H, s, PhCH2),
4.67e4.48 (5H, m, PhCH2), 4.10e3.72 (10H, m, H-2, H-3, H-4, H-5, H-
6, POCH2CH3), 1.27e1.21 (6H, m, POCH2CH3) 31P NMR (CDCl3,
Compound 2 (0.188 g, 0.54 mmol) was dissolved in dichloro-
methane (5.4 mL), and the solution was stirred and cooled to 0 ꢁC.
Triethylamine (0.28 mL, 2.2 mmol) and diethyl chlorophosphite
(0.16 mL, 1.1 mmol) were added to the solution, and the mixture
was warmed to room temperature. After 3 h, more diethyl chlor-
ophosphite (0.16 mL, 1.1 mmol) was added and the mixture was
stirred for 3 h. The solution containing compound 6 was cooled to
121.5 MHz)
d
ꢀ2.6.
MALDI-TOF MS: m/z calcd for C38H45NaO9P [MþNa]þ 699.27;
found 699.16.
4.9. Compound 15
0
ꢁC again and then (þ)-(8,8-dichlorocamphorylsulfonyl)oxazir-
Compound 13 (98.9 mg, 0.2 mmol) was coevaporated with
pyridine and toluene, and then dissolved in dichloromethane
(2.0 mL). The solution was stirred and cooled to 0 ꢁC, and then 2-
idine (0.480 g, 1.61 mmol) was added. The solution was warmed to
room temperature, stirred for 1 h, and then diluted with chloroform
(10 mL). The solution was washed twice with a saturated aqueous
solution of NaHCO3. The organic layer was dried over Na2SO4,
filtered, and concentrated. The crude product was purified by silica
gel column chromatography (hexane-ethyl acetate (1:1, v/v)) to
afford 10 as a pale-yellow oil (0.1596 g, 0.33 mmol, 61%).
cyanoethyl
N,N-diisopropylchlorophosphoramidite
(90
mL,
0.4 mmol) and N,N-diisopropylethylamine (140
mL, 0.6 mmol) were
added. The mixture was warmed to room temperature, and ethanol
(10 mL) was added after 2.5 h. The solution was diluted with
chloroform (20 mL) and washed with a saturated aqueous solution
of NaHCO3 (15 mL ꢂ 2). The organic layer was dried over Na2SO4,
1H NMR (CDCl3, 300 MHz)
d
5.63 (1H, dt, J ¼ 1.5, 6.9 Hz, H-1),

4564
R.I. Hara et al. / Tetrahedron 73 (2017) 4560e4565
filtered, and concentrated. The crude product was purified by
alumina column chromatography (hexane-ethyl acetate (9:1, v/v))
to afford 15 as a colorless solid (116 mg, 0.17 mmol, 86%).
4.12. Compound 19
Compound 16 (0.956 g, 1.2 mmol) and compound 17 (0.818 g,
1.4 mmol) were coevaporated with acetonitrile, and then dissolved
in acetonitrile (2.0 mL). To this solution, a solution of 5-ethylthio-
1H-tetrazole (0.818 g, 1.4 mmol) in acetonitrile (10 mL) was added
and the mixture was stirred for 5 min tert-Butyl hydroperoxide
(2.25 mL, 12 mmol) was added to the solution after cooling to 0 ꢁC,
and then the mixture was warmed to room temperature. After
25 min, the solution was diluted with ethyl acetate (10 mL) and
washed with a saturated aqueous solution of NaHCO3 (15 mL ꢂ 2).
The organic layer was dried over Na2SO4, filtered, and concentrated.
The crude product was purified by silica gel column chromatog-
raphy (hexane-ethyl acetate (3:2, v/v)) to afford 19 as a colorless
solid (1.23 g, 0.949 mmol, 77%).
1H NMR (CDCl3, 300 MHz)
d 8.06e7.92 (m, 6H, Ph), 7.58e7.33
(m, 9H, Ph), 5.93 (q, J ¼ 9.6 Hz, H-4), 5.80e5.61 (m, 1H, H-3),
5.58e4.49 (m, 1H, H-1), 5.07e4.80 (m, 1H, H-2), 4.66e4.42 (3H, m,
H-5, H-6), 3.94e3.66 (4H, m, OCH2CH2CN, OCH(CH3)2), 2.69, 2.54
(2H, 2t, J ¼ 6.3 Hz OCH2CH2CN, diastereomers), 1.32e1.17 (12H, m,
OCH(CH3)2).
13C NMR (CDCl3, 75.5 MHz)
d 165.8, 165.5, 165.4, 165.1, 165.0,
133.2, 132.9, 132.8, 129.6, 129.5, 129.4, 128.6, 128.2, 128.1, 117.3, 92.7,
92.4, 92.3, 92.0, 88.9, 88.8, 88.6, 86.4, 86.2, 70.5, 70.2, 69.6, 69.2,
66.3, 66.1, 62.9, 62.8, 58.8, 58.7, 58.6, 58.5, 43.6, 43.5, 43.4, 43.3,
24.4, 24.3, 24.2, 20.1, 20.0, 19.9.
31P NMR (CDCl3, 121.5 MHz)
d 153.4, 149.6.
1H NMR (CDCl3, 300 MHz)
d 8.14e7.77 (m, 14H, Ph), 7.60e7.17
(m, 28H, Ph), 6.22e5.76 (m, 8H), 5.00e4.90 (br, 1H), 4.76e4.15 (m,
4.10. Compound 16
7H), 2.71, 2.61 (2H, t, J ¼ 6.3 Hz, m) 31P NMR (CDCl3, 121.5 MHz)
d
ꢀ3.1, ꢀ3.6 MALDI-TOF MS: m/z calcd for C70H58NNaO20PS
Compound 14 (0.179 g, 0.3 mmol) was coevaporated with
pyridine and toluene, and then dissolved in dichloromethane
(3.0 mL). The solution was stirred and cooled to 0 ꢁC, and then 2-
[MþNa]þ 1318.29; found 1318.50.
4.13. Compound 20
cyanoethyl N,N-diisopropylchlorophosphoramidite (134
mL,
0.6 mmol) and N,N-diisopropylethylamine (204 L, 0.9 mmol)
m
Compound 18 (0.259 g, 0.2 mmol) was dissolved in a mixed
solvent of dichloromethane and triethylamine (5:7, v/v, 0.48 mL)
and stirred for 16 h. The mixture was concentrated and dissolved in
ethanol (6.0 mL). To the solution, 25% aqueous ammonia (20 mL)
was added and the mixture was stirred for 5 h at 55 ꢁC. The solution
was cooled to room temperature and ammonia was removed under
reduced pressure. The aqueous solution was washed with diethyl
ether (10 mL ꢂ 5) and concentrated. Triethylammonium hydro-
gencarbonate buffer (1.0 M) was added to the mixture, which was
and then concentrated again. The crude product was lyophilized,
and then one tenth of it was purified by RP-HPLC. The purified
product was converted to the sodium salt by addition of DowexⓇ
were added. The mixture was warmed to room temperature, and
ethanol (10 mL) was added after 2.5 h. The solution was diluted
with chloroform (20 mL) and washed with a saturated aqueous
solution of NaHCO3 (15 mL ꢂ 2). The organic layer was dried over
Na2SO4, filtered, and concentrated. The crude product was puri-
fied by alumina column chromatography (hexane-ethyl acetate
(9:1, v/v)) to afford 16 as a colorless solid (0.1737 g, 0.22 mmol,
69%).
1H NMR (CDCl3, 300 MHz)
d 8.13e7.82 (m, 8H, Ph), 7.61e7.24 (m,
12H, Ph), 6.23e6.13 (m, H-4), 5.98e5.93 (m,1H, H-3), 5.68e5.50 (m,
2H, H-1, H-2), 4.76e4.45 (m, 3H, H-5, H-6), 4.14e3.71 (4H, m,
OCH2CH2CN, OCH(CH3)2), 2.75, 2.61 (2H, dt, t, J ¼ 6.3, 2.7, 6.3 Hz,
OCH2CH2CN, diastereomers), 1.36e1.21 (12H, m, OCH(CH3)2) 13C
50 W X8 (Naþ form) to afford 20 (2.7 mg, 5.3
mmol, 27%).
7.62e7.60 (2H, m), 7.46e7.41 (3H, m),
1H NMR (D2O, 400 MHz)
d
NMR (CDCl3, 75.5 MHz)
d 166.0, 165.5, 165.4, 165.3, 133.4, 133.1,
5.54 (1H, t, J ¼ 7.2 Hz), 5.48 (1H, s), 4.82e4.68 (0.5H, s), 4.35e4.33
(1H, m), 4.24e4.23 (1H, q, J ¼ 1.6 Hz), 4.16e4.07 (2H, m), 3.99e3.85
(2H, m), 3.80e3.68 (1H, m, 5H).
133.0, 129.7, 129.6, 129.1, 128.9, 128.8, 128.5, 128.4, 128.3, 128.2,
117.5, 117.4, 93.0, 92.9, 92.7, 92.6, 71.3, 71.1, 71.0, 69.9, 69.8, 69.7,
69.4, 66.7, 66.4, 62.7, 62.6, 59.1, 59.0, 58.8, 58.7, 43.8, 43.7, 43.6, 24.5,
31P NMR (D2O, 162.0 MHz)
d
d
ꢀ1.3.
20.3, 20.2.31P NMR (CDCl3, 121.5 MHz)
d 152.7, 150.1.
13C NMR (D2O, 100.8 MHz)
135.6, 134.8, 132.1, 131.2, 96.0, 95.7,
93.2, 93.1, 91.4, 91.3, 91.0, 76.2, 75.0, 74.9, 74.0, 73.6, 71.8, 71.6, 69.3,
68.7, 67.5, 67.4, 62.7.
4.11. Compound 18
19F NMR (D2O, 376.3 MHz) e(204.8e205.1).
MALDI-TOF MS: m/z calcd for C18H25FO12PS [M]- 515.08; found
515.11.
Compound 15 (0.734 g, 1.1 mmol) and compound 17 (0.757 g,
1.3 mmol) were coevaporated with acetonitrile, and then dissolved
in acetonitrile (2.0 mL). To this solution, a solution of 5-ethylthio-
1H-tetrazole (1.4 g, 11 mmol) in acetonitrile (10 mL) was added and
the mixture was stirred for 10 min tert-butyl hydroperoxide
(2.1 mL, 11 mmol) was added to the solution after cooling to 0 ꢁC,
and then the mixture was warmed to room temperature. After
50 min, the solution was diluted with ethyl acetate (10 mL) and
washed with a saturated aqueous solution of NaHCO3 (15 mL ꢂ 2).
The organic layer was dried over Na2SO4, filtered, and concentrated.
The crude product was purified by silica gel column chromatog-
raphy (hexane-ethyl acetate (3:2, v/v)) to afford 18 as a colorless
solid (1.10 g, 0.92 mmol, 85%).
4.14. Compound 21
Compound 19 (0.139 g, 0.2 mmol) was dissolved in a mixed
solvent of dichloromethane and triethylamine (5:7, v/v, 0.48 mL)
and stirred for 16 h. The mixture was concentrated and dissolved in
ethanol (6.0 mL). To the solution, 25% aqueous ammonia (20 mL)
was added and the mixture was stirred for 5 h at 55 ꢁC. The solution
was cooled to room temperature and ammonia was removed under
reduced pressure. The aqueous solution was washed with diethyl
ether (10 mL ꢂ 5) and concentrated. Triethylammonium hydro-
gencarbonate buffer (1.0 M) was added to the mixture, which was
and then concentrated again. The crude product was lyophilized,
and then one tenth of it was purified by RP-HPLC. The purified
product was converted to the sodium salt by addition of DowexⓇ
1H NMR (CDCl3, 300 MHz)
d 8.10e7.81 (m, 12H, Ph), 7.60e7.20
(m, 24H, Ph), 6.06e5.57 (m, 7H, H-1, H-2, H-3, H-4, H-10, H-30, H-4’),
5.17e4.81 (m, 2H, H-20), 4.61e4.10 (m, 7H), 2.66, 2.53 (2H, t, q,
J ¼ 6.3, 6.3 Hz, OCH2CH2CN, diastereomers) 31P NMR (CDCl3,
121.5 MHz)
d
ꢀ3.1, ꢀ3.9.
50 W X8 (Naþ form) to afford 21 (4.2 mg, 8.1
1H NMR (D2O 400 MHz)
7.65e7.62 (2H, m), 7.49e7.42 (3H, m),
5.51 (1H, d, J ¼ 1.2 Hz), 5.42 (1H, dd, J ¼ 2.0, 7.6 Hz), 4.38e4.34 (1H,
mmol, 41%).
ESI MS: m/z calcd for C63H54FNO18PS [MþH]þ 1194.28; found
d
1194.28.

R.I. Hara et al. / Tetrahedron 73 (2017) 4560e4565
4565
b) Frasch CE. Expert Opin Biol Ther. 2005;5:273e280.
5. Frasch CE. Adv Biotechnol Process. 1990;13:123e145.
6. a) Teodorovic P, Slattegård R, Oscarson S. Org Biomol Chem. 2006;4:
4485e4490;
b) Gao Q, Zaccaria C, Tontini M, Poletti L, Costantino P, Lay L. Org Biomol Chem.
2012;10:6673e6681.
7. Nikolaev AV, Ivanova IA, Shibaev VN. Carbohydr Res. 1993;242:91e107.
8. Hoekstra CJ, Paglianiti I, Hoekstra OS, et al. Eur J Nuc Med. 2000;27:731e743.
9. a) McCarter JD, Yeung W, Chow J, Dolphin D, Withers SG. J Am Chem Soc.
1997;119:5792e5797;
m), 4.26, (1H, q, 1.6 Hz), 4.14 (2H, dd, J ¼ 4.0, 6.0 Hz), 3.99 (1H, t,
J ¼ 2.4 Hz), 3.92e3.68 (7H, m).
ꢀ
€
(2H, m), 3.99e3.85 (2H, m), 3.80e3.68 (1H, m, 5H).
31P NMR (D2O, 162.0 MHz)
d
ꢀ1.0.
13C NMR (D2O, 100.8 MHz)
d
135.7, 134.9, 132.2, 131.2, 98.9, 98.8,
91.1, 76.4, 75.1, 75.0, 74.1, 73.7, 73.2, 73.1, 72.6, 69.3, 68.9, 67.4, 67.3,
63.3.
MALDI-TOF MS: m/z calcd for C18H26O13PS [M]- 513.08; found
b) Lairson LL, Henrissat B, Davies GJ, Withers SG. Annu Rev Biochem. 2008;77:
521e555;
513.08.
c) Rempel BP, Withers SG. Org Biomol Chem. 2014;12:2592e2595.
10. a) Oka N, Sato K, Wada T. Trends Glycosci Glycotech. 2012;24:152e168;
b) Berkin A, Coxon B, Pozsgay V. Chem Eur J. 2002;8:4424e4443.
11. a) Stick RV, Watts AG. Monatsh für Chem. 2002;133:541e554;
b) Utille JP, Priem B. Carbohydr Res. 2000;329:431e439;
c) Bucher C, Gilmour R. Angew Chem Int Ed. 2010;49:8724e8728;
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2017.06.015.
ꢀ
d) Charbonnier F, Penades S. Eur J Org Chem. 2004:3650e3656.
12. a) Ugi I, Jacob B, Landgraf B, Ruppf C, Lemmen P, Verfürth U. Nucl Nucl. 1988;7:
References
605e608;
b) Wada T, Mochizuki A, Sato U, Sekine M. Tetrahedron Lett. 1998;39:
7123e7126;
1. a) Joshi VS, Bajaj IB, Survase SA, Singhal RS, Kennedy JF. Carbohydr Polym.
2009;75:553e565;
b) Trotter CL, McVernon J, Ramsay ME, et al. Vaccine. 2008;26:4434e4445.
2. (a) Mendonça-Previato L, Todeschini AR, Heise N, Agrellos OA, Dias WB,
Previato JO. Curr Org Chem. 2008;12:926e939;
c) Patrone JD, Yao J, Scott NE, Dotson GD.
J Am Chem Soc. 2009;131:
16340e16341.
13. a) Adinolfi M, Di Napoli L, Di Fabio G, Iadonisi A, Montesarchio D. Tetrahedron.
2002;58:6697e6704;
b) Matsumura F, Tatsumi S, Oka N, Wada T. Carbohydr Res. 2010;345:
1211e1215.
(b) Mendonça-Previato L, Todeschini AR, Heise N, Previato JO. Curr Org Struct
Biol. 2005;15:499e505;
14. Sato K, Oka N, Fujita S, Matsumura F, Wada T. J Org Chem. 2010;75:2147e2156.
15. Christensen MK, Meldal M, Bock K. J Chem Soc Perkin Trans. 1993;1:1453e1460.
16. a) Enkoji T, Lieber E. J Org Chem. 1961;26:4472e4479;
b) Welz R, Müller S. Tetrahedron, Lett. 2002;43:795e797.
17. Hayakawa Y, Uchiyama M, Noyori R. Tetrahedron Lett. 1986;27:4191e4194.
(c) Hansson J, Oscarson S. Curr Org Chem. 2000;4:535e564.
3. Ilg T, Handman E, Ng K, Stierhof Y-D. Bacic A Trend Glycosci Glycotech. 1999;11:
53e71.
4. a) Rogers ME, Sizova OV, Feguson MAJ, Nikolaev AV, Bates PA. J Infect Dis.
2006;194:512e518;