Solid-Phase Synthesis of Fluorinated Analogues of Glycosyl 1-Phosphate Repeating Structures from Leishmania using the Phosphoramidite Method

Article abstract of DOI:10.1002/open.201800030

Bacterial and protozoan sugar chains contain glycosyl 1-phosphate repeating structures; these repeating structures have been studied for vaccine development. The fluorinated analogues of [β-Gal-(1→4)-α-Man-(1→6)-P-]_n, which are glycosyl 1-phosphate repeating structures found in Leishmania, were synthesised using the solid-phase phosphoramidite method. This method has been less extensively studied for the synthesis of glycosyl 1-phosphate units than H-phosphonate chemistry. A stepwise synthesis of a compound containing five such repeating units has been conducted using the phosphoramidite method herein, which is the longest glycosyl 1-phosphate structures to be chemically constructed in a stepwise manner.

Full text of DOI:10.1002/open.201800030

DOI: 10.1002/open.201800030
Solid-Phase Synthesis of Fluorinated Analogues of
Glycosyl 1-Phosphate Repeating Structures from
Leishmania using the Phosphoramidite Method
Rintaro Iwata Hara,[a] Aya Yaoita,[a] Katsuya Takeda,[b] Hiroaki Ueki,[a] Ayumu Ishii,[b]
Hideyuki Imoto,[b] Satoshi Kobayashi,[b] Michi Sano,[a] Mihoko Noro,[b] Kazuki Sato,[b] and
Takeshi Wada*[a]
Bacterial and protozoan sugar chains contain glycosyl 1-phos-
phate repeating structures; these repeating structures have
been studied for vaccine development. The fluorinated ana-
logues of [b-Gal-(1!4)-a-Man-(1!6)-P-]n, which are glycosyl 1-
phosphate repeating structures found in Leishmania, were syn-
thesised using the solid-phase phosphoramidite method. This
method has been less extensively studied for the synthesis of
glycosyl 1-phosphate units than H-phosphonate chemistry. A
stepwise synthesis of a compound containing five such repeat-
ing units has been conducted using the phosphoramidite
method herein, which is the longest glycosyl 1-phosphate
structures to be chemically constructed in a stepwise manner.
1. Introduction
Monosaccharides such as glucose, mannose, and galactose
and oligo and polysaccharides are well-known biomolecules.
These compounds play a wide range of biological roles, from
an energy source to recognition between cell surfaces.[1] The
sugar units in oligo and polysaccharides are linked by means
of glycosidic bonds. Condensation between anomeric hydroxy
groups and hydroxy groups in sugars leads to the formation of
these bonds. In contrast, many bacterial and protozoan sugar
chains contain phosphodiester linkages between an anomeric
carbon and a carbon from another sugar.[2] Compounds con-
taining the repeating structures of these glycosyl 1-phosphate
units are known as phosphoglycans. For example, a-d-manno-
pyranosyl 1-phosphate repeating structures ([a-Man-(1!6)-
P-]n) are the constituents of Hansenula capsulata Y-1842 exo-
phosphomannan,[3] and a-d-galactopyranosyl 1-phosphate re-
peating structures ([a-Gal-(1!3)-P-]n) were found in capsular
antigens of E. coli.[4] The phosphoglycans from Leishmania con-
tain disaccharide 1-phosphate repeating units ([b-Gal-(1!4)-a-
Man-(1!6)-P-]n, Figure 1a), and many such oligosaccharide 1-
phospahte repeating structures have also been reported.[2,5]
These glycosyl 1-phosphate repeating structures are known to
play important roles in immunological responses and infec-
tions;[6] in addition, some phosphoglycans have been studied
for vaccine development.[7]
Figure 1. a) Structure of disaccharide 1-phosphate repeating units found in
Leishmania. b) The fluorinated analogues of the repeating units studied
herein.
[a] Dr. R. I. Hara, A. Yaoita, H. Ueki, M. Sano, Prof. T. Wada
Faculty of Pharmaceutical Sciences
Tokyo University of Science
2641 Yamazaki, Noda, Chiba 278–8510 (Japan)
E-mail: twada@rs.tus.ac.jp
The chemical synthesis of phosphoglycans is essential for
the elucidation of their functions. Using this method, these
compounds can be supplied in both large quantity and homo-
genous quality for their application in vaccines and therapeu-
tics. Owing to the chemical instability and high polarity of gly-
cosyl 1-phosphates and their derivatives, constructing their
structures and synthetic intermediates is challenging. Due to
these difficulties, the synthesis of glycosyl 1-phosphate repeat-
ing structures is limited in the number of repeating units possi-
ble. Vishwakarma et al. performed the chemical synthesis of
[b] K. Takeda, A. Ishii, H. Imoto, S. Kobayashi, M. Noro, K. Sato
Graduate School of Frontier Sciences
The University of Tokyo
5-1-5 Kashiwanoha, Kashiwa, Chiba 277–8562 (Japan)
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/open.201800030.
ꢀ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
compounds containing [b-Gal-(1!4)-a-Man-(1!6)-P-]19–22
,
which are the longest reported chemically synthesised glyco-
syl-1 phosphate repeating structures, to the best of our knowl-
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edge.[8] They followed a polycondensation approach using a H-
phosphonate building block; however, the length of the re-
peating structures could not be precisely regulated using this
approach. In this regard, performing a stepwise synthesis using
adequate building blocks could be advantageous. The exam-
ples of glycosyl 1-phosphate repeating structures which have
been synthesised in this manner are limited to be relatively
short oligomers. Vishwakarma et al. and Nikolaev et al. synthes-
ised the leishmanial [b-Gal-(1!4)-a-Man-(1!6)-P-]3 using the
H-phosphonate method.[8,9] Nikolaev et al. have also reported
the synthesis of a variety of other phospophoglycans such as
compounds containing [a-Man-(1!6)-P-]4 and [a-GlcNAc-(1!
3)-P-]3.[2,3] The synthesis of compounds containing [b-GlcNAc-
(1!3)-a-Gal-(1!4)-P-]2 and [b-GlcNAc-(1!3)-a-GalNAc-(1!4)-
P-]2 was reported by Oscarson et al.[10] In addition, Lay et al.
and Chhikara et al. respectively used the H-phosphonate
chemistry to construct [a-GlcNAc-(1!4)-P-]3 and [a-ManNAc-
(1!6)-P-]3 structures.[11] These studies suggest that the current
limitation for the length of the glycosyl 1-phosphate repeating
structures seem to be 2–4 repeating structures obtained by H-
phosphonate chemistry. In contrast, chemical synthesis of oli-
godeoxyribonucleotides (ODNs) and oligoribonucleotides
(ORNs) have been significantly developed over a few decades.
Despite containing multiple phosphodiester linkages, ODNs
with sizes of more than 20mers can now be easily synthesised
and are commercially available. Nowadays, ODNs and ORNs
are synthesised using the solid-phase phosphoramidite
method rather than the H-phosphonate method.[12] High effi-
ciency of condensation reaction and low polarity of phospho-
triester intermediates, which are particularly important in the
synthesis of long oligomers, can be achieved using the phos-
phoramidite method.[13] However, this method has not been
actively studied for the synthesis of phosphoglycans compared
to the oligonucleotide synthesis. As for the phosphoramidite
method, the most crucial difference between oligonucleotides
and glycosyl 1-phosphate repeating structures is the stability
of synthetic intermediates. Glycosyl 1-phosphate derivatives, in
contrast to ODNs and ORNs, can be easily degraded through
elimination of phosphorous-containing groups to generate ox-
ocarbenium cation.[14] Although the phosphoramidite chemis-
try has been applied to the synthesis of glycosyl 1-phosphate
structures,[15] long oligomers cannot be synthesised using this
method. In particular, the phosphotriester linkages are one of
the most labile functionalities in the intermediates.[16] In the
standard phosphoramidite method used for DNA and RNA syn-
thesis, trifluoroacetic and dichloroacetic acids are commonly
used for the removal of trityl type protecting groups. However,
glycosyl 1-phosphotriesters are unstable under acidic condi-
tions because the protonation of phosphotriester oxygen
atoms is expected to accelerate the elimination of the phos-
photriester group from the sugar.
We previously reported 2-deoxy-2-fluoromannopyranosyl 1-
phosphate derivatives as analogue molecules of [a-Man-1-P-]
structure.[14b] A fluorine atom is electronically equivalent to a
hydroxy group and its van der Waals radius is between hydro-
gen and oxygen atoms.[20] The usefulness of fluorinated sugar
analogues has been demonstrated by 2-deoxy-2-(18F)fluoro-d-
glucose, which has been used for positron emission tomogra-
phy.[21] Some studies have shown that fluorinated sugars serve
as a glycosidase substrate or inhibitor.[22] Therefore, fluorinated
sugar analogues have potential for sugar-based therapeutics
and biological tools. In our previous report, we demonstrated
the stability under acidic conditions of both mannosyl 1-phos-
photriesters and mannosyl 1-phosphodiesters could signifi-
cantly increase by a fluorine atom in place of 2-OH.[14b] The flu-
orine atom was expected to stabilise these compounds by pre-
venting the formation of oxocarbenium cation due to the
strong electron withdrawing property. Chemically stable ana-
logues of mannosyl 1-phosphodiesters are expected to be
useful as therapeutics in regarding to their handling and/or
drug duration. Here, we hypothesised a phosphoramidite
solid-phase synthesis could be applicable to the synthesis of
sugar analogues containing 2-deoxy-2-fluoro-glycosyl 1-phos-
phate structures. In this study, we aimed to synthesise the fluo-
rinated analogues of leishmainal glycosyl 1-phophate repeat-
ing structures (Figure 1b, [b-Gal-(1!4)-a-ManF-(1!6)-P-]n).
2. Results and Discussion
2.1. Synthesis of the Disaccharide Building Block
A disaccharide phosphoramidite was synthesised as shown in
Scheme 1. Compound 1 was prepared according to procedures
in the literature.[23] After deacetylation of compound 1, treat-
ment with approximately three equivalents of benzoyl chloride
(BzCl) afforded a protected fluorosugar bearing 4-OH 2. Com-
pound 2 was glycosylated with 3[24] to afford disaccharide 4.
The tert-butyldiphenylsilyl (TBDPS) group of 4 was removed by
One of the general strategies for resolving the instability of
natural compounds or their synthetic intermediates is the use
of their analogues.[17–19] Although some of these modifications
might have been effective for stabilisation of glycosyl 1-phos-
phate derivatives, the number of repeat units has currently
been limited to three.[17a]
Scheme 1. Synthesis of disaccharide phosphoramite 8. Reagents and condi-
tions: a) NaOMe, MeOH, RT (i); BzCl, pyridine, <M->408C, 60%, 2 steps (ii);
b) 3, TMSOTf, CH2Cl2, RT, 77%; c) TBAF, AcOH, THF, 08C to RT, 92%;
d) MMTrCl, pyridine, RT, 20 h, 91%; e) MeNH2, THF-MeOH, <M->308C, 83%;
f) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, 82%, a:b=97:3.
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treatment with tetramethylammonium fluoride (TBAF)[25] fol-
lowed by the protection of the newly generated hydroxy
group by a 4-methoxytrityl (MMTr) group to afford 6. The
anomeric benzoyl group in the 6 was selectively removed by
treatment with methylamine to afford compound 7,[26] then
phosphoramidite building block 8, with anomeric ratio of
a:b=97:3 was obtained by phosphitylation of 7 in 82% yield.
The anomeric isomers were partially resolved, and compound
8 with anomeric ratios of a:b=76:24 and 99:1 were used for
the following studies. It is noted that there were four diaste-
reomers derived from both the anomeric carbon atom in the
mannopyranose and the phosphorous atom, and their com-
plete separation was difficult.
2.2. Solid-Phase Synthesis of Compounds Containing
Disaccharide 1-Phosphate Repeating Units
We studied the solid-phase synthesis using phosphoramidite
unit 8 based on a standard synthetic procedure as shown in
Scheme 2.[13] Sugar elongation cycle contains mainly three
steps. The first step is the condensation reaction using phos-
phoramidite 8, in the presence of 4,5-dicyanoimidazole (DCI).
The second step is oxidation of the generated phosphite into
phosphotriester using (+)-(8,8-dichlorocamphorylsulfonyl)oxa-
ziridine (DCSO),[27] and the third is detritylation with trifluorace-
tic acid (TFA) to re-generate 6-OH for the next condensation.
After repeating of this cycle, removal of benzoyl groups and
cyanoethyl groups and release of compounds containing gly-
cosyl 1-phosphate repeating units can be simultaneously con-
ducted in the “deprotection and release” step. We chose a con-
trolled pore glass (CPG), which was commonly used for phos-
phoramidite DNA synthesis. A hydroquinone spacer was intro-
duced to CPG to enable us to quantitatively analyse the com-
pounds by UV absorption (see the Supporting Information).
We first conducted the synthesis of compounds containing
the two repeating units, [b-Gal-(1!4)-a-ManF-(1!6)-P-]2, ac-
cording to Scheme 2 using 8 with anomeric ratio of 99:1 (a:b),
and reaction conditions of the deprotection and release were
optimised. As shown in Figure 2, four-hour treatment with
methylamine was enough for removal of the protecting
groups and release of compounds from the solid support.
From MS analysis, however, there were multiple compounds
that were suggested to have the same molecular weight as
compound 10. The results suggested that stereo- and/or regio-
isomers were generated during solid-phase synthesis. One of
the most likely origins of the peaks was the generation of ste-
reoisomers because we used excess amounts of 8 containing a
small amount of the b anomer. To study stereoisomer genera-
Figure 2. Reverse-phase HPLC (RP-HPLC) profiles at 286 nm of crude prod-
ucts after the solid-phase synthesis according to Scheme 2 (n=2) after
a) 15 min, b) 30 min, c) 1 h, and d) 4 h treatment with methylamine.
10 equiv. of 8 (a:b=99:1) were used in each case. RP-HPLC was performed
with a linear gradient of 0–20% acetonitrile for an initial 60 min, and then
20–100% acetonitrile (between 60–65 min) in 0.1m triethylammonium ace-
tate buffer, and finally acetonitrile (between 65–75 min) at 308C.
tion, we synthesised compounds containing one repeating
unit 9 and two repeating units 10 by the same procedure
using compound 8 with two different anomeric ratios, one is
a:b=99:1 (Figure 3a,3b) and the other is 76:24 (Figure 3c,3d).
Comparing Figure 3a with Figure 3c, both of which illustrate
the synthesis of disaccharide 9, we found that there are two
peaks derived from disaccharides. It was clear that the later
peak was significantly larger in Figure 3c than that in Fig-
ure 3a. These results suggested the later peak was derived
from the b-isomer (i.e. [b-Gal-(1!4)-b-ManF-(1!6)-P-]). Fig-
ure 3b and 3d illustrate the synthesis of the tetrasaccharide
10. Four peaks were observed derived from tetrasaccharides
on HPLC profiles and the main peak should be derived from
compounds containing the [b-Gal-(1!4)-a-ManF-(1!6)-P-]2
structure. Other three peaks were expected to be of other
three isomers, that is, compounds containing [b-Gal-(1!4)-a-
ManF-(1!6)-P-b-Gal-(1!4)-b-ManF-(1!6)-P-], [b-Gal-(1!4)-b-
ManF-(1!6)-P-b-Gal-(1!4)-a-ManF-(1!6)-P-], and [b-Gal-(1!
4)-b-ManF-(1!6)-P-b-Gal-(1!4)-b-ManF-(1!6)-P-] structures,
and these peak areas are similar to that of 10. These results
clearly showed that the anomeric purities of mannosyl 1-phos-
phate products were lower than that of 8, which suggested
higher reactivity of the b-isomer of 8 (8b). To confirm the dif-
Scheme 2. Solid-phase synthesis of compounds containing glycosyl 1-phosphate repeating units by a phosphoramidite method.
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Scheme 3. Condensation reaction of 8 with isopropyl alcohol to form phos-
phite 11. The reaction was monitored by 31P NMR spectroscopy.
anomeric purity as shown in Figure 3a and 3b. Therefore, we
devised an improved strategy for the solid-phase synthesis to
solve these problems, and the overall procedures are shown in
Scheme 4. The only difference between Scheme 4 and the con-
ventional method (Scheme 2) is that phosphoramidite 8 had
initially been activated by DCI and reacted with a small
amount of isopropyl alcohol to consume 8b completely, before
addition to the reaction vessel containing solid support, in the
new strategy. The HPLC profiles in the synthesis of compounds
containing the two repeating units 10 by this pre-activation
method was shown in Figure 4b. In the HPLC profile no peak
derived from stereoisomers was observed, which contrasts
with the result from the conventional method in Figure 4a.
Therefore, we finally synthesised compounds containing the
one to five repeating units according to Scheme 4 and calcu-
lated the HPLC yields from the peak area derived from the hy-
droquinone spacer. It was clear from optimisation of reaction
conditions that the use of 20 equivalents of 8 was better for
each condensation reaction than using 10 equivalents. In these
conditions, compounds containing one repeating unit of 9 and
two repeating units of 10 could be synthesised with HPLC
yields of 98% and 93%, respectively (Figure 5a,5b; Table 1). As
these yields were high enough to study for the synthesis of
longer oligomers containing three to five repeating units,
these longer oligomers were also synthesised by the present
method. As shown in Figure 5c and Figure 5d, it was demon-
strated that compound containing the three and four repeat-
ing units 12 and 13 could be synthesised with high HPLC
yields, 95% and 91%, respectively, with trace amounts of
Figure 3. RP-HPLC profiles at 286 nm of crude products after the solid-phase
synthesis according to Scheme 2 after 4 h treatment with methylamine. a) 8
(a:b=99:1, 10 equiv) was used for the synthesis of compounds containing
the one repeating unit (n=1). b) 8 (a:b=99:1, 10 equiv) was used for the
synthesis of compounds containing the two repeating units (n=2). c) 8
(a:b=76:24, 10 equiv) was used for the synthesis of compounds containing
the one repeating unit (n=1). d) 8 (a:b=76:24, 20 equiv) was used for the
synthesis of compounds containing the two repeating units (n=2). RP-HPLC
was performed with a linear gradient of 0–8.7% acetonitrile for 52 min [in
(a) and (c)] or 0–16% acetonitrile for 48 min [in (b) and (d)] in 0.1m triethy-
lammonium acetate buffer at 308C. Whole HPLC profiles are provided in the
Supporting Information.
ference in reactivity of two isomers, condensation reaction was
conducted in solution and monitored by 31P NMR (see Support-
ing Information). In the experiments in Scheme 3, compound 8
with anomer ratio with 99:1 (a:b) was reacted with a quantity
of isopropyl alcohol equivalent to the amount of the b-isomer.
After the reaction, only b-mannosyl 1-phosphite triester 11b
was observed as glycosyl 1-phosphite derivatives. On the other
hand, when compound 8 was reacted with excess amount of
isopropyl alcohol, 11a was primarily observed. These results
also supported the higher reactivity of 8b.
This difference in reactivity was troublesome as considerable
b-isomers were produced even when using 8 with high
Scheme 4. Modified solid-phase synthesis containing a pre-activation step for phosphoramidite 8.
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shorter oligomers. Compared with these oligomers, the synthe-
sis of 14 containing five repeating units were slightly difficult.
As shown in Figure 5e, it was clear that the condensation of
octasaccharide was less efficient, even using 40 equiv. of 8,
than those of other shorter oligosaccharides. Although the
HPLC yields of the compounds containing the five repeating
units 14 were 39%, significantly lower than those of shorter
oligomers, this is the first example of the synthesis of com-
pounds containing five glycosyl 1-phosphate repeating units
except for a polycondensation method to the best of our
knowledge. All the compounds were isolated by HPLC and
identified by MS spectrometry (Table 1). The solid-phase phos-
phoramidite method could construct four and five repeating
units of [b-Gal-(1!4)-a-ManF-(1!6)-P-] with the pre-activation
process in this study. The results showed the potency of the
phosphoramidite method for the synthesis of compounds con-
taining acid-stable 2-deoxy-2-fluoro-mannopyranosyl 1-phos-
phate repeating units.
Figure 4. RP-HPLC profiles at 286 nm of crude products after the solid-phase
synthesis (n=2). a) The synthesis was conducted according to Scheme 2
except for condensation time of 20 min using 8 (a:b=99:1). b) The synthesis
was conducted according to Scheme 3 using 10 equiv of 8 (a:b=99:1). RP-
HPLC was performed with a linear gradient of 0–20% acetonitrile for 60 min
in 0.1m triethylammonium acetate buffer at 308C.
3. Conclusions
We synthesised compounds containing [b-Gal-(1!4)-a-ManF-
(1!6)-P-]5, the longest glycosyl 1-phosphate repeating units
that have been chemically synthesised in a stepwise manner,
to the best of our knowledge. We accomplished the synthesis
based on a solid-phase phosphoramidite method, and a prob-
lem concerning the high reactivity of the b isomer of the phos-
phoramidite was overcome by the consumption of the isomer
before solid-phase reaction with a pre-activation step. Investi-
gation for the synthesis of further longer oligomers and enzy-
matic activities of the synthesised analogues are now in
progress.
Experimental Section
General Procedure
Figure 5. RP-HPLC profiles at 286 nm of crude products after the solid-phase
synthesis (n=1–5) using 8 (a:b=99:1) according to Scheme 3. Condensa-
tion conditions and HPLC yields are on Table 1. RP-HPLC was performed
with a linear gradient of 0–20% acetonitrile for 60 min in 0.1m triethylam-
monium acetate buffer (pH 7) at 308C in (a) and (b). a) n=1. Compound 9
was eluted at 28.4 min. b) n=2. Compound 10 was eluted at 27.0 min. In
each case, N-methylbenzamide was eluted at 32 min. RP-HPLC was and with
a linear gradient of 0–10% acetonitrile for 60 min in 0.1m ammonium ace-
tate buffer (pH 7) at 308C in (c), (d), and (e). c) n=3. Compound 12 was
eluted at 23.6 min. d) n=4. Compound 13 was eluted at 20.8 min. e) n=5.
Compound 14 was eluted at 18.1 min. In each case, N-methylbenzamide
was eluted at 41–45 min.
All solution-phase reactions were conducted under an argon at-
mosphere. 1H NMR spectra were obtained at 300 MHz on a JEOL
Al300 spectrometer and at 400 MHz on a JEOL Lambda 400 spec-
trometer with tetramethylsilane (TMS) as an internal standard (d
0.0) in CDCl3. 31P NMR spectra were obtained at 162.0 MHz on a
JEOL Lambda 400 spectrometer with 85% H3PO4 (d 0.0) as an ex-
ternal standard. 19F NMR spectra were obtained at 376.3 MHz on a
JEOL Lambda 400 spectrometer. Mass spectra were recorded on a
JMS-700 spectrometer (JEOL) with trifluoroacetic acid as an exter-
nal standard (dꢀ76.5). Mass spectra were recorded on a 910-MS
Table 1. Reaction conditions and results of the synthesis of compounds containing [b-Gal-(1!4)-a-ManF-(1!6)-P-]n structure (n=1–5) according to
Scheme 3.
Entry
Product
Condensation conditions
8 (a:b=99:1) [equiv]
ESI-MS
Calcd (m/z)
HPLC yield
[%]
Time [min]
Found
1
2
3
4
5
9 (n=1)
20
20
20
20
40
60
20
20
20
20
[C22H33FO16P]-
603.1496
504.1050
1415.2849
1825.3525
1113.2064
603.1496
504.1050
1415.2843
1825.3513
1113.2053
98
94
95
91
39
10 (n=2)
12 (n=3)
13 (n=4)
14 (n=5)
[C34H53F2O28P2]2-
[C46H73F3O40P3]-
[C58H93F4O52P4]-
[C70H112F5O64P5]2
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FTMS system (Varian) or a Voyager System 4327 (Applied Biosys-
tems). Analytical TLC was performed on Merck Kieselgel 60-F254
plates. Silica gel column chromatography was carried out using
silica gel 60n (63–210 mm or 40–50 mm) as a neutral silica gel, and
Chromatorex NH-DM1020 as an NH silica gel. Gel filtration chroma-
tography was conducted using Sephadexꢁ LH-20. RP-HPLC was car-
ried out using a mBondasphere 5 mm C18, 100 ꢂ, 3.9 mm ꢃ
150 mm (Waters). Organic solvents were purified and dried accord-
ing to the appropriate procedures. Anomeric ratio was calculated
from relative peak areas in 19F NMR and 31P NMR.
mixture was dissolved in dichloromethane and washed with water.
The water layer was back-extracted with dichloromethane. The or-
ganic layers were combined and washed with brine, and then
dried over Na2SO4, filtered, and concentrated. The crude product
was purified by silica gel column chromatography (hexane-ethyl
acetate (3:1, v/v)) to afford pure 2 as colourless solid (4.64 g,
9.38 mmol, 60%, a:b=98:2).
1H NMR (CDCl3, 300 MHz) d 8.14–8.08 (6H, m), 7.68–7.44 (9H, m),
6.60 (1H, dd, J=1.8, 6,6 Hz, H-1), 5.68–5.54 (1H, m, H-3), 5.14–4.90
(2H, m, H-2, H-5), 4.50 (1H, dd, J=1.8, 12.4 Hz, H-6a), 4.33–4.16
(2H, m, H-4, H-6b), 3.31 (1H, d, J=4.1 Hz, 4-OH). 19F NMR d
-(203.9–204.2) (a), -(220.1–220.4) (b). MALDI-TOF MS Calcd ms for
Standard Procedure for Solid-Phase Synthesis with Pre-
Activating Step
C27H23FNaO8 [M + Na]+ 517.13 Found 517.13.
+
The CPG bearing MMTr group via hydroquinone spacer (23.8 or
58.4 mmolgꢀ1) was used for the solid-phase synthesis. Manual
solid-phase synthesis of compounds glycosyl 1-phosphate repeat-
ing units was conducted using a small glass filter with a stopper at
the top and stopcock at the bottom as a reaction vessel, and all re-
actions were conducted under argon atmosphere. Before synthesis,
solvents were dried over molecular sieves. Solutions of 4,5-dicya-
noimidazole (DCI) and (+)-(8,8-dichlorocamphorylsulfonyl)oxaziri-
dine (DCSO) in acetonitrile were also dried over molecular sieves,
respectively. Disaccharide phosphoramidite 8 was dried under re-
duced pressure. CPG bearing MMTr group via a hydroquinone
spacer (23.8 mmolgꢀ1, 21.0 mg, see SI) was placed into the reaction
vessel. The resin was treated with 1% TFA in dichloromethane
(1 mL, 15 sec ꢃ 3).
Compound 4
Glycosyl donor 3 (9.85 g, 11.3 mmol) and glycosyl acceptor 2
(4.64 g, 9.38 mmol) were coevaporated with toluene and dissolved
in dichloromethane (188 mL). Trimethylsilyl trifluoromethanesulfo-
nate (0.51 mL) was added dropwise to the solution over 5 min and
it was stirred for 40 min. The reaction was quenched by addition
of triethylamine (10 mL) and the solution was washed with saturat-
ed aqueous solutions of NaHCO3. The water layers were combined
and back-extracted with dichloromethane. The organic layers were
combined and then dried over Na2SO4, filtered, and concentrated.
The crude product was purified by silica gel column chromatogra-
phy (neutral silica gel, hexane-ethyl acetate (5:1, v/v)) to afford
pure 4 as colourless foam (8.70 g, 7.20 mmol, 77%, a:b=93:7).
After the MMTr-deprotection, the resin was washed with dichloro-
methane (1 mL ꢃ 4) and acetonitrile (1 mL ꢃ 3) and then dried
over reduced pressure. A pre-activated solution that contains dis-
accharide phosphoramidite 8 (20 mmol, a:b=98:2), DCI (75 mmol),
and 2-propanol (0.2 mmol) in acetonitrile (150 mL), was added to
the resin. The mixture was mixed by a vortex oscillator for 20 min.
Then the resin was washed with acetonitrile (1 mL ꢃ 3) and then
dried under reduced pressure. A solution of DCSO (0.5 M) in aceto-
nitrile (150 mL) was added to the resin. After 5 min, the resin was
washed with acetonitrile (1 mL ꢃ 3) and dichloromethane (1 mL ꢃ
3). The resin was treated with 1% TFA in dichloromethane (1 mL,
15 sec ꢃ 4). The procedural steps in this paragraph were repeated
1–5 times.
1H NMR (CDCl3, 400 MHz) d 8.10–7.94 (6H, m), 7.84–7.72 (4H, m),
7.65–7.20 (25H, m), 7.08 (1H, t, J=7.4 Hz), 6.99 (2H, t, J=7.6 Hz),
6.86 (2H, t, J=7.7 Hz), 6.53 (1H, dd, J=1.9, 6.6 Hz, H-1 (=H1Man)),
5.92 (1H, d, J=3.2 Hz, H-4’ (=H-4Gal)), 5.74–5.61 (2H, m, H-3, H-2’),
5.48–5.42 (1H, m, H-3’), 5.04 (1H, td, J=2.2, 48.9 Hz, H-2), 4.87 (1H,
d, J=7.8 Hz, H-1’), 4.58–4.42 (3H, m), 4.10 (1H, J=9.8 Hz), 3.77
(1H, dd, J=5.9, 8.5 Hz), 3.56 (1H, dd, J=5.6, 10.0 Hz), 3.36 (1H, t,
J=9.4 Hz). 0.94 (9H, s). 19F NMR (CDCl3, 376 MHz) d -(203.9–204.2)
(a), -(220.0–220.4) (b). MALDI-TOF MS Calcd ms for C70H63FNaO16Si+
[M + Na]+ 1229.37 Found 1229.19.
Compound 5
Then the resin was washed with dichloromethane (1 mL ꢃ 3) and
transferred to an eggplant flask. The resin was treated with 40%
aqueous solution of methylamine (4 mL) for 4 h. The resulting solu-
tion was filtered and collected, and freeze-dried. Finally, the crude
product was analysed by RP-HPLC.
Disaccharide 4 (7.23 g, 6.0 mmol) was dissolved in tetrahydrofuran
(113 mL) and acetic acid (0.41 mL) was added. A solution of tetra-
butylammonium fluoride trihydrate (1.89 g) in tetrahydrofuran
(6 mL), which was preliminarily dried over molecular sieves, was
added to the disaccharide solution after cooling to 08C. The mix-
ture was warmed to RT and stirred for 4.5 h and then the reaction
was quenched by addition of a saturated aqueous solution of
NaHCO3. The mixture was diluted with toluene and washed with
saturated aqueous solutions of NaHCO3. The water layers were
back-extracted with toluene. The organic layers were combined
and then dried over Na2SO4, filtered, and concentrated. The crude
product was purified by silica gel column chromatography (neutral
silica gel, hexane-ethyl acetate (3:1 to 1:1, v/v)) to afford pure 5 as
colourless foam (5.37 g, 5.54 mmol, 92%, a:b>99:1).
Compound 2
Compound 1 (5.45 g, 15.6 mmol) was dissolved in methanol
(156 mL). Sodium methoxide (84.1 mg, 1.56 mmol) added to the
solution and it was stirred for 1 h. The reaction was quenched by
addition of Dowex 50W X8 (H+ form) and the mixture was filtered.
The filtrate was dried over Na2SO4, filtered, and concentrated. The
crude product containing 2-deoxy-2-fluoro-d-mannopyranse was
coevaporated with pyridine, and then dissolved in pyridine
(200 mL). The solution was stirred and cooled to ꢀ408C and the di-
luted solution of benzoyl chloride in pyridine (5.4 mL BzCl in
111 mL pyridine) was added dropwise to the cooled solution over
10 min. After 2 h, additional benzoyl chloride (0.5 mL) was added
and the mixture was stirred for 3 h. The reaction was quenched by
addition of methanol, and then the mixture was concentrated. The
1H NMR (CDCl3, 400 MHz) d 8.11–7.93 (10H, m), 7.78–7.19 (20H, m),
6.55 (1H, dd, J=2.1, 6.9 Hz, H-1), 5.86–5.75 (2H, m, H-3, H-2’), 5.63
(1H, d, J=3.2 Hz, H-4’), 5.41 (1H, dd, J=3.3, 10.4 Hz, H-3’), 5.06
(1H, td, 2.5, 48.5 Hz, H-2), 4.96 (1H, d, J=8.0 Hz, H-1’), 4.59–4.46
(3H, m, H4), 4.18 (1H, d, J=9.6 Hz), 3.65 (1H, t, J=6.9 Hz) 3.28–
3.20 (1H, m), 3.08–2.99 (1H, m), 2.39 (1H, t, J=6.9 Hz, OH).
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19F NMR (CDCl3, 376 MHz) d -(202.9–203.2) (a), -(218.5–218.9) (b,
ture was diluted with dichloromethane and washed with saturated
aqueous solutions of NaHCO3. The water layers were combined
and back-extracted with dichloromethane. The organic layers were
combined, and then dried over Na2SO4, filtered, and concentrated.
The crude product was purified by silica gel column chromatogra-
phy (NH-silica gel, hexane-ethyl acetate (2:1, v/v)) to afford pure 8
as colourless foam (1.03 g, 0.81 mmol, 81%, a:b=97:3).
trace). MALDI-TOF MS Calcd ms for C54H45FNaO16 [M + Na]+
+
991.26 Found 991.02.
Compound 6
Compound 5 (1.45 g, 1.5 mmol) was coevaporated with pyridine
and dissolved in pyridine (15 mL). 4-Methoxytrityl chloride (1.39 g,
4.5 mmol) was added to the solution and it was stirred for 20 h.
Additional 4-methoxytrityl chloride (1.39 g, 4.5 mmol) was added
and the solution was stirred for 7 h. The reaction was quenched by
addition of methanol and the mixture was concentrated. The
crude product was diluted with dichloromethane and washed with
saturated aqueous solutions of NaHCO3. The water layers were
combined and back-extracted with dichloromethane. The organic
layers were combined, and then dried over Na2SO4, filtered, and
concentrated. The crude product was purified by silica gel column
chromatography (neutral silica gel, hexane-ethyl acetate (3.5:1 to
3:1, v/v)) to afford pure 6 as colourless foam (1.70 g, 1.37 mmol,
91%, a:b=96:4).
1H NMR (CDCl3, 300 MHz) d 8.09–7.92 (6H, m), 7.78–7.65 (4H, m),
7.59–7.05 (25H, m), 6.95–6.85 (2H, m), 6.61 (2H, d, J=8.8 Hz), 5.90
(1H, s, H-4’), 5.66–5.38 (4H, m, H-1, H-3, H-2’, H-3’), 5.03–4.74 (2H,
m, H-2, H-1’), 4.60–4.37 (3H, m, H-4, H-5, H-6a), 4.11–3.98 (1H, m),
3.92–3.50 (8H, m), 3.43–3.32 (1H, m), 3.19 (1H, q, J=9.2 Hz), 2.63,
2.38 (2H, t, t, J=6.2, 6.4 Hz, OCH2CH2CN, diastereomers). 1.23–1.04
(12H, m). 19F NMR (CDCl3, 376 MHz) -(202.3–202.6), -(203.1–203.4)
(a, diastereomers), -(219.7–220.1), -(220.2–220.5) (b, diastereomers)
31P NMR (CDCl3, 162 MHz) d 154.4 (b), 152.6 (a), 151.5 (b), 149.5 (a).
MALDI-TOF MS Calcd ms for C76H74FN2NaO17 [M + Na]+ 1359.46
+
Found 1359.28.
Acknowledgements
1H NMR (CDCl3, 300 MHz) d 8.10–7.93 (8H, m), 7.76–7.07 (32H, m),
6.90 (2H, t, J=7.7 Hz), 6.63 (2H, d, J=7.8 Hz), 6.53 (1H, dd, J=2.1,
6.8 Hz, H-1), 5.85 (1H, d, J=2.9 Hz, H-4’), 5.73–5.58 (2H, m, H-3, H-
2’), 5.39 (1H, dd, J=3.1, 10.3 Hz, H-3’), 5.11 (1H, d, J=48.7 Hz, H-2),
4.84 (1H, d, J=8.0 Hz, H-1’), 4.70–4.42 (3H, m, H-4), 4.10 (1H, d, J=
9.5 Hz), 3.74–3.66 (4H, m), 3.41–3.34 (1H, m), 3.21 (1H, t, J=
8.6 Hz). 19F NMR (CDCl3, 376 MHz) d -(204.0–204.3) (a), -(219.9–
This work was supported by SUNBOR GRANT from the Suntrory
Institute for Bioorganic Research.
Keywords: glycosyl 1-phosphate repeating units
chromatography phosphoglycans phosphoramidite
method · solid-phase synthesis
· liquid
·
·
220.4) (b). MALDI-TOF MS Calcd ms for C74H61FNaO17 [M + Na]+
+
1263.38 Found 1263.15.
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Compound 7
Disaccharide 6 (1.61 g, 1.30 mmol) was dissolved in tetrahydrofuran
(8 mL) and cooled to ꢀ308C. The mixed solution of tetrahydrofur-
an (5.0 mL) and 40 wt% methylamine in methanol (1.46 mL) was
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then gel filtration chromatography (neutral silica gel, dichlorome-
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1H NMR (CDCl3, 300 MHz) d 8.06–7.90 (6H, m), 7.75–7.67 (4H, m),
7.53 (2H, t, J=7.5 Hz), 7.56–7.07 (23H), 6.92 (2H, t, J=7.5 Hz), 6.63
(2H, d, J=7.8 Hz), 5.85 (1H, d, 2.9 Hz, H-4’), 5.66–5.36 (4H, m, H-1,
H-3, H-2’, H-3’), 4.97 (1H, d, J=49.8 Hz, H-2), 4.82 (1H, d, J=8.1 Hz,
H-1’), 4.54–4.44 (3H, m, H-4), 4.20 (1H, d, J=9.5 Hz), 3.72–3.64 (4H,
m, OMe, H-5’), 3.40–3.33 (1H, m, 6-H’a), 3.19 (1H, t, J=8.6 Hz, 6-
H’b), 2.76 (1H, t, J=3.3 Hz, OH). 19F NMR (CDCl3, 376 MHz) d
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C67H57FNaO16 [M + Na]+ 1159.35 Found 1159.11.
+
Compound 8
Disaccharide 7 (1.16 g, 1.0 mmol) was coevaporated with toluene
and dissolved in toluene (10 mL). The solution was cooled to 08C
and N,N-diisopropylethylamine (514 mL, 3.0 mmol) was added. 2-cy-
anoethyl N,N-diisopropylchlorophosphoramidite (335 mL, 1.5 mmol)
was added and the solution was warmed to room temperature.
After stirring for 4 h, additional 2-cyanoethyl N,N-diisopropylchloro-
phosphoramidite (112 mL, 0.5 mmol) was added. After 1 h, the mix-
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Received: February 27, 2018
Version of record online && &&, 0000
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FULL PAPERS
R. I. Hara, A. Yaoita, K. Takeda, H. Ueki,
A. Ishii, H. Imoto, S. Kobayashi, M. Sano,
M. Noro, K. Sato, T. Wada*
&& – &&
Solid-Phase Synthesis of Fluorinated
Analogues of Glycosyl 1-Phosphate
Repeating Structures from Leishmania
using the Phosphoramidite Method
Step by step: A solid-phase phosphora-
midite method was employed for the
synthesis of a series of compounds con-
taining [b-Gal-(1!4)-a-ManF-(1!6)-P-]5.
These compounds are, to our knowl-
edge, the longest glycosyl 1-phosphate
repeating units to be chemically syn-
thesised in a stepwise manner reported
to date.
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&
These are not the final page numbers! ÞÞ