Stereoselective synthesis of P-modified α-glycosyl phosphates by the oxazaphospholidine approach

Article abstract of DOI:10.1021/ol402785h

α-Glycosyl phosphate derivatives are widely known as constituents of biomolecules. To date, several types of non-natural α-glycosyl phosphates including P-modified analogs have been synthesized to investigate their characteristics. Herein a new approach to the stereoselective modification of the intersugar phosphorus atom in α-glycosyl phosphates by use of the oxazaphospholidine method is presented. Via this approach, the dimers of α-glycosyl phosphorothioates and α-glycosyl boranophosphates were obtained efficiently and stereoselectively.

Full text of DOI:10.1021/ol402785h

ORGANIC
LETTERS
2013
Vol. 15, No. 23
5948–5951
Stereoselective Synthesis of P‑Modified
R‑Glycosyl Phosphates by the
Oxazaphospholidine Approach
Mihoko Noro,† Shoichi Fujita,† 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, and Department of Medicinal and Life Schience, Faculty of Pharmaceutical Sciences,
Tokyo University of Science 2641, Yamazaki, Noda, Chiba 278-8510, Japan
twada@rs.tus.ac.jp
Received September 26, 2013
ABSTRACT
R-Glycosyl phosphate derivatives are widely known as constituents of biomolecules. To date, several types of non-natural R-glycosyl phosphates
including “P-modified analogs” have been synthesized to investigate their characteristics. Herein a new approach to the stereoselective
modification of the intersugar phosphorus atom in R-glycosyl phosphates by use of the oxazaphospholidine method is presented. Via this
approach, the dimers of R-glycosyl phosphorothioates and R-glycosyl boranophosphates were obtained efficiently and stereoselectively.
R-Glycosyl phosphate derivatives are widely known as
constituents of capsular polysaccharides in pathogenic
bacteria such as Neisseria meningitidis and Streptococcus
pneumoniae,1,3 or the glycocalyx of parasitic protozoans
such as Leishmania and Trypanosoma.2,3They have repeat-
ing units of phosphoglycans, many of which are considered
to be important factors in biophenomena such as
immunological responses and infection. Therefore, inves-
tigation of their roles as biomolecules is being carried out
for the purpose of elucidating their biomechanisms and
applying them in drug development.1ꢀ3
With such aims, several types of non-natural R-glycosyl
phosphate analogs have been synthesized in previous
studies.4 Among them, we focused on the “P-modified
analogs,” where one of the nonbridging oxygen atoms is
replaced by a different atom or a substituent. The inter-
sugar phosphodiester linkage is considered to be an important
factor affecting several biophenomena. Thus, P-modified
analogs could become useful tools to clarify their biologi-
cal modes of action. Moreover, the phosphodiester moiety
isknowntomake alargecontribution tochemicalstability,
and proper modification of the phosphorus atom is re-
ported to further improve chemical stability.5 For these
reasons, P-modified glycosyl phosphate derivatives are
considered promising analogs.
† The University of Tokyo.
‡ Tokyo University of Science.
(1) (a) Trotter, C. L.; McVernon, J.; Ramsay, M. E.; Whitney, C. G.;
Mulholland, E. K.; Goldblatt, D.; Hombach, J.; Kieny, M.-P. Vaccine
2008, 26, 4434–4445. (b) Joshi, V. S.; Bajaj, I. B.; Survase, S. A.; Singhal,
R. S.; Kennedy, J. F. Carbohydr. Polym. 2009, 75, 553–565.
(2) (a) Mendonc-a-Previato, L.; Todeschini, A. R.; Heise, N.; Agrellos,
O. A.; Dias, W. B.; Previato, J. O. Curr. Org. Chem. 2008, 12, 926–939.
(b) Mendonc-a-Previato, L.; Todeschini, A. R.; Heise, N.; Previato, J. O.
Curr. Org. Struct. Biol. 2005, 15, 499–505. (c) Hansson, J.; Oscarson, S.
Curr. Org. Chem. 2000, 4, 535–564.
(3) Nikolaev, A. V.; Botvinko, I. V.; Ross, A. J. Carbohydr. Res.
2007, 342, 297–344.
(4) (a) Routier, F. H.; Higson, A. P.; Ivanova, I. A.; Ross, A. J.;
Tsvetkov, Y. E.; Yashunsky, D. V.; Bates, P. A.; Nikilaev, A. V.;
Ferguson, M. A. J. Biochemistry 2000, 39, 8017–8025. (b) Singh,
A. H.; Newborn, J. S.; Raushel, F. M. Bioorg. Chem. 1988, 16, 206–
214. (c) Cohen, S. B.; Halcomb, R. L. J. Org. Chem. 2000, 65, 6145–6152.
(d) Klinger, M. M.; McCarthy, D. J. Bioorg. Med. Chem. Lett. 1992, 2,
197–200. (e) Sheu, F. R.; Frey, P. A. J. Biol. Chem. 1978, 253, 3378–3380.
(f) Yamazaki, Y.; Nagatsuka, Y.; Oshima, E.; Suzuki, Y.; Hirabayashi,
Y.; Hashikawa, T. J. Immunol. Methods 2006, 311, 106.
These molecules have a chiral center at the intersugar
phosphorus atom. Because biological activity generally
differs between stereoisomers, stereochemically controlled
(5) (a) Prosperi, D.; Panza, L.; Poletti, L.; Lay, L. Tetrahedron 2000,
56, 4811–4815. (b) Matsumura, F.; Oka, N.; Wada, T. Org. Lett. 2008, 8,
1557–1560.
r
10.1021/ol402785h
Published on Web 11/13/2013
2013 American Chemical Society

study, as representative P-modified analogs, we selected
boranophosphates and phosphorothioates to be the syn-
thetic targets.
Table 1. Preparation of Monomer Units
First, we describe the synthesis of glycosyl boranopho-
sphates. We employed O-benzoyl-protected monomer
units (Rp)- and (Sp)-3bꢀc because the benzyl groups could
not be removed without associated deboronation of borano-
phosphodiester derivatives.8
Scheme 1. Synthesis of Glycosyl Boranophosphates
diastereo ratioa
Man/
Glc
yield
(%)
trans:
cis
entry
2
product
R
R:βb
1
2
3
4
5
6
L
D
L
D
L
D
(Rp)-3a Bn Man quant
(Sp)-3a Bn Man 74
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
95:5
(Rp)-3b Bz
(Sp)-3b Bz
(Rp)-3c Bz
Man 90
Man 83
Glc
Glc
82
65
(Sp)-3c
Bz
>99:1
a Estimated by 31P NMR. b For isolated product.
derivatives are required for assessment of their biological
properties. However, stereoselective synthesis of P-modified
glycosyl phosphates has not been accomplished yet.
Against such a background, we herein present a new
approach to the stereoselective modification of the intersugar
phosphorus atom with the “oxazaphospholidine method.”
This method has been used for the stereocontrolled synthesis
of phosphorothioate DNA,6a RNA,6b and boranophosphate
DNA6c for nucleic acid drugs. We applied this method to the
synthesis of glycosyl phosphate analogs and first accom-
plished the stereocontrol of the intersugar phosphorus atom.
We prepared monomer units (Rp)- and (Sp)-3aꢀc as
shown in Table 1. They were synthesized by the stereo-
controlled phosphitylation of sugar derivatives bearing
free anomeric hydroxyl groups with (4S,5R)- or (4R,5S)-
2-chloro-1,3,2-oxazaphospholidine derivatives (L- or D-2),
which were obtained by the reaction between PCl3 and the
corresponding 1,2-amino alcohols as previously reported.4a
Mannopyranosyl monomers (Rp)- and (Sp)-3aꢀb were
obtained in a highly R-selective manner because of the
stereoelectronic effect of their axial 2-OH. As for glucopyr-
anosyl derivatives, precursor 1c, which was a mixture of R
and β isomers, was recrystallized several times from AcOEt
or CH2Cl2/hexane to obtain R-rich isomers. The isomers
almost retained their stereochemical purity through the
following phosphitylation step even though the reaction
conditions could induce anomerization. This experimental
fact suggests that phosphitylation of 1 was substantially
more rapid than its anomerization.7
The P-modified dimers were synthesized via a three-step
reaction in one pot (Scheme 1). The monomer units (Rp)-
or (Sp)-3bꢀc were condensed with 1-O-thiophenyl-2,3,4-
tri-O-benzoyl-β-D-glucopyranoside 4a or 1-O-thiophenyl-
2,3,4-tri-O-benzoyl-R-D-mannopyranoside 4b, in the presence
of N-(cyanomethyl)pyrrolidinium triflate (CMPT), which
is an acid activator we developed for the stereospecific
condensation of oxazaphospholidine monomers.9 Then,
the resultant diastereopure glycosyl phosphite intermediates
(Sp)- or (Rp)-5bꢀc were boronated by treatment with
1 M BH3 THF in THF to give the glycosyl boranopho-
3
sphotriesters (Sp)- or (Rp)-6bꢀc.
In the last step, the removal of the chiral auxiliary was
attempted in the presence of several basic reagents (Table 2).
Each reaction was monitored by 31P NMR, and it appeared
that pyridine, 2,6-lutidine, and triethylamine (TEA) were
not sufficiently basic to activate the nitrogen atom of the
Using the synthesized monomer units, we attempted to
synthesize stereoregulated P-modified dimers. In this
(7) We observed that glucopyranosyl derivative 1 (R:β = 79:21) gave
monomer unit 3 (R:β = 80:20) through this phosphitylation step.
(8) Unpublished data.
(9) (a) Oka, N.; Wada, T.; Saigo, K. J. Am. Chem. Soc. 2002, 124,
4962–4967. (b) Oka, N.; Wada, T.; Saigo, K. J. Am. Chem. Soc. 2003,
125, 8307–8317.
(6) (a) Oka, N.; Yamamoto, M.; Sato, T.; Wada, T. J. Am. Chem.
Soc. 2008, 130, 16031–16037. (b) Oka, N.; Kondo, T.; Fujiwara, S.;
Maizuru, Y.; Wada, T. Org. Lett. 2009, 11, 967–970. (c) Wada, T.;
Maizuru, Y.; Shimizu, M.; Oka, N.; Saigo, K. Bioorg. Mol. Chem. Lett.
2006, 16, 3111–3114.
Org. Lett., Vol. 15, No. 23, 2013
5949

pyrrolidine ring of the chiral auxiliary and caused debor-
onation as a competitive side reaction. Although such a side
reaction was not observed when using N,N-diisopropylethy-
lamine (DIPEA), the intended reaction was not complete.
In contrast, the chiral auxiliary was successfully removed
by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in 5 min and
glycosyl boranophosphodiester 7b was quantitatively pro-
duced as a DBU salt. These results suggest that the main
reaction should be rapid enough compared to deboronation
in this case.
Scheme 2. Synthesis of Glycosyl Phosphorothioates
Table 2. Reaction Conditions for the Removal of Chiral
Auxiliaries
reaction conditions
entry
base
equiv
time
6b:7b:5ba
b
1
2
3
4
5
pyridine
2,6-lutidine
TEA
10
20
30
10
10
overnight
overnight
overnight
overnight
1 h
ꢀ
85:0:15
8:77:15
29:71:0
0:100:0
Table 3. Reaction Conditions for Sulfurization
DIPEA
DBU
a Estimated by 31P NMR. b Not monitored.
Next, we describe the synthesis of glycosyl phosphor-
othioates (Scheme 2). It was performed by condensation of
monomer units and sugar derivatives bearing free 6-OH,
and the chiral auxiliaries were removed by the previously
reported procedure.
Inthesulfurization reaction of benzyl-protectedglycosyl
phosphites using N,N0-dimethylthiuram disulfide (DTD)10
as a sulfurizing reagent, the formation of undefined bypro-
ducts was observed. Because it seemed to be caused by the
decomposition of the P-activated intermediate, we changed
the protecting group of the sugar hydroxyl moiety from a
benzyl group to a benzoyl group, which destabilized the
oxocarbenium cation. In addition, we tested other types of
sulfurizing reagents such as POS11 and S8 whose sulfurizing
reactions are expected to be more rapid than that of DTD.
The results are shown in Table 3. As we expected, the
benzoyl-protected glycosyl phosphites were more efficiently
sulfurizing
reagents
product ratio
diastereo
ratioa
entry
R3
of 8aꢀba
b
1
2
3
4
5
6
Bn
Bn
Bn
Bz
Bz
Bz
DTD
POS
S8
46%
ꢀ
b
80%
ꢀ
quant
87%
>99:1
b
DTD
POS
S8
ꢀ
quant
quant
>99:1
>99:1
a Estimated by 31P NMR. b Not determined.
transformed into the corresponding glycosyl phosphor-
othioate triesters than the benzyl-protected compounds. In
addition, both of the alternative sulfurizing reagents improved
(10) Song, Q.; Wang, Z.; Sanghvi, Y. S. Nucleosides, Nucleotides,
Nucleic Acids 2003, 22, 629–633.
(11) Roy, S. K.; Tang, J. Y. U.S. Patent 6, 500, 944, 2002.
5950
Org. Lett., Vol. 15, No. 23, 2013

On the basis of these results, we synthesized eight types
of P-modified glycosyl phosphate dimers. The structure of
R-D-Glc-(1-P-6)-D-Man is a fragment of glycocalyx lipo-
phosphoglycans of Leishmania. The results are shown in
Table 4.
In conclusion, we successfully accomplished an efficient
and highly stereoselective synthesis of glycosyl boranopho-
sphates and glycosyl phosphorothioates in different com-
binations of sugar moieties. These results suggest that this
method could be a versatile approach to the synthesis of a
wide variety of P-modified glycosyl phosphate analogs. In
addition, this method is expected to be applicable to solid-
phase synthesis, which can enable the synthesis of oligo-
glycosyl phosphate analogs.
Table 4. Stereocontrolled Synthesis of Dimers of
Glycosyl Boranophosphate and Phosphorothioate
Derivatives
diastereo
ratioa
entry
target compounds
(Rp)-Man-PB-Glc
yield
(Rp:Sp)
1
2
3
4
5
6
7
8
(Rp)-7b
(Sp)-7b
(Rp)-7c
(Sp)-7c
(Rp)-9b
(Sp)-9b
(Rp)-9c
(Sp)- 9c
79%
76%
77%
96%
82%
80%
77%
73%
>99:1
>1:99
>99:1
>1:99
>99:1
>1:99
>99:1
>1:99
(Sp)-Man-PB-Glc
(Rp)-Glc-PB-Man
(Sp)-Glc-PB-Man
(Rp)-Man-PS-Glc
(Sp)-Man-PS-Glc
(Rp)-Glc-PS-Man
(Sp)-Glc-PS-Man
Supporting Information Available. Experimental de-
tails and characterization data, including 1H, 13C, and 31
a Estimated by 1H or 31P NMR.
P
spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
this step and the formation of byproducts was reduced
compared to the reaction using DTD.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 23, 2013
5951