Selective diphosphorylation, dithiodiphosphorylation, triphosphorylation, and trithiotriphosphorylation of unprotected carbohydrates and nucleosides

Article abstract of DOI:10.1021/ol0521432

(Chemical Equation Presented) Aminomethyl polystyrene resin-bound linkers of p-acetoxybenzyl alcohol were subjected to reactions with diphosphitylating and triphosphitylating reagents to yield the corresponding polymer-bound diphosphitylating and triphosphitylating reagents, respectively. A number of unprotected carbohydrates and nucleosides were reacted with the polymer-bound reagents. Oxidation with tert-butyl hydroperoxide or sulfurization with Beaucage's reagent, followed by removal of cyanoethoxy group with DBU and the acidic cleavage, respectively, afforded only one type of monosubstituted nucleoside and carbohydrate diphosphates, dithiodiphosphates, triphosphates, and trithiotriphosphates with high regioselectivity.

Full text of DOI:10.1021/ol0521432

ORGANIC
LETTERS
2005
Vol. 7, No. 25
5589-5592
Selective Diphosphorylation,
Dithiodiphosphorylation,
Triphosphorylation, and
Trithiotriphosphorylation of Unprotected
Carbohydrates and Nucleosides
Yousef Ahmadibeni and Keykavous Parang*
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy,
The UniVersity of Rhode Island, Kingston, Rhode Island 02881
kparang@uri.edu
Received September 5, 2005
ABSTRACT
Aminomethyl polystyrene resin-bound linkers of p-acetoxybenzyl alcohol were subjected to reactions with diphosphitylating and triphosphitylating
reagents to yield the corresponding polymer-bound diphosphitylating and triphosphitylating reagents, respectively. A number of unprotected
carbohydrates and nucleosides were reacted with the polymer-bound reagents. Oxidation with tert-butyl hydroperoxide or sulfurization with
Beaucage’s reagent, followed by removal of cyanoethoxy group with DBU and the acidic cleavage, respectively, afforded only one type of
monosubstituted nucleoside and carbohydrate diphosphates, dithiodiphosphates, triphosphates, and trithiotriphosphates with high regioselectivity.
The selective diphosphorylation, dithiodiphosphorylation,
triphosphorylation, and trithiotriphosphorylation of unpro-
tected carbohydrates are challenging goals for organic
chemists. Thus, the chemical syntheses of carbohydrate
diphosphates, dithiodiphosphates, triphosphates, and trithio-
triphosphates have been rarely reported, and their biological
properties remain unknown. On the other hand, nucleosides
are intracellularly converted to nucleoside monophosphates,
diphosphates, and triphosphates, respectively, in the presence
of kinases.1 Diphosphorylation and triphosphorylation are
required for the synthesis of nucleotides and nucleic acids,
thus producing biological activity of all nucleosides, as
shown in several antiviral drugs.2,3 Phosphorothioate and
phosphorodithioate oligonucleotides have been used as
diagnostic reagents and in therapeutics.4
drates. However, a number of chemical strategies have been
previously reported for diphosphorylation and triphosphory-
lation of nucleosides in solution,5-7 such as the reaction of
nucleoside phosphoramidates (e.g., morpholidate) or phos-
phorodichloridates with bis(tri-n-butylammonium) pyrophos-
phate derivatives5 or the reaction of nucleoside diphosphates
and their derivatives (e.g., morpholidate, imidazolidate) with
phosphoric acid.6 These synthetic strategies have been
hampered by one or more of the following difficulties. (i)
The reactions generally must be carried out in anhydrous
organic solvents. Because of the poor solubility of most pre-
(1) Van Rompay, A. R.; Johansson, M.; Karlsson, A. Pharmacol. Ther.
2000, 87, 189-198.
(2) Furman, P. A.; Fyfe, J. A.; St. Clair, M. H.; Weinhold, K.; Rideout,
J. L.; Freeman, G. A.; Lehrman, S. N.; Bolognesi, D. P.; Broder, S.; Mitsuya,
H.; Barry, D. W. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8333-8337.
(3) Arts, E. J.; Wainberg, M. A. Antimicrob. Agents Chemother. 1996,
40, 527-540.
Currently, there is no universal method for the selective
diphosphorylation, dithiodiphosphorylation, triphosphoryla-
tion, and trithiotriphosphorylation of unprotected carbohy-
(4) Ravikumar, V. T.; Kumar, R. K.; Capaldi, D. C.; Turney, B.; Rentel,
C.; Cole, D. L. Org. Proc. Res. DeV. 2003, 7, 259-266.
10.1021/ol0521432 CCC: $30.25
© 2005 American Chemical Society
Published on Web 11/11/2005

cursor phosphates in the reaction mixture, the yield is low
in most cases. (ii) For triphosphorylation in solution, the
phosphoramidates and phosphorodichloridates are needed to
be synthesized first. (iii) Extensive purification of interme-
diates and final products from the reagents are required. (iv)
These strategies involve protection and deprotection reactions
for carbohydrates and lead in most cases to low overall yield
due to the lack of regioselectivity. (v) The synthesis of
dithiodiphosphate and trithiotriphosphate derivatives from the
corresponding diphosphate and triphosphate derivatives in
solution phase often leads to the incorporation of two sulfur
atoms (disulfurization) on the terminal phosphorus atom.
To solve one or more of these problems, we report the
solid-phase diphosphorylation, dithiodiphosphorylation, tri-
phosphorylation, and trithiotriphosphorylation of unprotected
carbohydrates and nucleosides. This strategy offered several
advantages. (i) The main advantage of this chemical proce-
dure was that it produced one type of monosubstituted
derivatives. Similar reactions in solution phase yield a mix-
ture of polysubstituted products. (ii) The alcohols (unpro-
tected nucleosides and carbohydrates) were mixed with an
immobilized reagent and were thereby “captured” as an im-
mobilized compound. Washing the support allowed for re-
moval of unreacted reagents and guaranteed that no unreacted
starting materials remained. (iii) This approach made use of
the presence of reagents on a rigid solid support having a
hindered structure, thereby allowing for the regioselective
reaction. The most reactive hydroxyl group of carbohydrates
and nucleosides reacted selectively with hindered polymer-
bound reagents when an excess of carbohydrates and
nucleoside was used. (iv) Reactions using this strategy
offered the advantage of facile isolation and the recovery of
products. The linkers remained trapped on the resins, which
facilitated the separation of the monosubstituted final prod-
ucts by filtration. (v) This method was used for synthesis of
four classes of compounds, carbohydrate and nucleoside
diphosphates, triphosphates, dithiodiphosphates, and trithiot-
riphosphates, from the same polymer-bound linker.
Scheme 1. Synthesis of Diphosphitylating (6) and
Triphosphitylating Reagents (7)
were determined by nuclear magnetic resonance spectra (1H
NMR, 13C NMR, 31P NMR) and high-resolution time-of-
flight electrospray mass spectrometry. Stability studies using
spectroscopic methods showed that the compounds remained
stable even after 2 weeks storage at -20 °C.
Our research on the development of polymer-bound
linkers8 and the synthesis of monophosphates and mono-
thiophosphates9 revealed that the p-acetoxybenzyl alcohol
is a good linker for attachment to solid-phase resins and
application in a variety of reactions. Two polymer-bound
linkers containing the p-acetoxybenzyl alcohol were selected
and synthesized from aminomethyl polystyrene resin in
multiple-step reactions.9 The polymer-bound linkers included
aminomethyl polystyrene resin linked through amide bond
with p-acetoxybenzyl alcohol (8A) and aminomethyl poly-
styrene resin linked through reduced amide bond with
p-acetoxybenzyl alcohol (8B) (Scheme 2).
Two classes of aminomethyl polystyrene resin-bound
linkers of p-acetoxybenzyl alcohol, 8A (3.05 g, 0.87 mmol/
g) and 8B (3.75 g, 0.72 mmol/g), were subjected to reactions
with the diphosphitylating reagent (6, ∼10 mmol) in the
presence of 1H-tetrazole to produce the corresponding
polymer-bound diphosphitylating reagents (9A and 9B).
Several unprotected nucleosides (e.g., thymidine (a), uridine
(b), 3′-azido-3′-deoxythymidine (c), adenosine (d)) and
carbohydrates (e.g., R,â-D-mannose (e), â-D-galactopyranose
(f), â-D-fructopyranose (g), melibiose (h)) (1.28 mmol) were
reacted with the polymer-bound reagents (9A and 9B) in the
presence of 1H-tetrazole to yield 11a-h and 12a-h,
respectively. Oxidation with tert-butyl hydroperoxide or
sulfurization with Beaucage’s reagent (3H-1,2-benzotrithiole-
3-one-1,1-dioxide),10 followed by removal of the cyano-
ethoxy group with DBU, afforded the corresponding polymer-
bound diphosphodiesters, 23a-h and 24a-h, or diphospho-
dithioesters, 25a-h and 26a-h. The cleavage of polymer-
bound compounds was carried out under acidic conditions
(TFA). The crude products had a purity of 69-91% and were
purified by using small C18 Sep-Pak cartridges and appropri-
ate solvents to afford nucleoside and carbohydrate diphos-
Scheme 1 illustrates the synthesis of diphosphitylating and
triphosphitylating reagents (6 and 7). Phosphorus trichloride
(1, 20 mmol) was reacted with 3-hydroxypropionitrile (2, 1
equiv) to yield 2-cyanoethylphosphorodichloridite (3). No
base was required in this reaction since generated HCl was
insoluble in acetonitrile and was bubbled out under dry
nitrogen at atmospheric pressure. The subsequent reaction
of 3 with diisopropylamine (20 mmol, 1 equiv) afforded
2-cyanoethyldiisopropylphosphoramidochloridite (4). Addi-
tion of water (1 equiv) gave the intermediate 5 that was
reacted with 4 (1 equiv) or 3 (0.5 equiv) to yield the
diphosphitylating (6, 97%) and triphosphitylating (7, 94%)
reagents, respectively. The chemical structures of 6 and 7
(5) (a) Ludwig, J. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16,
131-133. (b) Burgess, K.; Cook, D. Chem. ReV. 2000, 100, 2047-2059.
(c) De´saubry, L.; Shoshani, I.; Johnson, R. A. Tetrahedron Lett. 1995, 36,
995-996. (d) Shoshani, I.; Laux, W. H. G.; Perigaud, C.; Gosselin, G.;
Johnson, R. A. J. Biol. Chem. 1999, 274, 34742-34744.
(6) Bettendorff, L.; Nghiem, H.-O.; Wins, P.; Lakaye, B. Anal. Biochem.
2003, 322, 190-197 and references cited therein.
(8) (a) Ahmadibeni, Y.; Parang, K. Org. Lett. 2005, 7, 1955-1958. (b)
Parang, K. Bioorg. Med. Chem. Lett. 2002, 12, 1863-1866. (c) Parang,
K.; Fournier, E. J.-L.; Hindsgaul, O. Org. Lett. 2001, 3, 307-309.
(9) Ahmadibeni, Y.; Parang, K. J. Org. Chem. 2005, 70, 1100-1103.
(10) Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L. J. Am. Chem.
Soc. 1990, 112, 1253-1254.
(7) Wu, W.; Meyers, C. L. F.; Borch, R. F. Org. Lett. 2004, 6, 2257-
2260.
5590
Org. Lett., Vol. 7, No. 25, 2005

Scheme 2. Selective Synthesis of Nucleoside and
Carbohydrate Diphosphates, Dithiodiphosphates, Triphosphates,
and Trithiotriphosphates on Solid Phase Using Polymer-Bound
Linkers 8A and 8B
Table 1. Overall Isolated Yields and Purity of Crude Products
for Carbohydrate and Nucleoside Diphosphates (31a-h),
Diphosphodithioates (32a-h), Triphosphates (33a-h), and
Triphosphotrithioates (34a-h)
overall
overall
purity
of crude
products (%)
using 8A
purity
of crude
products (%)
using 8B
yield (%)
calcd from
9A or 10A
yield (%)
calcd from
9B or 10B
No.
31a
31b
31c
31d
31e
31f
31g
31h
32a
32b
32c
32d
32e
32f
32g
32h
33a
33b
33c
33d
33e
33f
33g
33h
34a
34b
34c
34d
34e
34f
77
75
84
68
60
66
61
51
69
72
78
63
70
72
53
57
74
71
79
63
64
58
55
43
63
69
81
67
73
65
45
47
80
77
80
64
68
60
51
54
74
77
82
72
74
61
55
57
76
73
77
60
64
51
47
42
77
70
75
59
71
54
46
50
81
83
90
82
77
89
86
69
83
88
91
75
80
87
75
74
80
78
91
79
81
76
71
64
75
86
90
77
79
76
67
71
91
89
87
83
87
79
84
72
91
85
89
87
86
76
81
79
90
87
91
79
83
72
80
67
89
86
90
76
82
69
77
67
phates (31a-h) and diphosphodithioates (32a-h) (Scheme
2) in 51-84% overall yield (calculated from 9A and 9B in
four-step reaction sequence) (Table 1). Only one type of
monosubstituted compounds was produced with high selec-
tivity as a result of this sequence.
34g
34h
The synthetic approach for the selective solid-phase syn-
thesis of triphosphates and triphosphotrithioates was similar
to the diphosphorylation and dithiophosphorylation protocols
(Scheme 2), respectively, except for the use of a triphos-
phitylating reagent (7), instead of a diphosphitylating reagent
(6). The reaction of polymer-bound linkers 8A and 8B with
the triphosphitylating reagent (7) in the presence of 1H-
tetrazole afforded the polymer-bound triphosphitylating
reagents 10A and 10B. A number of unprotected nucleosides
(e.g., a-d) and carbohydrates (e.g., e-h) were reacted with
the polymer-bound reagents. The resulting polymer-bound
compounds, 13a-h and 14a-h, underwent oxidation and
deprotection reactions to afford the polymer-bound triphos-
phodiesters, 27a-h and 28a-h, or triphosphotrithiodiesters,
29a-h and 30a-h. Cleavage from the resins under acidic
conditions and purification of crude products (64-91%
purity) by using small C18 Sep-Pak cartridges afforded the
nucleoside and carbohydrate triphosphates (33a-h) and
triphosphotrithioates (34a-h) with high selectivity in 42-
79% overall yield (calculated from 10A and 10B in four
steps) (Table 1).
For a typical example (Scheme 2), â-D-galactopyranose
(f, 1.21 mmol) and 1H-tetrazole (68 mg, 0.96 mmol) were
added to 10A (0.59 mmol) in anhydrous THF (2 mL) and
DMSO (3 mL). The mixture was shaken for 24 h at room
temperature. The resin was collected by filtration and washed
with DMSO, THF, and MeOH, respectively, and dried under
vacuum to give 13f. tert-Butyl hydroperoxide in decane (5-6
M, 1.28 mmol) was added to the resin (13f) in THF. After
1 h shaking at room temperature, the resin was collected by
filtration and washed with THF and MeOH, respectively,
and was dried overnight at room temperature under vacuum
to give 19f. To the swelled resin 19f in THF was added DBU
(0.64 mmol). After 48 h shaking of the mixture at room
temperature, the resin was collected by filtration and washed
with THF and MeOH, respectively, and dried overnight at
room temperature under vacuum to give 27f. To the swelled
resin (27f) in anhydrous DCM was added DCM/TFA/water
(24:74:2 v/v, 3 mL). After the mixture was shaken for 25
min at room temperature, the resin was collected by filtra-
tion and washed with DCM, THF, and MeOH, respectively.
The solvents of filtrate solution were immediately evaporated
Org. Lett., Vol. 7, No. 25, 2005
5591

Scheme 3. Cleavage Mechanisms of Nucleoside and Carbohydrate Diphosphates, Dithiodiphosphates, Triphosphates, and
Trithiotriphosphates 32-34 (a-h) from 23-30 (a-h)
at -20 °C. The residue was mixed with Amberlite AG-
50W-X8 (100-200 mesh, hydrogen form, 500 mg) in
water:dioxane (75:25 v/v, 3 mL) for 15 min. After filtration,
the solvents were evaporated, and the crude product was
purified using C18 Sep-Pak to yield 33f.
The cleavage mechanisms of final products from 23-30
(a-h) using polymer-bound linkers A and B are shown in
Scheme 3. The linkers remained trapped on the resins in both
methods, which facilitated the separation of the final products
by filtration. In general, the yield and purity of nucleoside
products were higher than those of carbohydrate derivatives,
but there was no significant difference between polymer-
bound linkers 8A and 8B.
In conclusion, these solid-phase strategies offer the
advantages of monosubstitution, high selectivity, facile
isolation and purification of products, and trapping byprod-
ucts on resins. To the best of our knowledge, this is the first
time that carbohydrate and nucleoside diphosphates, diphos-
phodithioates, triphosphates, and triphosphotrithioates have
been synthesized by using solid-phase reagents. Washing of
the resins allows for the removal of an excess of alcohols
and unreacted reagents. This strategy ensures, as much as
possible, that no unreacted reagent is present in the cleaved
product. Another advantage of this chemical procedure is
that, in principle, it can produce selectively monosubstituted
derivatives. Additionally, for the dithiodiphosphorylation and
trithiotriphosphorylation reactions in the solid phase, the
terminal oxygen of the phosphorus is linked to the resin and
does not react with Beaucage’s reagent; therefore, only
monosulfurization occurs on the terminal phosphorus atom.
This procedure allows the synthesis of carbohydrate diphos-
phates, dithiodiphosphates, triphosphates, and trithiotriphos-
phates. Carbohydrate phosphate derivatives are known to
serve as important intermediates in metabolism or biosyn-
thesis of complex carbohydrates in several organisms, such
as bacteria.11,12 Synthesized carbohydrate diphosphates or
triphosphates may have application in biosynthesis or
metabolic studies of carbohydrates.
Acknowledgment. We acknowledge the financial support
from National Center for Research Resources, NIH, Grant
Number 1 P20 RR16457.
Supporting Information Available: Experimental pro-
cedures and characterization of resins with IR and final
1
compounds with H NMR, 13C NMR, 31P NMR, and high-
resolution mass spectrometry. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL0521432
(11) Shibaev, V. N.; Danilov, L. L. Biochem. Cell Biol. 1992, 70, 429-
437.
(12) Russell, J. B.; Bond, D. R.; Cook, G. M. Res. Microbiol. 1996,
147, 528-535.
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Org. Lett., Vol. 7, No. 25, 2005