Solid-phase reagents for selective monophosphorylation of carbohydrates and nucleosides

Article abstract of DOI:10.1021/jo048113e

(Chemical Equation Presented) Two classes of aminomethyl polystyrene resin-bound linkers of p-acetoxybenzyl alcohol were subjected to reactions with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to produce the corresponding polymer-bound phosphitylating reagents. These were reacted with a number of unprotected nucleosides and carbohydrates in the presence of 1H-tetrazole. Oxidation with tert-butyl hydroperoxide followed by removal of the cyanoethoxy group with 1,8-diazabicyclo-[5.4.0]undec-7-ene afforded the corresponding polymerbound phosphate diesters. Acidic cleavage of the p-acetoxybenzyl alcohol linker yielded monophosphorylated products with high regioselectivity and trapped linkers on the resins that can be reused.

Full text of DOI:10.1021/jo048113e

logical processes and pathways such as molecular recog-
nition and signal transduction. Organic chemists inves-
tigating these fields are required to prepare many kinds
of pure phosphorylated alcohols in sufficient quantities.
Currently there is no universal method for regioselec-
tive monophosphorylation of multihydroxylated com-
pounds and creating nucleoside and carbohydrate mono-
phosphate libraries. Several strategies have been estab-
lished for phosphorylating of alcohols in solution such as
Solid-Phase Reagents for Selective
Monophosphorylation of 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
reaction of alcohols with an activated P(IV) species,10
a
kparang@uri.edu
mixed ester,11 or a P(III) species followed by oxidation.12
These strategies can involve protection and deprotection
reactions leading in most cases to low overall yield. In
the case of unprotected carbohydrates, multiply phos-
phorylated compounds are usually formed and the puri-
fication of monophosphorylated products from multi-phos-
phorylated compounds is required. Although regioselec-
tive phosphorylation of nucleosides have been previously
reported in solution,13-16 these methods are rather cum-
bersome to be used for generating nucleoside phosphate
libraries needed in screening assays since extensive
purifications of final products from remaining reagents
are required.
Two versatile solid-phase approaches (methods A and
B) for selective monophosphorylation of unprotected
nucleosides and carbohydrates alcohols are now de-
scribed. In these two strategies, the linkers were attached
through amide or reduced amide bonds, respectively, to
aminomethyl polystyrene resin. Both strategies present
a general approach with initial immobilization of phos-
phitylating reagents on solid support using appropriate
linkers and subsequent reaction with alcohols. Washing
of the phosphitylated supports guaranteed the removal
of unreacted reagents and that no unreacted alcohol
remained in next steps. Oxidation and dealkylation
reactions followed by cleavage led to release of phospho-
rylated products. The linkers were susceptible to in-
tramolecular reactions in acidic conditions and remained
trapped on resins. The resins-attached linkers can be
reused with reaction with phosphitylating reagents and
subsequent reaction with unprotected alcohols (see the
Supporting Information). This work complements our
earlier efforts with capture phosphorylation and meth-
ylphosphorylation of carbohydrates and nucleosides.17-19
In the earlier work, the linkers did not remain trapped
on resins and were released along with phosphorylated
compounds in solution leading to contamination; there-
Received October 25, 2004
Two classes of aminomethyl polystyrene resin-bound linkers
of p-acetoxybenzyl alcohol were subjected to reactions with
2-cyanoethyl N,N-diisopropylchlorophosphoramidite to pro-
duce the corresponding polymer-bound phosphitylating re-
agents. These were reacted with a number of unprotected
nucleosides and carbohydrates in the presence of 1H-
tetrazole. Oxidation with tert-butyl hydroperoxide followed
by removal of the cyanoethoxy group with 1,8-diazabicyclo-
[5.4.0]undec-7-ene afforded the corresponding polymer-
bound phosphate diesters. Acidic cleavage of the p-acetoxy-
benzyl alcohol linker yielded monophosphorylated products
with high regioselectivity and trapped linkers on the resins
that can be reused.
Ready access to phosphorylated alcohols such as carbo-
hydrate phosphates (e.g., mannose-6-phosphate,1 glycosyl
phosphatidylinositols2,3), nucleosides (e.g., 2′,3′-dideoxy-
nucleosides monophosphates and triphosphates),4-7 and
phosphopeptides composed of phosphoserine, phospho-
threonine, and/or phosphotyrosine residues8,9 is an impor-
tant requirement for studying several fundamental bio-
* To whom correspondence should be addressed. Tel: +1-401-874-
4471. Fax: +1-401-874-5048.
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(14) Mungall, W. S.; Greene, G. L.; Miller, P. S.; Letsinger, R. L.
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10.1021/jo048113e CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/12/2005
1100
J. Org. Chem. 2005, 70, 1100-1103

SCHEME 1. Synthesis of Resin-Bound Linkers (4 and 8) and Resin-Bound Phosphitylating Reagents (5
and 9)a
a Reagents: (i) 5-formylsalicylic acid, DMF, HOBt, DIC; (ii) Ac2O, pyridine; (iii) (a) sodium borohydride, isopropyl alcohol, (b) NaOH (1
N), 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, THF; (iv) (i-Pr)2NP(Cl)OCH2CH2CN, DIEA, THF; (v) BH3, THF, 55-65 °C; (vi) Boc2O, DCM;
(vii) NaOH (1 N), 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, THF.
fore, further purification of final products was needed.
Additionally the overall yields were modest for unpro-
tected carbohydrates. These two polymer-bound phos-
phorylating reagents were designed to circumvent these
problems. The linkers remained trapped on resins upon
cleavage, which facilitated the separation of final prod-
ucts with filtration. The yield and purity of final products,
regioselectivity of phosphorylation, and monophospho-
rylation vs multiphosphorylation were compared for these
reagents.
Scheme 1 shows the procedure for the preparation of
resin-bound p-acetoxybenzyl alcohol linkers 4 and 8 and
resin-bound phosphitylating reagents 5 and 9. The reac-
tion of 5-formylsalicylic acid with aminomethyl polysty-
rene resin in the presence of HOBt and DIC as coupling
reagents afforded 2. The reaction of 2 with acetic anhy-
dride was used to cap the resin and to yield acetylated
polymer-bound linker 3. The reduction reaction with
sodium borohydride followed by selective acetylation of
phenolic group (produced from reduction of acetoxy) with
1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine gave the resin-
bound linker 4 (Scheme 1). The resin-bound linker with
reduced amide bond, 8, was synthesized according to
previously reported procedure as shown in Scheme 1.20
Reactions of 4 and 8 with 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite gave the phosphite donors 5 and
9, respectively, in 87% and 91% yields (Scheme 1).
Schemes 2 and 3 demonstrate some examples of the
use of 5 (method A) and 9 (method B) as phosphitylating
reagents for unprotected nucleosides, thymidine (a),
uridine (b) and adenosine (c), and unprotected monosac-
charide and disaccharides carbohydrates, R-D-mannose
(d) and 6-O-R-D-galactopyranosyl-D-glucose (melibiose)
(e), to yield monophosphorylated products 14f-j. The
polymer-bound phosphitylated precursors 5 and 9 were
subjected to reactions with a variety of unprotected
nucleosides and carbohydrates (a-e) in the presence of
1H-tetrazole to produce appropriate polymer-bound phos-
phite triesters 10a-e (Scheme 2) and 16a-e (Scheme
3), respectively. The resulting polymer-bound phosphite
triesters were washed with solvents and then oxidized
with tert-butyl hydroperoxide (tBuOOH) to give the
polymer-bound phosphate triesters of the corresponding
compounds 11a-e (Scheme 2) and 17a-e (Scheme 3),
respectively. The removal of cyanoethoxy group in 11a-e
and 17a-e with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
afforded 12a-e (Scheme 2) and 18a-e (Scheme 3),
respectively.
The linkers are attached to aminomethyl polystyrene
resins through amide or reduced amide bonds in these
two strategies. Although the mechanisms for intramo-
lecular final cleavage of phosphorylated alcohols from
12a-e and 18a-e are different, both reactions released
the monophosphorylated products (14f-j) in the presence
of 50% TFA, DCM (Schemes 2 and 3) without the
liberation of the reactive quinone methide byproducts in
solution. Acetic acid produced in the cleavage reaction
of the ester group in 12a-e (Scheme 2) was easily sepa-
rated from the monophosphorylated products by evapora-
tion and the linker remained trapped as quinone methide
on the resin (see 15). The Boc protecting group in 18a-e
(Scheme 3) was removed by treating with TFA/DCM to
yield monophosphate derivatives 14f-j through the
intramolecular cleavage of 19a-e and without the lib-
(20) Chitkul, B.; Atrash, B.; Bradley, M. Tetrahedron Lett. 2001,
42, 6211-6214.
J. Org. Chem, Vol. 70, No. 3, 2005 1101

SCHEME 2. Synthesis of Carbohydrate and Nucleoside Monophosphates (14f-j) from 5 (Method A)a
a Reagents: (i) ROH, THF, DMSO, 1H-tetrazole; (ii) t-BuOOH, THF, quant; (iii) DBU, THF; (iv) TFA/DCM (50%).
eration of the reactive quinone methide and byproducts
(Scheme 3). The side products remained trapped on the
resins (see 20a-e and 21a, Scheme 3). The chemical
structures of final products were confirmed by high-
resolution MS and NMR analysis.
immediately evaporated. The residue was mixed with
Amberlite AG-50W-X8 (100-200 mesh, hydrogen form)
in water/dioxane (70:30 v/v) for 30 min. After filtration,
the solvents were evaporated, and the crude product was
purified using C18 Sep-Pak to yield 14i.
For a typical example (Scheme 2), R-D-mannose (d) and
1H-tetrazole were added to 5 in anhydrous THF and
DMSO. The mixture was shaken for 24 h at room
temperature. The resin was collected by filtration, washed
with DMSO, THF, and MeOH, respectively, and dried
under vacuum to give 10d. tert-Butyl hydroperoxide in
decane (5-6 M) was added to the resin (10d) in THF.
After 1 h shaking at room temperature, the resin was
collected by filtration, washed with DMSO, THF, and
MeOH, respectively, and dried under vacuum to give 11d.
To the swelled resin 11d in THF was added DBU. After
the mixture was shaken for 48 h at room temperature,
the resin was collected by filtration, washed with THF
and MeOH, respectively, and dried under vacuum to give
12d. To the swelled resin (12d) in anhydrous DCM was
added DCM/TFA (50:50 v/v). After the mixture was
shaken for 30 min at room temperature, the resin was
collected by filtration and washed with DCM, THF, and
MeOH, respectively. The solvents of filtrate solution were
Products were compared for yield, purity, and regiose-
lectivity. No multiphosphorylated products were ob-
served, and both strategies afforded similar monophos-
phorylated alcohols with high regioselectivity. The total
isolated yields were slightly higher in the second strategy
than the first strategy for the phosphorylation of nucleo-
sides (Table 1). For example, the phosphorylation of
uridine (b) gave 53% (Scheme 2) and 65% (Scheme 3) of
5′-O-uridine monophosphate (14g), using methods A and
B, respectively. No product composed of 2′ or 3′-uridine
phosphates was formed. Phosphorylation of unprotected
monosaccharides and disaccharides using two strategies
gave comparable yields (Table 1). For example, these
strategies afforded melibiose monophosphate (6-O-R-D-
galactopyranosyl-6′-O-phosphate-D-glucose) (14j) in 41%
(Scheme 2) and 45% (Scheme 3) yields, respectively. In
general, monophosphorylated alcohols were generated in
higher yields from polymer-bound phosphitylating re-
agent having reduced amide (Scheme 3) (45-67%) com-
1102 J. Org. Chem., Vol. 70, No. 3, 2005

SCHEME 3. Synthesis of Carbohydrate and Nucleoside Monophosphates (14f-j) from 9 (Method B)a
a Reagents: (i) ROH, THF, DMSO, 1H-tetrazole; (ii) tert-butyl hydroperoxide, THF, quant.; (iii) DBU, THF; (iv) TFA/DCM (50%).
TABLE 1. Comparison of Percentage Yields for
In summary, two solid-phase phosphorylating regents
were introduced that were capable of trapping the linkers
and avoiding the contamination of final phophorylated
products. Phosphorylation using these solid-phase strate-
gies offered the advantages of facile isolation and the
recovery of monophosphorylated alcohols. The phospho-
rylation procedures exhibited high regioselectivity for
multi-hydroxylated compounds and only one monophos-
phorylated product was formed. These solid-phase phos-
phorylating reagents have the potential to be used for
monophosphorylation of unprotected multihydroxylated
compounds and generation of nucleoside and carbohy-
drate monophosphate libraries in a parallel format. Use
of these solid-phase methodologies can also allow for the
more expeditious development of valuable monophospho-
rylated alcohols.
Monophosphorylated Products Using Methods A and B
compd no.
method A (yield, %)
method B (yield, %)
14f
14g
14h
14i
14j
55
53
61
44
41
62
65
67
48
45
pared with the one with an amide bond (Scheme 2)
(41-61%).
The regioselectivity of these reactions is high as shown
in compounds 14f-j, and no unreacted alcohols remained
with the final products (all unreacted carbohydrates were
recovered in the conjugation reaction). Since in the final
cleavage the linkers remained trapped as quinone meth-
ide, the purification of final products was convenient.
This is an advantage to our previously reported proce-
dure17 that required purification of monophosphorylated
products from p-methoxyphenol and p-hydroxybenzyl
alcohol linker products released in solution. The phos-
phorylation of carbohydrates such as d and e using
solution-phase methods could potentially lead to different
phosphorylated products. These solid-phase strategies
afforded only monophosphorylated compounds with high
regioselectivity. Trapped solid-phase resins 15a and
21a can be reused for generating the solid-phase phos-
phitylating reagents (see the Supporting Information).
Acknowledgment. This research was supported in
part by NIH Grant No. P20 RR16457 from the BRIN/
INBRE program of the National Center for Research
Resources.
Supporting Information Available: Experimental pro-
cedures and characterization of resins with IR and final novel
compounds with 1H 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.
JO048113E
J. Org. Chem, Vol. 70, No. 3, 2005 1103