A solid phase reagent for the capture phosphorylation of carbohydrates and nucleosides.

Article abstract of DOI:10.1021/ol0069498

[figure: see text] A 1% cross-linked divinylbenzene-polystyrene copolymer, containing cyanoethoxy N,N-diisopropylamine phosphine was prepared as a phosphitylating agent. The polymer-bound phosphitylated precursor was subjected to reaction with alcohols in the presence of 1H-tetrazole to produce the corresponding polymer-bound phosphite triesters. These were then oxidized with tert-butyl hydroperoxide to give the polymer-bound monophosphate triesters. Removal of cyanoethoxy on the resin with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) followed by basic cleavage of the p-hydroxybenzyl linker products yielded monophosphate derivatives.

Full text of DOI:10.1021/ol0069498

ORGANIC
LETTERS
2001
Vol. 3, No. 2
307-309
A Solid Phase Reagent for the Capture
Phosphorylation of Carbohydrates and
Nucleosides
Keykavous Parang,*,†,‡ Eric J.-L. Fournier,† and Ole Hindsgaul†
Department of Chemistry, UniVersity of Alberta,
Edmonton, Alberta, T6G 2G2 Canada, and Department of Biomedical Sciences,
College of Pharmacy, UniVersity of Rhode Island, Fogarty Hall,
41 Lower College Road, Kingston, Rhode Island 02881-0809
kparang@uri.edu
Received December 1, 2000
ABSTRACT
A 1% cross-linked divinylbenzene−polystyrene copolymer, containing cyanoethoxy N,N-diisopropylamine phosphine was prepared as a
phosphitylating agent. The polymer-bound phosphitylated precursor was subjected to reaction with alcohols in the presence of 1H-tetrazole
to produce the corresponding polymer-bound phosphite triesters. These were then oxidized with tert-butyl hydroperoxide to give the polymer-
bound monophosphate triesters. Removal of cyanoethoxy on the resin with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) followed by basic cleavage
of the p-hydroxybenzyl linker products yielded monophosphate derivatives.
Crucial biological roles of carbohydrate phosphates have long
been recognized.1 In mammalian cells they are found in
phosphorylated inositols involved in protein-membrane at-
tachment and in signal transduction,1,2 and mannose 6-phos-
phate residues are required to target hydrolytic enzymes to
the lysosomes.3 In bacteria, they are much more abundant,
being present in many different classes of cell-surface
polysaccharides such as teichoic acids and polyribosylphos-
phates. In both types of cells, oligosaccharides are biosyn-
thesized from sugar-nucleotides requiring sugar 1-phosphate
to couple to nucleoside 5′-phosphates via their phosphotri-
esters.4
The direct phosphorylation of alcohols is generally ac-
complished by reaction with an activated P(IV) species5 or
mixed ester6 or by coupling with a P(III) species followed
by oxidation.7 For example, several solution phase synthetic
strategies have been reported in the preparation of 5-fluoro-
2′-deoxyuridine monophosphate.8,9 Solution monophospho-
rylation of diols and triols can involve a complicated process
of protection and deprotection reactions leading in most cases
to low overall yield and the need for purification. While
several examples exist with significant regioselectivity in
(5) Uchiyama, M.; Asoy, N. R.; Hayakawa, Y. J. Org. Chem. 1993, 58,
373.
(6) Marugg, J. E.; McLaughlin, L. W.; Piel, N.; Tromp, M.; van der
Marel, G. A.; van Boom, J. H. Tetrahedron Lett. 1983, 24, 3989.
(7) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 1925.
(8) Lorey, M.; Meier, C.; De Clercq, E.; Balzarini, J. Nucleosides
Nucleotides 1997, 16(5&6), 789.
(9) Lorey, M.; Meier, C.; De Clercq, E.; Balzarini, J. Nucleosides
Nucleotides 1997, 16(7-9), 1307.
(10) Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds, D. R.;
Fisher, E. F.; Mcbride, L. J.; Matteucci, M.; Stabinsky, Z.; Tang, J. Y.
Methods Enzymol. 1987, 154, 287.
† University of Alberta.
‡ University of Rhode Island.
(1) Watanabe, Y.; Yamamoto, T.; Ozaki, S. J. Org. Chem. 1996, 61,
14.
(2) Low, M. G.; Saltiel, A. R. Science 1988, 239, 268.
(3) Olson, L. J.; Zhang, J.; Lee, Y. C.; Dahms, N. M.; Kim, J. J. P. J.
Biol. Chem. 1999, 274, 29889.
(4) Fisher, W.; Behr, T.; Hartmann, R.; Peterkatalinic, J.; Egge, H. Eur.
J. Biochem. 1993, 215, 851.
10.1021/ol0069498 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/04/2001

Scheme 1a
a Reagents: (i) oxalyl chloride, toluene, 120 °C, 80%; (ii) p-hydroxybenzaldehyde, Et3N, DMAP, CH2Cl2, ∼98%; (iii) NaBH4, 2-propanol,
THF, 90%; (iv) (i-Pr)2NP(Cl)OCH2CH2CN, DIPEA, THF, 56%.
phosphorylation reactions, multiply phosphorylated com-
pounds are usually formed.
5. Commercial carboxylpolystyrene (1) was converted to the
acid chloride (2) in 80% yield on the basis of chloride content
determined by elemental analysis. This was reacted with
p-hydroxybenzaldehyde to produce 3 (98% based on recov-
ery of base cleavable aldehyde). Reduction of 3 with sodium
borohydride gave 4 (∼90% based on base-cleavable p-
hydroxybenzyl alcohol), which contained the cleavable linker
required for attachment of the phosphitylating reagent.
Reaction of 4 with 2-cyanoethyl N,N-diisopropylchlorophos-
phoramidite finally gave the target reagent 5 in 56% yield
on the basis of elemental analysis of nitrogen.
Scheme 2 shows the general protocol for the use of 5 as
a phosphorylating reagent. Mixing of an alcohol (ROH) with
5 in the presence of 1H-tetrazole in an appropriate solvent
(THF or THF/DMSO based on solubility of the alcohol)
gives the phosphite triester A which can then be oxidized to
the immobilized phosphotriester B using tert-butyl hydro-
peroxide. The cyanoethyl protecting group is then cleaved
conventionally using DBU, and the resulting phosphodiester
We present here preliminary results on a method designed
to produce carbohydrate and nucleoside monophosphates
using a phosphorylating reagent that is immobilized on the
solid phase. The novelty of the method lies in its simplicity.
The substrate alcohol (or polyol) is mixed with the reagent
which is an immobilized phosphitylating reagent. The alcohol
is thereby “captured” as an immobilized phosphite, and
washing the support guarantees that no unreacted alcohol
remains. Intermolecular reactions are suppressed on the solid
phase even when swelling resins are used and can in principle
be completely eliminated using rigid resins, potentially
resulting in mono-phosphitylation of polyols when the
phosphitylation reagent is immobilized. Using chemistry
developed for solid phase synthesis,10 the immobilized
phosphite is then oxidized, deprotected, and cleaved from
the resin.
Scheme 1 displays the synthesis of the solid phase reagent
Scheme 2a
a Procedure and reagents: (i) ROH, THF or THF/DMSO, 1H-tetrazole; (ii) tert-butyl hydroperoxide, THF, quant.; (iii) DBU, THF,
quant.; (iv) NaOMe, dioxane, MeOH, 90%.
308
Org. Lett., Vol. 3, No. 2, 2001

(C) is cleaved with sodium methoxide. The cleavage
proceeds with the concomitant release of a methylene
quinone which reacts with either methanol or adventitious
water to produce p-hydroxymethyl- or p-methoxymethylphe-
nol.
of 5 with uridine (8) under similar conditions produced
exclusively uridine 5′-monophosphate (8E, 67%) shown to
be identical to the commercial product (Sigma). Preparation
of uridine 5′-monophosphate in solution phase is laborious
without protection of 2′- and 3′-hydroxy groups.
Scheme 3 demonstrates some examples of the use of 5 as
a phosphorylating reagent. Mixing 1,2:3,4-di-O-isopro-
Reagent 5 proved very convenient also for the preparation
of sugar 1-phosphates. Stirring D-galactose pentaacetate (7,
333, 0.9 mmol) with 0.9 equiv of benzylamine in THF for
24 h resulted in the selective removal of the anomeric
O-acetyl group to produce the reducing sugar 7F. Reagent
5 was then added directly to this solution to capture the
alcohol. The sequence of oxidation, deprotection, and cleav-
age from the resin with NaOMe, which simultaneously
cleaved the remaining O-acetyl esters, then furnished R-D-
galactopyranosyl 1-phosphate (7E) in 78% yield on a 37 mg
scale. The preparation of R-sugar 1-phosphate donors in
solution often require many steps including purification and
give anomeric mixtures.11 The solid phase method was found
to afford specifically R-D-galactopyranosyl 1-phosphate when
a solution approach11 reported a major â-phosphorylated
product.
Scheme 3. Structure of Starting Materials and Productsa
a (i) THF, Bn-NH2.
In summary, reagent 5 was shown to be a practical reagent
for the capture-phosphorylation of alcohols. The primary
advantage of the reagent is that it ensures that no unphos-
phorylated alcohol is present in the cleaved product and that
polyphosphorylation is suppressed.
pylidene-D-galactopyranose (6, 234 mg, 0.9 mmol) with 5
for 24 h in THF:DMSO 4:1 immobilized the alcohol.
Oxidation (t-BuOOH/THF, 1 h) was followed by cleavage
of the cyanoethyl group (DBU/THF, 48 h). After each step,
the resin was washed sequentially with THF, MeOH, THF,
and Et2O. The phosphorylated product was then cleaved by
reaction with NaOMe/dioxane for 24 h. Water was then
added along with Amberlite-50W (H+) until the solution
became acidic (pH ∼ 4-5). After filtration and washing of
the Amberlite with dioxane and water, the solution was
lyophilized to leave a white powder (6E, 62 mg, 79%) that
was characterized by 1H NMR and electrospray MS. Reaction
Supporting Information Available: Experimental pro-
cedures and characterization of compounds. This material
is available free of charge via the Internet at htttp://
pubs.acs.org.
OL0069498
(11) Sim, M. M.; Kondo, H.; Wong, C.-H. J. Am. Chem. Soc. 1993,
115, 2260.
Org. Lett., Vol. 3, No. 2, 2001
309