Synthesis of aminophosphonate haptens for an aminoacylation reaction between methyl glucoside and a β-alanyl ester

Article abstract of DOI:10.1016/S0960-894X(00)00328-0

Two 2-aminophosphonate haptens derived from methyl α-D-glucopyranoside were synthesized to mimic the transition-state of a transesterification reaction between methyl α-D-glucopyranoside and 4-nitrophenylester of tert-BOC-β-alanine. Two sets of monoclonal

Full text of DOI:10.1016/S0960-894X(00)00328-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1749±1750
Synthesis of Aminophosphonate Haptens for an Aminoacylation
Reaction Between Methyl Glucoside and a ꢀ-alanyl Ester
Timo Lintunen and Jari T. Yli-Kauhaluoma*
VTT Technical Research Centre of Finland, Chemical Technology, PO Box 1401, FIN-02044 VTT Espoo, Finland
Received 31 March 2000; accepted 6 June 2000
AbstractÐTwo 2-aminophosphonate haptens derived from methyl a-d-glucopyranoside were synthesized to mimic the transition-state
of a transesteri®cation reaction between methyl a-d-glucopyranoside and 4-nitrophenylester of tert-BOC-b-alanine. Two sets of
monoclonal antibodies were generated against these haptens. # 2000 Elsevier Science Ltd. All rights reserved.
Only a few reports of catalytic monoclonal antibodies with
aminoacylase properties have hitherto been published. In
one example, an antibody raised against a charged benzyl-
phosphonate monoester catalysed the transesteri®cation
reaction between vinyl ester and various alcohols.1 Anti-
bodies generated against a neutral thymidylphosphonate
diester hapten catalysed the aminoacylation, i.e., the
transesteri®cation reaction of the 30-hydroxyl group of
thymidine with activated phenyl esters of phenylethyl
carbamate-protected d- and l-alanine.2 We have been
interested in modifying carbohydrates with amino acids
by the transesteri®cation reaction of mono-, di- and
oligosaccharides with activated N-protected amino acid
esters. We chose the transesteri®cation reaction between
methyl a-d-glucopyranoside 1 and 4-nitrophenyl ester of
tert-BOC-b-alanine 2 as a model system for the antibody
catalysis study (Scheme 1). The aminophosphonate
haptens 5 and 6 were designed and synthesized to elicit the
monoclonal antibodies. The tribenzoyl hapten 5 incorpo-
rates the charged phosphonate monoester moiety as a
transition-state analogue and three benzoyl groups for
additional immunogenicity. The trihydroxy hapten 6 con-
tains not only the phosphonate transition-state element
but also the unprotected 2-, 3- and 4- hydroxyl groups
providing structural similarity with the substrate 1.
HBF4 in acetonitrile) in 54% total yield.4 The tribenzoyl
glucopyranoside 7 was allowed to react with N-benzyloxy-
carbonyl (CBZ) and O-benzyl-protected (2-aminoethyl)-
phosphonochloridate 8 in the presence of Hunig's base
in CH2Cl2 to produce the neutral phosphonate diester 9.
The CBZ as well as the benzyl protecting groups were
removed by catalytic hydrogenation in 1,4-dioxane,
using palladium on activated carbon as a catalyst, to
give the (2-aminoethyl)phosphonic acid 10 as the key
intermediate in 81% yield. The bifunctional N-hydroxy-
succinimide glutaryl chloride linker 11 was subsequently
attached to the monophosphonic acid 10 in the presence
of Hunig's base in CH2Cl2 to give the tribenzoyl hapten
5 in 64% yield.
The intermediate phosphonochloridate 8 was conveniently
prepared in ®ve steps from ethanolamine 12, starting
with the protecting of its amino portion with a benzyl-
oxycarbonyl group (Scheme 3). The CBZ-protected
ethanolamine 13 was converted to tosylate 14, which
was subsequently allowed to react with sodium dibenzyl
phosphinate in THF at room temperature to provide
the CBZ-protected O,O-dibenzyl (2-aminoethyl)phos-
phonate 15 in 78% yield.5 Next, dibenzyl phosphonate
15 was monodeprotected with quinuclidine in re¯uxing
toluene. The resulting quinuclidinium salt 16 was chlori-
nated with oxalyl chloride using a catalytic amount of
DMF to yield the CBZ-protected (2-aminoethyl)phos-
phonochloridate 8 in 56% yield (®ve steps).6,7
Preparation of the tribenzoyl hapten 5 was rather
straightforward (Scheme 2).3 The starting material methyl
2,3,4-tri-O-benzoyl-a-d-glucopyranoside 7 was synthe-
sized from 1 in three steps (DMAP-catalysed tritylation
in DMF, benzoylation in pyridine and detritylation with
Finally, the trihydroxy hapten 6 was synthesized by cou-
pling phosphonochloridate 8 and methyl 2,3,4-O-tris
(phenylmethyl)-a-d-glucopyranoside8 17 in the presence of
Hunig's base in CH2Cl2 (Scheme 2). The CBZ- and benzyl-
protected phosphonate 18 were subjected to catalytic
*Corresponding author. Tel.: +358-9-456-5295; fax: +358-9-456-
7026; e-mail: jari.yli-kauhaluoma@vvt.®
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00328-0

1750
T. Lintunen, J. T. Yli-Kauhaluoma / Bioorg. Med. Chem. Lett. 10 (2000) 1749±1750
Scheme 1.
alanyl ester 2 in an aqueous medium. However, we are
currently studying the same aminoacylation reaction in
organic solvents, using the obtained KLH-5 and KLH-6
antibodies in reverse micelles and antibodies immobilised
to the solid supports.11 13 The results of these studies will
be reported in due course.
Acknowledgements
We are pleased to acknowledge ®nancial support provided
by TEKES, The National Technology Agency and VTT
Research Programme on Chemical Reaction Mechanisms.
We thank Professor Anneli Hase for helpful discussions
and Mrs. Anja Salakari for excellent technical assistance.
Scheme 2. Reagents and conditions: (a) phosphonochloridate 8,
Hunig's base, CH2Cl2, 0 ꢀC, 76% for 9, 79% for 18; (b) H2, Pd-C, 1,4-
dioxane, 81% for 10, 89% for 19; (c) 5-[(2,5-dioxo-1-pyrrolidinyl)oxy]-
5-oxopentanoyl chloride 11, Hunig's base, CH2Cl2, 64% for 5, 40%
for 6. Bn=benzyl, Bz=benzoyl, CBZ=benzyloxycarbonyl.
References and Notes
1. Wirsching, P.; Ashley, J. A.; Benkovic, S. J.; Janda, K. D.;
Lerner, R. A. Science 1991, 252, 680.
2. Jacobsen, J. R.; Prudent, J. R.; Kochersperger, L.; Yonkovich,
S.; Schultz, P. G. Science 1992, 256, 365.
3. All the compounds were characterised by 1H NMR,
13C NMR, FTIR and mass spectra.
Scheme 3. Reagents and conditions: (a) BnOCOCl, Hunig's base,
CH2Cl2, 93%; (b) TsCl, py, 82%; (c) (BnO)2PNa, THF, 78%; (d)
quinuclidine, PhMe, Á; (e), (COCl)2, DMF cat., 94%.
4. Kovac, P.; Glaudemans, C. P. J. J. Carbohydr. Chem. 1988,
7, 317.
5. Duggan, M. E.; Karanewsky, D. S. Tetrahedron Lett. 1983,
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6. Saady, M.; Lebeau, L.; Mioskowski, C. Tetrahedron Lett.
1995, 36, 4785.
hydrogenation to a€ord monophosphonic acid 19 in
89% yield. The bifunctional linker 11 was subsequently
attached to 19 to give the desired trihydroxy hapten 6 in
40% yield.
7. CAUTION. Phosphonochloridates are poisonous organo-
phosphorus compounds.
The aminophosphonate haptens 5 and 6 were conjugated
to keyhole limpet hemocyanin (KLH) and bovine serum
albumin (BSA) for the purposes of immunisation and
ELISA screening, respectively. Standard hybridoma
methodology was used to elicit monoclonal antibodies
of IgG class against KLH-5 and KLH-6.9 A total of 11
and 6 antibody-secreting hybridomas were isolated speci-
®cally for BSA-5 and BSA-6, respectively. After rigorous
puri®cation (sodium ammonium sulphate precipitation,
anion exchange chromatography with DEAE, cation
exchange chromatography with mono Q and anity
chromatography with protein G), the monoclonal anti-
bodies were investigated for their ability to enhance the
rate of the transesteri®cation reaction between methyl
a-d-glucopyranoside 1 and 4-nitrophenyl ester of tert-
BOC-b-alanine 2.10
8. Methyl 2,3,4-O-tris(phenylmethyl)-a-d-glucopyranoside was
obtained from methyl a-d-glucopyranoside in three steps: (a)
TBDPSCl, imidazole, DMF; (b) BnBr, NaH, DMF; (c)
TBAF, THF; a total yield of 59%. See: Reitz, A. B.; Tuman,
R. W.; Marchione, C. S.; Jordan, A. D.; Bowden, C. R.;
Maryano€, B. E. J. Med. Chem. 1989, 32, 2110.
9. Yli-Kauhaluoma, J.; Janda, K. D. Bioorg. Med. Chem.
1994, 2, 521.
10. Assay conditions: antibody 5 mM, substrates 500 mM,
phosphate-bu€ered saline PBS (10 mM, pH 7.40, 150 mM
NaCl), 5 v/v-% DMF. All reactions were carried out at 25 ꢀC
and monitored at 240 nm by HP Series 1050 HPLC equipped
with an HP autosampler and an ODS Hypersil RP-18 column
(100Â4.6 mm, 5 mm beads) using acetonitrile/water gradient at
1
1.5 mL min
.
11. Durfor, C. N.; Bolin, R. J.; Sugasawara, R. J.; Massey, R. J.;
Jacobs, J. W.; Schultz, P. G. J. Am. Chem. Soc. 1988, 110, 8713.
12. Janda, K. D.; Ashley, J. A.; Jones, T. M.; McLeod, D. A.;
Schloeder, D. M.; Weinhouse, M. I. J. Am. Chem. Soc. 1990,
112, 8886.
None of the antibodies elicited against KLH-5 and KLH-
6 were found to be catalysts for the aminoacylation reac-
tion between methyl glucoside 1 and the activated b-
13. Ashley, J. A.; Janda, K. D. J. Org. Chem. 1992, 57, 6691.