Synthesis of carbohydrate mimetics of an acceptor substrate for the Leishmania elongating α-D-mannopyranosylphosphate transferase

Article abstract of DOI:10.1016/S0040-4039(00)00438-X

Two mimetics of dec-9-enyl β-D-galactopyranosyl-(1→4)-α-D- mannopyranosyl phosphate containing the phosphodiester (3) or phosphonate (4) groups have been synthesized. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00438-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 3665–3668
Synthesis of carbohydrate mimetics of an acceptor substrate for
the Leishmania elongating α-D-mannopyranosylphosphate
transferase
Dmitry V. Yashunsky, Yury E. Tsvetkov and Andrei V. Nikolaev ∗
Department of Chemistry, University of Dundee, Dundee DD1 4HN, UK
Received 31 January 2000; accepted 13 March 2000
Abstract
Two mimetics of dec-9-enyl β-D-galactopyranosyl-(1→4)-α-D-mannopyranosyl phosphate containing the phos-
phodiester (3) or phosphonate (4) groups have been synthesized. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: carbohydrate mimetics; phosphoric acid derivatives; phosphonic acid derivatives.
The surface antigenic lipophosphoglycan (LPG) produced by the infectious promastigote stage of
all species of the Leishmania parasite contains a polymeric section consisting of (1→6)-linked β-D-
galactosyl-(1→4)-α-D-mannosyl phosphate repeating units. The importance of the LPG for parasite
infectivity and survival1 makes the enzymes responsible for the biosynthesis of this glycoconjugate
of great interest. The polymeric phosphoglycan region of the LPG was shown2 to be assembled in
vitro by the sequential action of the Leishmania α-D-Manp-phosphate transferase (MPT) and β-D-Galp
transferase (GT) using GDP-Man and UDP-Gal, respectively, as substrate donors. Since there are no other
mannosylphosphate transferases in mammals, the Leishmania MPT seems to be a good drug target. A
fine substrate specificity of the MPT has been recently studied using synthetic oligo-phosphosaccharide
substrates3 and substrate analogues.4 The results5,6 showed that: (1) the phosphodisaccharide 1 repre-
senting one repeating unit of the phosphoglycan is an efficient acceptor substrate for the MPT; (2) the
negative charge of the phosphodiester group is essential for the enzyme recognition; (3) both the C-6
and C-60 primary hydroxyls of 1 are essential for substrate recognition as well as the C-60 hydroxyl (the
acceptor site) is essential for catalysis; and (4) C-2, C-3, C-20, C-30 and C-40 hydroxyls of 1 make little,
or no contribution to the substrate recognition.
∗
Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00438-X
tetl 16718

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Here, we report the synthesis of two structural mimetics 3 and 4 of the MPT substrate 1 containing
just the elements of the structure, which were shown to be necessary for the enzyme recognition (i.e.
both primary hydroxyls and the phosphate anion). To secure chemical stability of the mimetics, the
intersaccharidic oxygen (O-5 in the hypothetical mimetic analog 2) was replaced with the CH2-group and
the O–C–O–P segment (O-8–C-9–O-10–P in the structure 2) was replaced with either C-8–C-9–O-10–P
(in 3), or O-8–C-9–C-10–P (in 4) fragments. The data obtained from testing 3 and 4 as acceptor substrates
for the MPT will be used to gain more comprehensive information about the substrate specificity of the
enzyme and to design potential inhibitors. Both compounds contain either a dec-9-enyl, or a n-decyl
moiety that assists biochemical assays.
The phosphoric diester 3 was obtained from the alcohol 8 (Scheme 1) and dec-9-en-1-ol using the
H-phosphonate method.7 The phosphonic ester 4 was synthesized using the Arbusov reaction8 from
the alkyl bromide 12 (Scheme 2) and tri-n-decyl phosphite. A common intermediate for both targeted
compounds, the alcohol 6, was prepared starting from commercially available 2-benzyloxyethanol and 5-
bromopent-1-ene. Their reaction in DMF in the presence of sodium hydride gave the ether 5. Successive
osmium tetroxide catalyzed hydroxylation9 of the double bond yielded the corresponding diol, which was
then selectively protected with benzyl bromide via a stannylene intermediate10 to afford the secondary
alcohol 6 in 46% overall yield (based on 2-benzyloxyethanol).
Scheme 1. Reagents: (i) (a) OsO4 cat., NMO, acetone–water; (b) Bu2SnO, PhCH3, ∆; (c) BnBr, n-Bu4NI; (ii) (a) n-Pr4NRuO4
cat., NMO, MS 4 Å, CH2Cl2; (b) NaH, (MeO)2POCH2COOMe, THF; (c) LiAlH4, THF; (iii) (a) TBDMSCl, imidazole, DMF;
(b) H2, Ra–Ni; (c) BzCl, pyridine; (d) n-Bu4NF, THF; (iv) (a) tri-imidazolylphosphine, MeCN; (b) Et3NHHCO3, water (pH 7);
(v) (a) dec-9-en-1-ol, Me3CCOCl, pyridine; (b) I2, pyridine–water; (vi) NaOMe, MeOH
For the preparation of 3, the derivative 6 was first oxidized11 to the corresponding ketone with N-
methylmorpholine N-oxide (NMO) in the presence of n-Pr4NRuO4 followed by the Horner–Emmons
olefination12 with trimethyl phosphonoacetate and reduction with LiAlH4 to produce the allylic alcohol

3667
Scheme 2. Reagents: (i) (a) NaH, allyl bromide, DMF; (b) OsO4 cat., NMO, acetone–water, then Na2SO3, then NaIO4; (c)
NaBH4, EtOH; (ii) (a) MeSO2Cl, Et3N, CH2Cl2; (b) n-Bu4NBr, PhCH3, ∆; (iii) P(OEt)3, ∆, for 13; P(OC10H21-n)3, ∆, for 14;
(iv) (a) H2, Pd(OH)2/C, MeOH; (b) BzCl, pyridine; (v) NaOH, dioxane–water, ∆; (vi) TMSI, CH2Cl2; (vii) H2, Pd(OH)2/C,
MeOH; (viii) Et3N, MeOH
7 (87%) as a mixture of two isomers. It was then converted to the alcohol 8 (89%) (representing the
disaccharide fragment in the mimetic structure 3) by consecutive O-protection with TBDMS–chloride,
hydrogenation of the double bond and both benzyl ethers over a nickel catalyst, conventional O-
benzoylation and desilylation with TBAF. Phosphitylation3,4,7 of 8 with tri-imidazolylphosphine (prepar-
ed in situ from PCl3, imidazole and Et3N) followed by mild hydrolysis gave the H-phosphonate 9, which
produced the protected phosphoric diester 10 (73%, δP −0.46) upon condensation3,4,7 with dec-9-en-1-ol
in pyridine in the presence of trimethylacetyl chloride and oxidation of the resulting phosphorous diester
with iodine in aq. pyridine. Debenzoylation of 10 with 0.1 M methanolic sodium methoxide provided the
phosphodiester 313 quantitatively.
For the preparation of 4 (Scheme 2), the alcohol 6 was first allylated with allyl bromide in the presence
of sodium hydride to give the corresponding allyl ether, which was then transformed to the alcohol
11 (93% based on 6) by consecutive hydroxylation with OsO4, periodate cleavage of the resulting
diol and reduction of the intermediate aldehyde with NaBH4. Conventional mesylation of the alcohol
11 followed by bromination of the mesylate with n-Bu4NBr led to the key 2-alkoxy-ethyl bromide 12
(95%). The Arbusov reaction of the bromide 12 with tri-n-decyl phosphite14 (neat, 170°C) afforded the
phosphonic diester 14 (73%, δP 28.7), which was selectively hydrolyzed15 by refluxing with 2% NaOH
in dioxane–water to produce (after neutralization with 0.5 M HCl) the n-decyl phosphonate 17 (H+-
form, δP 30.8) in 56% yield (93% based on recovered 14). Final debenzylation of 17 by hydrogenation
over Pd(OH)2/C (→18)16 followed by neutralization with triethylamine provided the phosphonic acid
monoester 417 quantitatively.
Alternatively, we attempted to prepare the ester 4 via the phosphonic acid 16. The Arbusov reaction of
the bromide 12 with triethyl phosphite18 (neat, 150°C) led to the diethyl phosphonate 13 (δP 28.8) in 92%
yield. The dibenzyl ether 13 was then O-reprotected by catalytic hydrogenation followed by conventional
benzoylation to give the dibenzoate 15 (88%, δP 28.5). Subsequent P-deprotection of 15 with TMSI18 in
CH2Cl2 at 0°C afforded the phosphonic acid 1619 (100%). Regrettably, all attempts to convert it to the
corresponding mono-n-decyl phosphonate by esterification20 with n-decanol in the presence of DCC, or
other condensing agents (TPS-NT, MS-NT, HATU, etc.) failed.

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The carbohydrate mimetics 3 and 4 are being tested as substrates for the Leishmania α-D-Manp-
phosphate transferase and the results will be published elsewhere in due course.
Acknowledgements
This work was supported by two Wellcome Trust International Grants (for D.V.Y. and Y.E.T.). The
research of A.V.N. was supported by an International Research Scholar’s award from the Howard Hughes
Medical Institute.
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13. Compound 3: δH (D2O) 0.90–1.80 (21H, m, 10×CH2 and CH), 0.96 (9H, t, J=7.3, 3×CH3CH2N), 2.88 (6H, q,
3×CH3CH2N), 3.17–3.30 (6H, m, 3×CH2O), 3.38 (2H, m, CH2O), 3.53 and 3.59 (4H, 2×q, J=6.3, 2×CH2CH2OP),
4.63 (1H, br d, Jcis=10.2, cis-H from CH2_CH), 4.70 (1H, br d, Jtrans=17.8, trans-H from CH2_CH) and 5.52 (1H, ddt,
JH,CH2=6.7, CH2_CHCH2); δC (D2O) 8.6 (NCH2CH3), 25.8, 26.4, 27.1, 29.0, 29.2, 29.3 and 29.5 (7×CH2), 30.5 and 31.9
(2×d, JC,P=6.5, 2×CH2CH2OP), 33.9 (CH2_CHCH2), 36.8 (CH), 47.0 (NCH2), 60.8 and 64.2 (2×CH2OH), 64.3 and 66.4
(2×d, JC,P=5.1, 2×CH2OP), 71.6 and 71.8 (CH2OCH2), 114.5 (CH2_CH) and 139.7 (CH2_CH); δP (D2O) 0.70; ES-MS
(−) data: m/z 409.21 (100%, [M–Et3N–H]−), (C25H54NO7P requires M, 511.36).
14. Petrov, K. A.; Nifant’ev, E. E.; Shchegolev, A. A.; Butilov, M. M.; Rebus, I. F. J. Gen. Chem. USSR (Engl. Transl.) 1963,
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16. Compound 18: δC (CDCl3) 14.5 (CH3), 23.1, 25.8, 29.6, 29.7, 29.9 (2C) and 32.3 [(CH2)7CH3], 25.9 and 28.1
[OCH2(CH2)2CHO], 27.8 (d, JC,P=142.1, CH2P), 30.9 (d, JC,P=5.1, CH2CH2OP), 62.1 and 64.5 (2×CH2OH), 64.0 and
65.8 (2×br, CH2CH2POCH2), 71.5 and 72.4 (CH2OCH2) and 81.3 (CHO); δP (CDCl3) 31.1.
17. Compound 4: δH (CD3OD) 0.92 (3H, t, J=6.9, CH3 from n-decyl), 1.28–1.80 [29H, m, (CH2)8CH3, 3×CH3CH2N and
OCH2(CH2)2CHO], 1.94 (2H, dt, JH,H=7.8, JH,P=17.3, OCH2CH2P), 3.21 (6H, q, J=7.1, 3×CH3CH2N), 3.30–3.90 (11H,
m, 5×CH2O and CHO) and 3.85 (2H, q, J=6.2, CH2CH2OP); δP (CD3OD) 21.9; ES-MS (−) data: m/z 411.24 (100%,
[M–Et3N–H]−), (C25H56NO7P requires M, 513.38).
18. Nicotra, F.; Ronchetti, F.; Russo, G. J. Chem. Soc., Chem. Commun. 1982, 470.
19. Compound 16: δC (CDCl3) 25.7 and 28.6 [OCH2(CH2)2CHO], 28.2 (d, JC,P=137.0, CH2P), 64.3 and 65.7 (2×CH2OBz),
65.5 (br, CH2CH2P), 68.8 and 71.3 (CH2OCH2), 78.1 (CHO), 128.4–133.3 (Ph), 166.7 and 167.0 (2×C_O); δP (CDCl3)
24.25.
20. Burger, A.; Anderson, J. J. J. Am. Chem. Soc. 1957, 79, 3575.