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Synthesis of peptidylglycophospholipids, novel derivatives of muramyl-dipeptide
Tetrahedron Letters, Vo1.22, Printed in Great Britain
No.24,
SYNTHESIS NOVEL
Rakesh
pp 2317 - 2320, 1981
OF
PEPTIDYLGLYCOPHOSPHOLIPIDS,
DERIVATIVES K.Jaic,
OW-4039/01/242317-04$02.00/O 01981 Pergamon Press Ltd.
Chhitar
OF
MURAMYL-DIPEPTIDI+ M.Gupta*,
Nitya Ansnd
Division of Medicinal Chemistry, Central Drug Research Institute, Lucknow (India) Abetr&
Synthesis of 6-deoxy-6-L-a-dipalmitoylphosphatidylethanolamin~N-acetylmuramyl-L-alanyl-D-isoglutamine7-methylester is described.
N-Acetylmuramyl-L-alanyl-D-iso&utamine (MDP) has been shown to be the minimal acljuvsnt-activestructure capable of replacing whole mycobacterial cells in Freud's complete adjuvent (FCA) for inCreasinE levels of humoral antibodies against a given antien and for InducinP delayed hyper*ensitivity2'3. It has been reported that owl monkeys are effectively protected against a human parasite after Immunization with an appropriate antipen in liposomes oontaininp:6-O-stearoyl-MDP, in a way similar to that obtained with FCA4. These observations prompted UB to undertake the synthesis of MDP derivatives whoee adjuvant activity may not require any oily vehicle, such as liposomes or mineral oil. This report describes the synthesis of one of such novel MDP derivatives in which phosphatidylethanolaminehas been linked to C-6 of the 6deoxy muramyl residue via C-N linkasre(I). 1-0-BenlJrl-4,6-d-0-benzylidine-N-acetylmuramic add (A) was prepared followina the published procedure5. Reaction of ;Lwith g-nitrobenzyltosylate6 in anhydrous acetone, under reflux for 2 h, Rave the nitrobeneyl ester p (m.p.162-163'C from CHC13-hexsne; [a]E5 + 13.1 (c 1.2, CHc13); Qmax (KBr): 1750 and 1640 (GO), 1520 and 1340 cm" (N02); 6 (CDC13)7: 1.35 (a, J='/Hz,3H), 1.95 (s, 3H), 5.15 (s, 2H), 5.27 (d, J=2.5Hz, lH), 5.5 (a, IX) In 75Y$yield'. Heatino of 2 in 60$ aqueous acetic acid on a boiline water bath for about 1 h yielded 2. The latter, without purification, was reacted with p-tosyl chloride (5 fold excess) in dry pyridine at O°C for 1 h. The mixture obtained after removal of the solvent was chronratoeraphedover ailicic acid column. Elution of the column with 56 CH OH in CHCl furnished the monotosyl derivativeg4 %5 + 7.6 (C32.5, CHCl3); L' (m.p.76-79'C; [a]D ,=(KBr): 1750 and 1640 (CXO),
2318
RO
I-- -0
NO2
(L@ = H
(Z> R= H
WR = _p-02NC6H4a2
(4) R = p-H3CC6H4S02
0
-'c'-o C151f31 C15H 31770 0
19 O-pa/ OH
H
(5) ~1 = PhC'H2,R2 = _p-02NC6H4CH2 (6) Rl = Rp = H 8 C15"31-g-0 R 0 -L o--P\ I OH
0
2319
1520 and 1340 (N02), 1360 and 1170 cm-1 (S03); 6 (CDC131: 1.35 (a, J=7Hz, 3H), 1.87 (s, 3H), 2.30 (8, 3H), 5.02 (a, J=205Hz, u), 5.13 (8, 2H) in 50r overall yield from 2. A gxture of the monotosylate 4, L-a-dipalmitoylphosphatidylethanolamine , and triethylamine in anhydrous CHC13 wa8 heated at 80-90°C in a sealed tube for 12-15 h. ChromatoPraphy of the reaction mixture, after removal of the solvent, on a eilicic acid column afforded 1, which was slightly contaminated with some non-phosphorus impurity. An snalytioelly pure sample of 5 was obtained aft!;:tirther ohromatoaraphy of the impure material over Sephadex LH-20 oolumn . Yield: 55s (m.p.54-56'C; [a]i5 + 3.8 (C 0.8, CHC13); ?max(KBr): 1750, 1740 and 1640 ('LO), 1520 and 1340 cm-I (N02)). Catalytic hydrogenation of 5.over Pa-black, in glacial acetic acid, gave 5 (m.p.165-167'C; Smax(KBr): 1740 and 1645 cm-' (GO)) in over 80% yield. The acid 6 was coupled to L-alsnyl-D-isodutamlne-y-methyl eater in THF/DMF (4:l) mixture using 12 DCC/N-hydroxysuccinimideprocedure . The peptldylalycophospholipid 1. (m.p.144-146'C; [a]:' -0.8 (C 1.1, CH30H)) was isolated in nearly 5896 yield after chromatography of the reaction mixture over Sephadex LH-20 column. Havina obtained the peptidylglycophospholipid2.by this method, we are currently preparing analops of 2, with unsaturated fatty acyl chains in the phosphollpld moiety. Attempts are also beinp made to convert 1 into the corresponding phosphatldylcholine analog. The aajuvant activity of 1 has been tested in minea pigs using egp albumin as antigen. The new MDP derivative when administered with the antigen In saline Induced more delayed type hypersensitivity as compared to MDP given in water-in-oil emulsion. The detail6 of biolodcal activity of j!and Its analogs will be published in due cour8e of time.
We thank Council of Scientific and Industrial Research, New Delhi, for award of Junior Research Fellowship to RKJ ena Drs.K.C.Saxena and V.Bhatia for biolodcal screeninp of z.
2320
Reference8 c1Note@ 1. 2.
CDRI Communication No.2918. F.Ellouz, A.Adam, R.Ciorbaru and E.Lederer, Blochem. Biophys. Res. Commun., 59, 1317 (1974).
3.
S.Kotani, Y.Watanabe, F.Kinoshita, T.Shimono, I.Morisaki, T.Shiba, S.Kusumoto, Y,Ta.rumiand K.Ikenaka, Biken J., u, 105 (1975).
4.
W.A.Siddlqul, D.W.Tsylor, S.Ch.Kan, K.Kramer, S.&Richmond-Crum, S.Kotani, T.Shlba and S.Kusumoto, Science, a, 1237 (1978).
5.
T.Osawa and R.W.Jeanloz, J. Org. Chem., z,
6.
D.Theodoropoulos and J.Tsamaris, J. Org. Chem., a, 2272 (1964). 1 H-nmr spectra were recorded in a Perkin-Elmer R-32 instrument using tetramethylsilane as Internal standard.
7.
448 (1965).
8.
Yields refer to analytically pure material. The satisfactory data from elemental snalysis have been obtained for all the oompounds.
9.
T.Shiba, S.Okada, S.Kusumoto, I.Azuma and Y.Yamamura, Bull. Chem. Sot., Japan, a, 3307 (1978).
10.
L-a-Dipalmltoylphosphatidylethsnolaminewas purchased from Sigma Chemical Company.
ll.
Sephadex m-20 (25-100 urnbeads) chromatopraphy was performed on a 2.5~100 cm column with CHcl /CH OH (1~1) as the eluant. 3 3 S.Kusumoto, Y.Tarumi, K.Ikenaka and T.Shiba, Bull. kern, Sot., Japan, 499 533 (1976).
12.
(Received in UK
7 April1981)
No.24,
SYNTHESIS NOVEL
Rakesh
pp 2317 - 2320, 1981
OF
PEPTIDYLGLYCOPHOSPHOLIPIDS,
DERIVATIVES K.Jaic,
OW-4039/01/242317-04$02.00/O 01981 Pergamon Press Ltd.
Chhitar
OF
MURAMYL-DIPEPTIDI+ M.Gupta*,
Nitya Ansnd
Division of Medicinal Chemistry, Central Drug Research Institute, Lucknow (India) Abetr&
Synthesis of 6-deoxy-6-L-a-dipalmitoylphosphatidylethanolamin~N-acetylmuramyl-L-alanyl-D-isoglutamine7-methylester is described.
N-Acetylmuramyl-L-alanyl-D-iso&utamine (MDP) has been shown to be the minimal acljuvsnt-activestructure capable of replacing whole mycobacterial cells in Freud's complete adjuvent (FCA) for inCreasinE levels of humoral antibodies against a given antien and for InducinP delayed hyper*ensitivity2'3. It has been reported that owl monkeys are effectively protected against a human parasite after Immunization with an appropriate antipen in liposomes oontaininp:6-O-stearoyl-MDP, in a way similar to that obtained with FCA4. These observations prompted UB to undertake the synthesis of MDP derivatives whoee adjuvant activity may not require any oily vehicle, such as liposomes or mineral oil. This report describes the synthesis of one of such novel MDP derivatives in which phosphatidylethanolaminehas been linked to C-6 of the 6deoxy muramyl residue via C-N linkasre(I). 1-0-BenlJrl-4,6-d-0-benzylidine-N-acetylmuramic add (A) was prepared followina the published procedure5. Reaction of ;Lwith g-nitrobenzyltosylate6 in anhydrous acetone, under reflux for 2 h, Rave the nitrobeneyl ester p (m.p.162-163'C from CHC13-hexsne; [a]E5 + 13.1 (c 1.2, CHc13); Qmax (KBr): 1750 and 1640 (GO), 1520 and 1340 cm" (N02); 6 (CDC13)7: 1.35 (a, J='/Hz,3H), 1.95 (s, 3H), 5.15 (s, 2H), 5.27 (d, J=2.5Hz, lH), 5.5 (a, IX) In 75Y$yield'. Heatino of 2 in 60$ aqueous acetic acid on a boiline water bath for about 1 h yielded 2. The latter, without purification, was reacted with p-tosyl chloride (5 fold excess) in dry pyridine at O°C for 1 h. The mixture obtained after removal of the solvent was chronratoeraphedover ailicic acid column. Elution of the column with 56 CH OH in CHCl furnished the monotosyl derivativeg4 %5 + 7.6 (C32.5, CHCl3); L' (m.p.76-79'C; [a]D ,=(KBr): 1750 and 1640 (CXO),
2318
RO
I-- -0
NO2
(L@ = H
(Z> R= H
WR = _p-02NC6H4a2
(4) R = p-H3CC6H4S02
0
-'c'-o C151f31 C15H 31770 0
19 O-pa/ OH
H
(5) ~1 = PhC'H2,R2 = _p-02NC6H4CH2 (6) Rl = Rp = H 8 C15"31-g-0 R 0 -L o--P\ I OH
0
2319
1520 and 1340 (N02), 1360 and 1170 cm-1 (S03); 6 (CDC131: 1.35 (a, J=7Hz, 3H), 1.87 (s, 3H), 2.30 (8, 3H), 5.02 (a, J=205Hz, u), 5.13 (8, 2H) in 50r overall yield from 2. A gxture of the monotosylate 4, L-a-dipalmitoylphosphatidylethanolamine , and triethylamine in anhydrous CHC13 wa8 heated at 80-90°C in a sealed tube for 12-15 h. ChromatoPraphy of the reaction mixture, after removal of the solvent, on a eilicic acid column afforded 1, which was slightly contaminated with some non-phosphorus impurity. An snalytioelly pure sample of 5 was obtained aft!;:tirther ohromatoaraphy of the impure material over Sephadex LH-20 oolumn . Yield: 55s (m.p.54-56'C; [a]i5 + 3.8 (C 0.8, CHC13); ?max(KBr): 1750, 1740 and 1640 ('LO), 1520 and 1340 cm-I (N02)). Catalytic hydrogenation of 5.over Pa-black, in glacial acetic acid, gave 5 (m.p.165-167'C; Smax(KBr): 1740 and 1645 cm-' (GO)) in over 80% yield. The acid 6 was coupled to L-alsnyl-D-isodutamlne-y-methyl eater in THF/DMF (4:l) mixture using 12 DCC/N-hydroxysuccinimideprocedure . The peptldylalycophospholipid 1. (m.p.144-146'C; [a]:' -0.8 (C 1.1, CH30H)) was isolated in nearly 5896 yield after chromatography of the reaction mixture over Sephadex LH-20 column. Havina obtained the peptidylglycophospholipid2.by this method, we are currently preparing analops of 2, with unsaturated fatty acyl chains in the phosphollpld moiety. Attempts are also beinp made to convert 1 into the corresponding phosphatldylcholine analog. The aajuvant activity of 1 has been tested in minea pigs using egp albumin as antigen. The new MDP derivative when administered with the antigen In saline Induced more delayed type hypersensitivity as compared to MDP given in water-in-oil emulsion. The detail6 of biolodcal activity of j!and Its analogs will be published in due cour8e of time.
We thank Council of Scientific and Industrial Research, New Delhi, for award of Junior Research Fellowship to RKJ ena Drs.K.C.Saxena and V.Bhatia for biolodcal screeninp of z.
2320
Reference8 c1Note@ 1. 2.
CDRI Communication No.2918. F.Ellouz, A.Adam, R.Ciorbaru and E.Lederer, Blochem. Biophys. Res. Commun., 59, 1317 (1974).
3.
S.Kotani, Y.Watanabe, F.Kinoshita, T.Shimono, I.Morisaki, T.Shiba, S.Kusumoto, Y,Ta.rumiand K.Ikenaka, Biken J., u, 105 (1975).
4.
W.A.Siddlqul, D.W.Tsylor, S.Ch.Kan, K.Kramer, S.&Richmond-Crum, S.Kotani, T.Shlba and S.Kusumoto, Science, a, 1237 (1978).
5.
T.Osawa and R.W.Jeanloz, J. Org. Chem., z,
6.
D.Theodoropoulos and J.Tsamaris, J. Org. Chem., a, 2272 (1964). 1 H-nmr spectra were recorded in a Perkin-Elmer R-32 instrument using tetramethylsilane as Internal standard.
7.
448 (1965).
8.
Yields refer to analytically pure material. The satisfactory data from elemental snalysis have been obtained for all the oompounds.
9.
T.Shiba, S.Okada, S.Kusumoto, I.Azuma and Y.Yamamura, Bull. Chem. Sot., Japan, a, 3307 (1978).
10.
L-a-Dipalmltoylphosphatidylethsnolaminewas purchased from Sigma Chemical Company.
ll.
Sephadex m-20 (25-100 urnbeads) chromatopraphy was performed on a 2.5~100 cm column with CHcl /CH OH (1~1) as the eluant. 3 3 S.Kusumoto, Y.Tarumi, K.Ikenaka and T.Shiba, Bull. kern, Sot., Japan, 499 533 (1976).
12.
(Received in UK
7 April1981)