Four compounds corresponding to partial structures of Re-type lipopolysaccharide (LPS) were synthesized for precise investigation of immunological properties of the 3-deoxy-D-manno-2-octulosonic acid (Kdo)-containing inner core and lipid A regions of LPS. The synthetic compounds lack all the ester-bound acyl groups which are present in natural LPS but known to be not essential for the antigenicity. The simplest compound is N,N'-diacylated glucosamine β1-6 disaccharide 4'-monophosphate 2 which corresponds to the des-O-acyl derivative of 1-dephospho lipid A. The other three contain one or two Kdo moieties linked to 2. The most complex one is a tetrasaccharide equivalent to the des-O-acylated 1-dephosphorylated derivative of LPS from Escherichia coli Re-mutant. Syntheses were performed by stepwise condensation of appropriately protected sugar moieties and final hydrogenolytic deprotection. The synthetic des-O-acyl LPS analogs were effectively purified by means of reversed phase HPLC to give homogeneous preparations.
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References
1.
Parts of this work were presented at: (a) Kurosawa M., Fukase K., Kusumoto S.61 st National Meeting of the Chemical Society of Japan. Tokyo (Japan), April 1991, abstr. no. 1C136; (b) Kurosawa M., Fukase K., Kusumoto S.63rd National Meeting of the Chemical Society of Japan. Higashi-Osaka (Japan), March 1992, abstr. no. 1P64; (c) Fukase K., Kurosawa M., Nakayama H., Yamada N., Kusumoto S.14th Japanese Carbohydrate Symposium. Tokyo (Japan), July-August 1992, abstr. no. BII-01; (d) Kusumoto S., Fukase K., Kurosawa M.2nd Conference of the International Endotoxin Society. Vienna (Austria), August 1992, abstr. no. 78.
Rietschel E Th, Kirikae T., Schade F.U. et al. The chemical structure of bacterial endotoxin in relation to bioactivity. Immunobiol1993; 187: 169-190.
4.
Imoto M., Yoshimura H., Sakaguchi N., Kusumoto S., Shiba T.Total synthesis of Escherichia coli lipid A. Tetrahedron Lett1985; 26: 1545-1548.
5.
Imoto M., Yoshimura H., Shimamoto T., Sakaguchi N., Kusumoto S., Shiba T.Total synthesis of Escherichia coli lipid A, the endotoxically active principle of cell-surface lipopolysaccharide. Bull Chem Soc Jpn1987; 60: 2205-2214.
6.
Imoto M., Yoshimura H., Yamamoto M., Shimamoto T., Kusumoto S., Shiba T.Chemical synthesis of a biosynthetic precursor of lipid A with a phosphorylated tetraacyl disaccharide structure. Bull Chem Soc Jpn1987; 60: 2197-2204.
7.
Galanos C., Lüderitz O., Rietschel E Th et al. Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur J Biochem1985; 148: 1-5.
8.
Takada H., Kotani S.Structure-function relationships of lipid A. In: Morrison D C, Ryan J L, eds. Bacterial endotoxic lipopolysaccharides, vol. 1, Boca Raton: CRC Press, 1992: 107-134.
9.
Zähringer U. , Lindner B., Seydel U. et al. Structure of de-O-acylated lipopolysaccharide from the Escherichia coli Re mutant strain F515. Tetrahedron Lett1985; 51: 6321-6324.
10.
Kusumoto S. , Kusunose N., Kamikawa T., Shiba T.Chemical synthesis of I-dephospho derivative of Escherichia coli Re lipopolysaccharide. Tetrahedron Lett1988; 29: 6325-6326.
11.
Imoto M. Kusunose N, Kusumoto S., Shiba T.Synthetic approach to bacterial lipopolysaccharide, preparation of trisaccharide part structures containing KDO and 1-dephospho lipid A. Tetrahedron Lett1988; 29: 2227-2230.
12.
Rietschel E Th, Brade L., Schade U. et al. Bacterial endotoxins: properties and structure of biologically active domains. In: Schrinner E, Richmond M H, Seibert G, Schwarz U, eds. Surface structures of microorganisms and their interactions with mammalian host. Weinheim: VCH Verlagsgesellschaft , 1988: 1-41.
13.
Brade L., Brandenburg K., Kuhn H-M. et al. The immunogenicity and antigenicity of lipid A are influenced by its physicochemical state and environment. Infect Immunol1987; 55: 2636-2644.
14.
Rozalski A. , Brade L., Kuhn H-M. et al. Determination of the epitope specificity of monoclonal antibodies against the inner core region of bacterial lipopolysaccharides by use of 3-deoxy-D-manno-octulosonate-containing synthetic antigens. Carbohydr Res1989; 193: 257-270.
15.
Appelmelk B.J. , Verweij-van Vught A M J J, Maaskant J J, Schouten W F, Thijs L G, MacLaren D M. Monoclonal antibodies detecting novel structures in the core region of Salmonella minnesota lipopolysaccharide. FEMS Microbiol Lett1987; 40: 71-74.
16.
Ziegler E.J. , Fisher Jr C.J., Sprung C.L. et al. Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. N Engl J Med1991; 324: 429-436.
17.
Kuhn H-M., Brade L., Appelmelk B.J., Kusumoto S., Rietschel E Th, Brade H.Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid A. Infect Immunol1992 ; 60: 2201-2210.
18.
Fu Y., Baumann M., Kosma P., Brade L., Brade H.A synthetic glycoconjugate representing the genus-specific epitope of chlamydial lipopolysaccharide exhibits the same specificity as its natural counterpart . Infect Immunol1992; 60: 1314-1321.
19.
Kosma P., Strobl M., März L. et al. Synthesis and immunoreactivity of poly(acrylamide) copolymers containing C-3- and C-7-modified, carboxyl-reduced, 4-O- and 5-O- phosphorylated Kdo residues. Carbohydr Res1993; 238: 93-107.
20.
Szábo P., Sarfati S R., Diolez C., and Szábo L.Synthesis of O-[2-deoxy-2-[(3R)-3-hydroxytetradecanamido]-β-D-glucopyranosyl 4-phosphate]-(1→6)-2-deoxy-2-[(3R)-3-hydroxytetradecanamido]-D-glucose. The monosaccharide route. Carbohydr Res1983 ; 111: C9-C12.
21.
Oltvoort J.J. , van Boeckel C A A., de Koning J H., van Boom J H.Use of the cationic iridium complex 1,5-cyclooctadienebis[methyldiphenylphosphine]iridium hexafluorophosphate in carbohydrate chemistry: smooth isomerization of allyl ethers to 1-propenyl ethers. Synthesis1981 ; 305-308.
22.
Nashed M A. , Anderson L.Iodine as a reagent for the ready hydrolysis of prop-1-enyl glycosides, for their conversion into oxazolines. J Chem Soc Chem Commun1982; 1274-1276.
23.
Fukase K., Kamikawa T., Iwai Y., Rietschel E Th, Shiba T., Kusumoto S.Synthesis of allyl 3-deoxy-D-manno-2-octulopyranosidic acid 4- and 5-phosphates . Bull Chem Soc Jpn1991; 64: 3267-3273.
24.
Matsumoto T. , Maeta H., Suzuki K., Tsuchihashi G.New glycosidation reaction 1. Combinational use of Cp2ZrCl2-AgClO4 for activation of glycosyl fluorides and application to highly β-selective glycosidation of D-mycinose. Tetrahedron Lett1988; 29: 3567-3570.
25.
Kunz H., Sager W.Stereoselective glycosylation of alcohols and silyl ethers using glycosyl fluorides and boron trifluoride etherate. Helv Chim Acta1985; 68: 283-287.
26.
The glycosidation by using a little excess of acceptor 17 against Kdo fluoride 18 (18:17 = 0.8:1) was also examined in order to save 18. In this case, however, the yield of the trisaccharide 19 was lower (35% from Kdo fluoride 18). In addition, undesired β-ketoside 19b was formed in a ratio of α:β = 6.2:1. The diagnostic Δ3e-a value of 0.39 p.p.m. for β-ketoside 19b (270 MHz 1H NMR; H-3e: 2.42 p.p.m., H-3a: 2.03 p.p.m.) was in the range of 0.26-0.64 p.p.m. so far reported for β-anomers.27
27.
Unger F.M.The chemistry and biological significance of 3-deoxy-D-manno-2-octulosonic acid (KDO). Adv Carbohydr ChemBiochem1981 ; 38: 323-388.
28.
28. All proton signals were assigned by 1H-1H COSY.
29.
29. THF used for 1-phosphorylation was dried by distillation after being refluxed in the presence of Na. The use of benzophenone and Na as drying reagents should be avoided since a trace of contaminating benzophenone reacted with the phosphorylating reagent.
