Chemical Synthesis of Lipid A for the Elucidation of Structure-Activity Relationships

Kusumoto, Shoichi; Inage, Masaru; Chaki, Haruyuki; Imoto, Masahiro; Shimamoto, Takuya; Shiba, Tetsuo

doi:10.1021/bk-1983-0231.ch011

Cited by 23 publications

(19 citation statements)

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“…Lipopolysaccharide (LPS), or 'endotoxin', the principal constituent of the outer membrane of Gram-negative bacteria [5][6][7], play a key role in the pathogenesis of Gram-negative sepsis via the activation of systemic inflammatory cascades [8][9][10][11]. The toxically active moiety of LPS is a negatively charged, amphipathic glycolipid termed Lipid A [12], which is composed of a hydrophilic, bis-phosphorylated (bis-anionic) diglucosamine backbone, and a hydrophobic domain of six (E. coli) or seven (Salmonella) acyl chains in amide and ester linkages [13][14][15]. For the reasons that lipid A is the toxic moiety of LPS specifically recognized by receptors on LPS-responsive cells [16][17][18][19][20][21][22][23], and is highly structurally conserved amongst all Gramnegative bacteria [24], it represents a logical molecular target for purposes of developing therapeutic strategies for the therapy and prophylaxis of Gram-negative sepsis.…”

Section: Introductionmentioning

confidence: 99%

Anti-Endotoxin Agents. 2. Pilot High-Throughput Screening for Novel Lipopolysaccharide-Recognizing Motifs in Small Molecules

Wood¹,

Miller²,

David³

2004

CCHTS

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Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are an integral part of the outer leaflet of the outer-membrane of Gram-negative bacteria. Lipopolysaccharides play a pivotal role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient, worldwide. The sequestration of circulatory endotoxin may be a viable therapeutic strategy for the prophylaxis and treatment of Gram-negative sepsis. We have earlier shown that the pharmacophore necessary for small molecules to bind LPS is simple, comprising of two protonatable cationic functions separated by about 15Å, permitting the simultaneous interaction with the negatively charged phosphates on lipid A, the toxically active center of endotoxin. In this report, we employ highthroughput screening methods, using a novel fluorescent probe displacement method. Searches in three-dimensional structure databases yielded about ~ 4000 commercially available small molecules, each possessing two cationic functions spaced approximately 15Å apart. Approximately 400 such compounds have been screened in an effort to validate the method by which high-affinity endotoxin binders can be identified. We show that the IC 50 values that are obtained from the fluorescence-based primary screen are correlated both to the enthalpy of binding, as measured by isothermal titration calorimetry, as well as to biological potency in vitro assays. By performing rapid toxicity screens in tandem with the bioassays, lead compounds of interest can be easily identified for further systematic structural modifications and SAR studies.

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Section: Introductionmentioning

confidence: 99%

Anti-Endotoxin Agents. 2. Pilot High-Throughput Screening for Novel Lipopolysaccharide-Recognizing Motifs in Small Molecules

Wood¹,

Miller²,

David³

2004

CCHTS

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“…The extract structure of the hydrophobic region is not known: when cleaved off by mild acid treatment, it appears as a heterogeneous mixture, insoluble in water, usually referred to as 'lipid A'. Structures derived from the glucosaminyl-P-1,6-glucosamine disaccharide identified Qureshi et al, 1982;Rosner et al, 1979;Strain et al, 1983;Takayama et al, 1983) in 'lipid A' preparations, when produced by chemical synthesis (Charon et al, 1983;Kiso et al, 1981 ;Kusumoto et al, 1983;Imoto et al, 1984), so far have failed to reproduce the complete set of biological properties of 'lipid A' preparations (Galanos et al, 1984;Kotani et al, 1983;Luderitz et al, 1983;Matsuura et al, 1983;Yasuda et al, 1982Yasuda et al, , 1984. It has been suggested that the nature and type of linkage of the fatty acids attached to the phosphorylated glucosamine disaccharide might be critical for the elicitation of the biological properties of the 'lipid A' preparations.…”

Section: Introductionmentioning

confidence: 99%

The Fatty Acid Content of the Bordetella pertussis Endotoxin

Starkloff¹,

Szabó

1986

Microbiology

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The fatty acid content of Bordetella pertussis endotoxin has been estimated by several methods. Expressed as 3-hydroxytetradecanoic acid, it was 0.74 mumol (mg lyophilized material)-1, 0.38 mumol being ester-bound, and 0.32 mumol in amide linkage. Reported molar ratios of ester-bound to amide-bound fatty acids in endotoxins of various bacterial species range from 2.4 to 2 in B. pertussis, to 5 to 2 in Salmonella minnesota; according to these figures large differences must exist in the degree of substitution, and the substitution pattern of the glucosaminyl-beta-1,6-glucosamine unit present in the hydrophobic region of endotoxins. When fatty acids, released by acid and alkaline hydrolyses of the B. pertussis endotoxin, were extracted into chloroform, unidentified chromogenic substances appearing in the extract interfered with their colorimetric estimation; no interference was observed when hexane was used instead of chloroform.

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“…According to our composition analysis, the purified lipid A contains total six fatty acids, i.e., four (R)-3-hydroxyteteradecanoic acids and each one of dodecanoic and tetradecanoic acids. From this information we concluded the structure of a previously isolated biosynthetic precursor of lipid A, which contains only four 3-hydroxytetradecanoic acids, should rationally be Chemical synthesis of newly elucidated structure of the biosynthetic precursor 1 was successfully achieved as shown in Scheme 1 (12,13), where knowledge accumulated in our preceding synthesis was utilized (10). The synthetic preparation 1, as immediately tested by our collaborating groups, showed definite endotoxic activity including both beneficial and detrimental ones described for natural lipid A (14,15).…”

Section: B Structure and Synthesis Of Lipid A As The Endotoxic Princmentioning

confidence: 99%

“…Before the start of our chemical synthesis, we had to know the precise structure of the target lipid A including the exact location of acyl groups, because our preliminary synthetic works based on the above information did not reproduced endotoxic activities (10). For the purpose of reliable analysis, a homogeneous molecular species had to be isolated: lipid A preparation from Escherichia coli Re mutant cells was selected as the substrate because this species was known to produce rather homogeneous lipid A.…”

Section: B Structure and Synthesis Of Lipid A As The Endotoxic Princmentioning

confidence: 99%

Synthesis and Functional Study of Bacterial Glycoconjugates Triggering the Innate Immune System of Higher Animals

Kusumoto

2010

TIGG

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Typical bacterial glycoconjugates, lipopolysaccharide (LPS) and peptidoglycan (PGN), which ubiquitously occur in a wide range of bacterial cells as important components of their cell envelope, have long been known to enhance the immunological responses of higher animals. Synthetic works and related biological investigations on LPS are reviewed which were performed to understand the molecular basis of this phenomena mainly by the author's and collaborating groups. Structural study followed by chemical synthesis of glycolipid part of LPS, named lipid A, proved it to exhibit all the biological activity described for LPS. Synthetic homogeneous lipid A and its derivatives were utilized for precise studies of their biological functions and interaction with receptor proteins. Similar research on PGN led to conclude its minimum active structure as muramyl dipeptide. Chemical synthesis gave unequivocal evidences for the concept that definite small molecular parts of LPS and PGN are recognized by its specific receptors and trigger our defense system which is now recognized as innate immunity.

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Chemical Synthesis of Lipid A for the Elucidation of Structure-Activity Relationships

Cited by 23 publications

References 0 publications

Anti-Endotoxin Agents. 2. Pilot High-Throughput Screening for Novel Lipopolysaccharide-Recognizing Motifs in Small Molecules

Anti-Endotoxin Agents. 2. Pilot High-Throughput Screening for Novel Lipopolysaccharide-Recognizing Motifs in Small Molecules

The Fatty Acid Content of the Bordetella pertussis Endotoxin

Synthesis and Functional Study of Bacterial Glycoconjugates Triggering the Innate Immune System of Higher Animals

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