Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative

Abstract

N-tert-butyloxycarbonyl (t-Boc) protected 6-aminocaproic (Cap) anhydride was reacted with unprotected hexaacyl-4'-O-monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Escherichia coli J5 to yield t-Boc-Cap-MLA. After a column purification step, the t-Boc group was removed by incubating the sample at low temperature in the presence of acid to yield Cap-MLA. This product was analyzed by californium plasma desorption mass spectrometry (PDMS). Purified t-Boc-Cap-MLA was further fractionated by reverse-phase high-performance liquid chromatography as its methyl ester and characterized by laser desorption mass spectrometry, PDMS, and proton nuclear magnetic resonance spectroscopy. These analyses revealed that the Cap group was selectively introduced into the 6'-position of MLA. To demonstrate that Cap-MLA can be conjugated to other compounds, it was reacted with biotin-Cap N-hydroxysuccinimide ester to yield biotin-(Cap)2-MLA. Analysis of this product by PDMS confirmed its expected molecular weight of 2171 and showed the presence of fragments containing the biotin and Cap groups. Monoclonal antibodies and streptavidin were used to show the presence of both lipid A and biotin in this conjugated product. These two novel lipid A derivatives were then tested for their bioactivities. Although both Cap-MLA and biotin-(Cap)2-MLA showed mitogenic activity using murine splenocytes, they were about 4-8 times less active than MLA at 20 micrograms/mL or less and only one-half as active at 100 micrograms/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Function of the Escherichia coli msbB Gene, a Multicopy Suppressor of htrB Knockouts, in the Acylation of Lipid A

“…1. These structural proposals are based on what is known about lipid A released from E. coli lipopolysaccharide by acid hydrolysis (11, [25][26][27][28]. The proposed structures also are in accord with the molecular weights of (Kdo) 2 -lipid IV A acylation products generated previously with crude cell extracts (7).…”

“…No decanoate is found in the position normally occupied by laurate on lipid A under ordinary growth conditions in living cells (Fig. 1), and very little laurate is incorporated in place of myristate (11,(25)(26)(27)(28).…”

“…The possibility that HtrB can function on the (Kdo) 2 -[myristoyl]-lipid IV A generated at a slow rate by MsbB from myristoyl-ACP and (Kdo) 2 -lipid IV A has not been examined (as indicated by the question mark). The proposed structures of the lipids generated by HtrB and MsbB are surmised based on what is known about the lipid A structure of E. coli (11,(25)(26)(27)(28). Unequivocal analyses to establish the structures of these acylation products of (Kdo) 2 -lipid IV A generated in vitro have not yet been carried out.…”

Function of the htrB High Temperature Requirement Gene of Escherichia coli in the Acylation of Lipid A

“…For instance, minor lipid A species are observed in E. coli in which the myristate residue is missing entirely (8) or is replaced with laurate (not shown in Fig. 1) (9). An additional palmitate residue may also sometimes be present in acyloxyacyl linkage on the proximal glucosamine unit (not shown in Fig.…”

A Special Acyl Carrier Protein for Transferring Long Hydroxylated Fatty Acids to Lipid A in Rhizobium

“…The two R-3-hydroxy moieties of the distal unit are further acylated with laurate and myristate ( Fig. 1), forming acyloxyacyl groups (1,3,5,(11)(12)(13)(14)(15). The latter are structural hallmarks of lipid A moieties from diverse sources, and they are critical for the immunostimulatory activity of endotoxins (3,5,16,17).…”