Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative.
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)
Myers
K R
KR
Ribi ImmunChem Research, Inc., Hamilton, Montana 59840.
Ulrich
J T
JT
Qureshi
N
N
Takayama
K
K
Wang
R
R
Chen
L
L
Emary
W B
WB
Cotter
R J
RJ
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000276:immunology; D002240:Carbohydrate Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D008050:Lipid A / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008809:Mice, Inbred C3H; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D016339:Spectrometry, Mass, Fast Atom Bombardment; D019809:Streptavidin; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000906:Antibodies; D001426:Bacterial Proteins; D008050:Lipid A; D014409:Tumor Necrosis Factor-alpha; C078686:6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A; D001710:Biotin; D019809:Streptavidin