Preparation and Characterization of Biologically Active 6'-O-(6-Aminocaproyl)-4'-O-O-Monophosphoryl Lipid a and its Conjugated Derivative

Kent R. Myers; J. Terry Ulrich; Nilofer Qureshi; Kuni Takayama; Nilofer Qureshi; Kuni Takayama; Rong Wang; Ling Chen; W. Bart Emary; Robert J. Cotter

doi:10.1021/bc00018a013

Preparation and Characterization of Biologically Active 6'-O-(6-Aminocaproyl)-4'-O-O-Monophosphoryl Lipid a and its Conjugated Derivative

Kent R. Myers, J. Terry Ulrich, Nilofer Qureshi, Kuni Takayama, Nilofer Qureshi, Kuni Takayama, Rong Wang, Ling Chen, W. Bart Emary, Robert J. Cotter

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6 Scopus citations

Abstract

N-Start-butyloxycarbonyl (f-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 μ g/_mL or less and only one-half as active at 100 μ g/_mL. In the induction of tumor necrosis factor release by RAW 264.7 murine macrophage cell line, the biotin-(Cap)₂-MLA showed 7-9-fold lower activity than MLA at the concentration range of 0.1-1.0 μ g/_mL. These results showed that Cap-MLA is a biologically active lipid A derivative that can be conjugated to other compounds through its free amino group to form new and active derivatives. It should thus be a useful reagent to study the biological properties of lipid A.

Original language	English (US)
Pages (from-to)	540-548
Number of pages	9
Journal	Bioconjugate Chemistry
Volume	3
Issue number	6
DOIs	https://doi.org/10.1021/bc00018a013
State	Published - 1992
Externally published	Yes

ASJC Scopus subject areas

Biotechnology
Bioengineering
Biomedical Engineering
Pharmacology
Pharmaceutical Science
Organic Chemistry

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@article{716f6ff9d96a4e42b5d8a5fa2596fbb5,

title = "Preparation and Characterization of Biologically Active 6'-O-(6-Aminocaproyl)-4'-O-O-Monophosphoryl Lipid a and its Conjugated Derivative",

abstract = "N-Start-butyloxycarbonyl (f-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 μ g/mL or less and only one-half as active at 100 μ g/mL. In the induction of tumor necrosis factor release by RAW 264.7 murine macrophage cell line, the biotin-(Cap)2-MLA showed 7-9-fold lower activity than MLA at the concentration range of 0.1-1.0 μ g/mL. These results showed that Cap-MLA is a biologically active lipid A derivative that can be conjugated to other compounds through its free amino group to form new and active derivatives. It should thus be a useful reagent to study the biological properties of lipid A.",

author = "Myers, {Kent R.} and Ulrich, {J. Terry} and Nilofer Qureshi and Kuni Takayama and Nilofer Qureshi and Kuni Takayama and Rong Wang and Ling Chen and Emary, {W. Bart} and Cotter, {Robert J.}",

year = "1992",

doi = "10.1021/bc00018a013",

language = "English (US)",

volume = "3",

pages = "540--548",

journal = "Bioconjugate Chemistry",

issn = "1043-1802",

publisher = "American Chemical Society",

number = "6",

}

TY - JOUR

T1 - Preparation and Characterization of Biologically Active 6'-O-(6-Aminocaproyl)-4'-O-O-Monophosphoryl Lipid a and its Conjugated Derivative

AU - Myers, Kent R.

AU - Ulrich, J. Terry

AU - Qureshi, Nilofer

AU - Takayama, Kuni

AU - Qureshi, Nilofer

AU - Takayama, Kuni

AU - Wang, Rong

AU - Chen, Ling

AU - Emary, W. Bart

AU - Cotter, Robert J.

PY - 1992

Y1 - 1992

N2 - N-Start-butyloxycarbonyl (f-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 μ g/mL or less and only one-half as active at 100 μ g/mL. In the induction of tumor necrosis factor release by RAW 264.7 murine macrophage cell line, the biotin-(Cap)2-MLA showed 7-9-fold lower activity than MLA at the concentration range of 0.1-1.0 μ g/mL. These results showed that Cap-MLA is a biologically active lipid A derivative that can be conjugated to other compounds through its free amino group to form new and active derivatives. It should thus be a useful reagent to study the biological properties of lipid A.

AB - N-Start-butyloxycarbonyl (f-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 μ g/mL or less and only one-half as active at 100 μ g/mL. In the induction of tumor necrosis factor release by RAW 264.7 murine macrophage cell line, the biotin-(Cap)2-MLA showed 7-9-fold lower activity than MLA at the concentration range of 0.1-1.0 μ g/mL. These results showed that Cap-MLA is a biologically active lipid A derivative that can be conjugated to other compounds through its free amino group to form new and active derivatives. It should thus be a useful reagent to study the biological properties of lipid A.

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DO - 10.1021/bc00018a013

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AN - SCOPUS:0026952291

SN - 1043-1802

VL - 3

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EP - 548

JO - Bioconjugate Chemistry

JF - Bioconjugate Chemistry

IS - 6

ER -

Preparation and Characterization of Biologically Active 6'-O-(6-Aminocaproyl)-4'-O-O-Monophosphoryl Lipid a and its Conjugated Derivative

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