Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative.
Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative.
复制标题
生物活性6-O-(6-氨基己酰基)-4-O-单磷酰脂质A及其共轭衍生物的制备和表征。
DOI：
10.1021/bc00018a013
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发表时间：
1992
影响因子：
4.7
通讯作者：
Cotter,RJ
中科院分区：
文献类型：
--
作者：
Myers,KR;Ulrich,JT;Qureshi,N;Takayama,K;Wang,R;Chen,L;Emary,WB;Cotter,RJ
2V-叔丁氧羰基 (f-Boc) 保护的 6-氨基己酸 (Cap) 酸酐与从大肠杆菌 J5 脂多糖中获得的未保护的六酰基-4'-0-单磷酰脂质 A (MLA) 反应，得到 f-Boc-Cap-MLA。经过柱纯化步骤后，通过在酸性存在下低温孵育样品除去 f-Boc 基团，产生 Cap-MLA。该产品通过锎等离子体解吸质谱（PDMS）进行分析。纯化的f-Boc-Cap-MLA通过反相高效液相色谱进一步分离为其甲酯，并通过激光解吸质谱、PDMS和质子核磁共振波谱进行表征。这些分析表明，Cap 基团被选择性地引入 MLA 的 6'-位。为了证明Cap-MLA可以与其他化合物缀合，将其与生物素-Cap iV-羟基琥珀酰亚胺酯反应以产生生物素-(Cap) 2-MLA。通过 PDMS 对该产物进行分析，证实其预期分子量为 2171，并显示存在含有生物素和 Cap 基团的片段。使用单克隆抗体和链霉亲和素证明该缀合产物中存在脂质 A 和生物素。然后测试了这两种新型脂质 A 衍生物的生物活性。尽管使用小鼠脾细胞时，Cap-MLA 和生物素-(Cap) 2-MLA 都显示出促有丝分裂活性，但它们在 20 µg/mL 或更低时的活性比 MLA 低约 4-8 倍，在 100 µg/mL 时活性仅为 MLA 的一半。在 RAW 264.7 鼠巨噬细胞系诱导肿瘤坏死因子释放过程中，生物素-(Cap) 2-MLA 在 0.1-1.0 µg/mL 浓度范围内的活性比 MLA 低 7-9 倍。这些结果表明Cap-MLA是一种具有生物活性的脂质A衍生物，可以通过其游离氨基与其他化合物缀合形成新的活性衍生物。因此它应该是研究脂质 A 生物学特性的有用试剂。
2V-ĢerĢ-butyloxycarbonyl (f-Boc) protected 6-aminocaproic (Cap) anhydride was reacted with unprotected hexaacyl-4'-0-monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Escherichia coli J5 toyield f-Boc-Cap-MLA. After a column purification step, the f-Boc group was removed by incubating the sample at low temperaturein the presence of acid toyield Cap-MLA. This product was analyzed by californium plasma desorption mass spectrometry (PDMS). Purified f-Boc-Cap-MLA was further fractionatedby 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 iV-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 ĩg/mL or less and only one-half as active at 100 ĩg/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 ĩg/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.