Walsh J P, Caldwell K K, Majerus P W
Washington University School of Medicine, Division of Hematology-Oncology, St. Louis, MO 63110-1093.
Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9184-7. doi: 10.1073/pnas.88.20.9184.
We have synthesized phosphatidylinositol 3-phosphate from phosphatidylinositol 4-phosphate by using diisopropylcarbodiimide to promote migration of the 4-phosphate via a cyclic phosphodiester intermediate. The product was isolated by a thin-layer chromatographic method that depends on the ability of phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate, to form complexes with boric acid. The final yield of the procedure was 8% phosphatidylinositol 3-phosphate, which was approximately 80% pure. The product was shown to be phosphatidylinositol 3-phosphate by the following criteria: (i) cochromatography with an authentic standard on borate thin-layer chromatography, (ii) cochromatography of the deacylated product with glycerophosphoinositol 3-phosphate on high-performance liquid chromatography, (iii) conversion of the product to phosphatidylinositol by homogeneous phosphatidylinositol 3-phosphate 3-phosphatase, and (iv) deacylation and deglyceration of the product to a compound that comigrates with inositol 1,3-bisphosphate on high-performance liquid chromatography. The availability of mass amounts of phosphatidylinositol 3-phosphate will allow further elaboration of reactions in this recently discovered pathway of phosphatidylinositol metabolism.
我们通过使用二异丙基碳二亚胺，促使4-磷酸经由环状磷酸二酯中间体迁移，从而由磷脂酰肌醇4-磷酸合成了磷脂酰肌醇3-磷酸。产物通过一种薄层色谱法进行分离，该方法依赖于磷脂酰肌醇4-磷酸而非磷脂酰肌醇3-磷酸与硼酸形成复合物的能力。该方法的最终产率为8%的磷脂酰肌醇3-磷酸，其纯度约为80%。通过以下标准证明产物为磷脂酰肌醇3-磷酸：(i) 在硼酸盐薄层色谱上与正品标准品共色谱；(ii) 脱酰基产物在高效液相色谱上与甘油磷酸肌醇3-磷酸共色谱；(iii) 产物通过均一的磷脂酰肌醇3-磷酸3-磷酸酶转化为磷脂酰肌醇；(iv) 产物脱酰基和脱甘油生成一种在高效液相色谱上与肌醇1,3-二磷酸共迁移的化合物。大量磷脂酰肌醇3-磷酸的可得性将有助于进一步阐明磷脂酰肌醇代谢这一最近发现的途径中的反应。
