Pathare P M, Wilbur D S, Heusser S, Quadros E V, McLoughlin P, Morgan A C
Department of Radiation Oncology, University of Washington, Seattle 98195, USA.
Bioconjug Chem. 1996 Mar-Apr;7(2):217-32. doi: 10.1021/bc9600022.
Six cobalamin-biotin conjugates have been prepared. The cobalamin-biotin conjugates were prepared to evaluate the effect that the location of attachment had on the binding with transcobalamin II (TCII), the cobalamin binding protein in plasma, and to evaluate their potential use for in vitro and in vivo applications. This study focused only on the effect of binding with TCII. To decrease the possibility of steric problems in binding of the cobalamin conjugates with TCII, and biotin's binding with streptavidin or avidin, moieties of 11-18 atoms in length were used as linkers. Four biotin conjugates were prepared which were attached to the corrin ring of the cobalamin molecule (on b-, c-, d-, and e-side chains). One conjugate was attached to the 5'-OH of the ribose moiety, and another conjugate was attached at the cobalt metal (in place of the cyanide moiety of cyanocobalamin). Competitive binding studies were conducted where various amounts of the cobalamin-biotin conjugates and their precursor cobalamin derivatives competed with [57Co]cyanocobalamin for binding of recombinant human TCII (rhTCII). Evaluation of cobalamin derivatives which were conjugated at the 5'-OH of ribose or the cobalt metal center indicated that conjugation at either of these positions had little effect on binding with rhTCII. However, conjugates where the attachment was made on the corrin ring substituents had a large variation in binding with rhTCII. Conjugates on the e-propionamide side chain had little effect (relative affinity was equal to or decreased less than a factor of 3) on binding with rhTCII, conjugates of the b-isomer had decreased binding (relative affinity decreased less than a factor of 10), conjugates of the d-propionamide had further decreased binding (relative affinity decreased between 44 and 69 times), and conjugates on the c-acetamide group had poor binding to rhTCII (relative affinity decreased between 295 and 1160 times). The significance of the side chains on the corrin ring in providing specificity and high-affinity binding with rhTCII is discussed.
已制备了六种钴胺素 - 生物素缀合物。制备这些钴胺素 - 生物素缀合物是为了评估连接位置对与转钴胺素II（TCII，血浆中的钴胺素结合蛋白）结合的影响，并评估它们在体外和体内应用的潜力。本研究仅关注与TCII结合的效果。为了降低钴胺素缀合物与TCII结合以及生物素与链霉亲和素或抗生物素蛋白结合时空间位阻问题的可能性，使用了长度为11 - 18个原子的部分作为连接子。制备了四种连接到钴胺素分子咕啉环（在b -、c -、d - 和e - 侧链上）的生物素缀合物。一种缀合物连接到核糖部分的5'-OH，另一种缀合物连接到钴金属（取代氰钴胺的氰基部分）。进行了竞争性结合研究，其中不同量的钴胺素 - 生物素缀合物及其前体钴胺素衍生物与[57Co]氰钴胺竞争结合重组人TCII（rhTCII）。对连接在核糖5'-OH或钴金属中心的钴胺素衍生物的评估表明，在这两个位置中的任何一个位置进行缀合对与rhTCII的结合影响很小。然而，连接在咕啉环取代基上的缀合物与rhTCII的结合有很大差异。e - 丙酰胺侧链上的缀合物对与rhTCII的结合影响很小（相对亲和力等于或降低不到3倍），b - 异构体的缀合物结合减少（相对亲和力降低不到10倍），d - 丙酰胺的缀合物结合进一步减少（相对亲和力降低44至69倍），c - 乙酰胺基团上的缀合物与rhTCII的结合很差（相对亲和力降低295至1160倍）。讨论了咕啉环上侧链在提供与rhTCII特异性和高亲和力结合方面的意义。
