Kuefner U, Esswein A, Lohrmann U, Montejano Y, Vitols K S, Huennekens F M
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Biochemistry. 1990 Nov 20;29(46):10540-5. doi: 10.1021/bi00498a017.
The L,L diastereomer of methotrexate-alpha-alanine (L,L-MTX-Ala) was synthesized by reaction of alpha-L-glutamyl-L-alanine di-tert-butyl ester with 4-amino-4-deoxy-10-methylpteroic acid, followed by removal of the blocking groups. It was identified by HPLC (C18 reversed-phase silica gel; acetic acid/CH3OH) as the slower of two closely spaced components in DL,L-MTX-Ala prepared previously by a different route [Kuefner et al. (1989) Biochemistry 28, 2288-2297]. The L,L diastereomer was hydrolyzed by pancreatic carboxypeptidase A (to yield MTX and Ala) twice as rapidly as the DL,L mixture. Analysis of the latter by HPLC established that the slower component was hydrolyzed to MTX and that the unreactive, faster component was D,L-MTX-Ala. DL,L-MTX-Arg was resolved by HPLC (NH4OAc/CH3CN) into two closely spaced components, and the diastereomers were partially separated by chromatography on DEAE-Trisacryl (H2O----2% NH4HCO3). Serum carboxypeptidase N hydrolyzed only the slower HPLC component (to yield MTX and Arg), thereby identifying it as the L,L diastereomer. When tested for cytotoxicity against L1210 cells, L,L-MTX-Arg (ID50 = 1.6 X 10(-8) M) was more effective than the D,L diastereomer (ID50 = 2.2 X 10(-7) M). Treatment of MTX with dicyclohexylcarbodiimide and N-hydroxysuccinimide (NHS), followed by hydrolysis of the NHS ester, led to racemization in the L-glutamate moiety of MTX as shown by the fact that the product was hydrolyzed by carboxypeptidase G2 (at the pteroate-Glu bond) only to the extent of ca. 50% compared to the untreated control. These observations have a broad significance, since coupling agents are employed extensively in the derivatization of MTX for attachment to affinity supports and monoclonal antibodies.
甲氨蝶呤-α-丙氨酸（L,L-MTX-Ala）的L,L非对映异构体通过α-L-谷氨酰-L-丙氨酸二叔丁酯与4-氨基-4-脱氧-10-甲基蝶酸反应合成，随后除去保护基团。通过HPLC（C18反相硅胶；乙酸/甲醇）鉴定，其为先前通过不同途径制备的DL,L-MTX-Ala中两个紧密相邻组分中较慢的那个组分[库夫纳等人（1989年），《生物化学》28卷，2288 - 2297页]。L,L非对映异构体被胰羧肽酶A水解（生成MTX和Ala）的速度是DL,L混合物的两倍。通过HPLC对后者进行分析表明，较慢的组分被水解为MTX，而无反应性、较快的组分是D,L-MTX-Ala。DL,L-MTX-Arg通过HPLC（醋酸铵/乙腈）分离为两个紧密相邻的组分，并且通过在DEAE-三羟甲基氨基甲烷聚丙烯酰胺（水→2%碳酸氢铵）上进行色谱分离，非对映异构体得到部分分离。血清羧肽酶N仅水解较慢的HPLC组分（生成MTX和Arg），从而将其鉴定为L,L非对映异构体。当测试其对L1210细胞的细胞毒性时，L,L-MTX-Arg（半数抑制浓度 = 1.6×10⁻⁸ M）比D,L非对映异构体（半数抑制浓度 = 2.2×10⁻⁷ M）更有效。用二环己基碳二亚胺和N-羟基琥珀酰亚胺（NHS）处理MTX，随后水解NHS酯，导致MTX的L-谷氨酸部分发生消旋化，这一事实表明，与未处理的对照相比，产物被羧肽酶G2（在蝶酸 - 谷氨酸键处）水解的程度仅约为50%。这些观察结果具有广泛的意义，因为在MTX与亲和载体和单克隆抗体连接的衍生化过程中广泛使用了偶联剂。
