Occurrence and significance of diastereomers of methotrexate alpha-peptides.
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.
Kuefner
U
U
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Esswein
A
A
Lohrmann
U
U
Montejano
Y
Y
Vitols
K S
KS
Huennekens
F M
FM
D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis*; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D007939:Leukemia L1210; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D051379:Mice; D013237:Stereoisomerism
D000970:Antineoplastic Agents; D007202:Indicators and Reagents; C066904:methotrexate-alpha-alanine; C066905:methotrexate-alpha-arginine; D008727:Methotrexate