Carbon-13 nuclear magnetic resonance study of protonation of methotrexate and aminopterin bound to dihydrofolate reductase

Abstract
Methotrexate, aminopterin, and folate were synthesized with 90% enrichment of C-2 with 13C. 13C NMR was used to examine the state of protonation of the pteridine ring of these compounds under various conditions and gives much more clear-cut results than most other methods. For the free compounds the following pK values were obtained: methotrexate, 5.73 .+-. 0.02 (N-1); aminopterin, 5.70 .+-. 0.03 (N-1); folic acid, 2.40 (N-1) and 8.25 .+-. 0.05 (N-3, O-4 amide group). The state of protonation of these compounds when complexed to dihydrofolate reductase (isoenzyme 2 from Streptococcus faecium var. durans) was also studied over the pH range 6-10. The resonance from bound methotrexate showed a constant chemical shift over the whole pH range studied, and it is inferred that in the complex the pteridine ring remains protonated to at least pH 10. The same result was obtained for the binary complex of aminopterin with the reductase and for either methotrexate or aminopterin in ternary complex with reductase and NADPH4. The latter is an inhibitor of the reductase competitive with NADPH. Folate bound to the reductase in either the binary or the ternary complex shows the same protonation behavior as in the free state. Apparently, the association constant for binding of methotrexate is increased enough when protonation of N-1 occurs to account for the enhanced binding of methotrexate as compared with folate.