Methotrexate, aminopterin, and folate have been synthesized with 90% enrichment of C-2 with 13C. 13C nuclear magnetic resonance has been 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) 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. However, folate bound to the reductase in either the binary or the ternary complex shows the same protonation behavior as in the free state. The data indicate that 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.

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Several fluorescence properties of two enantiomers, NSC 613862 (S)-(-) and NSC 613863 (R)-(+), have been compared. Even though the two isomers showed the same fluorescence behavior in solution in different solvents, drastic differences were observed after binding to purified calf brain tubulin. Binding measurements for the two compounds were performed both by fluorescence spectroscopy and by column gel permeation, a direct method of measurement. For both isomers, the binding was characterized by the presence of one high-affinity binding site with an apparent association constant of (3.2 f 0.5) X lo6 M-I and (4.1 f 0.9) X lo6 M-' for the Rand S-isomer, respectively, and by several low-affinity sites.Both isomers were also shown to induce GTPase activity in tubulin. The high-affinity binding site seems to be the same for the two isomers. Moreover, fluorescence competition experiments suggest at least a partial overlap of the colchicine and podophyllotoxin site. To explain the differences in fluorescence behavior after binding to tubulin, we hypothesize that the R-isomer is positioned differently in its binding locus as compared with the S-isomer.Initially synthesized by Elliott et al. (1968) to act as analogues of folic acid, a number of compounds that possess an aromatic nucleus with a l-deaza-7,8-dihydropteridine structure have been reported to inhibit mitosis and produce an accumulation of cells in metaphase (Wheeler et al., 1981). Among them, NSC 181928, ethyl (5-amino-1,2-dihydro-3-[ (N-methy1anilino)methyll pyrido [3,4-b]pyrazin-7-yl)carbamate has been studied in particular. Hamel and Lin (1982) demonstrated that this drug was an active antitubulin agent which inhibited both polymerization and binding of colchicine to tubulin and stimulated tubulin-dependent GTP hydrolysis.Bowdon et al. (1987) showed that another member of the same series, NSC 370147, ethyl (5-amino-1,2-dihydro-2methyl-3-phenylpyrido [ 3,4-b] pyrazin-7-yl)carbamate, was able to competitively inhibit the binding of [3H]c~lchicine and slightly enhance the binding of [3H]vincristine to purified tubulin.However, NSC 370147 is a racemic compound. Its two chiral isomers, NSC 613862 (S)-(-) and NSC 613863 (R)- (+) (see Chart I), have displayed significant differences in Abstract published in Advance ACS Abstracts, September 1, 1993. I Abbreviations: BSA, bovine serum albumin; PG buffer, 10 mM sodium phosphate and 0.1 mM GTP, pH 7.0; MDL 27048, trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylam~o)phenyl]-2-methyl-2-pro~n-1 -one; MTC, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6cyclohep~~en-1-one; SDS, sodium dodecyl sulfate; TME, tropolone methyl ether; AS,,, difference between theapparent sedimentation coefficient of tubulin alone and liganded.