Acetonitrile → Methotrexate

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Small samples (1 mg) of the end products of Examples 1 and 2 were treated at room temperature in 1M NaOAc (10 mL) containing ZnCl2 (10 mg) with freshly thawed carboxypeptidase G1 enzyme solution (0.3 μL, 4500 U/mL). In less than 15 min, glutamate hydrolysis was nearly complete according to HPLC analysis (20% MeCN 0.1M NH4OAc, pH 7.5), which showed the disappearance of >95% of the starting material (Example 1, 2.5 min; Example 2, 2.89 min) and the appearance of new peaks (3.0 and 3.3 min) assumed to be the 2-desamino and 2-desamino-2-methyl derivates of 4-amino-4-deoxypteroic acid, respectively. Under identical conditions, clinical grade methotrexate (mainly the L-form, 3.0 min) was cleaved to 4-amino-4-deoxy-N10 -methylpteroic acid (4.0 min), whereas D MTX (4.0 min, pre-formed from clinical grade MTX by carboxypeptidase G1 treatment) was resistant tc further treatment with the enzyme. Since the enzyme carboxypeptidase G1 is well known to cleave L-MTX to 4-amino-4-deoxy-N10 -methylpteroic acid under conditions that leave D-MTX unaffected, these results show that my desamino analogues are essentially pure L-enantiomers.

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