The Physical Binding of Several Phenylalanine Derivatives of Nitrogen Mustard with Rat Plasma

In this investigation, the binding of a number of phenylalanine derivatives of nitrogen mustard with rat plasma was studied using the ultrafiltration method. N-formylsarcolysine (NF) was used as a model to study the factors influencing this binding. NF and other N-acylated phenylalanine mustard derivatives were found to be bound to an extent of more than 60% to rat plasma, while sarcolysine and other related compounds with a free amino-group were found to be 24% bound. The former compounds exhibited a lower toxicity, while the latter ones possessed higher toxicity. This finding led us to advance the assumption that the difference in the degrees of binding between the two series of compounds contributes, at least in part, to their differences in toxicity. When the chloroethyl groups in the molecule were hydrolyzed to hydroxyethyl groups, the bonding capacity of the compound was shown to be lost almost entirely. When the carboxyl group of NF was esterified, however, the degree of binding was similar to its parent compound. It appears that the chloroethyl group is necessary for this binding, while the carboxyl group is not as important. Since the degree of binding is not affected by changes of room temperature, it is suggested that the binding is a physical process rather than alkylation. When the acidity of the rat plasma was adjusted to pH values of 2 to 8, the highest degree of binding of NF was shown to be at pH 3.6. At this pH, the highest solubility of NF in organic solvent was obtained. This result is in accord with the assumption that it is the unionic form which participates in the binding. It was found that, within a certain limit, the degree of NF binding decreased as the degree of dilution of the plasma was increased. The binding of NF with various tissue homogenates of normal rats and tumor-bearing animals was also investigated. The binding capacity may be listed in decreasing order as follows: liver, 81.5%; plasma, 71.9%; Kidney, 69.7%; spindle cell carcinoma B₂₂(NF-resistant), 60.9%. Yoshida solid carcinoma(NF-sensitive), 57.9%. It seems that the difference of binding between the NF-sensitive and NF-resistant tumors was insignificant.