Rathod P K, Khatri A
Department of Biology, Catholic University of America, Washington, D.C. 20064.
J Biol Chem. 1990 Aug 25;265(24):14242-9.
Fluorinated compounds play an important role in enzymology as well as clinical medicine. Based on the stereochemical preferences of dihydroorotate oxidase and enzymes that use fluoroaspartate, it was anticipated that threo-5-fluoro-L-dihydroorotate (t-FDHO) would have the properties of an antimetabolite. Thus, t-FDHO was synthesized via the reduction of 5-fluoroorotate using NADH and dihydroorotate dehydrogenase that was free of dihydroorotase. When the product was purified and studied by high field proton and carbon 13 NMR, the fluorine, the five carbons, and all the nonexchangeable protons were readily observed. Confirmation of threo configuration was obtained by examining the vicinal coupling constants between the substituents on carbon 5 and carbon 6 of the newly synthesized compound. Additionally, t-FDHO could be reoxidized to 5-fluoroorotate in the presence of dihydroorotate dehydrogenase and NAD+. Treatment of t-FDHO with dihydroorotase generated N-carbamyl-threo-3-fluoro-L-aspartate (CTF-ASP) which was also purified and characterized by NMR. The antiproliferative activity of t-FDHO was determined against a diploid human fibrosarcoma cell line (HT-1080). Fifty microM t-FDHO caused 50% inhibition of HT-1080 cell proliferation. During the 48-h toxicity study, extracellular t-FDHO underwent significant hydrolysis to CTF-ASP. Further extracellular degradation to fluoroaspartate was not seen. The antiproliferative activity of t-FDHO was not due to extracellular degradation since CTF-ASP itself was essentially nontoxic.
含氟化合物在酶学以及临床医学中发挥着重要作用。基于二氢乳清酸氧化酶和使用氟天冬氨酸的酶的立体化学偏好，预计苏式-5-氟-L-二氢乳清酸（t-FDHO）具有抗代谢物的特性。因此，通过使用不含二氢乳清酸酶的NADH和二氢乳清酸脱氢酶还原5-氟乳清酸来合成t-FDHO。当产物被纯化并用高场质子和碳13 NMR进行研究时，氟、五个碳以及所有不可交换的质子都很容易被观察到。通过检查新合成化合物碳5和碳6上取代基之间的邻位偶合常数，获得了苏式构型的确认。此外，在二氢乳清酸脱氢酶和NAD+存在的情况下，t-FDHO可以被重新氧化为5-氟乳清酸。用二氢乳清酸酶处理t-FDHO产生N-氨甲酰基-苏式-3-氟-L-天冬氨酸（CTF-ASP），其也被纯化并用NMR进行了表征。测定了t-FDHO对二倍体人纤维肉瘤细胞系（HT-1080）的抗增殖活性。50μM的t-FDHO导致HT-1080细胞增殖受到50%的抑制。在48小时的毒性研究中，细胞外的t-FDHO发生了显著水解生成CTF-ASP。未观察到进一步的细胞外降解为氟天冬氨酸。t-FDHO的抗增殖活性不是由于细胞外降解，因为CTF-ASP本身基本上是无毒的。
