Baggott J E, Johanning G L, Branham K E, Prince C W, Morgan S L, Eto I, Vaughn W H
Department of Nutrition Sciences, University of Alabama, Birmingham 35294, USA.
Biochem J. 1995 Jun 15;308 ( Pt 3)(Pt 3):1031-6. doi: 10.1042/bj3081031.
10-Formyl-7,8-dihydrofolic acid (10-HCO-H2folate) was prepared by controlled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate). The UV spectra of the 10-HCO-H2folate preparation has lambda max. 234, 333 nm and lambda min. 301 nm at pH 7.4, and lambda max. 257, 328 nm and lambda min. 229, 307 nm at pH 1. 1H-NMR spectroscopy of 10-HCO-H2folate (in 2H2O; 300 MHz) suggested a pure compound and gave resonances for one formyl group proton, two protons on C-7 and C-9, and no evidence for a C-6 proton, which is consistent with the structure proposed. The spectral properties indicated that the 10-HCO-H2folate preparation is not appreciably contaminated with 10-HCO-H4folate, 5,10-methenyltetrahydrofolic acid (5,10-CH = H4folate) or 10-formylfolic acid (10-HCO-folate). The above data establish that the 10-HCO-H2folate prepared here is authentic. In contrast, a folate with a UV spectrum having lambda max. 272 nm and lambda min. 256 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H4folate and reported to be 97% pure [Baram, Chabner, Drake, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H2folate. 10-HCO-H2folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazolecarboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-amino-imidazole-4-carboxamide ribotide (AICAR) resulting in the appearance of approximately 1 mol of H2folate product for each mol of AICAR formylated. The present 10-HCO-H2folate preparation had a kinetic advantage over 10-HCO-H4folate resulting from a difference of approx. 5-fold in K(m) values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H2folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, EC 2.1.2.2). The existence in vivo of 10-HCO-H2folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10-HCO-folate in bile and urine after administration of radiolabelled folic acid.
通过对10-甲酰基-5,6,7,8-四氢叶酸（10-HCO-H4叶酸）进行可控的空气氧化制备了10-甲酰基-7,8-二氢叶酸（10-HCO-H2叶酸）。在pH 7.4条件下，10-HCO-H2叶酸制剂的紫外光谱的最大吸收波长λmax为234、333 nm，最小吸收波长λmin为301 nm；在pH 1条件下，λmax为257、328 nm，λmin为229、307 nm。10-HCO-H2叶酸的1H-核磁共振光谱（在2H2O中；300 MHz）表明该化合物纯净，给出了一个甲酰基质子、C-7和C-9上两个质子的共振信号，且没有C-6质子的信号，这与所提出的结构一致。光谱性质表明，10-HCO-H2叶酸制剂未明显受到10-HCO-H4叶酸、5,10-亚甲基四氢叶酸（5,10-CH = H4叶酸）或10-甲酰基叶酸（10-HCO-叶酸）的污染。上述数据证实此处制备的10-HCO-H2叶酸是真实的。相比之下，通过2,6-二氯靛酚氧化10-HCO-H4叶酸制备的一种叶酸，在pH 7时紫外光谱的最大吸收波长λmax为272 nm，最小吸收波长λmin为256 nm，据报道其纯度为97%[巴拉姆、查布纳、德雷克、菲茨休、肖拉尔和阿莱格拉（1988年）《生物化学杂志》263, 7105 - 7111]，显然不是10-HCO-H2叶酸。在过量的5-氨基咪唑-4-甲酰胺核苷酸（AICAR）存在的情况下，Jurkat细胞（人T细胞白血病）和鸡肝氨基咪唑甲酰胺核糖核苷酸转甲酰酶（AICAR T'ase；EC 2.1.2.3）可利用10-HCO-H2叶酸，每摩尔被甲酰化的AICAR会产生约1摩尔的H2叶酸产物。当两种叶酸都用作Jurkat细胞和大鼠骨髓AICAR T'ase的辅因子时，由于米氏常数（K(m)）值相差约5倍，目前制备的10-HCO-H2叶酸制剂相对于10-HCO-H4叶酸具有动力学优势。使用鸡肝AICAR T'ase时未观察到明显的动力学优势。10-HCO-H2叶酸对Jurkat细胞或鸡肝甘氨酰胺核糖核苷酸转甲酰酶（GAR T'ase，EC 2.1.2.2）几乎没有活性。在给哺乳动物注射放射性标记的叶酸后，有几份报告称在胆汁和尿液中可检测到放射性标记的10-HCO-叶酸，这表明哺乳动物体内存在10-HCO-H2叶酸。
