dihydrofolate | 15623-14-0

• 分子结构分类: 有机化合物 - 有机酸及其衍生物 - 羧酸及其衍生物
• 英文名称: dihydrofolate
• 英文别名: DHF；7,8-dihydrofolate；H2F；(4S)-4-[[4-[(2-amino-4-oxido-7,8-dihydropteridin-6-yl)methylamino]benzoyl]amino]-5-hydroxy-5-oxopentanoate
• CAS: 15623-14-0
• 化学式: C₁₉H₁₉N₇O₆
• 分子量: 441.403
• InChiKey: OZRNSSUDZOLUSN-LBPRGKRZSA-L

计算性质

• 辛醇/水分配系数(LogP)：
  0.4
• 重原子数：
  32
• 可旋转键数：
  7
• 环数：
  3.0
• sp3杂化的碳原子比例：
  0.26
• 拓扑面积：
  213
• 氢给体数：
  5
• 氢受体数：
  9

反应信息

• 作为反应物：
  描述：

  dihydrofolate 在 Escherichia coli dihydrofolate reductase 、 还原型辅酶II(NADPH)四钠盐 作用下， 以 水 为溶剂， 生成 tetrahydrofolate
  参考文献：
  名称：

  The effect of active-site isoleucine to alanine mutation on the DHFR catalyzed hydride-transfer

  摘要：

  对二氢叶酸还原酶野生型和活性位点突变体（I14A）中氢化物转移性质的比较表明，第 14 位侧链的大小会调节 H 通道。

  DOI：

  10.1039/c0cc02988b
• 作为产物：
  描述：

  N^5,10-methylenetetrahydrofolate 、 dCMP 生成 5-Methyldeoxycytidine 5'-phosphate 、 dihydrofolate
  参考文献：
  名称：

  Enzymatic synthesis of deoxy-5-methyl-cytidylic acid replacing deoxycytidylic acid in Xanthomonas oryzae phage Xp12 DNA

  摘要：

  黄单胞菌上的 PHAGE Xp12 是从稻田灌溉水中分离出来的。在这种噬菌体的 DNA 中，胞嘧啶完全被 5-甲基胞嘧啶1 所取代。对这种不同寻常的嘧啶的生物合成进行的同位素追踪研究表明，这种胞嘧啶的甲基化与大多数动植物 DNA 中微量存在的 5-甲基胞嘧啶的甲基化不同。后者被蛋氨酸甲基化。另一方面，Xp12 的 5-甲基胞嘧啶残基的甲基来自丝氨酸的 3-碳，而不是蛋氨酸的硫代甲基碳2。在这项研究中，发现脱氧-5-甲基胞苷酸（d5MCMP）的合成是在核苷酸水平上进行的，以脱氧胞苷酸（dCMP）和甲醛为底物，在 5,6,7,8-四氢叶酸（THFA）和一种来自感染了 Xp12 噬菌体的 X. oryzae 的酶制剂的存在下进行。这一反应似乎解释了噬菌体 Xp12 DNA 中独特的嘧啶--5-甲基胞嘧啶的合成过程。

  DOI：

  10.1038/263615a0

文献信息

• Tetrahydrofolate biosynthesis in plants: Molecular and functional characterization of dihydrofolate synthetase and three isoforms of folylpolyglutamate synthetase in <i>Arabidopsis</i> <i>thaliana</i>
  作者：Stéphane Ravanel、Hélène Cherest、Samuel Jabrin、Didier Grunwald、Yolande Surdin-Kerjan、Roland Douce、Fabrice Rébeillé

  DOI：10.1073/pnas.261585098

  日期：2001.12.18

  Tetrahydrofolate coenzymes involved in one-carbon (C1) metabolism are polyglutamylated. In organisms that synthesize tetrahydrofolate de novo, dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) catalyze the attachment of glutamate residues to the folate molecule. In this study we isolated cDNAs coding a DHFS and three isoforms of FPGS from Arabidopsis thaliana. The function of

  与一碳（C1）代谢有关的四氢叶酸辅酶被聚谷氨酰化。在从头合成四氢叶酸的生物中，二氢叶酸合成酶（DHFS）和叶酰聚谷氨酸合成酶（FPGS）催化谷氨酸残基与叶酸分子的连接。在这项研究中，我们从拟南芥中分离了编码DHFS和FPGS的三种同工型的cDNA。通过补充缺乏DHFS或FPGS活性的酵母突变体，以及通过测量体外向二氢叶酸或四氢叶酸中掺入的谷氨酸盐，可以证明每种酶的功能。DHFS仅存在于线粒体中，使该区室成为植物细胞中二氢叶酸合成的唯一场所。相反，FPGS以不同的同工型存在于线粒体，细胞质和叶绿体中。每个同工型由一个单独的基因编码，这种情况在真核生物中是独特的。FPGS亚型的分隔与γ-谷氨酰胺基共轭的四氢叶酸衍生物的优势以及胞质溶胶，线粒体和质体中丝氨酸羟甲基转移酶和C1-四氢叶酸相互转化酶的存在是一致的。因此，FPGS与这些叶酸介导的反应的结合可以为每个区室提供C1代谢反应所需的多谷氨酰化
• Human AICAR Transformylase:  Role of the 4-Carboxamide of AICAR in Binding and Catalysis
  作者：Mark Wall、Jae Hoon Shim、Stephen J. Benkovic

  DOI：10.1021/bi0007268

  日期：2000.9.1

  mammalian enzyme (K(i) = 0.05 +/- 0.02 microM for 4-N-allyl-AlCAR). It is proposed that the conformation of the carboxamide moiety required for binding to AICAR Tfase is different than the conformation required for binding to IMP CHase, which is supported by inhibition studies of purine ribonucleotides. It is shown that 5-formyl-AICAR (FAICAR) is a product inhibitor of AICAR Tfase with K(i) of 0.4 +/- 0

  我们准备了5-氨基咪唑-4-羧酰胺核糖核苷酸（AICAR）的4-取代类似物，以研究AICAR转化酶（AICAR Tfase）的特异性和机理。在研究的AICAR的9个类似物中，只有一个类似物5-氨基咪唑-4-硫代羧酰胺核糖核苷酸是底物，然后被转化为6-巯基嘌呤核糖核苷酸。其他类似物没有结合或者是竞争性抑制剂，最有效的是5-氨基-4-硝基咪唑核糖核苷酸，K（i）为0.7 +/- 0.5 microM。结果表明，AICAR的4-羧酰胺对于催化是必不可少的，并建议通过介导加成化合物来协助介导质子转移，催化反应。AICAR类似物，其中4-羧酰胺的氮被甲基或烯丙基基团衍生，不结合AICAR Tfase，这是由稳态前爆发动力学确定的；但是，这些化合物是IMP环水解酶（IMP CHase）的有效抑制剂，IMP环水解酶是双功能哺乳动物酶的第二种活性（4-N-烯丙基-AlCAR的K（i）= 0.05 +/- 0
• Thymidylate synthetase purified to homogeneity from human leukemic cells.
  作者：A Lockshin、R G Moran、P V Danenberg

  DOI：10.1073/pnas.76.2.750

  日期：1979.2

  from neoplastic tissue. A ratio of 1.7 mol of 5-fluoro-2'-deoxyuridylate binds per mol of enzyme in the presence of 5,10-methylenetetrahydrofolate. The ternary complex so formed migrates intact on denaturing gels and can be precipitated with trichloroacetic acid; however, urea dissociates the ternary complex. The human thymidylate synthetase is composed of two subunits of 33,000 daltons each. It contains

  来自人白血病细胞系的胸苷酸合成酶（5,10-亚甲基四氢叶酸：dUMP C-甲基转移酶，EC 2.1.1.45）已通过一步亲和柱色谱纯化至均一。纯化的酶的比活性为每毫克蛋白质3.8微米/分钟，对应于250的周转数。这是来自肿瘤组织的胸苷酸合成酶的最高报道值。在5，10-亚甲基四氢叶酸存在下，每摩尔酶结合1.7摩尔5-氟-2′-脱氧尿苷酸。如此形成的三元复合物完整地迁移到变性凝胶上，可以用三氯乙酸沉淀。然而，尿素解离了三元复合物。人胸苷酸合成酶由两个亚基组成，每个亚基为33,000道尔顿。它包含更多的半胱氨酸，甘氨酸残基，
• Evidence for the hypothesis that 10-formyldihydrofolate is the <i>in vivo</i> substrate for aminoimidazolecarboxamide ribotide transformylase
  作者：Joseph E Baggott、Tsunenobu Tamura

  DOI：10.1258/ebm.2009.009151

  日期：2010.3

  We postulate that 10-formyl-7,8-dihydrofolate (10-HCO-H₂folate), not 10-formyl-5,6,7,8-tetrahydrofolate (10-HCO-H₄folate), is the predominant in vivo substrate for mammalian aminoimidazolecarboxamide ribotide (AICAR) transformylase, an enzyme in purine nucleotide biosynthesis de novo, which introduces carbon 2 (C₂) into the purine ring. 10-HCO-H₂folate exists in vivo as labeled 10-formyl-folic acid (10-HCO-folic acid: an oxidation product of 10-HCO-H₄folate and 10-HCO-H₂folate) and is found after doses of labeled folic acid in humans or laboratory animals. The bioactivity of the unnatural isomer, [6 R]-5-formyltetrahydrofolate, in humans is explained by its in vivo conversion to 10-HCO-H₂folate. The structure and active site of AICAR transformylase are not consistent with other enzymes that utilize 10-HCO-H₄folate. Because 10-HCO-H₄folate is rapidly oxidized in vitro to 10-HCO-H₂folate by cytochrome C alone and in mitochondria, it is hypothesized that this process takes place in vivo. In vitro data indicate that 10-HCO-H₂folate is kinetically preferred over 10-HCO-H₄folate by AICAR transformylase and that this enzyme may not have access to sufficient supplies of 10-HCO-H₄folate. Methotrexate blockage of the AICAR transformylase process in patients with rheumatoid arthritis suggests that dihydrofolate (H₂folate) reductase is involved and is consistent with H₂folate and 10-HCO-H₂folate being the product and substrate for AICAR transformylase. The labeling of purine C₂ by an oral dose of [6 RS]-5-H[¹³C]O-H₄folate in a human subject is consistent with 10-H[¹³C]O-H₂folate formation from unnatural isomer, [6 R]-5-H[¹³C]O-H₄folate, and it being a substrate for AICAR transformylase. In vitro exchange reactions of purine C₂ using H₄folate coenzymes are not duplicated in vivo and is consistent with H₂folate coenzymes being used in vivo by AICAR transformylase.

  我们假设10-甲酰-7,8-二氢叶酸（10-HCO-H2叶酸），而不是10-甲酰-5,6,7,8-四氢叶酸（10-HCO-H4叶酸），是哺乳动物氨基咪唑甲酰核苷酸（AICAR）转甲基酶在体内主要的底物，该酶介导嘌呤核苷酸新生，并将碳2（C2）引入嘌呤环。10-HCO-H2叶酸在体内存在为标记的10-甲酰叶酸（10-HCO-叶酸：10-HCO-H4叶酸和10-HCO-H2叶酸的氧化产物），并在人类或实验动物注射标记叶酸后发现。人类体内不自然异构体[6R]-5-甲酰四氢叶酸的生物活性可以通过其在体内转化为10-HCO-H2叶酸来解释。AICAR转甲基酶的结构和活性位点与利用10-HCO-H4叶酸的其他酶不一致。由于10-HCO-H4叶酸在体外仅通过细胞色素C或线粒体迅速氧化为10-HCO-H2叶酸，因此假设这个过程在体内发生。体外数据表明，AICAR转甲基酶对10-HCO-H2叶酸的动力学选择性优于10-HCO-H4叶酸，并且这种酶可能无法获得足够的10-HCO-H4叶酸供应。甲氧苄氨甲酸阻断风湿性关节炎患者的AICAR转甲基酶过程表明，二氢叶酸还原酶参与其中，并且与H2叶酸和10-HCO-H2叶酸是AICAR转甲基酶的产物和底物一致。在人体内，口服[6RS]-5-H[13C]O-H4叶酸的口服剂量标记嘌呤C2与不自然异构体[6R]-5-H[13C]O-H4叶酸形成的10-H[13C]O-H2叶酸的一致，且它是AICAR转甲基酶的底物。在体外使用H4叶酸辅酶进行嘌呤C2的交换反应在体内不会复制，并且与AICAR转甲基酶在体内使用H2叶酸辅酶一致。
• Cofactor role for 10-formyldihydrofolic acid
  作者：J E Baggott、G L Johanning、K E Branham、C W Prince、S L Morgan、I Eto、W H Vaughn

  DOI：10.1042/bj3081031

  日期：1995.6.15

  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.

  10-甲酰-7,8-二氢叶酸（10-HCO-H2folate）是通过控制空气氧化10-甲酰-5,6,7,8-四氢叶酸（10-HCO-H4folate）制备而成的。在pH 7.4下，10-HCO-H2folate制备物的UV光谱具有λ max. 234, 333 nm和λ min. 301 nm，而在pH 1下，λ max. 257, 328 nm和λ min. 229, 307 nm。在2H2O中，300 MHz的1H-NMR光谱表明10-HCO-H2folate是一种纯化合物，并给出了一个甲酰基质子、C-7和C-9上的两个质子的共振信号，没有C-6质子的证据，这与所提出的结构一致。光谱性质表明，10-HCO-H2folate制备物几乎没有受到10-HCO-H4folate、5,10-甲烯基四氢叶酸（5,10-CH=H4folate）或10-甲酰叶酸（10-HCO-folate）的污染。以上数据证实了在此制备的10-HCO-H2folate是真实的。相比之下，一种在pH 7下具有λ max. 272 nm和λ min. 256 nm的叶酸，通过2,6-二氯吲哚酚氧化10-HCO-H4folate制备，据报道其纯度达到97% [Baram, Chabner, Drake, Fitzhugh, Sholar和Allegra（1988）J. Biol. Chem. 263，7105-7111]，显然不是10-HCO-H2folate。在过量的5-氨基咪唑-4-甲酸核苷酸（AICAR）存在下，10-HCO-H2folate被Jurkat细胞（人T细胞白血病）和鸡肝氨基咪唑甲酸核苷酸转移酶（AICAR T'ase; EC 2.1.2.3）利用，每摩尔AICAR甲酰化会产生大约1摩尔H2folate产物。在Jurkat细胞和大鼠骨髓AICAR T'ase作为辅因子时，此处制备的10-HCO-H2folate具有动力学优势，其K(m)值与10-HCO-H4folate相比差异约为5倍。使用鸡肝AICAR T'ase时未观察到实质性的动力学优势。 10-HCO-H2folate在Jurkat细胞或鸡肝甘氨酸核苷酸转移酶（GAR T'ase, EC 2.1.2.2）中几乎没有活性。通过多个报告检测到放射标记叶酸酸给予后，哺乳动物体内存在10-HCO-H2folate的存在，其可在胆汁和尿液中检测到放射性标记的10-HCO-folate。