4033-27-6

Basic information

• Product Name: DIHYDROFOLIC ACID
• Synonyms: (S)-2-(4-(((2-aMino-4-oxo-1,4,7,8-tetrahydropteridin-6-yl)Methyl)aMino)benzaMido)pentanedioic acid;7,8-Dihydrofolic acid (DHF);Dihydrofolic acid 7,8-Dihydropteroyl-L-glutaMic acid;N-[4-[[(2-aMino-3,4,7,8-tetrahydro-4-oxo-6-pteridinyl)Methyl]aMino]benzoyl]-L-GlutaMic acid;Folinic Acid Impurity G (7,8-Dihydrofolic Acid);7,8-DIHYDROPTEROYL-L-GLUTAMIC ACID;FAH2;DIHYDROPTEROYL-L-GLUTAMIC ACID
• CAS NO: 4033-27-6
• Molecular Formula: C₁₉H₂₁N₇O₆
• Molecular Weight: 443.41
• Mol File: 4033-27-6.mol

Chemical Properties

• Boiling Point: °Cat760mmHg
• Flash Point: °C
• Appearance: /
• Density: 1.69g/cm³
• Refractive Index: 1.762
• Storage Temp.: −20°C
• Solubility: 0.1 M NaOH: 10 mg/mL, slightly hazy, orange
• PKA: pKa 1.38(H2O t=25 I=0.10) (Occasionally);3.84(H2O t=25 I=0.10) (Occasionally)
• Stability: Air Sensitive, Hygroscopic, Temperature Sensitive
• BRN: 69017
• CAS DataBase Reference: DIHYDROFOLIC ACID(CAS DataBase Reference)
• NIST Chemistry Reference: DIHYDROFOLIC ACID(4033-27-6)
• EPA Substance Registry System: DIHYDROFOLIC ACID(4033-27-6)

Safety Data

• Hazard Codes: Xi
• Statements: 36/37/38
• Safety Statements: 26-36
• WGK Germany: 3
• Hazardous Substances Data: 4033-27-6(Hazardous Substances Data)

Usage

Uses

1. Antidote to Methotrexate Toxicity:
Dihydrofolic Acid is used as an antidote for methotrexate toxicity, a condition that arises from the excessive accumulation of methotrexate in the body. Methotrexate is a drug that inhibits dihydrofolate reductase (DHFR), which is responsible for converting DHF to THF. By providing an alternative source of DHF, it helps to counteract the toxic effects of methotrexate.
2. Mammalian Metabolism:
Dihydrofolic Acid is used as an intermediate in the mammalian conversion of dietary folic acid to tetrahydrofolate by dihydrofolate reductase (DHFR). This process is essential for the synthesis of nucleotides, amino acids, and the maintenance of overall cellular function.
3. Bacterial Metabolism:
In the bacterial kingdom, Dihydrofolic Acid is used as a precursor in the generation of 7,8-dihydropteroate by dihydrofolate synthetase. DIHYDROFOLIC ACID is a crucial component in the bacterial metabolic pathway, contributing to the synthesis of essential biomolecules and cellular growth.

Biochem/physiol Actions

Folic acid (FA) and dihydrofolic acid (FAH2) are substrates of dihydrofolate reductase(s) which reduce them to tetrahydrofolate (THF), which in turn supports ‘one carbon′ transfer. Tetrahydrofolates are required for de novo synthesis of purines, thymidylic acid and various amino acids and for post-translational methylation (epigenetics).

Purification Methods

DHFA is best purified by suspending (1g mostly dissolved)) in ice-cold sodium ascorbate (300mL of 10% at pH 6.0, prepared by adjusting the pH of 30g of sodium ascorbate in 150mL of H2O by adding 1N NaOH dropwise using a gla

InChI:InChI=1/C19H21N7O6/c20-19-25-15-14(17(30)26-19)23-11(8-22-15)7-21-10-3-1-9(2-4-10)16(29)24-12(18(31)32)5-6-13(27)28/h1-4,12,21H,5-8H2,(H,24,29)(H,27,28)(H,31,32)(H4,20,22,25,26,30)/t12-/m0/s1

Upstream product

• 59-30-3 folic acid 
• 142811-51-6 N-<4-<<2(S)-amino-3-<(2,5-diamino-4(3H)-oxopyrimidin-6-yl)amino>propyl>amino>benzoyl>-L-glutamic acid 
• 4271-30-1 N-(4-aminobenzoyl)-L-glutamic acid 
• 3031-94-5 5-aminoimidazole-4-carboxamide ribonucleotide 
• 142811-49-2 N-<4-<<2(S)--3-<(2-amino-5-nitro-4(3H)-oxopyrimidin-6-yl)amino>propyl>amino>benzoyl>-L-glutamic acid 
• 142811-46-9 2-amino-6<<2'(R)-(benzylamino)-3'-hydroxypropyl>amino>-5-nitro-4(3H)-pyrimidinone 
• 142811-47-0 2-amino-6-<<2'(R)--3'-hydroxypropyl>amino>-5-nitro-4(3H)-pyrimidinone 
• 142811-48-1 [(R)-2-(2-Amino-5-nitro-6-oxo-1,6-dihydro-pyrimidin-4-ylamino)-1-formyl-ethyl]-benzyl-carbamic acid tert-butyl ester 
• 142811-50-5 N-<4-<<2(S)-(benzylamino)-3-<(2-amino-5-nitro-4(3H)-oxopyrimidin-6-yl)amino>propyl>amino>benzoyl>-L-glutamic acid 
• 2280-44-6 D-Glucose 
• 73-40-5 2-amino-1,9-dihydro-6H-purin-6-one 
• 86-01-1 Guanosine 5'-triphosphate 
• 1218-98-0 dihydro-7,8-D-neopterine 
• 3545-84-4 6-hydroxymethyl-7,8-dihydro-pterin pyrophosphate 

Downstream Products

• 58-05-9 folinic acid 
• 442634-22-2 5,10-methylidyne-5,6,7,8-tetrahydrofolic acid 
• 98814-61-0 (6RS,7RS)-<7-(2)H>-5-formyl-5,6,7,8-tetrahydrofolic acid 
• 59-30-3 folic acid 
• 1131-35-7 7,8-dihydroxanthopterin 
• 13214-44-3 6-formyl-7,8-dihydropterin 
• 135-16-0 (6S,S)-5,6,7,8-tetrahydrofolic acid 
• 71963-69-4 (6S,S)-5,6,7,8-tetrahydrofolic acid 
• 76937-22-9 (6S)-N[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-5-methyl-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid 
• 135-16-0 tetrahydrofolic acid 
• 65981-90-0 (6R)-5,10-methylylidenetetrahydrofolic acid chloride 
• 135-16-0 (6-R,S)-5,6,7,8-tetrahydrofolate 
• 82949-83-5 5,10-bis(uracil-5-ylmethyl)tetrahydropteroylglutamic acid 
• 82883-89-4 5,10-bis(uracil-5-ylmethyl)<6-2H₁>tetrahydropteroylglutamic acid 

Relevant articles and documents

Method for preparing L-5-calcium methyl tetrahydrofolate through enzymic method

The invention belongs to the technical field of enzymatic synthesis, and relates to a method for preparing L-5-calcium methyl tetrahydrofolate through an enzymic method. The method comprises the following steps that 1, folic acid serves as the raw materia

Chemoenzymatic Assembly of Isotopically Labeled Folates

Pterin-containing natural products have diverse functions in life, but an efficient and easy scheme for their in vitro synthesis is not available. Here we report a chemoenzymatic 14-step, one-pot synthesis that can be used to generate 13C- and 15N-labeled dihydrofolates (H2F) from glucose, guanine, and p-aminobenzoyl-l-glutamic acid. This synthesis stands out from previous approaches to produce H2F in that the average yield of each step is >91% and it requires only a single purification step. The use of a one-pot reaction allowed us to overcome potential problems with individual steps during the synthesis. The availability of labeled dihydrofolates allowed the measurement of heavy-atom isotope effects for the reaction catalyzed by the drug target dihydrofolate reductase and established that protonation at N5 of H2F and hydride transfer to C6 occur in a stepwise mechanism. This chemoenzymatic pterin synthesis can be applied to the efficient production of other folates and a range of other natural compounds with applications in nutritional, medical, and cell-biological research.

Binding pocket alterations in dihydrofolate synthase confer resistance to para-aminosalicylic acid in clinical isolates of Mycobacterium tuberculosis

The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor paraaminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug. Copyright

POSITRON EMISSION TOMOGRAPHY TRACER

The present invention relates to positron emission tomography tracers and methods of using these tracers.

Evidence that a ' dynamic knockoutg' in Escherichia coli dihydrofolate reductase does not affect the chemical step of catalysis

The question of whether protein motions play a role in the chemical step of enzymatic catalysis has generated much controversy in recent years. Debate has recently reignited over possible dynamic contributions to catalysis in dihydrofolate reductase, foll

Evaluation of the catalytic mechanism of AICAR transformylase by pH-dependent kinetics, mutagenesis, and quantum chemical calculations

The catalytic mechanism of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase) is evaluated with pH dependent kinetics, site-directed mutagenesis, and quantum chemical calculations. The chemistry step, represented by the burst rates, was not pH-dependent, which is consistent with our proposed mechanism that the 4-carboxamide of AICAR assists proton shuttling. Quantum chemical calculations on a model system of 5-amino-4-carboxamide imidazole (AICA) and formamide using the B3LYP/6-31G* level of theory confirmed that the 4-carboxamide participated in the proton-shuttling mechanism. The result also indicated that the amide-assisted mechanism is concerted such that the proton transfers from the 5-amino group to the formamide are simultaneous with nucleophilic attack by the 5-amino group. Because the process does not lead to a kinetically stable intermediate, the intramolecular proton transfer from the 5-amino group through the 4-carboxamide to the formamide proceeds in the same transition state. Interestingly, the calculations predicted that protonation of the N3 of the imidazole of AICA would reduce the energy barrier significantly. However, the pKa of the imidazole of AICAR was determined to be 3.23 ± 0.01 by NMR titration, and AICAR is likely to bind to the enzyme with its imidazole in the free base form. An alternative pathway was suggested by modeling Lys266 to have a hydrogen-bonding interaction with the N3 of the imidazole of AICAR. Lys266 has been implicated in catalysis based on mutagenesis studies and the recent X-ray structure of AICAR Tfase. The quantum chemical calculations on a model system that contains AICA complexed with CH3NH3+ as a mimic of the Lys residue confirmed that such an interaction lowered the activation energy of the reaction and likewise implicated the 4-carboxamide. To experimentally verify this hypothesis, we prepared the K266R mutant and found that its kcat is reduced by 150-fold from that of the wild type without changes in substrate and cofactor Km values. The kcat-pH profile indicated virtually no pH-dependence in the pH range 6-10.5. The results suggest that the ammonium moiety of Lys or Arg is important in catalysis, most likely acting as a general acid catalyst with a pKa value greater than 10.5. The H267A mutant was also prepared since His267 has been found in the active site and implicated in catalysis. The mutant enzyme showed no detectable activity while retaining its binding affinity for substrate, indicating that it plays a critical role in catalysis. We propose that His267 interacts with Lys266 to aid in the precise positioning of the general acid catalyst to the N3 of the imidazole of AICAR.

Large-scale Chemoenzymic Synthesis of Calcium (6S)-5-Formyl-5,6,7,8-tetrahydrofolate using the NADPH Recycling Method

Chemoenzymic large-scale synthesis of the calcium salt of (6S)-5-formyltetrahydrofolic acid was achieved from folic acid 1 via (6S)-tetrahydrofolic acid by using dihydrofolate reductase (DHFR) produced by Escherichia coli, harbouring a high-expression plasmid, pTP64-1.On the other hand, for the diastereoselective reduction of 7,8-dihydrofolic acid 2 to tetrahydrofolate (6S)-3, a new NADPH recycling system was constructed by coupling with glucose dehydrogenase from Gluconobacter scleroides.Having these enzymic systems to hand, compound 1 was reduced by zinc powder in alkaline solution to give compound 2 which, without isolation, was reduced enzymatically to afford tetrahydrofolate (6S)-3 (94 percent de).The pH adjustment of the reaction mixture containing dihydrofolate 2 was done with phosphoric acid in order to remove zinc ion which inhibited the following enzymic reduction.The formed tetrahydrofolate (6S)-3 was converted into entirely optically pure N-formyl compound (6S)-5 on a large scale.The specific rotation value of (-)-leucovorin was 20D -13.3 (c 1, water).For the comparison of pharmacological effects, a completely optically pure form of (+)-leucovorin was also prepared on a preparative scale.Compound (6S)-5 was 300-fold more active compared with the (6R)-diastereoisomer.

Synthesis of Tetrahydropteridine C6-Stereoisomers, Including N5-Formyl-(6S)-tetrahydrofolic Acid

Chiral N1-protected vicinal diamines derived from amino acids were condensed with 2-amino-6-chloro-5-nitro-4(3H)-pyrimidinone, the nitro group reduced, and the amine deprotected.Oxidative cyclization of the resulting triaminopyrimidinone via quinoid pyrimidine intermediates gave a quinoid dihydropteridine, which was then reduced to a tetrahydropteridine C6-stereoisomer.Thus, 6(R)- and 6(S)-propyltetrahydropterin were stereospecifically synthesized (99 percent enantiomeric purity) in good yield from D- and L-norvaline, respectively.Reductive alkylation of (p-aminobenzoyl)-L-glutamate with a niropyrimidine aldehyde derived from D- or L-serine similarly afforded, after cyclization and reduction, (6R)- or (6S)-tetrahydrofolic acid.The latter was then converted to the natural isomer of leucovorin by regioselective N5-formylation with carbonyl diimidazole / formic acid without loss of enantiomeric purity.

ASYMMETRIC REDUCTION OF DIHYDROFOLATE USING DIHYDROFOLATE REDUCTASE AND CHIRAL BORON-CONTAINING COMPOUNDS

The reduction of dihydrofolic acid to chiral tetrahydrofolic acid has been investigated by enzymic and non-enzymic means.With dihydrofolate reductase from E.coli as catalyst and recycling systems for NADPH, up to 1 g of optically pure stable tetrahydrofolate derivatives was obtained.The technique makes the possibility of synthesising chiral 5-formyltetrahydrofolate (leucovorin) for use in cancer rescue therapy attainable.In contrast, although dihydrofolate was reduced by a number of chiral boranes and borates built from amino acids and amino alcohols, enantiomeric excesses were minimal.

Stereochemistry of Reduction of the Vitamin Folic acid by Dihydrofolate Reductase

Reduction of the vitamin folic acid (6) to the coenzyme 5,6,7,8-tetrahydrofolic acid (1) by the enzyme dihydrofolate reductase is shown to involve transfer of the 4-pro R hydrogen of NADPH to the same face at both C-6 and C-7 of the pteridine system (the re face at C-6 and the si face at C-7).The orientations of the pteridine system of folic acid (6) and of dihydrofolic acid (5) when bound to the enzyme are different from the orientation of the pteridine ring of the anti-cancer drug methotrexate (11) when bound to this enzyme.