JPH059065B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides 6-(R)-L-erythro-5,6,7,
8-tetrahydrobiopterin (hereinafter referred to as 6-(R)
-L-BPH ₄ ). More specifically, 6-(R)-L- by enzymatic method.
Concerning improved methods of manufacturing BPH ₄ . Biopterin has, for example, D-series and L-series, depending on the relative configuration of the side chain of the carbon atom at the 6th position, and its tetrahydro form has two types, depending on the absolute configuration of the carbon atom at the 6th position: (R)−or
It is known that two diastereomers, (S)-, exist [Furrer, HJSHelu.Chim.
Acta, 62 , 2577 (1979)]. That is, the general formula () In the tetrahydropterin derivative represented by
R ₁ is

[Formula] is 6-(R)-L- _BPH4 . 6-(R)-L-BPH ₄ exists in living organisms and serves as a coenzyme for phenylalanine hydroxylase as well as for other aromatic amino acid hydroxylases. For that purpose, 6-(R)-L-
Deficiency of BPH ₄ results in a deficiency of neurotransmitters such as serotonin, dopamine, noradrenaline, and adrenaline, resulting in severe neurological symptoms. For example, malignant phenylketonuria is known, and in fact, pioneering therapeutic results have been reported by administering 6-(R,S)-tetrahydrobiopterin [Niederwieser.A. et al. Lancet 1]
550 (1979); Curtius, H-CH.5Clin.Chim.Acta
93 , 251-262 (1979)]. Many attempts have been made to chemically synthesize tetrahydrobiopterin; for example, there is a method in which L-biopterin is catalytically reduced in trifluoroacetic acid using Pt.Pd or the like to obtain a tetrahydrobiopterin. However, the tetrahydro compound obtained is a mixture of a set of diastereomers, which can be resolved into 6-(R)-L
-The only attempt to isolate BPH ₄ has been to resolve it using high-performance liquid chromatography. It has also been reported that L-biopterin is acetylated with acetic anhydride to form a triacetyl compound, followed by catalytic reduction, further acetylated, and resolved by fractional crystallization [Viscontini, M. et al. Helu.Chim.
Acta, 62 2577 (1979)], but the complexity of experimental operations,
It is not suitable for dividing a large amount of mixture due to poor yield. This is due to the product being very oxygen unstable. On the other hand, the general formula () (R ₁ is ) is an important metabolic intermediate of folic acid in the living body, and this substance is converted into dihydrofolate reductase (hereinafter abbreviated as DHFR) in the living body.
In the general formula (), R ₁ is It is reduced to tetradihydrofolic acid. Furthermore, when L-biopterin is chemically reduced using sodium hydroxide and zinc powder, it is reduced to 7,8-dihydro-L-biopterin (hereinafter abbreviated as L-BPH ₂ ) with good yield. . 6-(R) using DHFR for L-BPH ₂ in the same way as dihydrofolic acid
An attempt to obtain -L-BPH ₄ has been reported [Watanabe et al., Biochemistry, Vol. 53, No. 8, p1008 (1980)]
However, it is not practical to obtain a large amount of 6-(R)-L-BPH ₄ for the following reasons. That is, a trace amount of by-product that inhibits DHFR is produced in a reaction solution in which L-biopterin is reduced using zinc powder. Furthermore, when attempting to isolate L-BPH ₂ and use it in an enzymatic reaction, the amount of subsequent reaction solution increases because crystalline L-BPH ₄ is poorly soluble in water. Furthermore, because 6-(R)-L-BPH ₄ is unstable, there are many problems such as the difficulty in desalting the reaction solution, and the difficulty in desalting makes large-scale synthesis impossible. There is. As a result of extensive research into improving the production of L-BPH ₄ by this enzymatic method, the present inventors discovered that L-ascorbic acid and L-cysteine hydrochloride were added to the reaction solution as stabilizers after the reaction was completed. As a result, a stable and easily purified production method was discovered, and the present invention was completed. That is, the present invention reduces L-biopterin in the presence of an alkali using a reducing agent such as zinc powder, removes DHFR inhibitors as necessary, and reduces L-BPH ₂
was obtained, and without isolation, the reaction mixture was further added with reduced nicotinamide adenine nucleotide phosphate (NADPH), glucose-6-phosphate (G6P), glycose-6-phosphate reductase (G6PDH), and dihydrofolate reductase (DHFR). ) is added to carry out the enzymatic reduction reaction, and after the reaction is completed, L-ascorbic acid and L-cysteine are added as stabilizers to achieve stable purification. The ratio of adding the stabilizer may be L-ascorbic acid: L-cysteine = 2:1 to 3:1 in weight ratio, and it is sufficient that L-ascorbic acid is 1/10 or more to L-BPH ₄ . . After adding the stabilizer, the 6-(R)-L-BPH ₄ containing the stabilizer is purified by a commonly used purification method such as an activated carbon column or a CM-Sephadex column.
obtained, and then adsorbed on CM Cephadex,
Rinse the column with distilled water (containing the above-mentioned stabilizers if necessary) and add NADPH, G6P, G6PDH,
DHFR and unreacted L-BP and L-BPH ₂ are removed and eluted with hydrochloric acid containing a stabilizer to obtain the dihydrochloride of 6-(R))-L-BPH ₄ containing a stabilizer. In order to remove the stabilizer, column treatment such as CM-Sephadex is performed under anaerobic conditions, for example under an argon stream. 6-(R)-L- purified thereby
BPH _4.2 hydrochloride is obtained. Next, the present invention will be explained in detail by giving examples. Example 1 Method for producing 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (L-6R-BPH ₄ ) 15 g of biopterin was dissolved in 255 ml of 2N sodium hydroxide, and distilled water was added. Add 255ml zinc powder
Add 16.35g and stir for 2 hours under nitrogen flow. After the reaction, use Sephadex G-25 (diameter 3.5 cm, length 15
cm), then wash the column with 150 ml of distilled water. The effluent and washing liquid are combined and adjusted to pH 5.8 with 78 ml of 50% acetic acid in an ice bath to obtain an aqueous L-dihydrobiopterin solution. Transfer 57 g of glucose 6-phosphate to a 2-volume three-necked flask and add 300 g of distilled water.
1 g of β-nicotinamide adenine dinucleotide phosphate reduced form dissolved in 100 ml of 5% sodium acetate, 10000 U of glucose 6-phosphate reductase dissolved in 30 ml of distilled water, and then add distilled water. Add 100ml to make the total volume about 1.5
shall be. The reaction solution is replaced with argon using a vacuum pump, and 2000 U of dihydrofolate reductase dissolved in 20 ml of 10 mM phosphate buffer PH5.6 is added to start the enzyme reaction. After stirring for 14 hours on a hot water bath at 37°C under argon ventilation, 15 g of L-ascorbic acid and 7.5 g of L-cysteine hydrochloride were added and filtered under reduced pressure. 6cm length 30cm)
is passed through to adsorb L-6R- _BPH4 . The column was treated with 0.1% L-ascorbic acid and 0.05%
Distilled water containing L-cysteine hydrochloride (hereinafter referred to as VC)
After washing with solution 15, elute with eluent 20, which is a mixture of acetone and VC solution at a ratio of 4:1. The eluate is concentrated under reduced pressure to 1 to remove acetone and filtered under reduced pressure to obtain a crude L-6R-BPH ₄ solution. Add this solution to 0.025% L-ascorbic acid,
L-6R was passed through a column of CM-Cephadecsus C-25 (H ⁺ type, diameter 6 cm, length 30 cm) equilibrated with distilled water containing 0.0125% L-cysteine hydrochloride (hereinafter referred to as 0.25 VC liquid). - Adsorb BPH ₄ .
After washing the column with oxygen-free 0.25VC solution 20,
Elute with oxygen-free 0.25VC solution 3 containing 50mM hydrochloric acid. The eluate was dried under reduced pressure, dissolved in 20 ml of methanol containing 2 ml of 6N hydrochloric acid, and 250 ml of ether was added to precipitate. Furthermore, the precipitate was washed with 500 ml of ether and dried under reduced pressure to obtain 4% L-ascorbic acid and 2% L-ascorbic acid.
14.5 g of 6(R)-L-BPH ₄ dihydrochloride containing cysteine hydrochloride was obtained as a white powder. (yield
68%) L-6R-BPH ₄ 2 hydrochloride with stabilizer obtained
200 mg of CM-Sephadex C-25 (H ⁺
Pass through a column with a diameter of 1 cm and a length of 10 cm).
Argon-substituted distilled water at 100 °C under an argon stream
ml, and then elute with argon-substituted 30mM hydrochloric acid. After lyophilization, 150 mg of white powder is obtained. When [α] _D is measured in 0.1N HCl, it shows a value of [α] ²⁵ ° _D = −6.8° (C = 1.7; 0.1NHCl), and Viscontini (ibid.)
It was consistent with the reported value. Also, 0.1NHCl
The UV absorption inside showed a value of λ _nax265 ε=1.52±0.07×10 ⁴ mole ⁻¹ cm ⁻¹ . The pmr spectrum of this product in 0.1NDCl/D ₂ O is shown in FIG. PMR (360MHz), 1.31 (d, 3H), 3.62 (d, d,
1H), 3.77-3.93 (m, 4H)

[Brief explanation of the drawing]

Figure 1 shows the 6(R)-L- obtained in Example 1.
It is a pmr spectrum of BPH _4.2 hydrochloride in 0.1NDCl/D ₂ O.

Claims (1)

[Scope of Claims] 1 L-7,8-dihydrobiopterin (L-
BPH ₂ ) is reduced by dihydrofolate reductase (DHFR), then L-ascorbic acid and L-
A method for producing 6-(R)-L-erythro-5,6,7,8-tetrahydrobiopterin, which comprises adding cysteine hydrochloride for purification. 2 L-biopterin is reduced with zinc powder in the presence of an alkali, and the obtained L-7,8-dihydrobiopterin is treated with molecular sieves to remove dihydrofolate reductase (DHFR) inhibitors, and L-biopterin is reduced with zinc powder. 7,
8-dihydrobiopterin (L-BPH ₂ ) is reduced by dihydrofolate reductase (DHFR),
6-(R)-, which is then purified by adding L-ascorbic acid and L-cysteine hydrochloride.
Method for producing L-erythro-5,6,7,8-tetrahydrobiopterin.