JPH0533234B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a deuterium-labeled sugar compound used as a reagent for biochemical research.

More specifically, the present invention is a deuterated sugar in which only one hydrogen of the hydroxymethyl group at the 5-position of ribose, which is important as a constituent sugar of nucleic acids (RNA), is stereoselectively substituted with deuterium (2H or 3H). The content of this paper is a method for synthesizing the compound in high yield and at low cost using a photohalogenation reaction, and it has extremely high industrial value.

Currently, 600 types of deuterated label compounds, including sugars and amino acids, which occupy the main positions as reagents, are manufactured by Merck, Sharp & Dohme Canada Co., Ltd.
However, all of them are expensive, costing tens of thousands of yen per gram. Deuterated saccharides are particularly useful when using analysis methods such as mass spectrometry and nuclear magnetic resonance absorption spectroscopy in studies of the metabolism and stereochemistry of sugar compounds in vivo.

The deuterated ribose that is currently sold as a reagent has both hydroxymethyl groups deuterated, and the 5
Compounds with an asymmetric carbon in the position and a clear configuration are extremely useful as analytical reagents, but the optical purity of D-ribose is 30% ee.
Since only compounds with enantiomer excess (enantiomer excess) have been reported, it has been desired to establish an efficient synthetic method for highly pure deuterated (5-position) ribose.

Against this background, as a result of our research, the present inventors recognized the important fact that photohalogenation at the 5-position of 1,5-anhydro-ribose occurs stereoselectively, and furthermore, we developed this intermediate compound. It has been found that various useful compounds can be derived using this method.

That is, in the present invention, this intermediate compound is used and subjected to a reductive deuterium substitution method using deuterated tri-n-butyltin to obtain (5R)- and (5S)-(5-2H)-D-ribose. We have established a method for industrially producing .

The synthesis steps in the present invention are as follows.

The outline of this synthesis process is as follows.

(5S)-(5-2H)-D-ribose, D
- After protecting the 2- and 3-position hydroxyl groups of ribose 1,
A compound is obtained by 5-anhydration, and a compound in which only exo-type hydrogen is stereoselectively halogenated is obtained by a photohalogenation reaction. This was deuterated using tri-n-butyltin deuteride to obtain a compound, and then the protecting groups at the 2 and 3 positions were removed simultaneously with the cleavage of the 1,5-anhydro ring using trifluoroacetic acid ( 5S)-(5-2H)-D-ribose (compound) is obtained.

In the case of (5R)-(5-2H)-D-ribose,
A compound is obtained by cleaving the 1,5-anhydro ring of the above compound, and a compound in which the hydroxyl group at the 5-position of this compound is converted into a sulfonic acid ester is obtained. This was treated with a fatty acid alkali or an aromatic carboxylic acid alkali to cause Walden inversion to obtain a compound in which the steric configuration at the 5-position was inverted, and then the protecting group at the 5-position was removed with an alkali, and then the 1, 2, 3
By removing the protecting group at position (5R)-(5-2H)-D-
Obtain ribose (compound).

In the case of L-ribose, process in the same manner as above to obtain the corresponding (5S)-(5-2H)-L-ribose, (5R)
-(5-2H)-L-ribose is obtained.

Examples of the halogenating agent used in the photohalogenation reaction include chlorine, bromine, N-halogenosuccinimide, basic hydrogen trihalides such as pyridinium halide perhalide, and perhalides of tetraalkylammoniums such as phenyltrimethylammonium. Examples include perhalide. Further, bromination is carried out in an organic solvent by irradiation with light or by adding a radical reaction initiator such as azobisisobutyronitrile. Solvents used here include halogenated hydrocarbons, such as carbon tetrachloride, chloroform,
dichloromethane, ethers such as dioxane,
Examples include tetrahydrofuran. Furthermore, in the reduction reaction using deuterated tri-n-butyltin, it is desirable to add azobisisobutyronitrile as a radical reaction initiator, and the solvent used here is aromatic hydrocarbon, Examples include ethers. Examples of the sulfonyl group used in Walden inversion include alkylsulfonyl groups, such as methanesulfonyl groups, and arylsulfonyl groups, such as p-toluenesulfonyl groups and benzylsulfonyl groups.
Examples of fatty acids and alkali aromatic carboxylic acids include sodium acetate and sodium benzoate.

The method for producing the 1,5-anhydro-D-ribofuranose derivative used as a raw material in carrying out the present invention will be explained in reference examples.

The present invention will be explained below with reference to Examples and Reference Examples.

Example 1 (5S)-5-bromo-1,5-anhydro-
Production of 2,3-O-isopropylidene-β-D-ribofuranose () 1.5 g of 1,5-anhydro-2,3-O-isopropylidene-β-D-ribofuranose () was placed in a 200 ml eggplant flask. , 100 ml of CCl ₄ and 2.584 g (2 molar equivalents) of N-bromosuccimide were added. A reflux condenser was attached and the flask was heated to reflux using a 300W light bulb from below. I wrapped it in aluminum foil to prevent light from escaping. After 2 hours, confirm the completion of the reaction by thin layer chromatography, then filter, wash with 10% sodium thiosulfate, saturated aqueous sodium bicarbonate solution, and water, and dry the carbon tetrachloride layer over anhydrous magnesium sulfate. I let it happen. After filtration, the resulting syrup was concentrated under reduced pressure and applied to a silicic acid column (hexane:ether (3:
It was purified using the solvent of 1). The desired fractions were collected and concentrated under reduced pressure to obtain 0.649 g of white crystals (yield:
44.5%).

Melting point 137℃ [α] _D ²⁰ −152° (C=0.1, CHCl ₃ ) Elemental analysis value (%) C ₈ H ₁₁ O ₄ Calculated value as Br C38.27, H4.42, Br31.82 Actual value C38 .15, H4.47, Br32.43 Example 2 (5S)-( ^5-2H )-1,5-anhydro-2,
Production of 3-O-isopropylidene-β-D-ribofuranose () A 500 ml three-neck flask was equipped with a nitrogen gas inlet,
Build a device equipped with a reflux condenser, and ()
1.492 g (5.9 x 10 ^-3 mol) and 0.008 g of azobisisobutyronitrile were dissolved in 200 ml of distilled benzene.
Add 2.238 g of tri-n-butyltin deuteride (deuterated (2H) tri-n-butyltin) to this solution.
(1.3 molar equivalent) was added to the solution and refluxed with stirring. After 2 hours, it was confirmed by thin layer chromatography that the reaction had completely completed, and after air cooling, the reaction solution was concentrated and applied to a silicate column (30 g) (eluted with hexane:ethyl acetate (5:1)). Refined,
0.830 g (yield: 80.7%) of white crystals of () was obtained.

Melting point 61℃ [α] _D ²⁰ −62° (C=0.78, methanol) Elemental analysis value (%) C ₈ H ₁₁ ² HO ₄ Calculated value C55.48, H7.57 Actual value C55.32, H7. 64 Example 3 Production of (5S)-( ^5-2H )-D-ribose () 346 mg of () was dissolved in 2 ml of 90% trifluoroacetic acid and stirred at room temperature for 3 hours. The solvent was distilled off to obtain a syrup, which was subjected to column chromatography using silicic acid (5 g) and developed with chloroform:methanol (4:1) to obtain 0.186 g (75% yield) of ().

Melting point 95℃ [α] _D ²⁰ −23.7° (C=1.0, H ₂ O) Example 4 Methyl (5S)-(5- ² H)-2,3-O-isopropylidene-β-D-ribofuranoside Production of () () 0.260g, anhydrous methanol 25ml, 2,2-
1.60 g of p-toluenesulfonyl chloride was added as an acid catalyst to 25 ml of dimethoxypropane, and the mixture was stirred at room temperature for 48 hours. After confirming the completion of the reaction by thin layer chromatography, the mixture was neutralized with a saturated aqueous sodium bicarbonate solution and concentrated under reduced pressure (40°C, 100mmHg). The resulting syrup was purified by applying it to a silicic acid column (20 g) (eluted with benzene:ether (1:1)) to obtain 0.240 g (yield: 78.2%) of pale yellow syrup.

[α] _D ²⁵ −82.2° (C=1.0, CHCl ₃ ) Example 5 Methyl (5S)-(5- ² H)-2,3-O-isopropylidene-5-O-(p-toluenesulfonyl) -Production of β-D-ribofuranoside () 0.240 g of () and 0.029 g (1.5 molar equivalent) of p-toluenesulfonyl chloride were added to 5 ml of distilled pyridine, stirred overnight at room temperature, and the reaction was completed by thin layer chromatography. After confirming this, neutralize with saturated aqueous sodium bicarbonate solution and add chloroform (10ml).
x3), washed with water (50 ml), dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and crystallized the resulting syrup with methanol to obtain 0.320 g (yield 76.2%) of white crystals of (). Ta.

Melting point 84℃ [α] _D ²⁰ −34.8° (C=1.0, ethanol) Example 6 Methyl (5R)-(5- ² H)-2,3-O-isopropylidene-5-O-benzoyl-β -D-
Production of ribofuranoside () In 30 ml of N,N-dimethylformamide ()
0.300 g and 0.242 g (2 molar equivalents) of sodium benzoate were added and heated under reflux at 140°C for 2 hours. After air cooling,
The mixture was diluted with 30 ml of water, extracted with ether (50 ml x 2), washed with a saturated aqueous sodium bicarbonate solution and water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a syrup. This syrup was purified by column chromatography using silicic acid (10 g) and benzene:ether (12:1), and the syrup in () was purified.
0.250g (yield 96.5%) was obtained.

[α] _D ¹⁵ −52.08 (C=1.2, CHCl ₃ ) Elemental analysis value (%) C ₁₆ H ₁₉ ² HO ₆ Calculated value C62.12, H6.84 Actual value C62.25, H6.87 Example 7 Methyl (5R)-( ^5-2H )-2,3-O-isopropylidene-β-D-ribofuranoside ()
Production of () 0.155g and 20mg of metallic sodium 5
Sodium methoxide prepared by treatment with 10 ml of methanol was dissolved in 10 ml of methanol and stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure to obtain a syrup, which was subjected to column chromatography using silicic acid (5 g) and developed with hexane:ethyl acetate (2:1), the solvent was distilled off, and 0.094 g ( Yield: 92%).

[α] _D ²² −82.5°C (C = 1.0, CHCl ₃ ) Example 8 Production of (5R)-(5- ² H)-D-ribose () Dissolve 400 mg of () in 80 ml of 50% acetic acid, The mixture was heated to reflux for 1 minute. The reaction solution was concentrated under reduced pressure, and the remaining syrup was dissolved in 80 ml of warm ethanol and concentrated under reduced pressure.
370 mg of methyl (5R)-(5-2H)-β-D-ribofuranoside was obtained. 335 mg of this was dissolved in 5 ml of water, and Dowex 50W (H ⁺ type, 1 g, manufactured by Dow Chemical Company) was added and stirred at room temperature for 5 hours. After filtering the resin, the solvent was distilled off under reduced pressure to give 225 mg (yield: 84
%) was obtained.

Melting point 95℃ [α] _D ²⁰ −23.5° (C=1.0, H ₂ O) Reference example 1 Production of 1,5-anhydro-2,3-O-isopropylidene-D-ribofuranose () Under stirring and cooling , 18 g of p-toluenesulfonic acid as an acid catalyst and 50 g of D-ribose in 900 ml of acetone.
was added and reacted.

After confirming by thin layer chromatography that the reaction had progressed to 2,3-O-isopropylidene-D-ribofuranose, a reflux device was attached and 1.5 liters of benzene was added to azeotropically anhydride. The benzene layer from which acetone and water were removed was neutralized with a saturated aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting yellow syrup (1,5-anhydro-2,3-O-isopropylidene-β-D-ribofuranose as well as 1',5:
(containing 1,5'-dianhydro compound) was fractionated by sublimation using a distillation apparatus. 1,5-anhydro-
2,3-Isopropylidene-β-D-ribofuranose () was sublimed at 90-100°C/5mm and obtained as white crystals. When reconstituted from hexane ()
21.3g (yield 37.13%) of crystals were obtained.

Melting point 61℃ [α] _D ²⁰ -62° (C = 0.78, methanol) Elemental analysis value (%) As C ₈ H ₁₂ O ₄ Calculated value C58.81, H7.02 Actual value C55.97, H7.16

Claims (1)

[Claims] 1. General formula; [In the formula, R ₁ represents hydrogen or a hydroxyl group-protecting group (acyl group, alkyl group), but two R _1s may be combined to form an alkylidene group such as isopropylidene] 1, A stereoselective photohalogenation reaction is performed on the 5-anhydro-β-D-ribofuranose derivative, and the general formula; A (5S)-1,5-anhydro-5-halogeno-β-D-ribofuranose derivative represented by the formula (where R ₁ is the same as above and ) by reacting with tri-n-butyltin to reduce and dehalogenate the general formula; (5S) − (in the formula, R ₁ is the same as above)
Production of (5S)-(5-2H)-D-ribose, which is characterized by leading to a (5-2H)-1,5-anhydro-β-D-ribofuranose derivative, which is ring-opened and deprotected. Method. 2 General formula; [In the formula, R ₁ represents hydrogen or a hydroxyl group-protecting group (acyl group, alkyl group), but two R _1s may be combined to form an alkylidene group such as isopropylidene] 1, A stereoselective photohalogenation reaction is performed on the 5-anhydro-β-D-ribofuranose derivative, and the general formula; A (5S)-1,5-anhydro-5-halogeno-β-D-ribofuranose derivative represented by the formula (where R ₁ is the same as above and General formula obtained by reductive dehalogenation by the action of tri-n-butyltin ); (5S) − (in the formula, R ₁ is the same as above)
(5-2H)-1,5-anhydro-β-D-ribofuranose derivative is ring-opened and then sulfonylated to form a general formula; (In the formula, R ₁ is the same as above, R ₂ represents a hydroxyl protecting group such as alkyl, aryl, allyl or acyl group, and R ₃ represents methanesulfonyl group, p-toluenesulfonyl group or benzylsulfonyl group)
A (5S)-(5-2H)-5-O-sulfonyl-β-D-ribofuranoside derivative represented by A method for producing (5R)-(5-2H)-D-ribose, which comprises inverting the configuration and then deprotecting it.