JP3083780B2 - Method for producing labeled saccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a labeled saccharide. More specifically, the present invention relates to a novel method for producing a natural type labeled saccharide useful for measuring the behavior of various saccharides in a living body or the like.

[0002]

2. Description of the Related Art When producing a labeled saccharide in which the 1-position carbon is labeled with a radioactive isotope (eg, [ ¹¹ C] positron nuclide), alcohol sugars having alcohols at both ends are produced as intermediates. It is necessary to oxidize this to convert only the 1-position carbon to aldehyde. Conventionally, this step was performed by a chemical synthesis method. However, in this method, the radioactivity at the time of measurement decreases because the finally obtained labeled saccharide is a mixture of four types of isomers. The inconvenience existed.

In the case of D-glucose, D-mannose, etc., high performance liquid chromatography (HPLC)
However, since it is difficult to selectively fractionate a desired saccharide, there has been a long-awaited demand for a method for easily and reliably producing a natural saccharide in which only the 1-position carbon is labeled. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for producing a natural-type labeled saccharide in which only the 1-position carbon labeled with a radioisotope is converted to an aldehyde. The purpose is.

[0004]

SUMMARY OF THE INVENTION The present invention provides a method for producing a saccharide wherein the 1-position carbon is labeled with a radioisotope, comprising a precursor saccharide or a protected derivative thereof. Is labeled with a radioactive isotope, and an enzyme that oxidizes xylitol acts on the carbon to selectively react with the isotope.
Provided is a method for producing a labeled saccharide, characterized in that a natural saccharide in which the 1st carbon is labeled with a radioisotope is obtained by oxidizing the 1st carbon.

In the method of the present invention, the precursor saccharides are xylitol, D-glutitol, galactitol, D-mannitol and D-arabitol, and the natural saccharides obtained from these are D-xylose,
D-glucose, D-galactose, D-mannose,
Being D-arabinose, the radioisotope is
¹¹ C, ¹³ C or ¹⁴ that is C, oxidizing enzyme xylitol, some preferred embodiments it is a Xanthomonas (Xanthomonas) genus or Streptomyces (Streptomyces) derived from genus enzyme.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention is to label a carbon at position 1 of a precursor saccharide, which is a raw material for producing a target labeled saccharide, with a radioisotope, and then act on the xylitol oxidase. Then, only the 1-position carbon is oxidized to produce a natural-type labeled saccharide. The precursor when ¹¹ C is used as a radioisotope and the labeled saccharide produced by its selective oxidation are as follows:
For example:

[0007]

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[0008]

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[0009]

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[0010]

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In addition, labeled D-arabinose produced by selective oxidation of labeled D-arabitol can be included in the present invention. Enzymes that oxidize xylitol (xylitol oxidase)
Is an enzyme capable of oxidizing xylitol in the presence of oxygen to produce hydrogen peroxide and D-xylose, and is a bacterium that produces this enzyme, for example, Xanthomonas (X
anthomonas) genus or Streptomyces (Strepto
myces) isolated and purified from a culture solution of a bacterium of the genus Bacteria by a known enzyme purification method, or genomic DNA of these bacteria
Can be appropriately prepared and used by a genetic engineering technique using an enzyme gene isolated from E. coli. In addition, if the enzyme has the above ability, for example, Xanthomonas
(Xanthomonas) derived from genus sorbitol oxidase (JP-A-6-169764), Streptomyces (Strep
Sorbitol oxidase derived from the genus tomyces (collection of 1997 Annual Meeting of the Japanese Society of Agricultural Chemistry, p. 72) can also be used in the present invention.

[0012]

EXAMPLES Hereinafter, the method of the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples. Reference example: Preparation of xylitol oxidase Streptomyces sp. (Streptomyces sp.) Pre-cultured with dry broth (trade name "Nissui", manufactured by Nissui Pharmaceutical Co., Ltd.)
sp.) IKD472 (FERM P-14339) was obtained by polypeptone (1%), yeast extract (0.5%), MgSO ₄ -7H ₂ O (0.05%), KH
1% in 80 liters (pH 6.0) of medium consisting of ₂ PO ₄ (0.1%)
The cells were inoculated and cultured with aeration and stirring at 35 ° C. for 21.5 hours. After the cells were obtained by filtration, the cells were crushed with a Dynomill, and a supernatant (cell-free extract) was obtained from the crushed liquid by centrifugation. This is purified by ammonium sulfate fractionation (40: 60%), ion exchange chromatography, hydrophobic chromatography, gel filtration, etc., and the active ingredient is isolated by preparative electrophoresis to give a specific activity of 25.6 with a yield of 15.1%. 3 mg of U / mg xylitol oxidase were obtained. The total amount of protein, total activity, specific activity and yield in each purification step are as shown in Table 1.

[0013]

[Table 1]

Example: Production Example of D- [1- ¹¹ C] Glucose [ ¹¹ C] Methyl iodide synthesized from [ ¹¹ C] carbon dioxide was
O-Diol in which triphenylphosphine (4 mg, 0.017 mmol) and 2,3: 4,5-di-O-isopropylidene-D-arabinose (3 mg, 0.013 mmol) were dissolved by a nitrogen stream (30 ml / min). It was introduced into a 0.8 ml reaction vessel containing 0.3 ml of chlorobenzene. After heating at 150 ° C. for 2 minutes, epichlorohydrin (70 μl, 0.9 mmol) was added and the reaction mixture was heated for another 4 minutes. Dimethylformamide / water (25
/ 1) 70 μl of 4-methylformolin N-oxide (NM
O) (5 mg, 0.04 mmol) was added to remove unreacted triphenylphosphine. The alkene was separated and eluted using a known C-18 system.

After removing unreacted triphenylphosphine, the reaction mixture was treated with 200 μl of acetone;
g, 0.04 mmol) aqueous solution 50 μl; 4-methyl-2-quinolyl ether (DHQD-MQ) (7 mg, 0.015 mmol); Os
It was transferred to a 3 ml tube containing O ₄ (65 μl, 0.01 mmol, 4% aqueous solution). Asymmetric dihydrogenation
xylation: AD) was carried out at room temperature for 7 minutes with occasional stirring. The mixture is dissolved in 2.5 ml of dichloromethane and 3 ml of silica gel SPE (solid phase extraction)
Transferred to column. After washing with 0.6ml dichloromethane,
The labeled diol mixture was eluted in 1 ml of acetonitrile / dichloromethane (4/1). 3,4: 5,6-di-O-
Isopropylidene-D- [1- ¹¹ C] glutitol was separated and eluted using a known C-18 system.

The ¹¹ C-labeled diol was transferred to a hydrolysis vessel previously treated with 80 μl of 6 M hydrochloric acid and subjected to a nitrogen stream (140 ml /
) At 85 ° C for 5 minutes to hydrolyze the protecting group and remove the organic solvent. Using a Ca ²⁺ cation exchange column, D- [1- ¹¹ C] glutitol was eluted over 16.2 minutes. Each product in each of the above reactions is
As shown in Chemical formula 5. In addition, * shows a label position.

[0017]

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That is, the substances represented by the chemical formulas in Chemical Formula 5 are as follows. (1) 2,3: 4,5-di-O-isopropylidene-D-arabinose (starting material) (2) alkene (3) 3,4: 5,6-di-O-isopropylidene-D- [ 1- ¹¹ C]
Glutitol (4) D- [1- ¹¹ C] glutitol Then, the hydrolysis vessel was washed with potassium phosphate buffer (KPB), and the resulting solution was transferred to a tube.
The pH was adjusted to 7.4-8.0 with M potassium hydroxide. Catalase (10 μl, 3600 units) and xylitol oxidase (40 μl, 4 units) were added thereto, reacted at room temperature for 7 minutes, and denatured with 6 M hydrochloric acid (50 μl).

The reaction solution obtained as described above was filtered and analyzed by anion exchange chromatography.
The results are as shown in FIG. 1 and a fraction of D- [1- ¹¹ C] glucose was obtained. From this fraction, natural D- [1-C] was obtained.
^[11 C] glucose was purified. Incidentally, D- [1- ¹¹ C] conversion reaction from glucitol ^{(4) D- [1- 11 C} ] glucose (5) can be demonstrated as of 6.

[0020]

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As a result of the chromatographic analysis shown in FIG. 1, D- [1- ¹¹ C] glutitol was converted to D- [1- ¹¹ C] glucose, and D- [1- ¹¹ C] mannitol was converted to D- [1-C] mannitol. 1- ¹¹ C]
The change to mannose was clearly shown.

[0022]

As described above in detail, according to the present invention, it is possible to easily and reliably produce a natural labeled saccharide in which only the 1-position carbon labeled with a radioisotope is oxidized.

[Brief description of the drawings]

FIG. 1 shows the results of anion exchange chromatography analysis of a reaction product according to the method of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (72) Keigo Komura, Inventor 2-179-2, Minamimatsunaga-cho, Fukuyama-shi, Hiroshima (72) Inventor Yuji Furuya 20-9, Jusangenya, Kanbe-cho, Shenzhen-gun, Hiroshima 20-72 (72) Inventor Bengt Longstroke Sweden Uppsala S-751 85 Inside Uppsala University PET Center (72) Inventor Mathias Eglen Sweden Uppsala S-751 85 Inside Uppsala University PET Center (58) Fields studied (Int. Cl. ⁷ , DB) Name) C12P 19/02 A61K 51/00 C07H 1/00 BIOSIS (DIALOG)

Claims (4)

(57) [Claims] 1. A method for producing a saccharide wherein the 1-position carbon is labeled with a radioisotope, wherein the 1-position carbon of a precursor saccharide or a protected derivative thereof is labeled with a radioisotope and oxidizes xylitol therewith. A method for producing a labeled saccharide, characterized in that a natural saccharide in which the 1-position carbon is labeled with a radioisotope is obtained by selectively oxidizing the 1-position carbon by reacting the saccharide. 2. The precursor saccharide is xylitol, D-glutitol, galactitol, D-mannitol, D-arabitol, and the natural saccharide obtained therefrom is:
2. The method for producing a labeled saccharide according to claim 1, which is D-xylose, D-glucose, D-galactose, D-mannose, and D-arabinose, respectively. 3. The method according to claim 1, wherein the radioisotope is ¹¹ C, ¹³ C or ¹⁴ C.
C. The method for producing a labeled saccharide according to claim 1 or 2, wherein the saccharide is C. 4. The enzyme according to claim 1, wherein the enzyme oxidizing xylitol is derived from the genus Xanthomonas or the genus Streptomyces.
And a method for producing a labeled saccharide.