JPS5813394A - Preparation of 1-beta-d-ribofuranosyl-4,5-substituted- imidazole - Google Patents

Abstract

PURPOSE:To prepare 1-beta-D-ribofuranosyl-4,5-substituted-imidazole in high yield, by adding nucleoside phosphorylase to an aqueous solution containing 4,5-substitutd imidazole, a phosphoric acid salt, and nucleoside. CONSTITUTION:The 1-beta-D-ribofuranosyl-4,5-substituted-imidazole of formula II (X is carbamoyl or carboxyl; Y is amino or OH) is prepared by adding nucleoside phosphorylase existing in microbial cells to an aqueous solution containing the 4,5-substituted imidazole of formulaI, a phosphoric acid salt and nucleoside or nucleotide, and maintaining the mixture at 40-70 deg.C. The produced 1-beta-D- ribofuranosyl-4,5-substituted-imidazole is separated from the mixture. The microorganism capable of producing nucleoside phosphorylase is, e.g. Pseudomonas diminuta, Flavobacterium rhenanum, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 1-β-D-ribofuranosyl-4,5-substituted imidazole represented by the following general formula (2) using an enzyme of microbial origin.

4-Carbamoyl-1-β-D-ribofuranosyl-5-oleate in the general formula (2) r-, where X is a carbamoyl group and Y is a hydroxyl group, is a compound called Bredenine and has an immunosuppressive effect. It can be used as a medicine.

In addition to chemical synthesis methods, predenine can be produced using 4-
Carbamoyl-imidazolium-5-olei)C A method of causing specific microorganisms to act is known ( ゛)
l-1693). As a result of research aimed at developing an efficient manufacturing method for
A mixture of substituted imidazoles, phosphates and nucleosides or nucleotides 1 reacts with nucleoside phosphorylase to form 1-β-D-ribofuranosyl 4
．． It has been found that 5-substituted imidazoles are produced.

However, although this reaction proceeds well with purified enzymes, it has been found that when microbial cells or the like are used as the enzyme source, side reactions occur and the yield is reduced. Since it would be economically advantageous to use inexpensive microorganisms instead of purified enzymes, we further investigated ways to prevent side reactions and found that the above reaction could be
It was discovered that the Hatake 1 reaction was suppressed and the target compound was produced in high yield when carried out at a high temperature of 70C, leading to the completion of the present invention.

The present invention provides an aqueous solution containing a 4,5-substituted imidazole compound represented by the following general formula (1), a phosphate, and a nucleoside or a nucleoside. The nucleoside phosphorylase contained in this product is reacted with 40-70c to produce I-β-D-ribofuranosyl-
4,5-substituted imidazole is produced, separated and
This is a method for producing 1-β-D-ribofuranosyl-4,5-substituted imitasol, which comprises collecting the 1-β-D-ribofuranosyl-4,5-substituted imitasol.

The first aspect of the present invention will be explained below.

Nucleoside phosphorylase is an enzyme that phosphorolyzes purine nucleosides or pyrimidine nucleosides to produce pyrimidine or purine and lipose-1=phosphate. Contains nucleoside phosphorylase,
2 is known to be produced by microorganisms (Japanese Patent Application Laid-open No. 561/1999).

-8695). □ Nucleotide phosphorylase contained in a microorganism as used in the present invention refers to microorganism cells or a processed product of microorganisms obtained by culturing a microorganism capable of producing nucleoside phosphorylase primarily in a nutrient medium in a nutrient medium.

Pseudomonas is a microorganism that has the ability to produce nucleoside phosphorylase.
) genus, Flavobacterium A
ium) genus, Achromobacter (Achromobacter)
Ler) genus, Salmonella genus,
Genus Citrobaeter, Genus Escherichia, Genus Sporoaarcjna, Genus Alcaligenes, Genus Enterobacter (E
genus nterobacter, Aeromonas
Monas genus, Arthroba
cter) genus, Brevibacterium (Brevibac
terium) genus, Serratia (8srratla) genus,
Genus Erwinia (]i:rwlnia), Genus Proteus, 1 line/(Cterium (C
orynebacLeriu+n), Cellulomonas (
Cellulomonas genus, Xanthomonas (Xa
n thomonas ) genus, Talepsia (Klab
sialla), Microcococ
genus Bacillus (order BaC 1u8) or genus Bacterium, = 5 - Candida, +4 (Candjda), genus Saccharomyceg, genus Staphylococcus, genus Vibrio (
Vibrio genus, or Mycoplana
Microorganisms belonging to the genus Lana) are used, and more specifically, the following microorganisms may be mentioned.

Shigo-domonas self deiminuta ATCC115
68 (Pseudomonas diminota)
Flavobacterium I renanum CCM 29
B (Flavobacterium rhenanum
) Achromobacter raftium CCM 6
9 (Achromobacter Iactium) Salmonella war Scots Tom Elm 11 ATCC87
59 (Salmonella schottmuell
eri) Erwinia herbicola ATC
C14536 (Erwlnia herbicola
) Proteus vulgaris FERM-P
4795 (Proteus vulgaris) Bacterium [phase] Cadaveris ATCC976
0 (Baeteriun+ eadaveris) Xanthomonas-citri-FERM-P 3396 (Xan
thomonas cjtrl) 6- Mycoplana demonopha ATCC427
9 (Mycoplana dimorpha) Escherichia coli ATCC10798 (Esc
herichia coli) Enterobacter cloacani ATCCI: 1047 (Enterob
acter cloacae) Serratia Book Marcetus Sens TFO3046 (Serratia
marcescens) Klebsiella pneumonia ATCC9 (i21 (Klebsiell
a pneumoniae) Micrococeus luteus ATCC398 (Micrococeus
Iuteue) Corynebacterium myskanens ATCC7429 (Corynebacterium
michiganense) Bacillus plevis
AT(:C8]85(Bacillus
brevis) Cellulomonas flabigera ATCC111
83 (Cellulomonas flaviger
a) Arthrobacter φglobipormis ATCC8
010 (Arthrobacter globiho
rmls) Brevibacterium 7ammoniagenes A
'l'CC6871 (Brevibacterium
ammoniaganos) 1' 1゜alkaligenes-metallic regenes A']"C
C13270 (Alcaligenes metal
aligenes) Sporosarcina ureae
ATCC6473 (Sporoa arcins u
reae) Aeromonas salmonida AT
CC14174 (Aerononas salmo
nicida) Cancida tropicalis
ATCC14056 (Candida tropica
lis) Satsukaromyces cerevisiae ATC
C2601 (Saccharomyces cere
visiae) Staphylococcus evidermides
ATCC155 (Staphylococcus ep.
idermidia) Kurtia fist shifty
ATCC6900 (Kurthia zophi
i) Vibrio ΦMechnicobi ATCC7708 (
Vil)riOm6tahnikovii) These microorganisms can be cultured using a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and organic micronutrients such as vitamins if necessary; special methods are not required. Instead, conventionally known methods are adopted as appropriate.

As the nucleotide phosphorylase contained in microbial atpyn, the culture solution obtained by the above method can be used as it is, or the washed bacterial cells can be used, as well as the bacterial cell treated product (1) can be used. Acetone dried bacterial cells,
After extracting from 171 bacterial cells, including crushed bacterial cells, ultrasonicated bacterial cells, bacterial cells brought into contact with a surfactant or toluene, etc., and bacterial cells treated with an enzyme such as lysozyme, the cells were separated by salting out, etc. Any of the protein fractions of the microbial cells obtained above, as well as immobilized products of the above-mentioned microbial cells or processed bacterial cells using natural or synthetic polymers, etc. can be used.

4.5-Substituted imidazoles include imidazole derivatives represented by general formula (1), such as 4-carbamoyl-
5-Hydroxy-imidazole is used.

The ribocreosides used in the present invention include natural ribosides such as uridine, cytidine, inosine, adenosine, and guamazone, and the nucleotides include uridylic acid, inosinic acid, guanylic acid, etc. in which these nucleotides are bonded to phosphoric acid. is used. The amount added is 0.1% or more, preferably 1.0%; if it is less than 0.1%, the reaction is slow and the efficiency is low, making it impractical.

9- When purine nucleosides are used as nucleosides and purine nucleotides are used as nucleotides, the reaction proceeds with purine nucleoside phosphorylase alone, but when pyrimidine nucleosides or pyrimidine nucleotides are used as raw materials, in addition to purine nucleoside phosphorylase, pyrimine nucleoside phosphorylase is used. is necessary. In this case, microorganisms having the ability to produce both enzymes may be used, and both microbial descendants described above are included in a well-balanced manner.

When performing an enzymatic reaction? In this case, an aqueous solution containing the 4,5-substituted imidazole compound of general formula (1), a phosphate, and a nucleoside or nucleotide is prepared, and the pI (pI) is 4 to 10.
Microorganism cells having nucleoside phosphorylase activity, or a processed product of the cells, or unpurified enzymes are added thereto and maintained at a high temperature of 40 to 70C. During this period, if the reaction solution is maintained for 1 to 20 hours with occasional stirring, the desired 1-β-D-lipofuranone-4,5-substituted imidazole is accumulated in the reaction solution. Enzyme activity during reaction is 0.0
1-10- to IU/- may be used.

In the method of the present invention, it is necessary to set the reaction temperature in the range of 40 to 70υ, and if the temperature is less than 40C, side reactions will occur, and even if the reaction is carried out for a long time, the yield will be low, and the separation of impurities caused by the side reactions will be difficult. This is disadvantageous because it takes time and effort.

The product can be separated and collected from the reaction solution according to a known method, and after removing insoluble residues by centrifugation, etc., a method that takes advantage of the difference in solubility in a solvent such as water, or ion exchange chromatography. It can be separated by etc.

This will be explained in detail in Examples below.

Example 1 Yeast Extract 7, 0.5 f/dls Vep) 7 t,
ot/dis meat extract 1. oy/di and salt o,
Prepare a medium with a composition of 5t/ctp and add 1iI to pH 7.0.
11! It was knotted. This was dispensed into 500111 (100*/11 cm capacity shouldered flask) and heated at 120C for 10 minutes.This was inoculated with Klebsiella pneumonia ATCC9621 and cultured with shaking at 30C for 20 hours.The culture solution was centrifuged. Collect the bacterial cells and dilute to 0.05 M
The cells were suspended in phosphate buffer (p) 17.0)E (wet bacterial cell concentration song/ld). This bacterial cell suspension was cooled to 5 Ct8, irradiated with ultrasonic waves for 10 minutes to disrupt the bacterial cells, and centrifuged. Uridine phosphorylase activity and purine nucleoside phosphorylase activity were observed in the supernatant obtained. Next untreated bacterial cell suspension 1.0m/ln Urisif 10
4.0 mg of KH, Po and 2.011 g of 4-carbamoyl-imidazolium-5-oleate were added, the pH was adjusted to 7.0, and the mixture was maintained at 10 to 75 tl' for 3 hours, with occasional stirring during that time. . The amount of predenine produced in the reaction solution was quantified using high performance liquid chromatography (Hitachi @635W). The results are shown in Table 1.

Table 1 10 0.15 15 0.20 20 0.28 25 +1.30 30 0.35 35 0.45 40 0.50 45 0.70 s　o　　　　o,s　.

55 (1,85 601,00 650,95 700,52 75Q-13-Example 2 The liquid medium used in Example 1 was dispensed into large test tubes for 5.0 d and sterilized at 120C for 10 minutes.This medium The microorganisms shown in Table 2 were inoculated one platinum loop at a time, and cultured with shaking at 30C for 24 hours.The resulting culture solution was centrifuged and collected, washed with physiological saline and washed with a trowel. M phosphate buffer (pH 7,0) r-suspended (50 IIg-wet weight/d)
.

0.5 m of the above bacterial suspension was added to Urisif 2. Ot/di
, 4-carbamoyl-imidazolium-5-oley)
0.2 t/di1xH1lpo4o, s t/
It was added to 0.5 d of a reaction solution containing dl and adjusted to pH 7.0. Keep this for 60CE5 hours, stirring occasionally during that time,
Then, it was heated at 100C for 5 minutes.

When the products in the reaction solution were examined by high performance liquid chromatography, the results shown in Table 2 were obtained.

Table 2 also shows the results when inosine was used instead of uridine.

-14- Table 2 Production amount of predenine Substrate ATCC11568F15 20CCM
298 120 35CCM
69 135 5
5ATCC87597240 ATCC145361554O FFRM-P 4795 83 35
ATCC-97607530 FERM-P 3396 55 1
0ATCC809011050 ATCC42791510 ATCC1079871120 ATCC130479530 IF0 3049 28
5ATCC962110020 ATCC'398 52 10ATC
C74292510 ATCC818510050 ATCC818315025 ATCC801012030 ATCC68713510 ATCC132703010 ATCC64737525 ATCC141747, (1: 3° ATCC26011 20' 30ATCC
140563810 ATCC155111020 ATCC69,005525 ATCC77087810 Example 3 Example 1 100 ml of the above liquid medium was placed in a 500-capacity shoulder flask and sterilized. This one was inoculated with Escherichia coli ATCC 10798 and maintained at 30C.
Shake for 36 hours. Bacterial cells were collected by centrifugation, and 52 (wet weight) was suspended in Rod's phosphate buffer (pH 7,0) and sonicated for 15 minutes. 10ml of the supernatant obtained after centrifugation was diluted with 4-carbamoyl-imidazolium-
5-oleate toosy1 uridylic acid 500%, KH,
It was added to 90 aff of a reaction solution containing 350 mg of Po and adjusted to pH 7.0. This was maintained for 10 hours at 60CE. After the reaction, the reaction solution was treated at 100C for 5 minutes, the reaction solution was centrifuged to remove proteins, the supernatant was concentrated, the concentrated solution was charged to a Sephadex G-10 column, and water was added.
It eluted with the trowel. Concentrate the eluate and place it in a cool place to crystallize:
:.

was precipitated. The obtained crystals were re-crystallized from water to obtain 75 g of crystals.

This product was confirmed to be predenine because its NMR spectrum, infrared absorption spectrum, and UV spectrum matched those of the standard product of predenine.

Patent applicant: Ajinomoto Co., Inc. -17- Continuation of page 1 1/265 1/37 1/38 1/42 1/425 1/44 764 1/72 1/85 0 shots clear person Tadao Kobayashi 3-39-23 Mutsuura, Kanazawa-ku, Yokohama 0 shots: Shigeru Yamanaka 3-40-13 Ooka, Minami-ku, Yokohama

Claims (1)

Claims: A nucleoside phosphorylase contained in a microorganism is added to an aqueous solution containing a 4,5-substituted imidazole represented by the general formula (1), a phosphate, and a nucleoside or nucleotide to maintain 40 to 70 tr. -1-β-1) -1J characterized by producing a β-D-ribofuranosyl-4,5-substituted imidazole and separating and collecting the same.
Method for producing pofuranosyl-4,5-substituted imidazole.