Enzymatic Production of Antiviral Nucleosides by the Application of Nucleoside Phosphorylase KENZO YOKOZEKI, HYDEYUKI SHIRAE, AND KOJI KUBOTA Central Research Laboratories Ajinomoto Company, Incorporated 1-1 Suruki-cho. Kawasaki-ku Kawasaki 210, Japan Various chemical methods and several enzymatic methods have been studied on the synthesis of antiviral nucleosides. However, there remain some practical limitations to these approaches because they are laborious and they give unsatisfactory yields. Recently, we have successfully developed novel processes for the production of antiviral nucleosides from cheap nucleosides by the application of nucleoside phosphorylase. These methods seem to overcome the above problems.
BIOCONVERSION METHOD FIGURE1 shows the reaction scheme of a nucleoside production from another nucleoside with the combination of regular and reverse reactions of nucleoside phosphorylase. The substrate is phosphorolyzed to form sugar- I-phosphate in the presence of inorganic phosphate by the regular reaction of nucleoside phosphorylase. Then, sugar- 1 -phosphate is converted to another nucleoside in the presence of an appropriate base by the reverse reaction of nucleoside phosphorylase. The nucleoside phosphorylase enzymes reacting respectively on pyrimidine and purine nucleosides are shown in FIGURE1.
PRODUCIION OF ANTIVIRAL NUCLEOSIDES Ribavirin is a nucleoside having ribose as a sugar moiety and 1,2,4-triazole-3carboxamide (TCA) as a base moiety in the molecule. It is known as a potent antiviral agent and is extensively used as a drug for respiratory infectious disease and Lassa fever. It also is being clinically studied on the remedy of disease caused by AIDS, herpes, and hepatitis viruses. Utagawa et al.’ and ourselves2 have also reported the ribavirin production through two step reactions from inosine via ribosyl- 1-phosphate (R- 1-P) by bacteria having potent activity of purine nucleoside phosphorylase. However, ribavirin was scarcely produced directly from inosine and TCA. 757
ANNALS NEW YORK ACADEMY OF SCIENCES
758
Sugar
(Forward)
(Reverse)
(Pyrimidine) Pyrimidine n u c l e o s i d e p h o s p h o r y l a s e ... UR, TR, dUR Uridine p h o s p h o r y l a s e ........................ UR, dUR Thymidine p h o s p h o r y l a s e .................... TR (Purine) Purine n u c l e o s i d e p h o s p h o r y l a s e ......... GR, IR, etc. FIGURE 1. Scheme of nucleoside production from another nucleoside by the application of nucleoside phosphorylase.
100 mM Guanosine 100 mM TCA
250
-z E
v
200 mM Guanosine 200 mM TCA
300 mM Guanosine 300 mM TCA
f look
2oot
1501
50
1
0 1 2 3 4
1
1
1
1 2 3 4
I
I
I
I
1 2 3 4
Reaction time ( d a y s ) FIGURE 2. Time course of ribavirin production from guanosine and 1.2.4-triazole-3carboxamide by Brevibacterium aceryficum AJ-1442. The incubation mixture containing the indicated concentrations of guanosine and 1,2,4-triazole-3-carboxamide,300 mM potassium phosphate buffer (pH 7.0). and cells-50 mg/mL (0) or 100 mg/mL (0) with wet weight basis-were incubated at 60 "C.
YOKOZEKI et a/.: ANTIVIRAL NUCLEOSIDES
759
From these backgrounds, we screened various microorganisms and found the bacteria producing ribavirin directly from natural purine and pyrimidine nucleosides and TCA. They are Enterobacter aerogenes AJ-l 1 125,’ Envinia carotovora AJ-2992,4 and Brevibacterium acetylicum AJ- 1442,5etc., which were potent ribavirin producers from uridine, orotidine, and guanosine, respectively, in the presence of TCA. Among them, Brevibacterium acetylicum AJ- 1442 gave the best results. FIGURE2 shows the time course of ribavirin production from guanosine and TCA in the presence of 300 mM potassium phosphate buffer (pH 7.0) as the phosphate source by intact cells of Brevibacterium acetylicum AJ-1442. Only a small difference was observed between 50 mg/mL and 100 mg/mL of intact cells in regard to ribavirin production. The amounts of ribavirin produced were 82 mM, 162 mM, and 229 mM in the presence of 100 mM, 200 mM, and 300 mM guanosine and TCA, respectively, on 96-h reaction at 60 O C with 100 mg/mL of intact cells of AJ-1442. These reaction mechanisms producing ribavirin directly from a purine or pyrimidine nucleoside and TCA by these strains were considered to consist of the following two successive reactions via R-I-Pas an intermediate: ( I ) phosphorolysis of purine or pyrimidine nucleosides to form R-I-Pand the corresponding bases (regular reaction of purine or pyrimidine phosphorylase) and (2) transribosylation from R-1-P to TCA in order to form ribavirin (reverse reaction of purine nucleoside phosphorylase). These processes could be applied to the production of 2’,3’-dideoxynucleosides such as 2’,3’-dideoxyadenosine (DDA) and T.3’-dideoxyinosine (DDI) for anti-AIDS agents. This system consisted of the following two systems: T,3’-dideoxyuridine (DDU) production from uridine by the chemical method6 and conversion of DDU to DDA or DDI by the microbial method.’ REFERENCES 1.
2. 3. 4. 5. 6.
7.
UTAGAWA,T., H.MORISAWA, s. YAMANAKA, A. YAMAZAKI & Y.HIROSE. 1986. Agric. Biol. Chem. 5 0 121-124. SHIRAE, H., K . YOKOZEKI & K. KUBOTA.1988. Agric. Biol. Chem. 5 2 295-296. SHIRAE, H.,K . YOKOZEKI & K. KUBOTA.1988. Agric. Biol. Chem. 5 2 1233-1237. SHIRAE, H., K . YOKOZEKI & K.KUBOTA.1988. Agric. Biol. Chem. 5 2 1499-1504. S H I R A E , H., K . YOKOZEKI, M. UCHIYAMA & K . KUBOTA. 1988. Agric. Biol. Chem. 52: 1777-1783. S H I R A G A M I , H.,Y . IRIE, H.SHIRAE, K . YOKOZEKI & N. YASUDA. 1988. J. Org. Chem. 5 3 5 17&5 173. SHIRAE, H.,K. KOBAYASHI, H.SHIRAGAMI, Y . IRIE, N. YASUDA& K. YOKOZEKI. 1989. Appl. Environ. Microbiol. 55: 419-424.
BIOCONVERSION METHOD FIGURE1 shows the reaction scheme of a nucleoside production from another nucleoside with the combination of regular and reverse reactions of nucleoside phosphorylase. The substrate is phosphorolyzed to form sugar- I-phosphate in the presence of inorganic phosphate by the regular reaction of nucleoside phosphorylase. Then, sugar- 1 -phosphate is converted to another nucleoside in the presence of an appropriate base by the reverse reaction of nucleoside phosphorylase. The nucleoside phosphorylase enzymes reacting respectively on pyrimidine and purine nucleosides are shown in FIGURE1.
PRODUCIION OF ANTIVIRAL NUCLEOSIDES Ribavirin is a nucleoside having ribose as a sugar moiety and 1,2,4-triazole-3carboxamide (TCA) as a base moiety in the molecule. It is known as a potent antiviral agent and is extensively used as a drug for respiratory infectious disease and Lassa fever. It also is being clinically studied on the remedy of disease caused by AIDS, herpes, and hepatitis viruses. Utagawa et al.’ and ourselves2 have also reported the ribavirin production through two step reactions from inosine via ribosyl- 1-phosphate (R- 1-P) by bacteria having potent activity of purine nucleoside phosphorylase. However, ribavirin was scarcely produced directly from inosine and TCA. 757
ANNALS NEW YORK ACADEMY OF SCIENCES
758
Sugar
(Forward)
(Reverse)
(Pyrimidine) Pyrimidine n u c l e o s i d e p h o s p h o r y l a s e ... UR, TR, dUR Uridine p h o s p h o r y l a s e ........................ UR, dUR Thymidine p h o s p h o r y l a s e .................... TR (Purine) Purine n u c l e o s i d e p h o s p h o r y l a s e ......... GR, IR, etc. FIGURE 1. Scheme of nucleoside production from another nucleoside by the application of nucleoside phosphorylase.
100 mM Guanosine 100 mM TCA
250
-z E
v
200 mM Guanosine 200 mM TCA
300 mM Guanosine 300 mM TCA
f look
2oot
1501
50
1
0 1 2 3 4
1
1
1
1 2 3 4
I
I
I
I
1 2 3 4
Reaction time ( d a y s ) FIGURE 2. Time course of ribavirin production from guanosine and 1.2.4-triazole-3carboxamide by Brevibacterium aceryficum AJ-1442. The incubation mixture containing the indicated concentrations of guanosine and 1,2,4-triazole-3-carboxamide,300 mM potassium phosphate buffer (pH 7.0). and cells-50 mg/mL (0) or 100 mg/mL (0) with wet weight basis-were incubated at 60 "C.
YOKOZEKI et a/.: ANTIVIRAL NUCLEOSIDES
759
From these backgrounds, we screened various microorganisms and found the bacteria producing ribavirin directly from natural purine and pyrimidine nucleosides and TCA. They are Enterobacter aerogenes AJ-l 1 125,’ Envinia carotovora AJ-2992,4 and Brevibacterium acetylicum AJ- 1442,5etc., which were potent ribavirin producers from uridine, orotidine, and guanosine, respectively, in the presence of TCA. Among them, Brevibacterium acetylicum AJ- 1442 gave the best results. FIGURE2 shows the time course of ribavirin production from guanosine and TCA in the presence of 300 mM potassium phosphate buffer (pH 7.0) as the phosphate source by intact cells of Brevibacterium acetylicum AJ-1442. Only a small difference was observed between 50 mg/mL and 100 mg/mL of intact cells in regard to ribavirin production. The amounts of ribavirin produced were 82 mM, 162 mM, and 229 mM in the presence of 100 mM, 200 mM, and 300 mM guanosine and TCA, respectively, on 96-h reaction at 60 O C with 100 mg/mL of intact cells of AJ-1442. These reaction mechanisms producing ribavirin directly from a purine or pyrimidine nucleoside and TCA by these strains were considered to consist of the following two successive reactions via R-I-Pas an intermediate: ( I ) phosphorolysis of purine or pyrimidine nucleosides to form R-I-Pand the corresponding bases (regular reaction of purine or pyrimidine phosphorylase) and (2) transribosylation from R-1-P to TCA in order to form ribavirin (reverse reaction of purine nucleoside phosphorylase). These processes could be applied to the production of 2’,3’-dideoxynucleosides such as 2’,3’-dideoxyadenosine (DDA) and T.3’-dideoxyinosine (DDI) for anti-AIDS agents. This system consisted of the following two systems: T,3’-dideoxyuridine (DDU) production from uridine by the chemical method6 and conversion of DDU to DDA or DDI by the microbial method.’ REFERENCES 1.
2. 3. 4. 5. 6.
7.
UTAGAWA,T., H.MORISAWA, s. YAMANAKA, A. YAMAZAKI & Y.HIROSE. 1986. Agric. Biol. Chem. 5 0 121-124. SHIRAE, H., K . YOKOZEKI & K. KUBOTA.1988. Agric. Biol. Chem. 5 2 295-296. SHIRAE, H.,K . YOKOZEKI & K. KUBOTA.1988. Agric. Biol. Chem. 5 2 1233-1237. SHIRAE, H., K . YOKOZEKI & K.KUBOTA.1988. Agric. Biol. Chem. 5 2 1499-1504. S H I R A E , H., K . YOKOZEKI, M. UCHIYAMA & K . KUBOTA. 1988. Agric. Biol. Chem. 52: 1777-1783. S H I R A G A M I , H.,Y . IRIE, H.SHIRAE, K . YOKOZEKI & N. YASUDA. 1988. J. Org. Chem. 5 3 5 17&5 173. SHIRAE, H.,K. KOBAYASHI, H.SHIRAGAMI, Y . IRIE, N. YASUDA& K. YOKOZEKI. 1989. Appl. Environ. Microbiol. 55: 419-424.
