JPS6332325B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Applications] There are many plant virus diseases, many of which are the most contagious types. Statistics show that these diseases cost hundreds of millions of dollars each year. Such viral diseases are particularly difficult to control and virtually impossible to reverse. A single plant virus can attack many different plant species and can be carried by many different vectors (insects, nematodes, fungi, parasitic vascular plants, and agricultural tools). Viruses can be transmitted from one generation to another through seeds, and some viruses are born in the soil. In mammals, interferons are the most rapidly developed defense mechanism against viral infections. One activity induced by interferon in mammalian cells is the conversion of ATP into a series of unique 2',5'-oligoadenylates. (Pestka.S [ed] (1981) Methods in Enzymology Volumes 78 and 79, Academic Press,
New York). Similar but not identical plant oligonucleotides are AVF.
(antiviral factors or plant interferons) are introduced into tobacco plants after infection with tobacco mosaic virus (TMV) (Debatsch, Y et al. (1981) Virology 111:103-122). [Prior Art] Recently, natural 2',5'-oligoadenylates and chemically synthesized 2',5'-oligonucleotide analogs have been shown to inhibit TMV replication at nanomolar (10 ^-9 M) levels. It has been reported to be a strong inhibitor.
(Dabitsuyu, Y., Bitgus.S. and Sera.
Ai. (1982) Science 216:1415-1416; (1984) Jour.Biol.Chem.259:
3482−3486). [Problems to be Solved by the Invention] Attempts have been made to discover an antiviral agent containing a compound having antiplant virus activity equivalent to that of 2',5'-oligoadenylate. Means for Solving the Problem The present invention relates to the use of nucleotides conjugated by a pyrophosphoryl linkage as anti-plant viral agents. Specifically used are dinucleotide 5',5'- ^p1 , ^p2 -diphosphate, dinucleotide 2' to inhibit TMV replication in tobacco plants when applied through the leaves or roots of tobacco plants. , 5'- ^p1 , ^p2 -diphosphate and the dinucleotide 3',5'- ^p1 , ^p2 -diphosphate. Application of these antiviral compounds through the roots of tobacco plants or on the leaves of the plants results in inhibition of TMV replication. Conjugated nucleotides of the invention that can be used to inhibit plant virus replication are known or accessible compounds or compounds that can be readily prepared by procedures well known in the art. These conjugated dinucleotides have the formula where X is

[Formula] or

[Formula] or

[Formula], n is 2 or 3, one of R ₁ and R ₂ is an adenine residue, the other is selected from the group consisting of adenine, guanine, cytosine or uracil residue, R ₃ , R ₄ , R ₅ and R ₆ are OH or R ₃ and
R ₄ and R ₅ and R ₆ are represents. These purine and pyrimidine nucleotides are commercially available (Sikma Chemical Company, St. Louis, Mo.). Can be chemically condensed to pyrophosphorylated anti-plant viral dinucleotides using the methods disclosed herein. Mono-, di-, or triphosphates of nucleotides are suitable precursors for the synthesis of pyrophosphorylated anti-plant viral nucleotides within the formula. These nucleotides are commercially available (Sigma) and can be chemically prepared using the methods disclosed herein or other well-known methods to form the dinucleotide m,5'-p ¹ ,p ⁿ (m=2 ' or 3'; n=2-
3) Can be condensed to n-phosphate. Precursors for the organic synthesis of antiviral compounds, ie nucleotide 2'- or 3'- or 5'-monophosphates, are commercially available (Sigma). The integer n is the dinucleotide 2', 5'- or 3',
5'- or 5',5'-diester repeating internal bond. Pyrophosphoryl compounded anti-plant virus dinucleotides are available from Rice J. R. and Moffat I. G. (1965) J.Org.Chem 30,
3381−3387; Grunmut F. (1978) Proc.
Natl.Acad.Sci.USA 76 6081−6085; Dabreu et al. (1973) Biochemistry 12 4458-4463; Hoaro. Day. I-. and Otsuto Day. G. (1965) J. Amer.
Chem.Soc.87; 1785−1788; Hong et al. (1985) J.
Med.Chem.28 171-177; Deheras et al. (1985) J.
Med. Chem. 28 48-46; Black et al. (1985) Nucleosides and Nucleotides 4: 165-167. Using the chemical synthesis described above, a wide range of different phosphorylated dinucleotides p ¹ , p ⁿ (n=2 to
3) N-phosphates can be produced. Examples of such compounds are as follows. Diadenosine 5',5- ^p1 ,p2-diphosphate ( _Ap2A ), Diadenosine 5 ^' ,5- ^p1 , ^p3 -triphosphate ( _Ap3A ), (all available from Sigma) and Adenosine 5 ',5- ^p1 , ^p2 -diphospho-guanosine ( _Ap2G ), adenosine 5',5
-p ¹ , p ² -diphospho-uridine (Ap ₂ U), adenosine 5', 5-p ¹ , p ² -diphospho-cytidine (Ap ₂ C), 2', 3'-cyclic phosphodiadenosine 5', 5
^-p1 , ^p2 -diphosphate (p _<Ap2A> p),
Guanosine [5′] triphosphate adenosine (Gp ₃ A), adenosine 3′,5′-diphospho-adenosine (A3′pp5′A), adenosine 3′,5′-diphospho-cytidine (A3′pp5′C) , adenosine 3′,5′-diphospho-guanosine (A3′pp5′G), adenosine
2′,5′-diphospho-adenosine (A2′pp5′A), adenosine 2′,5′-diphospho-cytidine (A2′pp5′C), adenosine 2′,5′-diphospho-guanosine (A2′pp5′) G) etc. The most preferred compound is diadenosine 5',5-
p ¹ , p ² -diphosphate (Ap ₂ A), adenosine
5',5- ^p1 , ^p2 -diphospho-guanosine ( _Ap2G ), adenosine 5',5- ^p1 , ^p2 -diphospho-uridine ( _Ap2U ), adenosine 5',5-
p ¹ , p ² -diphospho-cytidine (Ap ₂ C), 2′, 3′-
It is cyclic phosphodiadenosine 5',5- ^p1 , ^p2 -diphosphate (p _<Ap2A> p). The second most preferred compound is adenosine 2′,
5′-diphosphoadenosine (A2′pp5′A), adenosine 2′,5′-diphospho-guanosine (A2′pp5′G),
They are adenosine 2',5'-diphospho-uridine (A2'pp5'U) and adenosine 2',5'-diphospho-cytidine (A2'pp5'C). The third most preferred compound is adenosine 3′,
5′-diphospho-adenosine (A3′pp5′A), adenosine 3′,5′-diphospho-guanosine (A3′pp5′G),
They are adenosine 3',5'-diphospho-uridine (A3'pp5'U) and adenosine 3',5'-diphosphocytidine (A3'pp5'C). All of the above compounds are available from PL Biochemicals, Milwaukee, Wyoming. When an anti-plant virus replication effective amount of phosphorylated dinucleotide is applied to plants susceptible to plant virus infection, the treated plants are able to resist virus attack. Although the method of the invention is exemplified herein by the use of tobacco plants and TMV, the method of the invention can be applied to a variety of plants such as tobacco, barley, beans, oats, beets, soybeans, sugar beets, sugar cane, It can be used to treat wheat, alpha alpha, cherries, citrus, grapes, melons, cucumbers, lettuce, pepper, potatoes, cocoa, coconut, etc. against a wide range of viruses, such as tobacco mosaic, vein. banding, striped mosaic, yellow doe wolf, yellow mosaic, common mosaic, curly top, soil bone mosaic, buckled bride, streak mosaic, reel roll, bean pot motsul virus, tobacco ring spot virus, peanut stunt virus, peanut bud vine virus, maize (corn)
It can be used for viruses susceptible to Douwouf mosaic disease complex, soybean mosaic virus, alpha mosaic virus, cucumber mosaic virus, tomato ringspot virus, and the like. Effective anti-plant virus replication amounts of phosphorylated dinucleotides can be readily determined for different plants and viruses using procedures disclosed herein or known in the art. This measurement can be easily made without extensive experimentation. The following is a disclosure of the materials and methods used in the present invention. 1 Plant Nicotiana glutenosa
glutinosa) and N. N. tabacum var. samsun plants were grown in pot media under greenhouse conditions (25±3° C. and constant lighting). Plants were fed with commercial fertilizer every two weeks. Plants were used at the 5- to 8-leaf stage (Debatsyu Y., Vitsugus S. and Sera
Ai. (1982) Science 216:1415-1416; (1984) Jour.Biol.
Chem.259:3482−3486). 2. Antiviral activity in TMV-infected leaf disks Antiviral activity in TMV-infected leaf disks was assayed in N. Tabakiyum bar. Samsan (N.
tabacum var. samsun). Leaves mechanically pure TMV (sodium phosphate buffer)
5 μg/ml in 0.01 M, pH 7.6, containing 0.1 g/ml carborundum as an abrasive agent) was inoculated. Immediately after infection, leaf disks (6.5 mm diameter) were punched out from the inoculated leaves and placed in a beaker containing 0.01 M sodium phosphate at pH 7.6. Calcium coprecipitation technique Groups of 20 discs were randomly selected from this common pool and placed in separate Petri dishes, each Petri dish containing 18ml of 10mM.
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH
Contained PH7.05 188mg/Na ₂ HPO ₄ 7H ₂ O. Add the compound to 2 ml of 600 mM _CaCl2
Discs are treated with antiviral dinucleotides for 1 hour by adding together with the antiviral dinucleotide. At the end of the 1 hour incubation, the buffer containing antiviral nucleotides is replaced with a buffer without antiviral dinucleotides for an additional 1 hour. Disk then 0.01M Sodium Phosphate PH
Wash with 7.6 and incubate in phosphate buffer for an additional 72 hours (Devassyu Y., Bitgus S.).
and Sera I. (1982) Science 216:
1415−1416., Davatshiyu Wai et al. (1984)
J.Biol.Chem.259:3482−3486). disk
Homogenize in 0.01 molar sodium phosphate buffer pH 7.6 and TMV content determined by enzyme-linked immunosorbent assay (TLISA) as described below. 3 Determination of antiviral activity using an infectivity test The activity of antiviral dinucleotides to inhibit TMV replication in intact N. glutinosa plants (containing the N gene for TMV localization) was determined by an infectivity test. did. 5μg/ml TMV, 0.1μ
A solution containing g/ml carborundum and antiviral dinucleotides was applied to five half leaves of N. gluteninosa. The opposite half of the leaf served as a control and was inoculated with an infection solution containing TMV and carborundum, but no dinucleotide. Infection was allowed to proceed for 48 hours. Inhibition of TMV replication was calculated as the % localized disease that occurred in the antiviral compound-treated half of the leaves compared to the control half of the leaves. 4 Antiviral activity of N. tabacium on intact plants Nicotiana tabacium (Nicotiana tabacium)
tabacum) plants were used as a source of plant tissue in which TMV was spread throughout. These plants did not contain the N-gene for TMV localization. (a) Application to roots Remove N. tabaquiyum plants from their pots and add 10 ml of 10 mM HEPES-KOH.
Transferred into a 50ml tube containing PH7.6 and antiviral dinucleotide. After 2 hours the plants were transferred to 50 ml tubes without antiviral compounds and inoculated with TMV as above. After 24 hours, leaves were homogenized in 0.01 M sodium phosphate buffer PH 7.6 (1 g leaf/3 ml buffer) and TMV content was determined by ELISA. (b) Foliar Application Antiviral activity was measured as described above, but using N. tabaquium plants. After 24 hours, half the leaves were removed, homogenized, and TMV content was determined by ELISA. 5 Enzyme-linked immunosorbent assay (ELISA) The TMV content of the homogenate was essentially determined by Clark and Adams (1977) J. Gen. Virol. 34:475-
(483) was determined by ELISA using the following layer order on a microtiter plate. (a) The γ-globulin fraction of serum obtained from chickens immunized against TMV was absorbed directly onto the plate. (b) The next layer consists of a test sample (various dilutions) or a series of purified samples at known concentrations (standard curve).
It was a TMV suspension. (c) The third layer is γ− from the serum of TMV-immunized rabbits.
It was globulin. (d) The final layer was goat antibody to rabbit immunoglobulin G conjugated to alkaline phosphatase (Sigma). Phosphate release from p-nitrophenyl phosphate for antibody-conjugated alkaline phosphatase was monitored spectrophotometrically using a microplate reader (Dynatech Diagnostics Inc., South Windham ME); This was proportional to TMV concentration in an approximately 2-log to log-linear manner. (<
2ng～＞200ng) Debatshu Y. , Bitgus S. and Sera I. [1982] Science 216:1415-1416) 6 Antibodies to TMV Gamma-globulin fractions of serum obtained from chickens and rabbits immunized against TMV were obtained from Professor Iran Serra, Department of Agriculture, Hebrew University, Jerusalem, Rehovot, Israel. It was a generous gift. The following example describes procedures, including the best mode, for carrying out the invention. These examples should not be construed as limiting. Example 1 In a disk of tobacco leaves infected with TMV
5',5'-p ¹ p ⁿ (n=2-3)-dinucleotide antiviral activity 5',5'-p ^{1 p} n -dinucleotide can inhibit TMV replication in TMV-infected discs. 2′, 5′−
It was as effective as the adenylate trimer core (Table 1). As demonstrated, the calcium phosphate co-precipitation technique is essential for dinucleotide synergism in the assay of TMV infection disks.
However, the calcium coprecipitation technique was only required in the disk assay and not in the other inhibitory activity measurements disclosed above. The results in Table 1 show that the calcium co-precipitation technique enhances antiviral compounds by increasing their uptake in the disc assay. Table 1 also demonstrates the importance of calcium coprecipitation in synergizing antiviral dinucleotides in the TMV-infected leaf disk assay. This result indicates that the coprecipitation technique increases the uptake of antiviral dinucleotides. 5',5'-p ¹ p ⁿ (n = 2-3) dinucleotides containing n = 4 or more initial phosphates have more antiviral activity compared to dinucleotides with 2 and 3 internal phosphates. It was small.
However, modification of the nucleotide moiety (i.e. replacing one of the adenosines with G, C, U, addition of an external phosphate or external 2',3'-
Adding a cyclic phosphate (e.g. p<
A5′pp5′A>p) did not reduce their antiviral ability.

[Table] Example 2 Intact N. 5′,5′− in Gluteinosa plants
Dinucleotide antiviral activity of p ¹ p ⁿ (n=2-3) 5′, for inhibiting TMV replication in intact plants.
N. the ability of 5'-dinucleotides. tested using Gluteinosa plants. 5',5'-dinucleotides suppressed TMV replication as demonstrated by controlling the formation of local lesions after infection (Table 2). The most interesting are A5′pp5′A, A5′ppp5′A,
Application of A5'pp5'C and A5'pp5'G at a concentration of 1 μM (10 ^-6 M) even in the untreated half of the leaves.
This means that resistance to TMV infection was induced. The number of local diseases on the untreated half leaves was reduced compared to the control. Therefore, antiviral compounds induced antiviral resistance in plant tissues even without direct application.

【table】 %.
(2′,5′〓ApApA is a comparative compound.)
Example 3 Application of 5',5'-p ¹ p ⁿ (n=2-3) dinucleotides to roots) N for virus localization of induced resistance
- A comparison was made between foliar and root application of antiviral dinucleotides to N. tabacium plants in order to establish the relationship to the gene and compound transport capacities. 10 ml of N. tabaquium plants
in a vial containing mMHEPES-KOH PH7.6 and 1 μM antiviral dinucleotide. Plant roots were left in the solution for 2 hours and then transferred to vials containing buffer without antiviral compounds. Plants were infected with TMV as follows (see section 4 (page 22), Antiviral activity of N. tabacium on intact plants, paragraph (a)). Another N. tabaculum plant population was tested as described in paragraph (b) of the section above. Exposure of plant roots to 1 μM (10 ⁻⁶ M) 2′,5′-ApApA, A5′pp5′A, or A5′pp5′G resulted in overall suppression (98 to 100% suppression) of the virus. (Table 3). Induced resistance in leaf areas not directly treated with antiviral dinucleotides
It was also observed in N. tabacium plants, so the N- gene is not responsible.

[Table] Example 4 In the disk of tobacco leaves infected with TMV
3′,5′ ^- p ^{1 p 2} -dinucleotide antiviral activity.
It was as effective as the 5'-adenylate trimer nucleus. (Table 4)

Table 4 The results in Table 4 demonstrate that at comparable concentrations, A3'pp5'A and 2',5'ApApA confer similar antiviral status. Example 5 A3'pp5'A antiviral activity in intact N. glutinosa plants The ability of A3'pp5'A to suppress TMV replication in intact plants was tested using N. glutinosa plants.
A3′pp5′A suppressed TMV replication as shown by suppressing the formation of local lesions after infection (Dabatsyu, Y. et al. (1984) J. Biol.
Chem.259:3482−3486). Application of A3'pp5'A at a concentration of 1 μM in the infection solution resulted in the introduction of resistance to TMV infection even in the untreated half of the leaves (Table 5).

[Table] % of protection.
(2′,5′〓ApApA is a comparative compound.)
Example 6 2', ^5' - ^p1p2 -diadenosine antiviral activity 2',5'- ^p1p2 -diadenosine was effective in inhibiting ^TMV replication in TMV-infected disks (Table 6). Table 6 Suppression of TMV replication in TMV-infected leaf discs with 2 ^' ,5'-p 1 p ² -diadenosine Inhibition % control O ^b Adenosine 2',5'- p ¹ ,p ² -diadenosine 10 ^{- 10} M 0 10 ^-9 M 21 10 ^-8 M 26.4 10 ^-7 M 37 10 ^-6 M 42 b = % inhibition relative to control The above result is (1) m ~ 5'-p ¹ , p ⁿ −(m=2′, 3′,
5'; n = 2-3)-dinucleotides, (2) advantages of root application (application through irrigation), due to the active transfer of phosphate-containing compounds; and (3)m
〜5′−p ¹ , p ⁿ −(m=2′, 3′, 5′; n=2−3)−
This demonstrates the ability of the dinucleotide to induce resistance to viral infection (due to activation of active rabid transport or triggering of an antiviral state) even in plant areas that were not directly treated. It is noteworthy that application of antiviral compounds in the range of 10 ^-9 M to ^{10 -6} M resulted in total suppression of the virus without any apparent phytotoxicity.

Claims (1)

[Claims] 1 formula [In the formula, X is [Formula] or [Formula] or [Formula], n is 2 or 3, one of R ₁ and R ₂ is an andene residue, and the other of R ₁ and R ₂ is an adenine residue ,
A group selected from the group consisting of guanine, cytosine, and lauyl residues, and R ₃ , R ₄ , R ₅ , and
An anti-plant virus composition comprising a compound in which R ₆ is OH, or R ₃ and R ₄ and R ₅ and R ₆ are [Formula]. 2 formulas A composition according to claim 1, comprising a 5',5'-dinucleotide of the formula: wherein n, R ₁ and R ₂ are as defined in claim 1. 3 The above 5',5'-nucleotide is diadenosine-
5',5- ^p1 , ^p2 -diphosphate (Ap2A), adenosine-5',5- ^p1 , ^p2 -diphospho-guanosine (Ap2G), adenosine-5',5- ^p1 , ^p2-
Diphosphouridine (Ap2U), adenosine-5′,
5- ^p1 , ^p2 -diphospho-cytidine (Ap2C), or 2',3'-cyclic phosphodiadenosine 5',5- ^p1 ,
The composition according to claim ² , which is p2-diphosphate (p<Ap2A>p). 4 The above 2′,5′-dinucleotide is adenosine 2′,
5′-diphospho-adenosine (A2′pp5′A), adenosine 2′,5′-diphospho-guanosine (A2′pp5′G),
Adenosine 2′,5′-diphospho-uridine (A2′pp5′U), or adenosine 2′,5′-diphospho-uridine (A2′pp5′U)
The composition according to claim 1, which is cytidine (A2'pp5'C). 5 The above 3',5'-dinucleotide is adenosine 3',
5′-diphospho-adenosine (A3′pp5′A), adenosine 3′,5′-diphospho-guanine (A3′pp5′G),
Adenosine 3′,5′-diphospho-uridine (A3′pp5′U), or adenosine 3′,5′-diphospho-uridine
The composition according to claim 1, which is cytidine (A3'pp5'C).