Proc. Natl. Acad. Sci. USA Vol. 76, No. 9, pp. 4262-4264, September 1979 Biochemistry

Synthesis of fluorescent nucleotide analogues: 5'-Mono-, di-, and triphosphates of linear-benzoguanosine, linear-benzoinosine, and linear-benzoxanthosine (phosphorylation/xanthine oxidase oxidation/quantum yield/lifetime/dimensional probes) NELSON J. LEONARD AND GENE E. KEYSER Roger Adams Laboratory, School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801 Contributed by Nelson J. Leonard, May 29, 1979

ABSTRACT The fluorescent nucleotide analogues (the 5'- 0 mono-, di-, and triphosphates of lin-benzoguanosine, ftn-ben- 8 9 zoxanthosine, and Iin-benzoinosine) have been prepared for use 7 N 1 as dimensional probes of enzyme binding sites. They have HN N > quantum yields in aqueous solution of 0.39,0.55, and 0.04 and fluorescent lifetimes of 6, 9, and t1.5 nsec, respectively. Un- H2N*J'N 3 Benzoinosine 5'-monophosphate is a substrate for xanthine 5 4 1 oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2), providing R lin-benzoxanthosine 5'-monophosphate, and lin-benzoinosine 5'-diphosphate is a substrate for polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8), giving poly(lin-benzoinosinic acid). The benzologues of the purine diphosphates are substrates for pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40), which is I.N used to prepare the triphosphates.

Fluorescent analogues or derivatives of adenine-containing O NN 'N nucleotides have been prepared to aid in the definition of en- H I H zyme binding sites and in the determination of interactions in 2 3 nucleic acids (1-6). Notably, the lin-benzoadenine series of analogues (lin = linear) (1-3) are unaltered at the typical loci of biological interactions, yet the terminal pyrimidine and imidazole rings are separated by an additional 2.4 A as a con- sequence of the formal insertion of a benzene ring. Accordingly, the members of the lin-benzoadenine series may serve as in- R= P 0 vestigative tools in view of their fluorescence properties and OH their potential for interaction in a wide range of biological systems (3). a, n = 1 Although fluorescent derivatives of guanine have been re- b, n = 2 ported, none to date possesses unaltered terminal rings and most c. n = 3 are modifications of guanine nuclei preexistent in the system under investigation. The naturally occurring Y bases (7), which are highly substituted guanosine derivatives found in tRNAs, MATERIALS AND METHODS have shown utility as fluorescent probes in connection with Ultraviolet absorption spectra were obtained on a Beckman conformational studies of tRNA (8, 9). Related heterocycles are Acta M VI spectrophotometer. Molecular fluorescence emission formed by reactions of the guanine nucleus with substituted and excitation spectra were measured on a Spex Fluorolog malondialdehydes (10, 11), glycidaldehyde (12), and chloroa- spectrofluorometer, with the quantum yield of 0.40 for lin- cetaldehyde (13). These modified analogues are subject to benzoadenosine triphosphate as a standard. A cross-correlation limited applicability in base-specific systems due to the external subnanosecond fluorometer interfaced with a Monroe 1880 alteration of the pyrimidine ring. We report here the prepa- programmable calculator was used to determine fluorescence ration of lin-benzoguanosine 5'-mono-, di-, and triphosphates lifetimes. The purity and identity of the nucleotides were es- (1), laterally extended versions of the natural nucleotides in tablished by combinations of chromatography and electro- which the substituted pyrimidine ring is not further modified. phoresis and by chemical and enzymatic transformations. These guanine ribotide analogues are strongly fluorescent, as Thin-layer chromatograms were run on EM silica gel f-254 are the corresponding xanthine ribotide analogues (2). The less plates, with isobutyric acid/concentrated ammonia/water, fluorescent lin-benzoinosine 5'-mono-, di-, and triphosphates (3) have also been prepared. Abbreviations: The prefix lin refers to the linear disposition of the three rings, as in 1; "benzo" in the trivial name refers to the additional ring The publication costs of this article were defrayed in part by page which, only when central, contains no nitrogen. This terminology is charge payment. This article must therefore be hereby marked "ad- in use for derivatives similarly related to all purines. The other parts vertisement" in accordance with 18 U. S. C. §1734 solely to indicate of the names follow accepted IUPAC-IUB nomenclature. TEAB, this fact. triethylammonium bicarbonate. 4262 Downloaded by guest on September 26, 2021 Biochemistry: Leonard and Keyser Proc. Natl. Acad. Sci. USA 76 (1979) 4263 56:1:43 (vol/vol) as solvent. Bio-Rad Dowex 50W-X8 and Sigma TEAB (pH 8.0) at which they were first eluted from a DEAE- DEAE-cellulose were used for ion-exchange chromatography. cellulose column (2.5 X 40 cm) were: la-3a, 0.12-0.15 M; All aqueous columns were maintained at 5-10°C. Electro- lb-3b, 0.20-0.25 M; and lc-3c, 0.30-0.35 M. phoresis was run on an LKB Servall with LKB Power Supply Poly(lin-benzoinosinic acid). The compound was prepared at lOOVand lOmA. in a reaction medium containing the following: lin-benzo-IDP Synthesis of lin-Benzo-GMP (la), lin-Benzo-XMP (2a), and (3b) (2 mM), MgCl2 (5 mM), Tris-HCl (100 mM, pH 8.0), 70 lin-Benzo-IMP (3a). The compounds were synthesized from units of polynucleotide phosphorylase (Micrococcus lysodeik- the corresponding ribosides (14) by the method of Imai et al. ticus, Sigma) (polyribonucleotide:orthophosphate nucleotid- (15), previously used in this laboratory to obtain lin-benzo-AMP yltransferase, EC 2.7.7.8), and guanylyl (3' 5')uridine (1 AM) (3). In a typical procedure, to a stirred suspension of lin-ben- in a total volume of 0.5 ml. The reaction mixture was incubated zoinosine (32 mg, 0.13 mmol) in m-cresol (2 ml) under argon at 370C for 48 hr and then extracted several times with chlo- at 00C was added pyrophosphoryl chloride (0.2 ml). Stirring roform/isoamyl alcohol, 3:1 (vol/vol). The aqueous layer was was continued at 00C for 4 hr, during which time a clear yellow applied to a column of Sephadex G-50 (2.0 X 100 cm) and solution was obtained. This solution was poured into ice water eluted with 10 mM TEAB (pH 8.0). The polymeric material (25 ml), thoroughly extracted three times with ether (50 ml), was excluded from the column just prior to the small protein and then added to 0.5 M aqueous triethylammonium bicar- fraction. Attempts to determine the molecular weight by gel bonate (TEAB) (100 ml). After removal of the solvent at re- electrophoresis resulted in precipitation of the polymer at and duced pressure at 0-5°C, methanol (50 ml, three times) was near the origin. Incubation of the polymeric material (0.5 A326) added and coevaporated at 0-5OC to decompose excess TEAB. with a mixture of micrococcal nuclease, snake venom phos- The white residue was dissolved in water (8 ml), applied to a phodiesterase, and alkaline phosphatase in Tris-HCl (pH 8.5) DEAE-cellulose column (2.5 X 40 cm), and eluted with a linear at 37°C for 30 min returned the fine structure to the long- gradient of 0.01 M (500 ml) to 0.25 M (500 ml) aqueous TEAB. wavelength bands. The hypochromicity at 326 nm exhibited Fractions containing the material (by A326) were reduced to by the polymeric material obtained under our experimental dryness at reduced pressure at 0-5°C and treated with meth- conditions was about 60%. anol as above. The clear glass obtained was dissolved in water RESULTS AND DISCUSSION (2 ml) and applied to a column of Li+ Dowex 5OW-X8 (200-400 Our experience with lin-benzoadenosine phosphate analogues mesh, 1 X 20cm). Elution with distilled water and evaporation has established the concept of using defined dimensional probes of fractions containing the material gave 42 mg of 3a (76%) as for enzyme active sites, aided by fluorescence methodology (3, clear plates. For 3a: UV 4Xjp72 (10mM phosphate) 326 (c 6300), 4, 17-19). We have prepared the benzologues (1-3) of the gu- 313 (6300), 298 (5600), 290 (sh). For la: UV X"'x72 (10 mM anosine, inosine, and xanthosine nucleotides to extend this phosphate) 335 (sh), 323 (5600), 288 (4100), 277 (5100). For 2a: concept and to provide additional fluorescent probes for the UV AX72 (10 mM phosphate) 324 (6300), 284 (4700), 270 study of the functional interactions of purine nuclei. The mono- (6900). The UV E values were obtained by alkaline phosphatase and diphosphate derivatives (la,b-3a,b) were prepared from hydrolysis to determined concentrations of the corresponding the nucleosides (14) by the procedure of Imai et al. (15) and nucleosides. Moffatt and Khorana (16) as modified in our laboratory (3, 4). Enzymatic Synthesis of lin-Benzo-XMP (2a). This ribonu- Enzymatic conversion of the diphosphates (lb-3b) to the tri- cleotide was prepared chemically (see above) and also by the phosphates (lc-3c) was accomplished with phosphoenolpy- enzymatic oxidation of 3a. To a solution of 50 mM aqueous ruvate and pyruvate kinase. The reaction was driven to com- TEAB (pH 8.0,9 ml, made by diluting 0.5 M aqueous TEAB) pletion by the use of lactate dehydrogenase, with NADH to was added a solution of lin-benzoinosine 5'-monophosphate (3a) consume the pyruvate formed in the reaction. Even though the (10 mg, 0.025 mmol) in 50 mM aqueous TEAB (0.9 ml, pH 8.0) enzymatic phosphorylation was slower than that of lin- and xanthine oxidase (xanthine:oxygen oxidoreductase; EC benzo-ADP (3), the outcome, >95% yield of corresponding 1.2.3.2) (Sigma, 2 units in 0.1 ml of 2.3 M aqueous ammonium triphosphate, was much better than the result of chemical sulfate). The solution was agitated vigorously and then allowed phosphorylation (3, 16). to stand for 18 hr. The solution was diluted with an equal vol- The exceptional fluorescence properties (Table 1) of the ume of methanol and immersed in a dry ice/acetone bath for lin-benzopurine ribosylphosphates make them potentially 30 min. Evaporation and chromatographic purification as de- useful for the investigation of enzyme-coenzyme interactions. scribed above gave 2a (10.2 mg, 98%). The wavelength of excitation of these compounds is well be- lin-Benzo-GDP (lb), lin-Benzo-XDP (2b), and lin-Benzo- yond the region of absorption of the natural ribonucleotides, IDP (3b). The 5'-phosphomorpholidates of la-3a were pre- permitting selective observation, and the wavelength of emis- pared according to the general procedure of Moffatt and sion can be monitored with equivalent selectivity. The satis- Khorana (16) from the free acids of la-3a and were converted factory quantum yields and fluorescence lifetimes of 1 and 2, to the 5'-diphosphates (lb-3b) in 70-75% yield by the proce- coupled with the environmental sensitivity and reactivity dure previously reported from this laboratory (3). demonstrated for the lin-benzoadenosine 5'-phosphate series lin-Benzo-GTP (ic). lin-Benzo-XTP (2c), and lin-Benzo-ITP (2-4, 17-19), make the lin-benzoguanosine and lin-benzo- (3c). The triphosphates were prepared enzymatically from xanthosine phosphate series particularly attractive as fluo- lb-3b as described for the synthesis of lin-benzo-ATP, with rescent, dimensional probes. phosphoenolpyruvate and pyruvate kinase (ATP:pyruvate Preliminary attempts to use guanosine monophosphate kinase 2-O-phosphotransferase, EC 2.7.1.40) (3). A typical conversion (EC 2.7.4.8) for the conversion of lin-benzo-GMP to lin- used 10-15 mg of the diphosphate with yields >95%, limited benzo-GDP have failed, perhaps as anticipated, due apparently only by handling procedures and chromatographic recovery. to the characteristic high specificity of the GMP active site (20). Characterization of the Nucleotide Analogues by Elec- Succinyl-CoA synthetase (EC 6.2.1.4) (21) failed to convert trophoresis and Chromatography. The electrophoretic mo- lin-benzo-GDP to lin-benzo-GTP under normal assay condi- bilities of the compounds in 100 mM sodium phosphate buffer tions. (pH 7.3), relative to GTP = 1.0, were: la, 0.5; 2a, 0.65; 3a, 0.5; Our report of the oxidation of lin-benzoinosine to lin-ben- lb, 0.8; 3b, 0.8; ic, 0.95; and 3c, 0.95. The concentrations of zoxanthosine (2, 14) was the first for a ribonucleoside by xan- Downloaded by guest on September 26, 2021 4264 Biochemistry: Leonard and Keyser Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Fluorescence data Compound* Excitation >260, nm Emission, nmt T, nsect PFt* lin-Benzo-GMP (la) 277, 288, 323 385 6 0.39 lin-Benzo-XMP (2a) 270, 284, 324 403 9 0.55 lin-Benzo-IMP (3a) 298, 313, 326 365 1.5 0.04 lin-Benzo-AMPI 320 (sh), 332, 348 385 3.7 0.44 * <0.1 A323 in 10 mM sodium phosphate buffer (pH 7.2). t +2 nm, Xex 323 nm. t The di- and triphosphates give essentially the same values. § Relative to lin-benzo-ATP, pH 8.5, 41 = 0.40. ¶ pH 8.5, 50 mM Tris-HCl buffer. See refs. 2 and 4.

thine oxidase. The oxidation of lin-benzo-IMP (3a) to lin- supported by Research Grant GM-05829 from the National Institutes benzo-XMP (2a) by this enzyme is even more surprising as the of Health, U.S. Public Health Service. G.E.K. was supported by a fel- first example of such a conversion occurring at the 5'-mono- lowship from Eli Lilly & Co. phosphate level. It suggests that the distance of the ribose or ribose 5'-phosphate moiety from the pyrimidine ring permits oxidation to occur at C-6 while maintaining steric protection 1. Leonard, N. J., Morrice, A. G. & Sprecker, M. A. (1975) J. Org. of the imidazole carbon, C-2 (see 1 for numbering system). Chem. 40, 356-363. Polynucleotide phosphorylase has been used previously in 2. Leonard, N. J., Sprecker, M. A. & Morrice, A. G. (1976) J. Am. our laboratory (3) to prepare poly(lin-benzo-AMP). Reaction Chem. Soc. 98,3987-3994. of lin-benzoinosine diphosphate (3b) in the presence of Mn2+, 3. Leonard, N. J., Scopes, D. I. C., VanDerLijn, P. & Barrio, J. R. used to decrease the specificity of the enzyme in a primer- (1978) Biochemistry 17,3677-3685. 4. Scopes, D. I. C., Barrio, J. R. & Leonard, N. J. (1977) Science 195, independent polymerization, was not feasible because addition 296-298. of Mn2+ caused nearly quantitative precipitation of 3b. Addi- 5. Thomas, R. W. & Leonard, N. J. (1976) Heterocycles 5, 839- tion of a GpU primer in the presence of Mg2+, however, al- 882. lowed the preparation of poly(lin-benzo-IMP). Our first at- 6. Leonard, N. J. & Tolman, G. L. (1975) Ann. N.Y. Acad. Sci. 255, tempts to characterize the extent of polymerization by gel 43-58. electrophoresis (22) have been frustrated thus far by the pre- 7. Stryer, L. (1968) Science 162,526-533. cipitation of the concentrated polymer on the gels. Polymer- 8. Beardsly, K. & Cantor, C. R. (1970) Proc. Natl. Acad. Sci. USA ization is accompanied by a strong hypochromic effect and loss 65,39-46. of UV fine structure (14), indicative of extensive r-ir overlap 9. Blumberg, W. E., Dale, R. E., Eisinger, J. & Zuckerman, D. M. (1974) Biopolymers 13, 1607-1620. in the polymeric material. The hypochromicity at 326 nm was 10. Moschel, R. C. & Leonard, N. J. (1976) J. Org. Chem. 41, observed to be about 60% by enzymatic hydrolysis of the 294-300. polymer. If the poly(lin-benzo-IMP) is helical, one can imagine 11. Work, D. R. (1976) Dissertation (Univ. Illinois, Urbana, IL). as a complement a ribotyl-N=C(NH2)R partner in which R 12. Goldschmidt, B. M., Blaiej, T. P. & Van Duuren, B. L. (1968) is, e.g., CH3, NH2, OR', or SR', that would provide normal Tetrahedron Lett., 1583-1586. Watson-Crick double helical dimensions. The result would be 13. Sattsangi, P. D., Leonard, N. J. & Frihart, C. R. (1977) J. Org. equivalent to complementary base pairing between I and C. Chem. 42, 3292-3296. The pKa values corresponding to ring deprotonation of the 14. Keyser, G. E. & Leonard, N. J. (1979) J. Org. Chem. 44, in conjugate acids of lin-benzo-GMP (la) and of lin-benzogua- press. nosine have been determined by UV spectroscopy to be 5.7 and 15. Imai, K., Fujii, S., Takanohashi, K., Furukawa, Y., Masuda, T. & Honjo, M. (1969) J. Org. Chem. 34, 1547-1550. 4.6, respectively. The pKa for the conjugate acid of the riboside 16. Moffatt, J. G. & Khorana, H. G. (1961) J. Am. Chem. Soc. 83, is similar to that of the free base (23), as expected. The dimin- 649-659. ished acidity of the ring-conjugate acid of la compared with 17. VanDerLijn, P., Barrio, J. R. & Leonard, N. J. (1978) Proc. Natl. lin-benzoguanosine is explained by the interaction of the Acad. Sci. USA 75,4204-4208. a-phosphate with +N(1)-H, providing intramolecular stabili- 18. Schmidt, M. J., Truex, L. T., Leonard, N. J., Scopes, D. I. C. & zation of the acquired positive charge. Barrio, J. R. (1978) J. Cyclic Nucleotide Res. 4, 201-207. Applications of the dimensional probes here described, and 19. Kauffman, R. F., Lardy, H. A., Barrio, J. R., Barrio, M. d. C. G. particularly of the fluorescent analogue of GTP (ic), in bio- & Leonard, N. J. (1978) Biochemistry 17,3686-3692. logical systems are anticipated that will use both the defined 20. Anderson, E. P. (1973) Enzymes (Academic, New York), 3rd Ed., Vol. 9, pp. 82-86. dimensional change from the natural GTP and the diagnostic 21. Bridger, W. A. (1974) Enzymes (Academic, New York), 3rd Ed., value of the fluorescence. Vol. 10, pp. 581-606. We appreciate the help of Dr. P. VanDerLijn and of J. B. Holtwick 22. Loening, U. E. (1969) Biochem. J. 113, 131-138. in determination of the fluorescence lifetimes and Dr. J. R. Barrio in 23. Keyser, G. E. & Leonard, N. J. (1976) J. Org. Chem. 41, valuable discussion and sustained interest in this project. This work was 3529-3532. Downloaded by guest on September 26, 2021