... be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate ... lin-Benzo-GTP (ic). lin-Benzo-XTP (2c), and lin-Benzo-ITP. (3c).
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
The fluorescent nucleotide analogues (the 5'ABSTRACT mono-, di-, and triphosphates of lin-benzoguanosine, ftn-benzoxanthosine, and Iin-benzoinosine) have been prepared for use as dimensional probes of enzyme binding sites. They have quantum yields in aqueous solution of 0.39,0.55, and 0.04 and fluorescent lifetimes of 6, 9, and t1.5 nsec, respectively. UnBenzoinosine 5'-monophosphate is a substrate for xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2), providing 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 used to prepare the triphosphates.
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Fluorescent analogues or derivatives of adenine-containing nucleotides have been prepared to aid in the definition of enzyme binding sites and in the determination of interactions in 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 consequence of the formal insertion of a benzene ring. Accordingly, the members of the lin-benzoadenine series may serve as investigative tools in view of their fluorescence properties and their potential for interaction in a wide range of biological systems (3). Although fluorescent derivatives of guanine have been reported, none to date possesses unaltered terminal rings and most 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, have shown utility as fluorescent probes in connection with conformational studies of tRNA (8, 9). Related heterocycles are formed by reactions of the guanine nucleus with substituted malondialdehydes (10, 11), glycidaldehyde (12), and chloroacetaldehyde (13). These modified analogues are subject to limited applicability in base-specific systems due to the external alteration of the pyrimidine ring. We report here the preparation of lin-benzoguanosine 5'-mono-, di-, and triphosphates (1), laterally extended versions of the natural nucleotides in which the substituted pyrimidine ring is not further modified. These guanine ribotide analogues are strongly fluorescent, as are the corresponding xanthine ribotide analogues (2). The less fluorescent lin-benzoinosine 5'-mono-, di-, and triphosphates (3) have also been prepared.
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MATERIALS AND METHODS Ultraviolet absorption spectra were obtained on a Beckman Acta M VI spectrophotometer. Molecular fluorescence emission and excitation spectra were measured on a Spex Fluorolog spectrofluorometer, with the quantum yield of 0.40 for linbenzoadenosine triphosphate as a standard. A cross-correlation subnanosecond fluorometer interfaced with a Monroe 1880 programmable calculator was used to determine fluorescence lifetimes. The purity and identity of the nucleotides were established by combinations of chromatography and electrophoresis and by chemical and enzymatic transformations. Thin-layer chromatograms were run on EM silica gel f-254 plates, with isobutyric acid/concentrated ammonia/water, 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 which, only when central, contains no nitrogen. This terminology is in use for derivatives similarly related to all purines. The other parts of the names follow accepted IUPAC-IUB nomenclature. TEAB, triethylammonium bicarbonate.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 4262
Biochemistry: Leonard and Keyser 56:1:43 (vol/vol) as solvent. Bio-Rad Dowex 50W-X8 and Sigma DEAE-cellulose were used for ion-exchange chromatography. All aqueous columns were maintained at 5-10°C. Electrophoresis was run on an LKB Servall with LKB Power Supply at lOOVand lOmA. Synthesis of lin-Benzo-GMP (la), lin-Benzo-XMP (2a), and lin-Benzo-IMP (3a). The compounds were synthesized from the corresponding ribosides (14) by the method of Imai et al. (15), previously used in this laboratory to obtain lin-benzo-AMP (3). In a typical procedure, to a stirred suspension of lin-benzoinosine (32 mg, 0.13 mmol) in m-cresol (2 ml) under argon at 00C was added pyrophosphoryl chloride (0.2 ml). Stirring was continued at 00C for 4 hr, during which time a clear yellow solution was obtained. This solution was poured into ice water (25 ml), thoroughly extracted three times with ether (50 ml), and then added to 0.5 M aqueous triethylammonium bicarbonate (TEAB) (100 ml). After removal of the solvent at reduced pressure at 0-5°C, methanol (50 ml, three times) was added and coevaporated at 0-5OC to decompose excess TEAB. The white residue was dissolved in water (8 ml), applied to a DEAE-cellulose column (2.5 X 40 cm), and eluted with a linear gradient of 0.01 M (500 ml) to 0.25 M (500 ml) aqueous TEAB. Fractions containing the material (by A326) were reduced to dryness at reduced pressure at 0-5°C and treated with methanol as above. The clear glass obtained was dissolved in water (2 ml) and applied to a column of Li+ Dowex 5OW-X8 (200-400 mesh, 1 X 20 cm). Elution with distilled water and evaporation of fractions containing the material gave 42 mg of 3a (76%) as clear plates. For 3a: UV 4Xjp72 (10 mM phosphate) 326 (c 6300), 313 (6300), 298 (5600), 290 (sh). For la: UV X"'x72 (10 mM phosphate) 335 (sh), 323 (5600), 288 (4100), 277 (5100). For 2a: UV AX72 (10 mM phosphate) 324 (6300), 284 (4700), 270 (6900). The UV E values were obtained by alkaline phosphatase hydrolysis to determined concentrations of the corresponding nucleosides. Enzymatic Synthesis of lin-Benzo-XMP (2a). This ribonucleotide was prepared chemically (see above) and also by the enzymatic oxidation of 3a. To a solution of 50 mM aqueous TEAB (pH 8.0,9 ml, made by diluting 0.5 M aqueous TEAB) was added a solution of lin-benzoinosine 5'-monophosphate (3a) (10 mg, 0.025 mmol) in 50 mM aqueous TEAB (0.9 ml, pH 8.0) and xanthine oxidase (xanthine:oxygen oxidoreductase; EC 1.2.3.2) (Sigma, 2 units in 0.1 ml of 2.3 M aqueous ammonium sulfate). The solution was agitated vigorously and then allowed to stand for 18 hr. The solution was diluted with an equal volume of methanol and immersed in a dry ice/acetone bath for 30 min. Evaporation and chromatographic purification as described above gave 2a (10.2 mg, 98%). lin-Benzo-GDP (lb), lin-Benzo-XDP (2b), and lin-BenzoIDP (3b). The 5'-phosphomorpholidates of la-3a were prepared according to the general procedure of Moffatt and Khorana (16) from the free acids of la-3a and were converted to the 5'-diphosphates (lb-3b) in 70-75% yield by the procedure previously reported from this laboratory (3). lin-Benzo-GTP (ic). lin-Benzo-XTP (2c), and lin-Benzo-ITP (3c). The triphosphates were prepared enzymatically from lb-3b as described for the synthesis of lin-benzo-ATP, with phosphoenolpyruvate and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) (3). A typical conversion used 10-15 mg of the diphosphate with yields >95%, limited only by handling procedures and chromatographic recovery. Characterization of the Nucleotide Analogues by Electrophoresis and Chromatography. The electrophoretic mobilities of the compounds in 100 mM sodium phosphate buffer (pH 7.3), relative to GTP = 1.0, were: la, 0.5; 2a, 0.65; 3a, 0.5; lb, 0.8; 3b, 0.8; ic, 0.95; and 3c, 0.95. The concentrations of
Proc. Natl. Acad. Sci. USA 76 (1979)
4263
TEAB (pH 8.0) at which they were first eluted from a DEAEcellulose column (2.5 X 40 cm) were: la-3a, 0.12-0.15 M; lb-3b, 0.20-0.25 M; and lc-3c, 0.30-0.35 M. Poly(lin-benzoinosinic acid). The compound was prepared in a reaction medium containing the following: lin-benzo-IDP (3b) (2 mM), MgCl2 (5 mM), Tris-HCl (100 mM, pH 8.0), 70 units of polynucleotide phosphorylase (Micrococcus lysodeikticus, Sigma) (polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8), and guanylyl (3' 5')uridine (1 AM) in a total volume of 0.5 ml. The reaction mixture was incubated at 370C for 48 hr and then extracted several times with chloroform/isoamyl alcohol, 3:1 (vol/vol). The aqueous layer was applied to a column of Sephadex G-50 (2.0 X 100 cm) and eluted with 10 mM TEAB (pH 8.0). The polymeric material was excluded from the column just prior to the small protein fraction. Attempts to determine the molecular weight by gel electrophoresis resulted in precipitation of the polymer at and near the origin. Incubation of the polymeric material (0.5 A326) with a mixture of micrococcal nuclease, snake venom phosphodiesterase, and alkaline phosphatase in Tris-HCl (pH 8.5) at 37°C for 30 min returned the fine structure to the longwavelength bands. The hypochromicity at 326 nm exhibited by the polymeric material obtained under our experimental conditions was about 60%. RESULTS AND DISCUSSION Our experience with lin-benzoadenosine phosphate analogues has established the concept of using defined dimensional probes for enzyme active sites, aided by fluorescence methodology (3, 4, 17-19). We have prepared the benzologues (1-3) of the guanosine, inosine, and xanthosine nucleotides to extend this concept and to provide additional fluorescent probes for the study of the functional interactions of purine nuclei. The monoand diphosphate derivatives (la,b-3a,b) were prepared from the nucleosides (14) by the procedure of Imai et al. (15) and Moffatt and Khorana (16) as modified in our laboratory (3, 4). Enzymatic conversion of the diphosphates (lb-3b) to the triphosphates (lc-3c) was accomplished with phosphoenolpyruvate and pyruvate kinase. The reaction was driven to completion by the use of lactate dehydrogenase, with NADH to consume the pyruvate formed in the reaction. Even though the enzymatic phosphorylation was slower than that of linbenzo-ADP (3), the outcome, >95% yield of corresponding triphosphate, was much better than the result of chemical phosphorylation (3, 16). The exceptional fluorescence properties (Table 1) of the lin-benzopurine ribosylphosphates make them potentially useful for the investigation of enzyme-coenzyme interactions. The wavelength of excitation of these compounds is well beyond the region of absorption of the natural ribonucleotides, permitting selective observation, and the wavelength of emission can be monitored with equivalent selectivity. The satisfactory quantum yields and fluorescence lifetimes of 1 and 2, coupled with the environmental sensitivity and reactivity demonstrated for the lin-benzoadenosine 5'-phosphate series (2-4, 17-19), make the lin-benzoguanosine and lin-benzoxanthosine phosphate series particularly attractive as fluorescent, dimensional probes. Preliminary attempts to use guanosine monophosphate kinase (EC 2.7.4.8) for the conversion of lin-benzo-GMP to linbenzo-GDP have failed, perhaps as anticipated, due apparently to the characteristic high specificity of the GMP active site (20). Succinyl-CoA synthetase (EC 6.2.1.4) (21) failed to convert lin-benzo-GDP to lin-benzo-GTP under normal assay conditions. Our report of the oxidation of lin-benzoinosine to lin-benzoxanthosine (2, 14) was the first for a ribonucleoside by xan-
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Biochemistry: Leonard and Keyser
Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Fluorescence data Excitation >260, nm Emission, nmt
Compound* lin-Benzo-GMP (la) 277, 288, 323 lin-Benzo-XMP (2a) 270, 284, 324 lin-Benzo-IMP (3a) 298, 313, 326 lin-Benzo-AMPI 320 (sh), 332, 348 *
