enzymatic synthesis of 5’-phosphate nucleotides of … · enzymatic synthesis of 5’-phosphate...

ENZYMATIC SYNTHESIS OF 5’-PHOSPHATE NUCLEOTIDES OF PURINE ANALOGUES*

BY JAMES L. WAYt AND R. E. PARKS, Ja.$

(From the Department of Pharmacology and Toxicology, i%iversity of Wisconsin Medical School, Madison, Wisconsin)

(Received for publication, September 23, 1957)

Analogues of natural purines and pyrimidines have been synthesized and examined for biological activity, and a number of these have been found to inhibit the growth and metabolism of various tissues. Since certain of these compounds are more inhibitory to the growth of neoplastic than of normal cells, it is of importance that we understand the biochemical mechanisms by which they act.

Much of the evidence suggests that analogues such as S-azaguanine (l-3) and 2,6-diaminopurine (4, 5) inhibit cell growth after conversion to more active subst.ances. The formation of “defective” nucleic acids due to incorporation of these compounds was considered as a possible explanation of t.he growth failure, and, indeed, some of these analogues are found to enter nucleic acids very readily (5-S). However, organisms and tissues which are not appreciably affected metabolically may incorporate the drugs as readily as those which are inhibited (6, 8, 9). This has encouraged the search for alt.ernative explanations for the actions of these analogues.

The importance in metabolism of nucleotides containing bases other than adenine has been fully appreciated only recently. Polyphosphate nucleotides of most of the natural purines and pyrimidines have been found in a number of tissues (lo-12), and major metabolic functions have now been assigned to some of these. For example, guanine and hypoxanthine nucleotides serve as coenzymes for such reactions as the incorporation of amino acids into microsome protein (13, 14), the “substrate level” phosphorylation of a-ketoglutarate oxidation (15), phosphoenolpyruvate car- boxylation (16), and the conversion of IMP to AMP (17) .I These obser-

* This work was supported in part by grants from the American Cancer Society and the National Institutes of Health, United States Public Health Service. Pre- liminary reports of this work were presented at the Fall meeting of the American Society for Pharmacology and Experimental Therapeutics in French Lick, Indiana, Nov., 1956, and at the annual meeting of the Federation of American Societies for Experimental Biology in Chicago, Illinois, Apr., 1957.

t Postdoctoral Fellow of the National Cancer Institute, United States Public Health Service (1955-56).

1 Scholar in Medical Science of the John and Mary R. Markle Foundation. 1 The following abbreviations are used: adenosine 5’-mono-, di-, and triphosphate,

AMP, ADP, and ATP, respectively; inosine 5’-phosphate, IMP; 8-azaguanosine

467

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468 MONONUCLEOTIDES OF PURISE ANALOGUES

vations raise the possibility that purine and pyrimidine analogues may block growth and metabolism by act,ing at t.he nucleotide rather than at the nucleic acid level, perhaps by the formation of “defective” coenzymes. With this in mind, n-e have undertaken the preparation of nucleotide coenzymes containing analogues in place of the natural bases.

This report is concerned with the enzymatic synthesis of nucleotide monophosphates containing various analogues. Two methods have been employed : phosphorylation of nucleosides by the transphosphatation procedure of Brawerman and Chargaff (18) and the interaction of the analogues with PRPP in the presence of nucleotide pyrophosphorylases (19-21).

Materials

AMP and ATP (crystalline disodium salt) were purchased from the Pabst Laboratories. The barium salt of ribose 5-phosphate (Schwarz Laboratories, Inc.) was converted to the potassium salt before use. 6- Mercaptopurine was obtained from the Nutritional Biochemicals Corpora- tion. Samples of 2,6-diaminopurine and 8-azaguanine were generously provided by the Lederle Laboratories. The adenine, guanine, and hypoxanthine analogues of the pyrazolo(3,Qd)pyrimidine series were a gift of Dr. R. K. Robins (22). The purities were tested by chemical analysis, dtraviolet spectrophotometry, and paper chromatography. Other chemi- cals were of the highest grade of purity commercially available.

Enzyme Preparations

Inorganic pyrophosphatase2 was partially purified by ammonium sulfate fractionation from bakers’ yeast, as described by Heppel and Hilmoe (23). 5’-Nucleotidase was kindly furnished by Dr. D. Gibson. This enzyme was obtained from rattlesnake venom and purified according to the procedure described by Hurst and Butler (24). Purine nucleoside phosphorylase was purified from beef liver acetone powder (25). Acet,one powders of liver were prepared by homogenizing fresh liver in 5 volumes of acetone (- 157, followed by filtration through a Biichner funnel. The residue was immediately rehomogenized in cold acetone, filtered, dried in air, and stored at -15”. Extracts were prepared by stirring acetone powders in 10 volumes of 0.01 M Tris-HCl buffer, pH 8.0, for 15 minutes at 0”. The residues were removed by centrifugation and, unless otherwise stated, the supernatant fluids were dialyzed against several changes of 0.01 M Tris-HCl buffer. These dialyzed extracts were either used immediately or lyophil-

5’-phosphate, 8-aza-GMP; guanosine triphosphate, GTP; uridine triphosphate’ UTP; diphosphopyridine nucleotide, DPN; 5-phosphoribosyl-1-pyrophosphate, PRPP; tris(hydroxymethyl)aminomethane, Tris; ribonucleic acid, RNA.

8 The preparation employed hydrolyzed 6.7 amoles of pyrophosphate per minute per ml. of enzyme.


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J. L. WAY AND R. E. PARKS, JR. 469

ized and stored at -15” for future use. Purine nucleotide pyrophosphorylase activity was obt.ained from a dialyzed, cent.rifuged autolysate of brewers’ yeast (20), as well as from hog and beef liver acetone powders. Extracts of barley malt were employed for transphosphorylation activity (18).

The enzyme for preparing PRPP was purified from pigeon liver (26). All the steps were carried out at O-5”. A homogenate was made by using a Potter-Elvehjem homogenizer to contain by volume 1 part of liver and 1.5 parts of a solution of 0.15 M KC1 and 0.01 M Tris-HCl buffer, pH 8.0. Extracts were prepared from the homogenat,e by centrifugation at 18,000 X g for 15 minutes. The residue was discarded and 95 per cent ethanol was added slowly to the supernatant fluid until a concentration of 15 per cent by volume was achieved. The precipitate was collected by centrifugation, suspended in 15 per cent ethanol, recentrifuged, and dissolved in 0.1 M

phosphate buffer, pH 7.4. The solution was centrifuged to remove insoluble particles, lyophilized, and st,ored at - 15”.

Determinations

Pentose was determined by the orcinol procedure (27)) a 40 minut.e heating period being employed with AMP as the standard. Orthophosphate was determined by the Fiske-Subbarow method (28). Acid-labile phosphate was measured as the orthophosphate liberated after heating at 100” for 10 minutes in 1 N H&O*. Total phosphate was determihed after heating at 150” for 1 hour in 10 N HzS04. Orotic acid and orotidine 5’-phosphate pyrophosphorylase were employed in the speetrophotometric measurement of PRPP (29).

All spectrophotometric determinations were carried out in a Beckman model DU spectrophotometer with a photomultiplier attachment which made possible measurements in the presence of crude protein solutions and other extraneous ultraviolet-absorbing materials. Nucleotides were sepa- rated from nucleosides and bases by paper chromatography with an ammonium acetate and ethanol solvent system (30).

Preparation of PRPP-PRPP was prepared and isolated by a modifica- tion of the method of Remy et al. (21). The incubation mixture consisted of 1.8 mmoles of ATP, 2.4 mmoles of ribose S-phosphate, 9.6 mmoles of MgC12e6Hz0, 18 mmoles of potassium phosphate buffer, pH 7.4, and lyophilized 0 to 15 per cent ethanol fraction of pigeon liver extract in a total volume of 600 ml. The incubation was carried out at 37”. The amount of enzyme employed and time of incubation necessary in each preparation were determined by a preliminary small scale experiment in which the rate of PRPP synthesis was measured. The reaction was terminated by chilling the incubation mixture to 0” and adding 26 gm. of Norit A. All


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470 MOSONUCLEOTIDES OF PURINE ANALOGUES

subsequent steps were carried out at temperatures below 5”. The mixture was agitated occasionally for 15 minutes, filtered through a layer of Celite, and 40 gm. of MgCL.6HzO were dissolved in the filtrate. The crude precipitate which formed upon the addition of 4 volumes of cold ethanol was collected by centrifugation. The precipitate was dried with alcohol and ether and stored at - 15’,

The precipitate was extracted three times with 30 ml. of cold distilled water. The extracts were combined and added to a Dowex l-formate column (10 cm. X 3 sq. cm., 200 to 400 mesh, 10 per cent cross-linked) at a rate of 2 ml. per minute. The column was washed with 100 ml. of water at a rate of 5 ml. per minute and eluted by t,he gradient procedure of Hurl- bert et al. (10). The mixing flask contained 500 ml. of distilled water and the reservoir contained 1.5 M ammonium formate, pH 5.0. This procedure resolved the incubation mixture into three ribose-containing fractions. The PRPP was eluted as the last ribose-containing peak which appeared after approximately 25 resin bed volumes of eluent. The fractions containing PRPP were pooled and neutralized to pH 7.5. Upon the addition of 20 gm. of MgC12.6H20 and 4 volumes of cold ethanol, the PRPP pre- cipitated as the magnesium salt. The Mg PRPP was washed with ethanol and ether and dried in vacua. This procedure resulted in the isolation of 0.40 to 0.54 mmole of Mg PRPP.

Results

Since various laboratories have reported the enzymatic synthesis of nucleotides from their respective bases (26, 31, 32), attempts were made to synthesize 8-aza-GMP directly from 8-azaguanine. Preliminary experi- ments in which ATP, ribose 5-phosphate, Mg++, 8-azaguanine, and phosphate buffer xere incubated with a pigeon liver enzyme syst,em (26) failed to result in appreciable conversion of 8-azaguanine to 8-aza-GMP. The apparent explanation of these failures will be discussed below.

8-aza-GMP Formation by Transphosphorylation of 8-Azaguanosine-In subsequent studies t,he preparation of 8-aza-GRIP was attempted by enzymatic phosphorylat,ion of the nucleoside, 8-azaguanosine. The isolation of beef liver purine nucleoside phosphorylase free from phosphoribomutase (25) permitted the syntheses of ribose 1-phosphat,e and 8-azaguanosine by procedures similar to those of Friedkin (33) as shown in Equations 1 and 2.

(1) Inosine + orthophosphate e ribose l-phosphate + hypoxant,hine (2) 8-Azaguanine + ribose l-phosphate e S-azaguanosine + orthophosphate

The 8-azaguanosine was prepared from a reaction mixture containing 0.4 mmole of ribose l-phosphate, 1.0 mmole of 8-azaguanine, and 20 mg. of beef liver purine nucleoside phosphorylase (Fraction III of Rowen and


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J. L. lV-4Y 9ND H. E. PARKS, JR. 471

Kornberg (25)) in 160 ml. The reaction mixture was incubated at pH 7.7 and 30” for 24 hours. The react,ion was terminated by heating the mixture in a boiling water bath and the residue was removed by centrifugation. The supernatant fluid was passed through a Dowex 50-H+ column (3 cm. X 3 sq. cm., 200 to 400 mesh, 12 per cent cross-linked) to remove the unchanged 8-azaguanine which was added in excess in t,he reaction. The eluate was placed on a Dowex 1-formate column (10 cm. X 3 sq. cm., 200 to 400 mesh, 10 per cent cross-linked), washed with 200 ml. of water, and eluted with 0.1 M formic acid. The fractions containing the nucleoside, %azaguanosine, which appear after 30 resin bed volumes, were pooled and lyophilized to a dry powder. By this procedure approximately 0.2 mmole of 8-azaguanosine was isolated.

In order t,o form t,he 5’-phosphate nucleotide, 8-azaguanosine was inter- acted with phenylphosphate, as in Equation 3, in the presence of barley malt phosphatase, according to the method of Brawerman and Chargaff (18).

(3) S-Aaaguanosine + phenylphosphate ---t phenol + orthophosphat,e + S-aza-GMP

The nucleotide was isolated by paper chromatography and detected on the paper as a fluorescent spot in ultraviolet light and by uranium fixation (34). Although the formation of 8-aza-GMP can be demonstrated by t,his step- wise synthesis, t,he nucleotide was produced in insufficient amounts to con- sider adoption of this approach for large scale preparations. However, recently Tunis and Chargaff (35) have reported the purification of a nucleoside phosphotransferase from carrots which carries out the phosphorylation of nucleosides in very good yield. This may make this type of nucleotide synthesis a practical one.

Reaction of S-Amguanine with Purine Nucleotide PyrophosphoryZase4n view of the isolation and characterization (21, 29) of a new intermediate, PRPP, which int.cracts with purine and pyrimidine bases to form their respect.ive 5’-phosphate nucleotides, t,hat method has now been employed in preparing nucleotides of purine analogucs in good yields by the reaction shown in Equation 4.

(4) 8-Azaguanine + PRPP t-------i S-aza-GMP + pyrophosphate (7)

It was found that the incubation of 8-azaguanine with PRPP in the presence of a dialyzed extract of beef liver acetone powder resulted in nucleotide formation. However, chromatographic and spectrophotometric examinations before and aft!er acid hydrolysis revealed the product to con- sist of a mixture of 8-aza-GMP and Sazaxanthine. Apparently the enzyme guanase, found in beef liver, brought about the deamination of 8-


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472 MOSONUCLEOTIDES OF PURINE ANALOGUES

azaguanine t.o 8-azaxanthine, resulting in gross contamination of the desired product. This difficulty was overcome by employing extracts of hog liver, since this tissue is virtually devoid of guanase activity (36). It was then possible to form 8-aza-GMP in essentially quantitative yields with respect to initially added PRPP.

Preparation and Isolation of 8-aza-GMP-The incubation mixture (800 ml.) contained 0.80 mmole of 8-azaguanine, 0.40 mmole of PRPP, 8.0 mmoles of MgC12*6HzO, 80 mmoles of Tris-HCl buffer, pH 7.0, and 700 mg. of the dialyzed, lyophilized hog liver acetone powder extract. After being incubated for 30 minutes at 37”, the mixture was heated to 95” in a boiling wat.er bath, maintained above this temperature for 2 minutes, immediately cooled, and centrifuged to remove the residue. The residue was resuspended in 100 ml. of distilled water and centrifuged. The supernatant fluids were pooled and passed through a Dowex 50-H+ column (5 cm. X 1 sq. cm., 200 to 400 mesh size, 12 per cent cross-linkage) to remove the unchanged 8-azaguanine which was added in excess to the reaction mixture. The eluate was neutralized t.o pH 8.0 with 1 M NH40H and added to a Dowex 1-formate column (10 cm. X 3 sq. cm., 200 to 400 mesh, 10 per cent cross-linked) at a rate of 2 ml. per minute. The column was washed with 150 ml. of water and eluted at a rate of 5 ml. per minute by a gradient procedure (10) with 500 ml. of distilled water in the mixing flask. The reservoir flask cont,ained initially 0.15 M ammonium formate, pH 5.0. After 250 ml. of the eluate were collected from the column with an auto- matic fraction collector, the solution in the reservoir flask was changed to 1.5 M ammonium formate, pH 5.0, to elute the 8-aza-GMP. The fractions containing the nucleotide were pooled and adsorbed on a column (3 cm. X 1 sq. cm.) containing a mixture of equal amounts of Celite and Darco S-51. The column was washed with water and eluted with a solvent system containing 50 per cent ethanol and 3 per cent NH*OH. About 70 to 85 per cent of the 8-aza-GMP can be recovered from the charcoal column. The solvent was evaporated and the nucleotide stored as a dry powder. This procedure resulted in t,he isolation of approximately 0.3 mmole of 8-aza- GMP.

Characterization and Properties of 8-aza-GMP-A sample of the isolated nucleotide was submitted to chemical and spectrophotometric analyses and found to have an approximately equimolar ratio of 8-azaguanine to ribose to phosphate. The phosphate group was shown to be on the 5’ position by use of the enzyme, 5’-nucleotidase, which liberated 8-azaguanosine from t,he nucleotide. After 1 hour incubation of 0.5 pmole of the nucleotide with Mg++ and 5’-nucleotidase at pH 8.5 and 37” (24), the reaction mixture was deproteinized by heating for 90 seconds and submitted to paper chromatography in an ethanol-ammonium acetate solvent.


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system (30). A single fluorescent spot was detected with an Rp of 0.73, corresponding to that of the riboside, 8-azaguanosine. In this system the nucleotide 8-aza-GMP migrates with an Rp of 0.28, which is similar to the Rp of other mononucleotides. The fluorescent spot was eluted with 0.01 M phosphate buffer, pH 7.4, and found to have an ultraviolet spectrum similar to that of 8-azaguanosine.

Fig. 1 shows the ultraviolet spectra of 8-aza-GMP at acid, neutral, and alkaline pH values. It is to be noted that these spectra are almost identical

FIG. 1 FIG. 2

‘7, /

/ ‘,B-Aza-GMP

\ BEFORE ‘, HYDFtOLWS

I I I , I I # Yrl

ii0 250 270 290 31 WAVELENGTH Cmp 1

FIG. 1. Ultraviolet absorption spectra of d-aza-GMP (0.035 pmole per ml.). -, 0.01 M potassium phosphate buffer, pH 7.5; . . , 0.01 M HCI; - - -, 0.01 M KOH.

FIG. 2. ELaza-GMP (0.1 pmole) was hydrolyzed with 0.6 N HCI by heating in a boiling water bath for 30 minutes. It was cooled, neutralized to pH 7.4, and the ultraviolet spectrum determined in a volume of 1.2 ml. For comparison, the spectra of 0.1 pmole of 8-aza-GMP (unhydrolyzed) and 8-azaguanine were similarly measured at pH 7.4 in a volume of 1.2 ml.

to those reported by Friedkin (33) for the riboside, 8-azaguanosine. In Fig. 2 are the spectra of 8-azaguanine and a sample of 8-aza-GMP before and after acid hydrolysis. This treatment causes the spectrum of the nucleotide to shift to one which resembles that of the base, 8-azaguanine.

Measurement of &uza-GMP Format&n-The marked spectral difference at 260 rnp between 8-azaguanine and its nucleotide (Fig. 2) has been made the basis of a method for determining the formation of 8-aza-GMP. Under the conditions employed (Fig. 3), 0.1 pmole of 8-azaguanine, being converted to the mononucleotide, causes an increase in ODzao of 0.610. This reaction has been used successfully in the measurement of PRPP concentrations as well as in the determination of the activity of 8-aza-GMP pyrophosphorylase (Fig. 3). It should be mentioned that the enzyme


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474 MONONUCLEOTIDES OF PURISE ANALOGUES

guanase similarly causes an increase in optical density at 260 rnp as 8- azaguanine is converted t,o 8-azaxanthine. Its presence may be noted by increases in OD260 which occur in the absence of PRPP. The above method has also been employed in the measurement of nucleotide formation from guanine and guanine pyrazolo(3 ,Qd)pyrimidine (Fig. 5).

It was occasionally noted t,hat during nucleotide synthesis the reaction mixtures became progressively turbid. This was apparently the result of

.4 I.5

$‘” 1.0

“0 .2

l?&&a

0.5

.I 0.25

O I TlM2E 1: MlGJTEs5

6 El -UANT ti20-&SAM-F-i.5M AM-F

FIG. 3 FIG. 4

FIG. 3. Assay of S-aza-GMP pyrophosphorylase activity. Optical density measurements were made at 260 mcc in Beckman DU spectrophotomet.er with photomultiplier attachment. Reaction mixtures consisted of the following: 8-uzaguanine, 1 X 10-4 M; PRPP, 2 X 1W M; Mg++, 0.01 M; Tris-HCI buffer, pH 7.0, 0.1 M; inorganic pyrophosphstase, 0.05 ml., and enzyme in the amount indicated in mg. on graphs in a final volume of 1 ml. The enzyme preparation used was dialyzed and lyophilized extract of acetone powder of hog liver. Temperat,ure 30”.

FIG. 4. Isolation of mononucleotide of guanine pyrazolo(3,4-d)pyrimidino by gradient elution from Dowex l-formate column. The chart represents a copy of a tracing from an Esterline-Angus recorder. The eluate from the column passed through ultraviolet absorption meter (Gilson Medical Electronics) before collection in consecutive 12 ml. fractions. Esterline-Angus (E.A.) units represent arbitrary galvanometer readings which depend upon the initial setting of the inst,rument.

the formation of an insoluble salt of pyrophosphate, since the inclusion of small amounts of inorganic pyrophosphatase in the reaction mixture usually abolished the effect.

Nucleotide Synthesis with Purine Analogues Other Than 8-Azaguanine- Since 8-azaguanine was found to react readily with PRPP, it, was of im- mediate interest to learn whether other purine analogues could be similarly convert,ed to their 5’-phosphate mononucleotides. This was first studied by incubating 1.2 Imoles of the particular analogue, 1 .O pmole of PRPP, and 5.0 pmoles of Mg++ with various relatively crude purine nucleot,ide pyrophosphorylase preparations in 0.1 M Tris buffer, pH 8.0, at 37” for 2 hours.


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The reaction mixtures were deproteinized by heat, were centrifuged, and the supernatant fluids submitted to paper chromatography. For comparison, an aliquot of the reaction mixt,ure inactivated immediat,ely after addition of the enzyme was similarly chromatographed. The synthesis of mononucleot.ides was detected by the appearance of new ultraviolet- absorbing or fluorescent spots with low RF values (0.2 to 0.35). The nucleotide spots were eluted from the paper and examined spectrophoto- metrically. When apparent nucleotide formation was observed, the experiment was repeated on a larger scale with 30 pmoles of the analogue and approximately 20 pmoles of PRPP. After incubation for 30 to 90 minutes, the heat-inactivated reaction mixture was added t,o a Dowex 1-formate column and &ted in a fashion similar to that employed wit,h 8-azaguanine. In each case the unchanged analogue was removed from the column during the water wash or immediat,ely after beginning gradient elution with 0.15 Ed ammonium formate, pH 5.0. When the acid concentration in the reservoir flask was increased to 1.5 M ammonium formate, pH 5.0, second peaks were eluted which contained the mononucleotides. An example of the elut.ion pat,tern observed with one of these analogues and its nucleotide is shown in Fig. 4. The nucleotides were concentrated by adsorption and elution wit,h Darco S-51 and were dried in vacua. Table I shows the results of these studies and some of the properties of t,he isolated nucleotides. It is notewort,hy that marked differences in substrate specificit,y were observed, depending upon the enzyme preparation used (Table I). Ex- haustively dialyzed hog liver acetone powder extracts formed nucleotides from guanine and hypoxant,hine analogues, while being inactive with adenine pyrazolo(3,4-d)pyrimidine, 6-mercaptopurine,’ and 2,6-diaminopurine. However, the hog liver, beef liver, and yeast preparations submitted to brief dialysis reacted with all of the analogues listed. In agreement with t,he findings of ot,hers (20, 37), this is evidence for the existence of at least two different purine nucleotide pyrophosphorylases, one of which is more labile, and t,he ot,her of which appears t,o require a hydroxyl group on car- bon 6 of the purine ring. However, purification studies will be needed to establish whether the latter is a single enzyme.

pH Optima and Inhibition StudiesAlthough information on such fact.ors as the Michaelis constants with different substrates, the reversibility of %aza-GMP pyrophosphorylase, et,c., is of obvious importance, it was not felt advisable to undertake these studies until a more purified enzyme is

8 Since the submission of this publication, Flaks, Erwin, and Buchanan (52) have reported the partial purification of the AMP-phosphorylase from beef liver and find that 6.mcrcaptopurine will not serve as a substrate. In our laboratory three sepa- rate attempts failed to synthesize the 5’-mononucleotide of 6-mercaptopurine by using a hog liver preparat.ion (exhaustively dialyzed) which was act,ive for guanine and 8-azaguanine but not for adenine.


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476 MONONtJCLEO!TIDES OF PURINE ANALOGUES

TABLE I

Properties of Nucleotides of Purine Andogues

Substance

8-Azaguanine

Cuanine pyraaolo(3,4d)pyrimidine

Adenine pyrazolo(3,4-d)pyrimidine

Hypoxanthine pyrazolo(3,4-d)- pyrimidine

2,6-Diaminopurine

6Mercaptopurine

-

.-

-

(1) Base (2) Nucleotide (1) Base (2) Nucleotide (1) Base (2) Nucleotide (1) Base (2) Nucleotide (1) Base (2) Nuoleotide (1) Base (2) Nucleotide

-

I

.-

-

Absorption naximum at

pH 7.0

mP

247, 275 257 251 253 261 261 252 252 242, 281 252 320 320

-

v,

.-

-

RF dl%’

0.76 0.28 0.69 0.25 0.88 0.31 0.85 0.29 0.65 0.31 0.61 0.33

H, B, Y

I‘ “ ‘L

B, Y

H, B, Y

B, Y

“ “

* Solvent system for paper chromatography: ethanol, 1.0 M ammonium acetate, pH 7.3,7:3, Whatman No. 1 filter paper.

t H = hog liver acetone powder extract dialyzed 24 hours against three changes of 40 volumes of 0.001 M Tris-HCl, pH 8.0; B = beef liver acetone powder extract dialyzed 6 hours against 40 volumes of 0.001 M Tris-HCl, pH 8.0; Y = autolysate of brewers’ yeast dialyzed 6 hours against 40 volumes of 0.001 M Tris-HCI, pH 8.0.

w ZOLO 3,4-d PYRIMIDINE

‘oI 160

E > 120

I 2 60

% cl 40

2 5.5 6 7 6 9

FIG. 5. pH optima for nucleotide formation from 8-azaguanine, guanine, and guanine pyrazolo(3,4-d)pyrimidine. Reactions measured as in Fig. 3; guanine or analogue concentration, 1 X 10e4 M. Buffers used at 0.1 M concentrations were as follows: pH 5.7 to 6.3, succinate; pH 6.6 to 9.4, Tris-HCl; pH 9.8, bicarbonate. pH values were determined immediately after assay procedures.

available. However, some evidence of interest on the pH optima and effects of inhibitors on the crude enzyme has been obtained. Fig. 5 shows the pH optima for nucleotide formation with hog liver acetone powder extract with 8-azaguanine, guanine, and guanine pyrazolo(3,4-d)pyrimi-


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dine as substrates. Marked differences in pH optima and rates of reaction were observed, with guanine pyrazolo(3,4+$pyrimidine reacting somewhat more rapidly (at its pH optimum) than does the normal purine guanine.

8-aza-GMP formation was profoundly inhibited by very low concentrations of ADP and ATP (Table II). The inhibitory effects of these materials were not overcome by increasing the concentrations of PRPP or Mg++. In the concentrations tested, ribose 5-phosphate was without effect, while inorganic orthophosphate causes marked inhibition. These interesting observations should be examined more fully with an enzyme of higher purity.

TABLE II

Inhibition of 8-aza-QMP Formation by Orthophosphate, ATP, and ADP

Compound

ATP

ADP Orthophosphate

Ribose &phosphate

C0lKelltrhXl

Y

5 x lo-’ 1 x lo-’ 1.5 x lo-’ 2 x lo-4 5 x lo-4 1 x lo-’ 2 x lo-” 1 x lo-’ 5 x lo-’

-

Inhibition

)M cent 47 57 72 85

100 66 17 81 0

8-aza-GMP pyrophosphorylase activities determined as in Fig. 3. Orthophos- phate added as potassium phosphate buffer, pH 7.0.

DISCUSSION

PRPP has been shown to react enzymatically with natural purines (20, 21)) pyrimidines (19), and pyridines (38) as well as with precursors in- volved in the de nova synthesis of purines (39,40). In the present study it is shown that this sugar phosphate will also react with analogues of natural purines, including those with non-purine ring structures; i.e., the triazolo- pyrimidines and pyrazolo(3,4-d)pyrimidines. It would be of interest to examine the activity of purine analogues with other ring types such as the 2-azapurines (41)) the l-deazapurines (42)) the pyrazolo(4,3-d)pyrimidines (43), and the benzimidazoles (44).

The fact that certain carcinostatic analogues react readily with purine nucleotide pyrophosphorylases raises the possibility that they may exert their antitumor action by competing with natural substrates for these enzymes. While this hypothesis warrants further investigation, it is also true that non-carcinostatic analogues such as hypoxanthine pyrazolo- (3 ,&d)pyrimidine also may serve as substrates (45).


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478 MOXONUCLEOTIDES OF PURINE ANALOGUES

Several considerations suggest t,hat these analogue-containing mononucleotides be tested for their chemotherapeutic effectiveness. There is a distinct possibility that these compounds may be dealt with by living tissues in ways differing from those of either the free bases or the ribosides, with the result of increa,sed therapeut.ic activity. Roll et al. (46), for example, have shown that guanine is incorporated into nucleic acids much more readily if administered as the nucleotide rat,her than as the nucleoside or purine base. Also, in the form of the nucleotide, an analogue may be less susceptible to attack by degradative enzymes. %Azaguanine, when administered parenterally, is rapidly converted to the non-carcinostatic compound, %azaxant,hine, presumably by the action of guanase (4749). The nucleotide 8-aza-GMP will not serve as a substrate for this enzyme and, therefore, should be less readily detoxified. Of course this may result in an analogue which is more toxic as well as more effective. A serious problem encountered in the testing of many purine analogues has been their slight solubility in water. Some are so poorly soluble as to preclude paren- teral administration of adequate amounts for therapeutic trial. In general, mononucleotides a.re significantly more soluble than the bases and may pro- vide an answer t,o this difficulty. The enzymatic procedures described in this report should lend themselves to the preparation of analogue-containing nucleotides in amounts adequate for preliminary chemotherapeutic tests. The principal difficulty encountered has been in the preparation and isolation of the extremely labile intermediate PRPP.

An explanation is now available for the low yields obtained when nucleotide synthesis was attempted in a reaction combining PRPP synthesis with nucleotide formation. Inorganic orthophosphate and ATP are both re- quired for t,he PRPP synthesizing step, while in turn acting as inhibitors of the 8-aza-GMP-forming reaction (Table II). The powerful inhibition shown by ATP and ADP suggests the existence of a metabolic mechanism which resembles a “negative feed-back.” Thus, if adequate concentra- t,ions of nucleotides are present, they would suppress the formation of new nucleotide from preformed purines. On the other hand, if free nucleotide concentrations fall, their synthesis would be promoted. Similar findings have been reported by ot,her laboratories. Saffran and Scarano (32) found that the addiCon of ATP alone to undialyzed pigeon liver preparations caused a decrease in the rate of conversion of adenine t.o its nucleotide. A marked inhibition of erotic acid incorporation into RXA has been observed wit,h increased ATP concentrations (50), and Yates and Pardee (51) found that ureidosuccinic acid formation is suppressed by cytidylic acid. These observations may have uncovered an important and widely occurring mechanism for the control of nucleotide and nucleic acid formation.

Since nucleotides of some purine analogues can now be prepared in good


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yield, work has been initiated on the synthesis and enzymatic test.ing of analogue-containing coenzymes, e.g. analogues of ATP, GTP, UTP, DPK, etc.

SUMMARY

1. 8-Azaguanine has been shown to interact with 5-phosphoribosyl-l- pyrophosphate and a hog liver extract to form 8-azaguanosine-5’-phosphate (8-aza-GMP) .

2. A method for measuring t,he enzymatic activity of 8-azaguanine nucleotide pyrophosphorylase has been described.

3. The preparation, purification, isolation in good yield, and the characterization of 8-aza-GMP are reported.

4. Ort,hophosphate and low concentrations of adenosine di- or triphosphate were’found to inhibit 8-aza-GMP formation.

5. 2,6-Diaminopurine, 6-mercaptopurine, and the adenine, hypoxanthine, and guanine analogues of the pyrazolo(3,4-d)pyrimidine series have been similarly converted to their respective 5’-nucleotides.

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James L. Way and R. E. Parks, Jr.PURINE ANALOGUES

5'-PHOSPHATE NUCLEOTIDES OF ENZYMATIC SYNTHESIS OF

1958, 231:467-480.J. Biol. Chem.

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