Volume 4 Number 3 March 1977

Nucleic Acids Research

Isolation and characterization of N6-succinyladenosine from human urine

Girish B.Chheda

Surgical Developmental Oncology, Dept. of General Surgery, Roswell Park Memorial Institute, Buffalo, NY 14263, USA Received 4 February 1977 ABSTRACT From the urines of colon carcinoma patients and normal subjects we have isolated a nucleoside in which an amino group of aspartic acid is attached to the six position of purine ribonucleoside. The structure, N6-succinyladenosine, N-(9-B-D-ribofuranosylpurin-6-yl)aspartic acid was assigned on the basis of spectral data, chemical degradation, and by synthesis. The ultraviolet and mass spectra, chromatographic and electrophoretic mobilities, and the chemical properties of the naturally occurring nucleoside were identical to those of the synthetic N6-succinyladenosine. In contrast to the methylated and hypermodified nucleosides which are products of RNA catabolism, this urinary nucleoside appears to be derived from adenylosuccinic acid, a key intermediate required in the biosynthesis of ubiquitous, natural purine nucleotide adenosine-

5'-monophosphate (AMP). INTRODUCTION; With an objective to look for the possible new markers in the urines of specific groups of cancer patients, we have been investigating the urinary substances related to nucleic acid anabolism and catabolism. Presence of more than twenty modified bases and nucleosides in normal and cancer urine has been documented (1). The modified nucleosides, N2-dimethylguanosine, 1-methylinosine and pseudouridine derived primarily from the catabolism of tRNA appear to be elevated in the urines of cancer patients as compared to those of the normals and non-cancer subjects (2, 3). The present paper deals with isolation and characterization of another type of modified nucleoside identified as N-succinyladenosine, from the urines of colon carcinoma patients (4). This nucleoside is also present in the urines of normal subjects, but in a smaller quantity. The N6-succinyladenosine represents a dephosphorylated product of adenylosuccinic acid, one of the important anabolic intermediates required in the biosynthesis of naturally occurring purine nucleotides, AMP and GMP

(5-7). 0 Infomaton Reeval Umited 1 Falconberg Court London W1 V 5FG England

739

Nucleic Acids Research MATERIALS AND METHODS Isolation The three day pooled urine (3800 ml.) from a colon carcinoma patient was adjusted to pH 2.5 with concentrated hydrochloric acid and passed through a column of charcoal-celite (200 g each) (5 x 80 cm) (8). The column was washed with water (8 litres) till chloride test was negative and the column bound material was eluted with 5 litres of 2N NH4OH in 50% EtOH. The ammonia eluate was concentrated to a small volume (100 ml) and then centrifuged to remove the insoluble precipitate. The concentrated clear solution was then passed through a column of Ag-l-x-8 (formate) (3.8 x 80 cm, 800 ml wet volume). The column was washed with six litres of water and the column bound material was eluted with three litres of 3N formic acid. The formic acid eluate was concentrated to a small volume at 20-25 C. The concentrate was then diluted with water and re-evaporated to a light brown liquid. This concentrated material was applied in narrow bands (0.5 x 47 cm) to 46 x 57 cm Whatman 3MM papers (20 papers). After ascending chromatography for 8 hrs at room temperature in solvent C, major ultraviolet absorbing areas corresponding to the marker nucleoside were cut out and eluted with distilled water. This combined eluate was concentrated and chromatographed successively in solvents G,A,B,D and H. This chromatographic purification gave the yellowish white material L(5.9 mg estimated by UV Amax, 268

(C,17,400)J. For mass spectral studies the material was further purified by applying a small sample on to CG-50 column (Amberlite, 100-200 mesh weakly acidic resin, polymethacrylic type, cation exchange, Rohm & Haas Co.) and then eluting with water. The aqueous solution containing the desired material was then lyophilized to white hygroscopic powder. In a normal batch, 55 ml of urine (5% of the 24 hr collection) was processed in a similar manner but with smaller size columns; charcoalcelite (3 g each 1.9 cm x 11.5 cm) (8) and Ag-l-x-8 formate (5 g.; 1.3 x 6 cm). The material was then purified by paper chromatography as described above to give 0.09 mg of the N6-succinyladenosine (1.8 mg/24 hour). Acid Hydrolysis of N -Succinyladenosine A solution of 20 O.D. units (A268) of urinary N -succinyladenosine in 400 ul of 1N HC1 was heated in a sealed tube at 1000c for 30 minutes. The solution was streaked on 3MM paper along with the markers of N-(purin -6-yl) aspartic acid (N -succinyladenine) and ribose. The paper was developed in solvent D for 7 hours. The single uv absorbing band cor740

Nucleic Acids Research responding to the authentic free base (9) was isolated by elution with water (14 O.D. units). The chromatographic and electrophoretic mobility and uv spectra of the hydrolysis pxoduct were determined (Table 1, and Fig. 2). Sugar was detected by aniline hydrogen pthallate and was found to be of the same chromatographic mobility as that of ribose. Preparation of N6-Succinyladenosine (a) Chemical Synthesis (Scheme I) (10) To a suspension of 285 mg (1.0 mmole) of 6-chloropurine riboside, in 2 ml of water was added 665 mg (5 mmoles) of L-aspartic acid in 4 ml of water (pre-adjusted to pH 9.5 with 20% aqueous potassium hydroxide). The solution was refluxed for two hours and then after cooling to room temper ature, ethanol (300 ml) was added. The resulting precipitate was filtered and washed with cold absolute ethanol and then dissolved in minimal amount of water. This solution was applied to a column of Ag-l-x-8 formate. The column was washed with 150 ml of 0.3N formic acid, (aspartic acid was eluted in this wash), and then eluted with 150 ml of 2N formic acid. This 2N solution was evaporated to dryness and the

residue was triturated with water and re-evaporated to dryness. To this residue ether was added and the mixture was the cooled, the product (Lit. (10) m.p., was collectedo7n a filter, 251 mg (65%) m.p. 240-245 268 (&, 17,400). 245-250°0)Amax .

(b) By Enzyme Hydrolysis of Adenylosuccinic Acid To 1.0 mg (2.2Amoles) of the nucleotide (Sigma Chemical Co.) in 20 jil of H20 was added, 20 4l of 1M tris-HCl buffer (pH 9.0) and 25 ul of a solution of 5'-nucleotidase (10 mg/ml, Sigma Chemical Co.). The solution was incubated at 370C for 3 hr. The solution was streaked on a single 8" wide sheet of the acid washed paper along with a marker of the synthetic nucleoside, and the paper was developed in solvent A. A single major UV absorbing product was isolated from paper by elution with water. The UV spectra and chromatographic and electrophoretic mobilities of this sample were identical to both the urinary nucleoside

(I) and the synthetic N6-succinyladenosine. RESULTS The nucleoside N -succinyladenosine, N-(9-B-D-ribofuranosylpurin-6yl)-L-aspartic acid was synthesized in good yield from 6-chloropurine riboside and L-aspartic acid as described above (Scheme I). The material was also prepared from the biosynthetic intermediate adenylosuccinic acid by treatment with 5'-nucleotidase. The uv spectra of the 741

Nucleic Acids Research COOH NH-CH-CH2-COOH

?OOH L-Asporti Acid..NH-CH-CH2-COOH

V*-Nuebofidon

N>zL-Asvlic Acidt N-'

_

s

44O0 4

HO4 HO OH N6-Succinylodenon (I) HCI I J,COOH

HOO 6-Chloropurine riboside

HOOH Adesnyloswccinic acid (l)

NH--CH-CH2-COOH N

N

N-(Purin-6-YL)-L-Asporic ocid (I)

SCHEME I material isolated from urine were identical to those of the synthetic sample and the enzymatically prepared material (Fig. 1). The chromatographic mobilities of the urinary nucleoside in 10 solvent systems were identical to those of the synthetic sample (Table 1). The electrophoretic behavior of the urinary nucleoside at pH 2 and 9.5 was also identical to the synthetic sample. The acid hydrolysis of the urinary material gave the product which was identical to the synthetic N-(purin-6-yl)-L-aspartic acid both in ultraviolet spectra (Fig. 2) and in the chromatographic and electrophoretic mobilities (Table I). The authentic N-(purin-6-yl)-L-aspartic acid was prepared

FROM URINE

0.6

0.6

~~~~~~~~~~~~~~~0.5

0.5 /

0.4

SYNTHETIC

/

0.4

d

0.3

~.

-j0.3

nm im

742

Nucleic Acids Research TABLE I

SOLVENT SYSTEMS

(Rf

x

100)

Electrophoresis I(Distance es)

A

B

C

D

E

F

G

H

I

J

(N6uclinylI adenosine)

12

49

49

10

0.1

0.9

49

60

12

44 +10.8 -3.2

N6-succinyladenosine (Synthetic) (I)

12

48

49

10

0.1

0.9

49

60

12

44 +10.8

Acid Hydrolysis Product Of Urinary Nucleoside (I)

9

59

41

9

0.1

0.1

42

47

16

+14.0 -5.4

N-(purin-6-yl)

9

59

41

9

0.1

0.1

43

47

17

+14.1

Compounds

K

L

Urinary

-3.2

-5.4

aspartic acid

Chromatograms developed in a descending manner on Whatman No. 1 paper for 16-20 hours in systems A,B,E,G,H and I and for 6-8 hours in systems C,D,F and J. U.V. absorbing spots were detected by viewing the chromatogram under a short wave lamp at 254 nm. The following solvent systems (Proportion by volume) were used:

A) B) C) D) E) F) G) H) I) J) K)

L) M)

7:2:1 2-Propanol : water : conc. NH40H 66:34 Iso-Butyric Acid : 2N NH40H 4:1:2 Ethyl Acetate,: 2-Ethoxyethanol 16% formic acid 6:1:2:1 Acetonitrile,: n-Butanol 0.1 Ammonium Acetate : conc. NH40H 86:14:5 n-Butanol, : water: conc. NH40H 4:1:2 Ethyl Acetate n-Propanol : water 55:10:35 n-Propanol : conc. NH40H : water 60:40 n-Propanol : water 4:1:2 n-Butanol : conc. Acetic Acid : water 1:1 Dimethylformamide 60% Ethanol 0.05 M Glycine was adjusted to pH 9.2 with NaOH, Electrophoresis was carried out at 52 volts/cm for 1 hour. 1:1 2M formic acid : 1.5 M acetic acid pH 2, 43 volts/cm, for 1 hour 7:3 Ethanol: 1M Ammonium Acetate (This solvent was used in preparative paper chromatography for isolation of the urinary nucleoside I)

SYNTHETIC

HYDROLYSIS PRODUCT

am

Fip

2-

thuwwhW

SPsh

d

N-

'

b.6in

ael ao

tic

OK o@*oiuy

pb_

WA

uho _c ,W hrio) 743

Nucleic Acids Research from 6-chloropurine and L-aspartic acid (9) . In addition to the free base, ribose liberated from the nucleoside was also characterized by chromatographic comparison with the authentic sample. The amino acid analysis of the 6N HCI hydrolysate of the urinary nucleoside I gave aspartic acid. The mass spectra of both, the natural sample and the synthetic N6-succinyladenosine (I) gave after trimethylsilylation, a molecular ion at 815 corresponding to the hexa TMS derivative of the N6-succinyladenosine. The fragmentation pattern of the two samples was also similar. From the above studies the urinary nucleoside is assigned the structure, N6-succinyladenosine (I). On the basis of the configurations in the naturally occurring adenylosuccinic acid, it would seem that in the urinary N6-succinyladenosine glycosidic linkage is (3and that the aspartic acid is in the L-configuration. These aspects of the structure, however, need further supporting studies. DISCUSSION Urinary N6-succinyladenosine appears to be formed by dephosphorylation of the purine biosynthetic intermediate, N6-succinyladenosine 5'-phosphate more commonly termed as adenylosuccinic acid. In biological systems, inosine 5'-monophosphate (IMP) from salvage or from the de-novo pathway reacts with aspartic acid and is converted into adenylosuccinic

acid. This reaction is mediated by the enzyme adenylosuccinate synthetase and GTP. The adenylosuccinic acid (II) is then cleaved by the enzyme adenylosuccinate lyase resulting in the formation of AMP and fumarate. Thus the biosynthesis and metabolism of this intermediate appears to be regulated primarily by the above two enzymes. Preliminary studies indicate that N-succinyladenosine is present in the range of 1 to 1.8 mg in 24 hour urine collection of normal subjects (three subjects), as compared to the levels of 2 to 5 mg in the urines of the patients with colon carcinoma (four subjects). In order to determine if the key nucleotide adenylosuccinic acid and the previously characterized free base N-(purin-6-yl)aspartic acid (11,12,13) were also excreted in urine along with the nucleoside N6-succinyladenosine, one urine sample from each category was re-investigated specifically for the above two compounds. Under the isolation conditions avoiding strong acids and alkalis, neither the nucleotide (II) nor the free base (III) could be detected in normal or colon carcinoma urine. Using high pressure liquid chromatography, additional samples are currently under 744

Nucleic Acids Resarch investigation for the presence of these two substances in urine. At this point it is not known as to the tissue source of the urinary N -succinyladenosine in the normal subjects. It is suggested that it is a dephosphorylated metabolite of the endogenously derived adenylosuccinic acid. The urinary N-succinyladenosine, in contrast to the methylated nucleosides such as N -dimethylguanosine, N6-methyladenosine (1,14) and the anticodon adjacent nucleosides 1-methylinosine (3) and N-(purin-6ylcarbamoyl)-L-threonine riboside (15,16) derived primarily from catabolism of tRNA, may well represent the anabolic picture of the nucleic acid synthesis. Increased levels of this nucleoside (I) in the urines of the patients with colon carcinoma could well be due to the elevated levels of adenylosuccinate lyase in the tumor. In order to fully evaluate the potential of this nucleoside as a possible marker of neoplastic activity further work involving measurement of the nucleoside in many more

samples is essential.

ACKNOWLEDGMENTS: The author thAnks Dr. A. Mittelman for useful discussions, Drs. E. Holyoke and G. Murphy for their keen interest, Mr. C.F. Piskorz for excellent technical assistance in this work and Dr. L. Baczynskyj for providing the mass spectra on synthetic and natural material. This work was supported by a U.S.P.H.S. grant CA 14185-03.

REFERENCES 1 2

3 4

5 6

7 8

9 10

Chheda, G.B. (1975) In The Handbook of Biochemistry, 3rd Edn. Fasman, Vol. I, Chemical Rubber Co. Cleveland, Ohio Chheda, G.B., Mittelman, A. and Grace, J.T. (1969) J. Pharm. Sci. 58, 75 Waalkes, T.P., Gehrke, C.W., Zumwalt, R.W., Chang, S.Y., Lakings, D.B., Tormey, D.C., Ahmann, D.I. and Moertel, C.G. (1975) Cancer 36, 390 Chheda, G.B. and Mittelman, A. (1976) Abstracts of the 172nd Meeting of the American Chemical Society, San Francisco, August Carter, C. and Cohen, L.H. (1956) J. Biol. Chem. 222, 17 and (1955) J. Am. Chem. Soc. 77, 499 Liberman, I. (1956) J. Biol. Chem. 223, 327 and (1956) J. Am. Chem. Soc. 78, 251 Mansurova, S.E., Shabarova, Z.A. and Kulaev, I.S. (1967) Vestn. Mosk. Univ., Khim. 22, 70 To a mixture of 200 g of neutral charcoal and 200 g of acid washed celite (Johns Manville Co.) was added 100 ml. of water and then mixed well. The moist mixture was packed tightly into a precision bore glass columm. Ward, D.N., Wade, J., Walborg, E.F. and Osdene, T.S. (1961) J. Q. Chem. 26, 5000 HAmpton, A. (1957) J. Am. Chem. Soc. 79, 503 745

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12 13 14 15 16

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Park, R.W., Holland, J.F. and Jenkins, A. (1962) Cancer Res. 22, 469 Weissman, B. and Gutman, A.B. (1957) J. Biol. Chem. 229, 239 McCloskey, J.A., Barnes, L.B., Crain, P.F., Lyman, K.J. and Bishop, S.H. (1975) Biomed. Mass Spettrometry 2, 90 Fink, K. and Adams, W.S. (1968) Arch. Biochem. and Biophys. 126, 27 Chheda, G.B. (1969) Life Sciences 8 (II), 979 Chheda, G.B., Hong, C.I., Dutta, S.P., De, N.C. and Parthasarathy, R., (1974) In Proceedings of the Symposium on the Recent Developments in Oligonucleotide Synthesis and the Chemistry of Minor Bases of tRNA, Polish Academy of Sciences, Poznan, Poland