This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution4.0 International License.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschungin Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung derWissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht:Creative Commons Namensnennung 4.0 Lizenz.

Selective Suecinylation of Adenosine Catalyzed by 4-Morpholine-N,N' -dicyclohexylcarboxamidine

C. Sauer and U. Schwabe* Institut für Pharmakologie und Toxikologie der Medizinischen Hochschule Hannover, Karl -Wiechert -Allee 9, D-3000 Hannover 61 Z. Naturforsch. 36b, 750-754 (1981); received February 2, 1981

Adenosine, Succinyl Adenosine Derivatives, 4-Morpholine-N,N'-dicyclohexylcarboxamidine Succinyl derivatives of adenosine are synthesized by direct reaction with succinic

anhydride catalyzed by 4-morpholine N,N'-dicyclohexylcarboxamidine under kinetic or thermodynamic conditions and partial hydrolysis. Derivatives obtained were N6-succinyl adenosine, N6,5'-0-disuccinyl adenosine, 3'(2')-0-succinyl adenosine, N6,3'(2')-0-di-succinyl adenosine and 3'(2'),5'-0-disuccinyl adenosine. The last three derivatives were crystallized and gave equilibrium mixtures of 2'-0- and 3'-0-isomers in aqueous solvents with the 3'-0-isomer prevailing. Differentiation of isomers was done by PMR. A mechanism for isomerisation is proposed. The products were purified by anion exchange chromato-graphy and desalting. The procedures are useful for pharmacological applications because of the purity and yield of products.

Introduction

The increasing interest for the nucleosides has initiated the search for selective acylation of sugar hydroxyls. Different methods of activation and protection of single sugar hydroxyls have been employed [1-3]. Recently a new method for the selective 2'-0-substitution of nucleosides has been introduced. Adenosine 3',5'-cyclic monophosphate (cyclic AMP) was succinylated in the 2'-0-position and the product 2'-0-succinyl cyclic AMP enzym-atically dephosphorylated [4]. This paper presents methods for the synthesis of further succinyl derivatives of adenosine employing 4-morpholine N,N'-dicyclohexylcarboxamidine as a catalyst. The products synthesized were: N6-succinyl adenosine (N6-SA), N6,5'-0-disuccinyl adenosine (N6,5'-SA), 2'-0-succinyl adenosine (2'-SA), 3'-0-succinyl ade-nosine (3'-SA), N6,2'-0-disuccinyl adenosine (N6,2'-SA), N6,3'-0-disuccinyl adenosine (N6,3'-SA), 2',5'-0-disuccinyl adenosine (2',5'-SA), 3' ,5'-0-di-succinyl adenosine (3',5'-SA). They give an equi-librium mixture with their 2'(3')-isomers in aqueous solvents. We have specially refined the conditions of obtaining N6-succinyl adenosine used for pharma-cological purposes.

Material and Methods Chemicals and thin layer chromatography

Adenosine, 5'-AMP and cyclic AMP were pur-chased from Boehringer, Mannheim. N6-Butyryl

cyclic AMP, N6 , 2 '-0-dibutyryl cyclic AMP, 2 ' -0-succinyl cyclic AMP and 4-morpholine-N,N'-dicyclo-hexylcarboxamidine (morpholine-DCC) were pur-chased from Sigma (München). Thin layer plates coated with cellulose and fluorescence indicator CEF were purchased from Riedel de Haen (Hanno-ver) and silica gel TLC plates 60 F 254 from Merck (Darmstadt). All other reagents were applied in p.a. quality. Chromatography was performed in the following solvent systems: A : 2-butanol/methanol/ ethylacetate/glacial acetic acid/water ( 7 : 3 : 4 : 2 : 4 ) ; B : ethanol/0.5 M ammonium acetate (5:2).

Instrumentation UV spectra were recorded on a Zeiss PMQ II or

an automatic DMR 22 spectrometer. Elution dia-grams of column chromatography were taken when-ever available with connected L K B uvicord UV meters at 254 nm and 280 nm. Mass spectra were recorded on type CH 4 of AEI, Manchester/England. PMR was applied to distinguish between 2 ' -0- and 3'-0-isomers [4]. The relative proportion of the 2 ' -0 - and 3'-0-isomers in equilibrated isomer mix-tures was estimated after the ratio of the heights of the low field (or high field) signals of the H(6) doublet [5],

Three standard procedures were employed for hydrolysis. Strong alkaline hydrolysis: 20 /A of the original fraction of the column chromatography were heated with 20 /u\ 5 N K O H for 20 min at 56 °C. Weak alkaline hydrolysis: 20 /u\ of one frac-tion and 20 /A 0.1 N NaOH were kept for 3 h at room temperature. Strong acidic hydrolysis: 20 /ul of one fraction were heated with 20 [A 9 8 % formic acid 1 h at 95 °C.

* Reprint requests to Prof. Dr. med. Ulrich Schwabe, Institut für Pharmakologie und Toxikologie der Univer-sität Bonn, Reuterstr. 2b, D-5300 Bonn 1. 0340-5087/81/0600-0750/$ 01.00/0

Table I. Reaction conditions of succinylation of adenosine.

Type Pyridine Succinic anhydride Reaction time, °C Centri- Column size Buffer [ml] [mg] fugation [cm] (M NH4AC)

I II III IV

5

5

1 5

9

150 80

2500 3000

10 min, 20 °C 24 h, 50 °C 20 min, 20 °C 16 h, 37 °C

+ 3 0 X 2 . 5

3 0 x 2 . 5

3 0 x 2 . 5

2 0 x 2 . 5

0.3 0.2

0.3 Gradient

Results

Succinylation of adenosine

The succinylation of adenosine by succinic an-hydride was performed with four variations of conditions to yield different sets of reaction products :

Type I: Equimolar amounts of adenosine and succinic anhydride under kinetic control.

Type II: Equimolar amounts of adenosine and succinic anhydride under thermodynamic control.

Type III: Surplus of succinic anhydride under kinetic control.

Type IV: Surplus of succinic anhydride under thermodynamic control.

250 mg adenosine and 300 mg morpholine-DCC were dissolved in different amounts of pyridine indicated in Table I with 1 % water added. Solution was completed by heating under reflux for 20 min. The different amounts of succinic anhydride were immediately added with thermodynamically con-trolled reactions. After the reaction was completed the solution was lyophilized. With kinetic control the reaction solutions were slowly cooled to room temperature to give oversaturation before succinic anhydride was added. After 10 or 20 min reaction the surplus succinic acid was centrifuged if neces-sary. The pellet was washed and the solution three

times lyophilized to get rid of the solvent pyridine. The lyophilisate dissolved in the starting buffer was applied to a QAE-Sephadex A-25 column and eluted with 2 1 buffer (4 min per fraction).

Fractions corresponding to single nucleosides were pooled and either applied as they were on the desalting column or lyophilized and dissolved in the smallest possible volume of water. Fractions con-taining mixtures of nucleosides from the overlap of different peaks were subjected to a desalting procedure under modified conditions since hydro-chloric acid allows separate elution of N6- and O'-substituted nucleosides because of base protonation at different pH values.

The yield of succinylated adenosine derivatives for the different types of reaction conditions is shown in Table II and Figure 1. The desalting procedure of crude N6 ,3 ' -0-succinyl adenosine (N6 ,3 '-SA) is reported as an sample for the working up. The crude N6 ,3 ' -SA was put in 1 ml volume on 30 X 2 cm QAE-Sephadex A-25 column. The column was rinsed with water and eluted with 700 ml 100 mM HCl in 8 h and 80 fractions. The small peak (fraction 14) with Amax = 2 6 0 n m contained 0- ,0-disubstituted nucleosides, the large peak (fractions 16 to 21) with /max = 272 nm contained N6 ,3 ' -SA and its isomer N 6 ,2 ' -SA (98.3% yield). N6 ,3 ' -SA crystallized from this isomer mixture in 7 2 % yield in aqueous ethanol.

Table II. Yields of succinylated adenosine derivatives after different types of reaction conditions (see Table I).

5'-SA Type Eluted Adenosine N6-SA 2'-SA N6,5'-SA 2',5' -SA N6 ,2' -SA Type

fractions 2'-SA 3'-SA 3',5' -SA N6 ,3 ' -SA 3'-SA [%] [%] [%] [%] [%] [%]

I 216 17 28 9 43 II 230 1 50 9 34 4 III 280 8 17 64 3 IV 280 see Fig. 1

Fig. 1. Yield of succinylated adenosine derivatives in presence of surplus of succinic anhydride under thermo-dynamic control (type IV). E Amax is plotted versus the eluted fractions. E Amax is the extinction at the Amax of the UV spectra taken of every single fraction. The left part of the diagram is enlarged. The column was eluted with a gradient of 1 1 125 m l ammonium acetate plus 10 mM phosphate buffer, pH 7 and 1 1 1 M ammonium acetate plus 10 mM phosphate buffer, pH 7. Result: Excess succinic anhydride and thermodynamic control favor polyacylated adenosines.

Desalting of 2',5'-SA resp. 3',5'-SA with hydrochloric acid gave isomer mixtures 70% 3',5'-SA was crystal-lized from aqueous ethanol not dependent if it was started from the 2'- or 3'-isomer. Problems arising from isomerisation during desalting can be avoided if lyophilisable ammonium acetate is used as eluent of the column chromatography. Peaks from acetate elution were not as sharp.

Preparation of NG-succinyl adenosine by deacylation of higher substituted adenosines

10 mg (21.4 /miol) of a mixture of several di-succinyl adenosine derivatives (N6,2'-SA; N6 ,3'-SA; N6 ,5'-SA) dissolved in 2 ml 50 mM sodium hydroxide were stirred for 15 min. The solution was neutralized and lyophilized. The recovery of N6-succinyl adenosine from column chromatography with 100 mM ammonium acetate as eluent was 80%. The other product eluted was adenosine.

Structure determination The structural features were determined by a

quickly applicable method combining data from column and paper chromatography and UV since some acylated nucleosides are known to rapidly establish an equilibrium of 2 ' -0- and 3'-0-isomers and to hydrolyse. Substitution of the sugar moiety

does not change the Amax (258-260 nm) whereas substitution of the base moiety shifts Amax to significant longer wavelengths (271-273 nm). The Rf values of the reaction products were compared with similar substances and products prepared in different synthetic ways [4] (Table III) .

Table III. Rf and Amax values of nucleosides and succinyl derivatives of adenosine.

Amax TLC- TLC-at pH 6 System A System B

2'SA 259 0.61 0.48 Adenosine 258 0.51 0.50 Adenine 0.54 0.52 Hypoxanthine 247 0.48 0.48 5'-SA 259 0.62 0.52 N6-SA 270 0.64 0.54 N6,5'-SA 270 0.69 0.28 3',5'-SA 260 0.76 0.23 2',5'-SA 260 0.76 0.25 N6,2'-SA 270 0.69 0.24 N6,3'-SA 270 0.76 0.24

Hydrolysis experiments under alkaline conditions gave deacylation, under strong acidic conditions deacylation and cleavage of the N-glycosidic bond. The succinates substituted at the base moiety were

N®SA CHG—CHG-COOH M-CH2O

CO 1 NH

\ HOCH^A^J

OH OH

M + = 367

-CH3-CH2-COOH

M—74

M —89

c=o I NH \ Kj

xy OH OH

293

OH 2-CH2-COOH

CO I NH >

1 CH Ii CH 1 H

278

NH2

OH OH 250

CO—CH2-CH2~COOH

NH > C H 2

248

CO-CH2-CH2-COOH

NH

R > B+H

236

222= 248-CN

CH2-CH2-COOH

CO

NH 1 1 > /\

OH H

264

208 « 235-HCN

+

OH

194

177 = 178-1

163 = B+H

Fig. 2. Degradation pattern of N6-succinyl adenosine in the mass spectrometer.

more resistent to hydrolysis than those substituted at the sugar moiety.

Up to ten peaks of the column chromatography had to be identified. The nucleosides with the strongest ester bond were expected to be eluted first under the conditions applied. N6-SA was expected to be the most resistant to hydrolysis similar to N6-succinyl cyclic A M P [6], 5'-SA as an ester of a primary alcohol contains a stronger ester bond than 2' -SA and 3'-SA. Elution of 5 ' -AMP prior to 3 ' -AMP [7], o f N6-succinyl cyclic A M P prior to 2 ' -0-succinyl cyclic A M P [6] and of 2 ' ,5 ' -0-disubstituted nucleo-sides prior to 3' ,5'-0-disubstituted nucleosides on ion exchange columns have been reported [8].

Two orders of elution appeared most likely: First, 5 ' -SA > N6 -SA > 2 ' -SA > 3 ' -SA and second, N 6 -SA > 5 ' -SA > 2'-SA > 3'-SA. The first interpre-tation was established by trying to match different

interpretations with the peaks considering their Amaxj their charges, the products they gave under different conditions, their Rf values. The products were compared with those of the three other types of succinylation and nucleosides prepared by other methods {e.g. 2 ' -SA and 3'-SA [4]).

The masspectrum of N 6 -SA contained several m/e typical for N6-substitution (relative intensity): 264 (3%) , 248 (7%) , 236 (7%) , 222 (10%) , 208 (10%) and further m/e: 293 (6%) , 278 (2%) , 250 (25%) , 194(3%) , 177(31%) , 163(100%) , 135 (99%) (Fig. 2).

Discussion The elution diagram of the column chromatography had shown the two disuccinylated isomer pairs (2',5'-SA, 3' ,5 '-SA and N6 ,2 ' -SA, N6 ,3 ' -SA) in four distinct peaks whereas the monosuccinylated 2 ' -SA and 3'-SA were eluted in just one peak. The neutral

HOCH2/°N HOCH

OH O

HOCH2

V I7" -* J. X ^ C = 0 H +

HOCH

O Fig. 3. Proposed mechanism of the H isomerisation of 2 ' -0- and 3 ' -0 -

acylated nucleosides

conditions of the column (strong buffer of pH 7, about 4 °C) seemed to have slowed down the iso-merisation rate of the disuccinylated sufficiently, whereas 2 ' -SA and 3' -SA did isomerise on the column. The assumption of a greater stability of higher acylated nucleosides is consistent with reports that tribenzoylated nucleosides had been separated by column chromatography without problems arising from stability of isomers (N6 ,2 ' -0,5 ' -0-tri -benzoylcytidine and N 6 ,3 ' -0 ,5 ' -0-tr ibenzoylcyti -dine) whereas monobenzylated derivatives were reported to isomerise fast [9]. The desalting proce-dure showed that hydrochloric acid under the applied conditions is strong enough to establish equilibria of disuccinylated nucleosides.

Only 3 ' - 0 isomers were found to crystallize from mixtures of 2 ' - 0 and 3 ' - 0 isomers. 3 ' - 0 acylated nucleosides have been reported to prevail over their 2 ' - 0 isomers in different solvent systems (solvent: Me2SO/() .2M phosphate buffer p H 7, 8 :1 [5]; sol-vent : water free pyridine [10]). The equilibrium constant K 2 / K I was about 2, where KI is for the isomerisation from 2' to 3' and K 2 for 3 ' to 2'. This is consistent with our findings. Isomerisation has been reported to be 80 to 2000 times faster than acyl hydrolysis for 3 ' -0 - formyl adenosine and 3 ' -0 -acetyl uridine. Therefore an isomerisation mech-

anism has to be faster than usual hydrolysis mech-anism. According to the finding of acid catalysis for isomerisation a " f a s t " intramolecular mechanism is proposed (Fig. 3).

Chheda claimed to have found N6-succinyl adenosine in the urine of a colon carcinoma patient [11], Two facts reported in his paper support it that his product is not N6-succinyl adenosine but N-(9-/?-0-ribofuranosylpurin-6-yl)-L-aspartic acid. The synthesis of the urinary nucleoside from 6-chloropurine riboside and L-aspartic acid was possible. Acid hydrolysis of the urinary nucleoside produced not the expected adenosine but N-(purin-6-yl)-L-aspartic acid. In that case all of the nucle-osides synthetisized in our laboratory have been prepared for the first time.

The chemistry o f succinylates provides several interesting applications for these derivatives of adenosine. The free carboxylic acid group of the succinyl nucleosides enables connection with hy-droxyl or amine groups of other molecules, e.g. synthetic polymers for the proof of adenosine receptors on the exterior surface of cell membranes. Additional uses are in pharmacology, e.g. testing their influence on enzyme like phosphodiesterase. Highly purified succinyl adenosines are necessary for these purposes.

[1] H. P. M. Fromageot, B. E. Griffin, and J. E. Sulston, Tetrahedron 23, 2315 (1967).

[2] O. Wagner, J. P. H. Verheyden, and J. G. Moffatt, J. Org. Chem. 39, 24 (1974).

[3] K. K. Ogilvic and D. J. Iwacha, Tetrahedron Lett. 4, 317 (1973).

[4] C. Sauer and U. Schwabe, Z. Naturforsch. 35c, 163 (1980).

[5] B. E. Griffin, M. Harman, C. B. Reese, J. E. Sulston, and D. R. Trentham, Biochemistry 5, 3638 (1966).

[6] H. L. Cailla and M. Delaage, Anal. Biochem. 48, 62 (1972).

[7] W. E. Cohn and E. Volin, Nature 167, 483 (1951). [8] M. Keren-Zur, R. Levy, and Y . Lapidot, Nucleic.

Acids Res. 2, 2289 (1975). [9] D. H. Rammler and H. G. Khorana, J. Am. Soc.

Chem. 84, 3112 (1962). [10] C. B. Reese and D. R. Trentham, Tetrahedron

Lett. 29, 2467 (1965). [11] G. B. Chhedda, Nucl. Acids Res. 4, 739 (1977).