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The parameters and the measuring principle of a urease electrode are described. The urease is physically entrapped in cellulose triacetate on the active surface of a pH glass electrode with an internal reference electrode. The Michaelis constant of immobilized urease was similar to that of soluble enzyme (KM = 2.40 mmol/L). The average apparent enzyme activity amounts to 2 IU per sensor preparation. An enzyme loading factor of 0.04 was determined. It indicates kinetically controlled reaction conditions of the sensor. A fixed-time measuring procedure provides a response time of 30 seconds and a linear response within a concentration range of 2 to 40 mmol/L urea in real biological samples (e.g., serum). INT.RODUCTI0NTwo distinct reaction conditions-control by mass transport or reaction kinetics-characterize the working parameters of enzyme electrodes. Enzyme electrodes with high immobilized enzyme activity have a diffusion-controlled overall reaction [ 11. These enzyme electrodes have a high, linear response and are little influenced by interferences [ 1, 21.Kinetically controlled conditions are characteristic for enzyme electrodes prepared with a low enzyme activity. Because of the low working stability and long response time as well as the influence of interfereii;z:q these conditions are generally considered unfavorable. This contribution will show that by modifying the enzyme membrane preparation and the measuring procedure, useful enzyme electrodes (even with enzyme membranes bearing low activity) can be achieved. For the urea determination with a rate-controlled potentiometric enzyme electrode, linear response characteristics, a measuring frequency, and a working stability similar to those of diffusion-controlled enzyme electrodes have been obtained. A detailed analysis of enzyme membrane parameters will be given. EXPERIMENTAL Urea ElectrodeThe pH glass electrode EGA 501 N from Forschungsinstitut, Meinsberg, is wetted and dipped into lyophylized urease (from VEB Arzneimittelwerk, Dresden) with an enzyme activity of 2 IU/mg. The urease-covered elecTo whom correspondence should be addressed 0 1989 VCH Publishers, Inc.trode is then immersed in a solution of 25 mg of cellulose triacetate (CTA) per milliliter of methylene chloride. The entrapped urease is dried for 1 minute in air. Before the measurement the enzyme sensor is equilibrated in the measuring solution for 1 hour. ApparatusThe enzyme electrode is arranged in a thermostated measuring cell with a volume of 5 mL (Figure 1). It is connected with a digital pH meter (MV 87, VEB Praecitronic, Dresden), and pH changes are registered with a recorder; 50 p L of sample is pipetted into the containing cell. Determination of Membrane ThicknessThe membrane is removed from the electrode and arranged on a glass rod. The membrane thickness is determined by means of a graduated microscope (VEB Carl Zeiss, Jena). The data can be measured to within plus or minus 0.2 pm. The determination of the substrate diffusion coefficient (D,) was carried out according to Bollm...

Several 9-furfuryl-6-substituted purines were synthesized by reacting 9-furfuryl-6-chloropurine (5) with the corresponding nucleophilic reagent. The mother compound (5) was prepared by direct cyclization of 4-furfurylamino-5-amino-6-chloropyrimidine (4) with a mixture of ethyl orthoformate and acetic anhydride. The diaminopyrimidine (5) was prepared by treatment of 4,6-dichloro-5-aminopyrimidine with furfurylamine in boiling water.Canadian Journal of Chemistry, 46, 419 (1968) In the synthesis of various potential antagonists of the natural purines 9-methyl-6-chloropurine (1) has shown the same order of activity against Adenocarcinoma 755 in C-57 black mice as 6-chloropurine3 (2), while two other 9-methyl-6-substituted purines have shown less activity against this tumor. 9-Ethyl-6-chloropurine (3) and 9-propyl-6-chloropurine (4), synthesized by Montgomery and Temple, have also shown the same kind of activity.Due to the structural similarity between naturally occurring metabolic purine ribonucleosides, deoxyribonucleosides, and 9-furfuryl-6-substituted purines, the investigation of the biological activity of 9-furfuryl-6-substituted purines could be of some interest. A comparison of the biological activity of 9-f~1rfuryl-6-chloropurine with that of 9-methyl-, 9-ethyl-, 9-propyl-, and 9-phenyl-6-chloropurine (5) would be especially desirable.Thirteen such derivatives of purine were synthesized through the method devised by Robins and Lin (1) and sent to the Cancer Chemotherapy National Service Center, National Institutes of Health, Bethesda, Maryland, for screening their biological activities. The mother compound 9-furfuryl-6-chloropurine (5) amino-5-amino-6-chloropyrimidine (4) with a mixture of ethyl orthoformate and acetic anhydride.Treatment of several aliphatic and aromatic amines with 5 in boiling amyl alcohol yielded corresponding amino derivatives in fairly good yield. Cyclization of 4-furfurylamino-5-amino-6-chloropyrimidine by refluxing with 97 % formic acid failed to yield 9-furfuryl-6-hydroxypurine (6). A black sticky polymeric residue was the only product that could be isolated. The ring cleavage of furfuryl ring in the boiling formic acid could cause the formation of this polymeric residue. 9-Furfuryl-6-hydroxypurine (6) was prepared easily by treatment of the mother coinpound (5) with 1 N sodium hydroxide in good yield. 9-Furfuryl-6-methoxypurine (9) was prepared by treatment of 5 with sodium inethoxide. 9-Furfuryl-6 -aminopurine (9-furfuryladenine) (8) was prepared by heating 5 with excess concentrated ammonium hydroxide at 130" in a steel Parr bomb. 9-Furfuryl-6-mercaptopurine (7) was prepared in high yield by treatment of 5 with thiourea. The ultraviolet absorption spectra of the 9-furfuryl-6-substituted purines are in general very similar to that of 9-methyl-6-substituted purines. Biological Activities9-Furfuryl-6-mercaptopurine (7) was found to be active at 112 mg/Kg (TIC, 31 %) against Adenocarcinoma 755. 4,6-Difurfurylamino-5-nitropyrimidine (1) was found to be non-toxic and inactive at 12...

Übersicht.

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