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Synthesis and chromatographic properties of Se-carboxymethyl-selenocysteamine (carboxymethyl, 2-aminoethyl selenide)
ANAL,YTlCAL
69, 289-293
BIOCHEMISTRY
Synthesis
and
( 1975)
Chromatographic
Properties
Se-Carboxymethyl-Selenocysteamine (Carboxymethyl, 2-Aminoethyl
of
Selenide)
In the course of a recent study on the oxidation of selenocystamine by pig kidney diamineoxidase, we needed to analyze for the production of selenocysteamine during the reaction. This was done by trapping selenocysteamine by monoiodoacetate, and then searching for Se-carboxymethyl-selenocysteamine. To identify this latter compound we needed the pure substance, and attempted to prepare it. In the present note we report the details for the synthesis of Se-carboxymethyl-selenocysteamine, CMSeC, and some chromatographic data useful for its identification. Previously we have reported the synthesis of S-carboxymethylcysteamine, CMC, prepared either from cysteamine and monochloroacetate (l), or from thioglycolic acid and ethyleneimine (2). Availability of CMSeC allows comparison of some of its properties with those of CMC, and this may be of some general interest in comparative studies of sulfur and selenium compounds. MATERIALS
AND
METHODS
Selenocystamine 2 HCl was obtained from Sigma Chemical Co., cysteamine HCl and thioglycolic acid from Fluka. Carboxymethylcysteamine was prepared as previously reported (2). All other reagents were analytical grade products obtained from Messrs. Merck. NMR spectra were recorded in a JEOL-INM-C-60 Hl spectrometer at 60 mCi/sec in D,O, using hexamethyldisiloxane (HMDS) as an internal standard. Paper chromatography was performed on Whatman no. 1 paper sheets, in the solvents specified below. Ion exchange chromatography was performed on a BIO-CAL 200 Amino Acid Analyzer; the long column, 54 X 0.9 cm, filled with Aminex A 6 resin from Bio Rad, was equilibrated with 0.2 M sodium citrate buffer pH 3.25 and then eluted with citrate buffer, 0.2 or 0.38 M, pH 4.25. The temperature was 5o”C, and the buffer flow rate 80 ml/hr. 289 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
290
SHORT
RESULTS
COMMUNICATIONS
AND
DISCUSSION
CMSeC has been prepared from selenocysteamine and monochloroacetate in alkaline medium at 50°C. Selenocysteamine was obtained by reduction of selenocystamine with sodium borohydride; it was not isolated from the solution, but allowed to react directly with chloroacetate. The overall procedure was as follows. 320 mg (1 mmole) of selenocystamine were dissolved in 15 ml lop3 N NaOH, nitrogen bubbled through the solution, and 370 mg (10 mmoles) of NaBH, added portionwise in 10 min. The solution was allowed to stand at room temperature with continuous bubbling of nitrogen. The reduction of selenocystamine was followed by the Folin-Marenzi reaction (3), performed as follows: a drop of the reaction mixture was put into 2 ml of 1 M acetate buffer, pH 5, the solution shaken for 2-3 min to allow the borohydride to be completely destroyed, and then 0.2 ml of the Folin-Marenzi reagent added. A blue color immediately appears if selenocysteamine is present in solution. Since selenocystamine reacts with the Folin-Marenzi reagent after addition of bisulfite, the reduction was assumed to be complete when the color obtained in the absence of bisulfite was equal to that obtained after its addition. This requires about 3 hr. Then, by dropwise addition of concentrated HCl the pH of the solution was brought to 2-3 to destroy the excess borohydride. 220 mg (2.2 mmoles) of monochloracetic acid are now added, the reaction mixture brought to pH 8-9 by dropwise addition of 40% NaOH, and the vessel placed in a water bath at 50°C always under bubbling of nitrogen. After about 30 min the Folin-Marenzi reaction becomes negative, indicating that all the selenocysteamine has reacted. The solution, which is now slightly turbid, is filtered and percolated through a 2 x 8 cm column of Dowex 50, H+. The column is washed with water, and then eluted with 1 N NH,OH, collecting fractions of about 10 ml. The first two to three alkaline fractions contain CMSeC, as checked by paper chromatography; these fractions are pooled and taken to dryness in a rotary evaporator at 70°C. The residue is suspended in boiling ethanol, dissolved by dropwise addition of water, and then allowed to cool in the refrigerator. Crystallization starts almost immediately. After a few hours the precipitate is collected by centrifugation, washed with absolute ethanol and dried with ether. 300 mg of white, plate shaped, birefringent crystals were obtained, which melted at 179-182°C (Kofler, uncorr.) Elemental analysis gave the following results. Anal. Calcd for C,H,NO$e. C 26.37, H 4.95, N 7.69. Found C 26.25, H 5.04, N 7.69.
SHORT
291
COMMUNICATIONS
The NMR spectrum showed a singlet centered at 3.54 ppm assigned to the -Se-CH,-COOD methylene group, and two multiplets centered at 3.64 and 3.25 ppm, associated, respectively, with the ND,-CH,and the -Se-CH,-CH,methylene groups. The assignments of the multiplets were made on the basis of the different values of electroaffinity of the Se and N atoms. The NMR spectrum of CMSeC is very similar to that of CMC (see Fig. 1 of ref. 2). On paper chromatography CMSeC gives only one well-defined spot reactive to ninhydrin and to iodoplatinate (4). The following Rf values have been obtained (in parentheses those for CMC): butanol-acetic acid-water (4: 1 : 5, upper phase) 0.4 (0.3); water saturated phenol 0.81 (0.77): collidine-lutidine (1: 1 saturated with water) 0.25 (0.20); butanol-formic acid-water (75 : 15 : 10) 0.17 (0.14): methanol-pyridinewater (80: 20: 4) 0.53 (0.56). Butanol-acetic acid-water is the only solvent in which a good separation between CMSeC and CMC may be observed. From the long column of the Amino Acid Analyzer, operating under the standard conditions for the analysis of acidic and neutral aminoacids, that is 0.2 M sodium citrate buffer, pH 3.25, followed by 0.2 M sodium citrate buffer, pH 4.25, CMSeC is eluted by this latter buffer and emerges at 160 min after the buffer change. It is well separated from CMC, which is eluted at 100 min, after the phenylalanine peak. A color constant, CHW, of 15 has been calculated for CMSeC, while that for CMC was 25. If the 0.38 M sodium citrate buffer, pH 4.25, is used after the pH 3.25 buffer, CMSeC is eluted 33 min, and CMC 26 min after the buffer change. In conclusion CMSeC is easily obtainable in a pure form and in discrete yield by the method described. It may be easily identified, and well differentiated from CMC by ion exchange chromatography. Therefore the carboxymethylation reaction may be exploited for trapping selenocysteamine, and for its identification. As will be reported elsewhere, CMSeC has been identified among the products of the enzymic oxidation of selenocystamine performed in the presence of monoiodoacetate.
ACKNOWLEDGMENTS This work has been supported in part by a Grant from the Consiglio Ricerche. Rome, Italy. The AA. are indebted to Dr. A. Lai of the Istituto University of Cagliari, for the NMR spectra.
Nazionale Chimico
delle of the
292
SHORT
COMMUNICATIONS
REFERENCES 1. 2. 3. 4.
De Marco, C.. Riva, F.. and Dupri, S. (1964) Anal. Biochem. 8, 269. De Marco, C.. Crifo, C., and Rinaldi, A. (1970) Ital. J. B&hem. 19, 337. Folin. 0. and Marenzi, A. D. (1929) J. Biol. Chem. 83, 109. Toennies, G. and Kolb, J. J. (I 95 1) Anal. Chem. 23, 823.
A. RINALDI S. DERNINI M. R. DESSY C. DE MARCO Institute of Biochemistry of the University of Cagliari, Cagliari, Italy and I” Cnttedra di Chimica Biologica delltr Facolta di Scienze M.F.N.. University of Rome, Rome, Italy Received January 31. 1975; accepted May 28, 1975
69, 289-293
BIOCHEMISTRY
Synthesis
and
( 1975)
Chromatographic
Properties
Se-Carboxymethyl-Selenocysteamine (Carboxymethyl, 2-Aminoethyl
of
Selenide)
In the course of a recent study on the oxidation of selenocystamine by pig kidney diamineoxidase, we needed to analyze for the production of selenocysteamine during the reaction. This was done by trapping selenocysteamine by monoiodoacetate, and then searching for Se-carboxymethyl-selenocysteamine. To identify this latter compound we needed the pure substance, and attempted to prepare it. In the present note we report the details for the synthesis of Se-carboxymethyl-selenocysteamine, CMSeC, and some chromatographic data useful for its identification. Previously we have reported the synthesis of S-carboxymethylcysteamine, CMC, prepared either from cysteamine and monochloroacetate (l), or from thioglycolic acid and ethyleneimine (2). Availability of CMSeC allows comparison of some of its properties with those of CMC, and this may be of some general interest in comparative studies of sulfur and selenium compounds. MATERIALS
AND
METHODS
Selenocystamine 2 HCl was obtained from Sigma Chemical Co., cysteamine HCl and thioglycolic acid from Fluka. Carboxymethylcysteamine was prepared as previously reported (2). All other reagents were analytical grade products obtained from Messrs. Merck. NMR spectra were recorded in a JEOL-INM-C-60 Hl spectrometer at 60 mCi/sec in D,O, using hexamethyldisiloxane (HMDS) as an internal standard. Paper chromatography was performed on Whatman no. 1 paper sheets, in the solvents specified below. Ion exchange chromatography was performed on a BIO-CAL 200 Amino Acid Analyzer; the long column, 54 X 0.9 cm, filled with Aminex A 6 resin from Bio Rad, was equilibrated with 0.2 M sodium citrate buffer pH 3.25 and then eluted with citrate buffer, 0.2 or 0.38 M, pH 4.25. The temperature was 5o”C, and the buffer flow rate 80 ml/hr. 289 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
290
SHORT
RESULTS
COMMUNICATIONS
AND
DISCUSSION
CMSeC has been prepared from selenocysteamine and monochloroacetate in alkaline medium at 50°C. Selenocysteamine was obtained by reduction of selenocystamine with sodium borohydride; it was not isolated from the solution, but allowed to react directly with chloroacetate. The overall procedure was as follows. 320 mg (1 mmole) of selenocystamine were dissolved in 15 ml lop3 N NaOH, nitrogen bubbled through the solution, and 370 mg (10 mmoles) of NaBH, added portionwise in 10 min. The solution was allowed to stand at room temperature with continuous bubbling of nitrogen. The reduction of selenocystamine was followed by the Folin-Marenzi reaction (3), performed as follows: a drop of the reaction mixture was put into 2 ml of 1 M acetate buffer, pH 5, the solution shaken for 2-3 min to allow the borohydride to be completely destroyed, and then 0.2 ml of the Folin-Marenzi reagent added. A blue color immediately appears if selenocysteamine is present in solution. Since selenocystamine reacts with the Folin-Marenzi reagent after addition of bisulfite, the reduction was assumed to be complete when the color obtained in the absence of bisulfite was equal to that obtained after its addition. This requires about 3 hr. Then, by dropwise addition of concentrated HCl the pH of the solution was brought to 2-3 to destroy the excess borohydride. 220 mg (2.2 mmoles) of monochloracetic acid are now added, the reaction mixture brought to pH 8-9 by dropwise addition of 40% NaOH, and the vessel placed in a water bath at 50°C always under bubbling of nitrogen. After about 30 min the Folin-Marenzi reaction becomes negative, indicating that all the selenocysteamine has reacted. The solution, which is now slightly turbid, is filtered and percolated through a 2 x 8 cm column of Dowex 50, H+. The column is washed with water, and then eluted with 1 N NH,OH, collecting fractions of about 10 ml. The first two to three alkaline fractions contain CMSeC, as checked by paper chromatography; these fractions are pooled and taken to dryness in a rotary evaporator at 70°C. The residue is suspended in boiling ethanol, dissolved by dropwise addition of water, and then allowed to cool in the refrigerator. Crystallization starts almost immediately. After a few hours the precipitate is collected by centrifugation, washed with absolute ethanol and dried with ether. 300 mg of white, plate shaped, birefringent crystals were obtained, which melted at 179-182°C (Kofler, uncorr.) Elemental analysis gave the following results. Anal. Calcd for C,H,NO$e. C 26.37, H 4.95, N 7.69. Found C 26.25, H 5.04, N 7.69.
SHORT
291
COMMUNICATIONS
The NMR spectrum showed a singlet centered at 3.54 ppm assigned to the -Se-CH,-COOD methylene group, and two multiplets centered at 3.64 and 3.25 ppm, associated, respectively, with the ND,-CH,and the -Se-CH,-CH,methylene groups. The assignments of the multiplets were made on the basis of the different values of electroaffinity of the Se and N atoms. The NMR spectrum of CMSeC is very similar to that of CMC (see Fig. 1 of ref. 2). On paper chromatography CMSeC gives only one well-defined spot reactive to ninhydrin and to iodoplatinate (4). The following Rf values have been obtained (in parentheses those for CMC): butanol-acetic acid-water (4: 1 : 5, upper phase) 0.4 (0.3); water saturated phenol 0.81 (0.77): collidine-lutidine (1: 1 saturated with water) 0.25 (0.20); butanol-formic acid-water (75 : 15 : 10) 0.17 (0.14): methanol-pyridinewater (80: 20: 4) 0.53 (0.56). Butanol-acetic acid-water is the only solvent in which a good separation between CMSeC and CMC may be observed. From the long column of the Amino Acid Analyzer, operating under the standard conditions for the analysis of acidic and neutral aminoacids, that is 0.2 M sodium citrate buffer, pH 3.25, followed by 0.2 M sodium citrate buffer, pH 4.25, CMSeC is eluted by this latter buffer and emerges at 160 min after the buffer change. It is well separated from CMC, which is eluted at 100 min, after the phenylalanine peak. A color constant, CHW, of 15 has been calculated for CMSeC, while that for CMC was 25. If the 0.38 M sodium citrate buffer, pH 4.25, is used after the pH 3.25 buffer, CMSeC is eluted 33 min, and CMC 26 min after the buffer change. In conclusion CMSeC is easily obtainable in a pure form and in discrete yield by the method described. It may be easily identified, and well differentiated from CMC by ion exchange chromatography. Therefore the carboxymethylation reaction may be exploited for trapping selenocysteamine, and for its identification. As will be reported elsewhere, CMSeC has been identified among the products of the enzymic oxidation of selenocystamine performed in the presence of monoiodoacetate.
ACKNOWLEDGMENTS This work has been supported in part by a Grant from the Consiglio Ricerche. Rome, Italy. The AA. are indebted to Dr. A. Lai of the Istituto University of Cagliari, for the NMR spectra.
Nazionale Chimico
delle of the
292
SHORT
COMMUNICATIONS
REFERENCES 1. 2. 3. 4.
De Marco, C.. Riva, F.. and Dupri, S. (1964) Anal. Biochem. 8, 269. De Marco, C.. Crifo, C., and Rinaldi, A. (1970) Ital. J. B&hem. 19, 337. Folin. 0. and Marenzi, A. D. (1929) J. Biol. Chem. 83, 109. Toennies, G. and Kolb, J. J. (I 95 1) Anal. Chem. 23, 823.
A. RINALDI S. DERNINI M. R. DESSY C. DE MARCO Institute of Biochemistry of the University of Cagliari, Cagliari, Italy and I” Cnttedra di Chimica Biologica delltr Facolta di Scienze M.F.N.. University of Rome, Rome, Italy Received January 31. 1975; accepted May 28, 1975