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Quantitative monitoring of carboxyl groups in polymers
Analytica &mica Actu, 219 (1989) 161-163 Elsevier Science Publishers B.V., Amsterdam -
161 Printed in The Netherlands
Short Communication
QUANTITATIVE POLYMERS M. BASSEDAS*,
MONITORING
OF CARBOXYL
GROUPS IN
F. ALBERICIO and F. LCPEZ-CALAHORRA
Dpt. Quimica Orghica, Divisi6 de Cihcies Experimental i Matemhtiques, Universitat de Barcelona, Marti i Franqu6 l-l I,08028 Barcelona (Spain) (Received 19th October 1988) Summary. A convenient, non-destructive spectrophotometric method for the quantitation of the substitution levels of carboxyl-containing polymers has been developed, based on the deprotection of 9-fluorenylmethyl esters under basic conditions.
One of the commonest problems in the chemistry of insoluble polymers is the determination of the functional groups attached to the resin. Several methods for the determination of the free amino groups on resins have been described, for example, titrations with perchloric acid [ 1] or picric acid [ 21 and the quantitative ninhydrin reaction [3]. However, there is no convenient method for the determination of carboxylic acid groups. This communication describes a simple, fast and non-destructive procedure for the measurement of free carboxyl groups of an insoluble polymer. The method is based on the use of 9-fluorenylmethyl (Fm) esters for protection of carboxyl groups [ 4,5], the subsequent deprotection being measured spectrophotometrically, similarly to the quantitative monitoring in solid-phase peptide synthesis with the N “-9-fluorenylmethoxycarbonyl (Fmoc ) group [ 61. The carboxyl-containing resin is treated with 9-fluorenylmethanol in the presence of dicyclohexylcarbodiimide (DCC ) and dimethylaminopyridine (DMAP) to form the corresponding ester. The resin is then treated with a solution of piperidine in dimethylformamide (DMF) in order to deprotect quantitatively the carboxyl group. The concentration of N- (9-fluorenylmethyl)piperidine is determined spectrophotometrically and reflects the carboxyl content of the resin (Scheme 1) . General procedure
Allow a weighed portion of dry resin (25-50 mg) to swell with addition of DMF (2 ml, 3 x 2 min). Add a solution of 9-fluorenylmethanol (FmOH) (ca. 5 equivalents, depending on the expected substitution level ), DCC ( 1 equivalent with respect to FmOH) and DMAP (0.1 equivalent with respect to FmOH) in 0.5-l ml of DMF (2 x 60 min), and wash with DMF (2 ml, 3 x 2 min). To deprotect, treat with a solution of piperidine in DMF (2:8; 0.5 ml, 1 x 1 min + 1 X 2 min + 1 x 5 min), wash with DMF (0.5 ml, 3 x 2 min), dilute with 0003-2670/89/$03.50
0 1989 Elsevier Science Publishers B.V.
162
WXlDMAP
QP 1-1 \/
DMF
CHZOH
c
w
1-1 \/
NH _
yn2
DMF
Scheme 1.
+
@nmoon
N
0
dichloromethane to a known volume in order to obtain an absorbance range between 0.3 and 0.6 and measure spectrophotometrically at 301 nm. The molar absorptivity of N- (9-fluorenylmethyl)piperidine at 301 nm = 7800 1 mol-l cm-‘); it is constant in the range 1 x 10m5-17 x lob5 M if the dichloromethane solution contains less than 10% DMF [ 71. Results and discussion
Both ester formation and deprotection times were established on the basis of our previous experience with the use of these esters in solution, and from the present observation that completeness of both the esterification and deprotection steps is not necessarily improved by longer reaction times. In order to prove the applicability and reproducibility of the method, a sample of N “-benzyloxycarbonyl+aspartyl (P-t-butyl ester)aminomethylpolystyrene resin (obtained by coupling of the corresponding protected amino acid and aminomethyl resin with DCC) with a known substitution level of 3.1 x low4 mol g-’ resin as calculated by amino acid analysis [B] was deprotected with trifluoroacetic acid in dichloromethane (1:l; 1 x 1 min + 1 x 30 min). Three consecutive determinations of the free /?-carboxylic groups by the general procedure gave 3.1 x 10T4, 3.0~ 10m4 and 3.3 x 10m4 eq g-’ resin. Likewise, four applications of the method to an unknown sample of tartaric acid attached to a polystyrene resin gave 1.0 X 10T4, 1.0 x 10m4,0.9 x 10m4and 1.1 x 10m4eq g-’ resin.
REFERENCES 1 2 3 4 5
S. Ehrlich-Rogozinski, Isr. J. Chem., 12 (1974) 31. B.F. Gisin, Anal. Chim. Acta, 58 (1972) 248. V.K. Sarin, S.B.H. Kent, J.P. Tam and R.B. Merrifield, Anal. Biochem., 117 (1981) 147. M.A. Bednarek and M. Bodanszky, Int. J. Pept. Protein Res., 21 (1983) 196. H. Kessler and R. Siegmeier, Tetrahedron I&t., 24 (1983) 281.
163 6 J. Meienhofer, M. Waki, E.P. Heimer, T.J. Lambros, R.C. Makofske and C.D. Chang, Int. J. Pept. Protein Res., 13 (1979) 35. 7 A. Grandas, E. Giralt and E. Pedroso, unpublished results. 8 F. Albericio, E. Nicolis, J. Josa, A. Grandas, E. Pedroso, E. Giralt, C. Granier and J. van Rietschoten, Tetrahedron, 43 (1987) 5961.
161 Printed in The Netherlands
Short Communication
QUANTITATIVE POLYMERS M. BASSEDAS*,
MONITORING
OF CARBOXYL
GROUPS IN
F. ALBERICIO and F. LCPEZ-CALAHORRA
Dpt. Quimica Orghica, Divisi6 de Cihcies Experimental i Matemhtiques, Universitat de Barcelona, Marti i Franqu6 l-l I,08028 Barcelona (Spain) (Received 19th October 1988) Summary. A convenient, non-destructive spectrophotometric method for the quantitation of the substitution levels of carboxyl-containing polymers has been developed, based on the deprotection of 9-fluorenylmethyl esters under basic conditions.
One of the commonest problems in the chemistry of insoluble polymers is the determination of the functional groups attached to the resin. Several methods for the determination of the free amino groups on resins have been described, for example, titrations with perchloric acid [ 1] or picric acid [ 21 and the quantitative ninhydrin reaction [3]. However, there is no convenient method for the determination of carboxylic acid groups. This communication describes a simple, fast and non-destructive procedure for the measurement of free carboxyl groups of an insoluble polymer. The method is based on the use of 9-fluorenylmethyl (Fm) esters for protection of carboxyl groups [ 4,5], the subsequent deprotection being measured spectrophotometrically, similarly to the quantitative monitoring in solid-phase peptide synthesis with the N “-9-fluorenylmethoxycarbonyl (Fmoc ) group [ 61. The carboxyl-containing resin is treated with 9-fluorenylmethanol in the presence of dicyclohexylcarbodiimide (DCC ) and dimethylaminopyridine (DMAP) to form the corresponding ester. The resin is then treated with a solution of piperidine in dimethylformamide (DMF) in order to deprotect quantitatively the carboxyl group. The concentration of N- (9-fluorenylmethyl)piperidine is determined spectrophotometrically and reflects the carboxyl content of the resin (Scheme 1) . General procedure
Allow a weighed portion of dry resin (25-50 mg) to swell with addition of DMF (2 ml, 3 x 2 min). Add a solution of 9-fluorenylmethanol (FmOH) (ca. 5 equivalents, depending on the expected substitution level ), DCC ( 1 equivalent with respect to FmOH) and DMAP (0.1 equivalent with respect to FmOH) in 0.5-l ml of DMF (2 x 60 min), and wash with DMF (2 ml, 3 x 2 min). To deprotect, treat with a solution of piperidine in DMF (2:8; 0.5 ml, 1 x 1 min + 1 X 2 min + 1 x 5 min), wash with DMF (0.5 ml, 3 x 2 min), dilute with 0003-2670/89/$03.50
0 1989 Elsevier Science Publishers B.V.
162
WXlDMAP
QP 1-1 \/
DMF
CHZOH
c
w
1-1 \/
NH _
yn2
DMF
Scheme 1.
+
@nmoon
N
0
dichloromethane to a known volume in order to obtain an absorbance range between 0.3 and 0.6 and measure spectrophotometrically at 301 nm. The molar absorptivity of N- (9-fluorenylmethyl)piperidine at 301 nm = 7800 1 mol-l cm-‘); it is constant in the range 1 x 10m5-17 x lob5 M if the dichloromethane solution contains less than 10% DMF [ 71. Results and discussion
Both ester formation and deprotection times were established on the basis of our previous experience with the use of these esters in solution, and from the present observation that completeness of both the esterification and deprotection steps is not necessarily improved by longer reaction times. In order to prove the applicability and reproducibility of the method, a sample of N “-benzyloxycarbonyl+aspartyl (P-t-butyl ester)aminomethylpolystyrene resin (obtained by coupling of the corresponding protected amino acid and aminomethyl resin with DCC) with a known substitution level of 3.1 x low4 mol g-’ resin as calculated by amino acid analysis [B] was deprotected with trifluoroacetic acid in dichloromethane (1:l; 1 x 1 min + 1 x 30 min). Three consecutive determinations of the free /?-carboxylic groups by the general procedure gave 3.1 x 10T4, 3.0~ 10m4 and 3.3 x 10m4 eq g-’ resin. Likewise, four applications of the method to an unknown sample of tartaric acid attached to a polystyrene resin gave 1.0 X 10T4, 1.0 x 10m4,0.9 x 10m4and 1.1 x 10m4eq g-’ resin.
REFERENCES 1 2 3 4 5
S. Ehrlich-Rogozinski, Isr. J. Chem., 12 (1974) 31. B.F. Gisin, Anal. Chim. Acta, 58 (1972) 248. V.K. Sarin, S.B.H. Kent, J.P. Tam and R.B. Merrifield, Anal. Biochem., 117 (1981) 147. M.A. Bednarek and M. Bodanszky, Int. J. Pept. Protein Res., 21 (1983) 196. H. Kessler and R. Siegmeier, Tetrahedron I&t., 24 (1983) 281.
163 6 J. Meienhofer, M. Waki, E.P. Heimer, T.J. Lambros, R.C. Makofske and C.D. Chang, Int. J. Pept. Protein Res., 13 (1979) 35. 7 A. Grandas, E. Giralt and E. Pedroso, unpublished results. 8 F. Albericio, E. Nicolis, J. Josa, A. Grandas, E. Pedroso, E. Giralt, C. Granier and J. van Rietschoten, Tetrahedron, 43 (1987) 5961.