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A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups
Journal of Biochemical and Biophysical Methods, 18 (1989) 323-330 Elsevier
323
BBM 00744
A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups R.K. Gaur, S. Paliwal +, P. Sharma and K.C. Gupta DNA Synthesis Laboratory, CSIR Centre for Biochemicals, V.P. Chest Institute Building, Delhi 110007, lndia
(Received 29 January 1989) (Accepted 24 February 1989)
Summary A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Bioehem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3"-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution, The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (c498 = 70000), is then determined spectrophotometrically. A comparative study of DTDS, N-succiuimidyl-3-(2-pyridyldithio)propionate and 4,4'-dimethoxytrityl chloride is also included. The method was found to be very usefulto determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionaliTation of solid supports for affinity chromatography and synthesis of biopolymers. Key words: Amino group; Polymer supported amine; Spectrophotometric method; 4,4'-Dimethoxytrityi cation
Introduction A large number of solid supports with different immobilized functional groups are of great interest in many biochemical and biotechnological problems [2-7]. In recent years, for example, solid supports containing amino groups have been widely
Correspondence address: K.C. Gupta, DNA Synthesis Laboratory, CSIR Centre for Biochemicals, V.P. Chest Institute Building, Delhi 110007, India. + Stayed for a summer course. 0165-022X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
324 used to prepare affinity matrices [8] for cell and enzyme immobilization [9-12] and solid supports for the synthesis of biopolymers [13-16]. There has been a continuing need for a simple, sensitive and rapid method, to determine the free amino groups on solid supports for various applications. The ninhydrin [17] and TNBS [18,19] methods are known for the quantitative determination of solid supported amino groups. However, for the correct use of a solid matrix, a quantitative determination of those amino groups which are available for functionalization of solid supports is required. The N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) method as developed by Ngo [1] for the quantitative determination of amino functionalities was found to be quite useful for routine use in biochemical laboratories, but the molar extinction coefficient of the liberated pyridine-2-thione being low (C343-~-8080//M) requires larger quantities (50 mg) of the polymer supports for analysis. We wish to report here a simple, sensitive and convenient spectrophotometric method for the quantitative determination of solid supported amino groups. The method involves the use of the 2,4-dinitrophenyl active ester of commercially available 5'-O-(4,4'-dimethoxytrityl)thymidine 3'-O-succinate (DTS). The higher molar extinction coefficient of the liberated 4,4'-dimethoxytrityl cation (c498 = 70 000) makes this method very sensitive and requires smaller quantities (1-2 mg) of the solid support for analysis. Moreover, the number of steps involved in the DTDS method as compared to the SPDP one are less. The utility of the method was further confirmed by monitoring the functionalization of some solid supports for DNA synthesis.
Materials and Methods
4,4'-Dimethoxytrityl chloride, cyanogen bromide and DTS were obtained from CSIR Centre for Biochemicals, Delhi, India; 2,4-dinitrophenol, dicyclohexylcarbodiimide (DCCI) and 4-dimethylaminopyridine (DMAP) were the products of Fluka AG, Buchs, Switzerland; long chain alkylamine-CPG (LCAA-CPG) was obtained from Pierce Chemical Co., Rockford IL, U.S.A.; 3-aminopropyl silica gel, ~aminohexyl Sepharose 4B, 1,6-diaminohexane, aminomethyl polystyrene resin, SPDP were procured from Sigma Chemicals Co., St. Louis, MO, U.S.A. Sepharose 4B, Sepharose 6B and Sepharose CL 4B were purchased from Pharmacia, Uppsala, Sweden. Kieselgel 60 (70-230 mesh) and Fractosil 200 were obtained from Merck, Darmstadt, F.R.G. Aminoethyl Bio-Gel was the product of Bio-Rad, Richmond, CA , U.S.A. Other reagents and chemicals used in this study were of analytical grade and purified, if necessary, before use. Sepharose derivatives containing amino groups were prepared by activation of the gel with CNBr [20] and subsequent reaction with 1,6-diaminohexane was carried out according to the published [21] procedure. The gels were washed with 0.1 N HCI, followed by water to neutrality, before analysis. The amount of gel is expressed as the volume of settled gel. Aminopropylation of Fractosil 200 and CPG
325 was carried out according to the method [22] reported earlier. A Gilford response spectrophotometer was used for UV measurements.
Reagent preparation (a) Reagent A: 5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS, 0.405 g, 0.5 mmol), DMAP (15.20 mg, 0.1 mmol) and triethylamine (TEA, 1 ml) were dissolved in dry N, N-dimethylformamide (DMF, 5 ml). (b) Reagent B (detritylating reagent): 70% perchloric acid, 51.4 ml + methanol, 46.0 ml. Synthesis of 5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) A solution of DTS (0.64 g, 1 mmol) and 2,4-dinitrophenol (0.20 g, 1.1 retool) in dry D M F (10 ml) containing dry pyridine (0.2 ml) was cooled in an ice-bath. DCCI (0.3 g, 1.5 mmol) was added with stirring. After 2 h of stirring at room temperature, precipitated dicyclohexylurea was removed by filtration and the filtrate was concentrated in vacuum to give the desired ester as an oil: a single spot was obtained on TLC. Determination of amino groups on solid supports Method A A weighed quantity of the dry solid support ( - 1 mg) was taken in a 10 ml calibrated centrifuge tube to which reagent A (0.25 ml) was added. The vial was screw capped and gently tumbled at room temperature for 30 min and then centrifuged at 2000 rpm for 1 min. After removing the supernatant, the solid support was washed successively with D M F (2 x 10 ml), methanol (2 x 10 ml) and finally with diethyl ether (2 × 10 ml). After being decanted, the centrifuge vial was filled up to 5 ml with reagent B and the liberated 4,4'-dimethoxytrityl cation was then determined spectrophotometrically at 498 nm against reagent B as blank. Method B For solid supports which are available in the wet/preswollen condition or do not swell completely in organic solvents, the above procedure was slightly changed. Dry solid support ( - 1 mg) or wet settled gel (0.5 ml) was taken in a calibrated centrifuge tube to which 0.5 M sodium bicarbonate buffer, pH 8.5 (0.5 ml), and reagent A (0.25 ml) were added. The solid matrix suspension was tumbled at room temperature for 30 min and the supernatant was removed by using a Pasteur pipet. The solid support was washed successively with D M F / w a t e r (1 : 1, 2 x 10 ml), methanol (2 X 10 ml) and finally with m e t h a n o l / w a t e r (20 : 80, 2 x 10 ml), and then method A was followed exactly.
Results and Discussion
Synthesis plan of the reagent D T D S is depicted in Fig. 1. The reagent was synthesized in quantitative yield starting from the commercially available reagent DTS.
326
T
DMTrOO ~
O=~ =O
NO2 HO~ N O 2 / D M F
DCCI
DMTrO~ )
O
0=~0
ON
( DTS)
O NO2 ~N02 (DTDS)
Fig. l. Synthesis scheme of the reagent DTDS.
Based on the principle shown in Fig. 2, a simple, sensitive and rapid spectrophotometric method is developed for the quantitative determination of those amino groups on solid supports which are available for functionalization of solid supports. The first step involves the acylation of the solid supported amino groups with an excess of the reagent D T D S in the presence of an efficient catalyst D M A P [23]. After removal of the excess reagent, solid support was treated with perchloric acid to liberate a 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has very high absorption (~49s = 70000) at 498 nm, was determined spectrophotometrically. The concentration of the solid support bound amino groups was then calculated from the following formula Amount of the amino groups (/~mol/g solid support) 14.3 X h498n m X Vol (ml) Qt. of solid support ( m g ) / V o l (ml) " The optimum time of acylation of solid supported amino groups with D T D S on different solid supports was found to be 30 min. The detritylation studies of solid supported amino groups acylated with D T D S were also performed according to the method reported earlier [24] and it was found that 70% detritylation occurred instantaneously and it was completed within 5 min. N o significant difference in detritylation time was observed with different matrices used in this study. The values of amino groups on different solid supports determined by the proposed method were found to be in good agreement with those determined by the SPDP and DMTrC1 methods (Table 1). DMTrC1 has been found to be equally good for a limited number of solid supports but it suffers from the following drawbacks (i) it cannot be used with hydroxyl bearing polymer supports, and (ii) is unsuitable for solid supports available in the wet/preswollen condition or requires aqueous conditions for swelling, due to its high-moisture sensitive nature. In order to overcome the limitations of DMTrC1, we synthesized the reagent DTDS, a derivative of DMTrC1, for the determination of solid support bound amino groups. The reagent D T D S was found to be suitable for a variety of solid supports including
327
DTDS + H2 N , - / ~ ' ~ Solidsupport boundominogroup
DMAP/DMF
TEA
T
)
DMTr0 ~ ~ O~
~ DMTr,O--~O~Q
0 = ~, NH..,-....
T~ Thymine,
t I)
Removol of excess reogent.
~
llll Perchl~c ocid.
OCH3
T +
H O - ~
,) OCH3 Dimethoxyteltylcotlon t~mox =4gSnm;E498=70,O00/M
I
Fig. 2. Determination of solid supported amino groups.
those available in the wet/preswollen form or which require an aqueous environment for swelling and even with the supports bearing hydroxyl groups. The amino loading on different solid supports determined by the proposed method were found to be lower than those determined by nitrogen estimation or the acid-base titration method (reported by the manufacturer). This may be attributed to the following reasons: (i) some of the amino groups may be sterically inaccessible
TABLE 1 D E T E R M I N A T I O N O F A M I N O L O A D I N G S ON SOLID SUPPORTS a Amino functionalized sofid support
1.3-Aminopropyl silica gel 2, Aminomethyl polystyrene 3. LCAA-CPG 4. Aminoethyl Bio-gel 5. ~-Aminohexyl (AH) Sepharose b,a 6. A H - S e p h a r o s e 4 B b'd 7. A H - S e p h a r o s e C L 4 B b,d 8. A H - S e p h a r o s e 6 B b,d
a b c d
Amino loadings in/~ m o l / g solid support
Reported by the
DTDS
SPDP
DMTrC!
manufacturer ¢
318.40 420,47 34.02 441.26 4.90
315.70 400.20 29.42 439.31 4.12
310.85 401.00 31.00 440.12 -
1000 1000 100 1000 6-10
0.27 0.35 0.82
0.25 0.30 0.79
-
---
The values presented in this table are the mean from three separate experiments. Amino loading determined using method B. Based on nitrogen estimation or acid-base titration methods. Loadings expressed in /tmoi/ml.
328 TABLE 2 MONITORING THE FUNCTIONALIZATION OF SOLID SUPPORTS FOR DNA SYNTHESIS Solid supports containing amino groups
Amino loading (/~mol/g)
Nucleoside loading a (/~mol/g)
1. Aminopropyl Fractosil 200 2. Aminopropyl-CPG 3. LCAA-CPG
66.50 49.24 34.02
64.90 49.10 33.30
a The nucleoside loadings are the mean from two separate experiments. for the reaction with D T D S and yet can still be titrated by acid and (ii) a part of the 4,4'-dimethoxytrityl cation released from the solid support m a y n o t be released in the surrounding solution but remained adsorbed to the solid support. The p r o p o s e d m e t h o d measures only those a m i n o groups which are accessible to D T D S . It does not measure those a m i n o groups which are sterically hindered or otherwise burried within the p o l y m e r matrix. Therefore, the n u m b e r of amino groups determined by the proposed m e t h o d would either be equal or less than the n u m b e r determined by nitrogen estimation or acid-base titration. However, the n u m b e r of amino groups determined by the p r o p o s e d m e t h o d would be an i m p o r t a n t parameter for calculating the a m o u n t of ligand required for immobilization. This was further confirmed by monitoring the functionalization of polymer supports for D N A synthesis. Three different p o l y m e r supports, viz, aminopropyl-Fractosil 200, aminopropylC P G and L C A A - C P G were chosen to test the new D T D S monitoring procedure. The amino loadings of all three p o l y m e r supports were determined b y the procedure mentioned in m e t h o d A. Suitably protected deoxynucleoside derivatives were then a n c h o r e d on a m i n o functionalized polymer supports [25]. Nucleoside loadings of all three functionalized p o l y m e r supports were then determined by the m e t h o d reported earlier [25]. The values of the amino loadings of all three polymer supports were found to be in good agreement with the nucleoside loadings of the corresponding functionalized p o l y m e r supports (Table 2). Thus, the solid support b o u n d a m i n o group determination by the p r o p o s e d m e t h o d could be a useful guide for the preparation of solid supports for the synthesis of oligonucleotides, peptides and matrices for affinity c h r o m a t o g r a p h y .
Simplified description of the method and its advantages A simple, sensitive and convenient spectrophotometric method for the determination of solid support bound amino groups is described. The method involves the use of the 2,4-dinitrophenyl ester of the commercially available 5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-succinate (DTS) and perchloric acid for the subsequent release of a strong chromophore of the bound DTDS, 4,4'-dimethoxytrityl cation. This method can be used to monitor the functionalization of all kinds of polymer supports including soft gels, for various applications in biotechnology. The method does not require any sophisticated instrumentation and the reagent DTDS can be synthesized in any laboratory without any problem.
329
Acknowledgements Financial support from the Department of Biotechnology, Government of India, N e w D e l h i is g r a t e f u l l y a c k n o w l e d g e d . T h a n k s a r e a l s o d u e t o M r . R a h i m f o r h i s t e c h n i c a l a s s i s t a n c e . O n e o f t h e a u t h o r s ( R . K . G . ) is t h a n k f u l t o t h e C S I R , N e w Delhi, for a research fellowship.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Ngo, T.T. (1986) J. Biochem. Biophys. Methods 12, 349-354 Anderson, J.K., Mole, J.E. and Baker, H.J. (1978) Biochemistry 17, 467-473 Glover, C.V.C. and Gorovsky, M,A. (1978) Biochemistry 17, 5705-5713 Letsinger, R.L. and Kalus, I. (1964) J. Am. Chem. Soc. 86, 3884-3885 Mitchell, A.R., Kent, S.B.H., Erickson, B.W. and Merrifield, R.B. (1976) Tetrahedron Lett. 3795-3798 Farral, M.J. and Frechet, J.M.J. (1976) J. Org. Chem. 41, 3877-3882 Seliger, H. and Gupta, K.C. (1985) Angew. Chem. Int. Ed. Engl. 24, 685-687 Wilchek, M., Miron, T. and Kohn, J. (1984) Enzyme Purification and Related Techniques, Methods in Enzymology, Vol. 104, Academic Press, New York Lowe, C.R. and Dean, P.D.G. (1974) Affinity Chromatography, John Wiley & Sons, New York Mosbach, K. (1976) Immobilized Enzymes, Methods in Enzymology, Vol. 44, Academic Press, New York Scouten, W.H. (1981) Affinity Chromatography. Bioselective Adsorption on Inert Matrices, John Wiley & Sons, New York Scouten, W.H. (1983) Solid Phase Biochemistry, Analytical and Synthetic Aspects, John Wiley & Sons, New York Letsinger, R.L. and Mahadevan, V. (1965) J. Am. Chem. Soc. 87, 3526-3527 Merrifield, R.B. (1963) J. Am. Chem. Soc. 85, 2149-2154 Frechet, J.M. and Schuerch, C. (1971) J. Am. Chem. Soc. 93, 492-496 Zehavi, U. and Patchornik, A. (1973) J. Am. Chem. Soc. 95, 5673-5677 Sarin, V.K., Kent, S.B.H., Tam, J.P. and Merrifield, R.B. (1981) Anal. Biochem. 117, 147-157 Antoni, G., Presentini, R. and Neri, P. (1983) Anal. Biochem. 129, 60-63 Miyagawa, A. and Okuyama, T. (1977) J. Biochem. 81, 1715-1720 Kohn, J. and Wilchek, M. (1982) Biochem. Biophys. Res. Commun. 107, 878-884 Shaltiel, S. (1974) Affinity Techniques, Methods in Enzymoiogy, Vol. 34, Academic Press Fritz, J.S. and King, J.N. (1976) Anal. Chem. 48, 570-572 Hofle, G., Steglich, W. and Vorbruggen, H. (1978) Angew. Chem. Int. Ed. Engl. 17, 569-583 Matteucci, M.D. and Caruthers, M.H. (1980) Tetrahedron Lett. 21, 719-722 Caruthers, M.H. (1982) In: Gassen, H.G. and Lang, A. (Eds.), Chemical and Enzymatic Synthesis of Gene Fragments, Verla8 Chemie, Basel, pp. 71-79.
323
BBM 00744
A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups R.K. Gaur, S. Paliwal +, P. Sharma and K.C. Gupta DNA Synthesis Laboratory, CSIR Centre for Biochemicals, V.P. Chest Institute Building, Delhi 110007, lndia
(Received 29 January 1989) (Accepted 24 February 1989)
Summary A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Bioehem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3"-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution, The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (c498 = 70000), is then determined spectrophotometrically. A comparative study of DTDS, N-succiuimidyl-3-(2-pyridyldithio)propionate and 4,4'-dimethoxytrityl chloride is also included. The method was found to be very usefulto determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionaliTation of solid supports for affinity chromatography and synthesis of biopolymers. Key words: Amino group; Polymer supported amine; Spectrophotometric method; 4,4'-Dimethoxytrityi cation
Introduction A large number of solid supports with different immobilized functional groups are of great interest in many biochemical and biotechnological problems [2-7]. In recent years, for example, solid supports containing amino groups have been widely
Correspondence address: K.C. Gupta, DNA Synthesis Laboratory, CSIR Centre for Biochemicals, V.P. Chest Institute Building, Delhi 110007, India. + Stayed for a summer course. 0165-022X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
324 used to prepare affinity matrices [8] for cell and enzyme immobilization [9-12] and solid supports for the synthesis of biopolymers [13-16]. There has been a continuing need for a simple, sensitive and rapid method, to determine the free amino groups on solid supports for various applications. The ninhydrin [17] and TNBS [18,19] methods are known for the quantitative determination of solid supported amino groups. However, for the correct use of a solid matrix, a quantitative determination of those amino groups which are available for functionalization of solid supports is required. The N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) method as developed by Ngo [1] for the quantitative determination of amino functionalities was found to be quite useful for routine use in biochemical laboratories, but the molar extinction coefficient of the liberated pyridine-2-thione being low (C343-~-8080//M) requires larger quantities (50 mg) of the polymer supports for analysis. We wish to report here a simple, sensitive and convenient spectrophotometric method for the quantitative determination of solid supported amino groups. The method involves the use of the 2,4-dinitrophenyl active ester of commercially available 5'-O-(4,4'-dimethoxytrityl)thymidine 3'-O-succinate (DTS). The higher molar extinction coefficient of the liberated 4,4'-dimethoxytrityl cation (c498 = 70 000) makes this method very sensitive and requires smaller quantities (1-2 mg) of the solid support for analysis. Moreover, the number of steps involved in the DTDS method as compared to the SPDP one are less. The utility of the method was further confirmed by monitoring the functionalization of some solid supports for DNA synthesis.
Materials and Methods
4,4'-Dimethoxytrityl chloride, cyanogen bromide and DTS were obtained from CSIR Centre for Biochemicals, Delhi, India; 2,4-dinitrophenol, dicyclohexylcarbodiimide (DCCI) and 4-dimethylaminopyridine (DMAP) were the products of Fluka AG, Buchs, Switzerland; long chain alkylamine-CPG (LCAA-CPG) was obtained from Pierce Chemical Co., Rockford IL, U.S.A.; 3-aminopropyl silica gel, ~aminohexyl Sepharose 4B, 1,6-diaminohexane, aminomethyl polystyrene resin, SPDP were procured from Sigma Chemicals Co., St. Louis, MO, U.S.A. Sepharose 4B, Sepharose 6B and Sepharose CL 4B were purchased from Pharmacia, Uppsala, Sweden. Kieselgel 60 (70-230 mesh) and Fractosil 200 were obtained from Merck, Darmstadt, F.R.G. Aminoethyl Bio-Gel was the product of Bio-Rad, Richmond, CA , U.S.A. Other reagents and chemicals used in this study were of analytical grade and purified, if necessary, before use. Sepharose derivatives containing amino groups were prepared by activation of the gel with CNBr [20] and subsequent reaction with 1,6-diaminohexane was carried out according to the published [21] procedure. The gels were washed with 0.1 N HCI, followed by water to neutrality, before analysis. The amount of gel is expressed as the volume of settled gel. Aminopropylation of Fractosil 200 and CPG
325 was carried out according to the method [22] reported earlier. A Gilford response spectrophotometer was used for UV measurements.
Reagent preparation (a) Reagent A: 5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS, 0.405 g, 0.5 mmol), DMAP (15.20 mg, 0.1 mmol) and triethylamine (TEA, 1 ml) were dissolved in dry N, N-dimethylformamide (DMF, 5 ml). (b) Reagent B (detritylating reagent): 70% perchloric acid, 51.4 ml + methanol, 46.0 ml. Synthesis of 5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) A solution of DTS (0.64 g, 1 mmol) and 2,4-dinitrophenol (0.20 g, 1.1 retool) in dry D M F (10 ml) containing dry pyridine (0.2 ml) was cooled in an ice-bath. DCCI (0.3 g, 1.5 mmol) was added with stirring. After 2 h of stirring at room temperature, precipitated dicyclohexylurea was removed by filtration and the filtrate was concentrated in vacuum to give the desired ester as an oil: a single spot was obtained on TLC. Determination of amino groups on solid supports Method A A weighed quantity of the dry solid support ( - 1 mg) was taken in a 10 ml calibrated centrifuge tube to which reagent A (0.25 ml) was added. The vial was screw capped and gently tumbled at room temperature for 30 min and then centrifuged at 2000 rpm for 1 min. After removing the supernatant, the solid support was washed successively with D M F (2 x 10 ml), methanol (2 x 10 ml) and finally with diethyl ether (2 × 10 ml). After being decanted, the centrifuge vial was filled up to 5 ml with reagent B and the liberated 4,4'-dimethoxytrityl cation was then determined spectrophotometrically at 498 nm against reagent B as blank. Method B For solid supports which are available in the wet/preswollen condition or do not swell completely in organic solvents, the above procedure was slightly changed. Dry solid support ( - 1 mg) or wet settled gel (0.5 ml) was taken in a calibrated centrifuge tube to which 0.5 M sodium bicarbonate buffer, pH 8.5 (0.5 ml), and reagent A (0.25 ml) were added. The solid matrix suspension was tumbled at room temperature for 30 min and the supernatant was removed by using a Pasteur pipet. The solid support was washed successively with D M F / w a t e r (1 : 1, 2 x 10 ml), methanol (2 X 10 ml) and finally with m e t h a n o l / w a t e r (20 : 80, 2 x 10 ml), and then method A was followed exactly.
Results and Discussion
Synthesis plan of the reagent D T D S is depicted in Fig. 1. The reagent was synthesized in quantitative yield starting from the commercially available reagent DTS.
326
T
DMTrOO ~
O=~ =O
NO2 HO~ N O 2 / D M F
DCCI
DMTrO~ )
O
0=~0
ON
( DTS)
O NO2 ~N02 (DTDS)
Fig. l. Synthesis scheme of the reagent DTDS.
Based on the principle shown in Fig. 2, a simple, sensitive and rapid spectrophotometric method is developed for the quantitative determination of those amino groups on solid supports which are available for functionalization of solid supports. The first step involves the acylation of the solid supported amino groups with an excess of the reagent D T D S in the presence of an efficient catalyst D M A P [23]. After removal of the excess reagent, solid support was treated with perchloric acid to liberate a 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has very high absorption (~49s = 70000) at 498 nm, was determined spectrophotometrically. The concentration of the solid support bound amino groups was then calculated from the following formula Amount of the amino groups (/~mol/g solid support) 14.3 X h498n m X Vol (ml) Qt. of solid support ( m g ) / V o l (ml) " The optimum time of acylation of solid supported amino groups with D T D S on different solid supports was found to be 30 min. The detritylation studies of solid supported amino groups acylated with D T D S were also performed according to the method reported earlier [24] and it was found that 70% detritylation occurred instantaneously and it was completed within 5 min. N o significant difference in detritylation time was observed with different matrices used in this study. The values of amino groups on different solid supports determined by the proposed method were found to be in good agreement with those determined by the SPDP and DMTrC1 methods (Table 1). DMTrC1 has been found to be equally good for a limited number of solid supports but it suffers from the following drawbacks (i) it cannot be used with hydroxyl bearing polymer supports, and (ii) is unsuitable for solid supports available in the wet/preswollen condition or requires aqueous conditions for swelling, due to its high-moisture sensitive nature. In order to overcome the limitations of DMTrC1, we synthesized the reagent DTDS, a derivative of DMTrC1, for the determination of solid support bound amino groups. The reagent D T D S was found to be suitable for a variety of solid supports including
327
DTDS + H2 N , - / ~ ' ~ Solidsupport boundominogroup
DMAP/DMF
TEA
T
)
DMTr0 ~ ~ O~
~ DMTr,O--~O~Q
0 = ~, NH..,-....
T~ Thymine,
t I)
Removol of excess reogent.
~
llll Perchl~c ocid.
OCH3
T +
H O - ~
,) OCH3 Dimethoxyteltylcotlon t~mox =4gSnm;E498=70,O00/M
I
Fig. 2. Determination of solid supported amino groups.
those available in the wet/preswollen form or which require an aqueous environment for swelling and even with the supports bearing hydroxyl groups. The amino loading on different solid supports determined by the proposed method were found to be lower than those determined by nitrogen estimation or the acid-base titration method (reported by the manufacturer). This may be attributed to the following reasons: (i) some of the amino groups may be sterically inaccessible
TABLE 1 D E T E R M I N A T I O N O F A M I N O L O A D I N G S ON SOLID SUPPORTS a Amino functionalized sofid support
1.3-Aminopropyl silica gel 2, Aminomethyl polystyrene 3. LCAA-CPG 4. Aminoethyl Bio-gel 5. ~-Aminohexyl (AH) Sepharose b,a 6. A H - S e p h a r o s e 4 B b'd 7. A H - S e p h a r o s e C L 4 B b,d 8. A H - S e p h a r o s e 6 B b,d
a b c d
Amino loadings in/~ m o l / g solid support
Reported by the
DTDS
SPDP
DMTrC!
manufacturer ¢
318.40 420,47 34.02 441.26 4.90
315.70 400.20 29.42 439.31 4.12
310.85 401.00 31.00 440.12 -
1000 1000 100 1000 6-10
0.27 0.35 0.82
0.25 0.30 0.79
-
---
The values presented in this table are the mean from three separate experiments. Amino loading determined using method B. Based on nitrogen estimation or acid-base titration methods. Loadings expressed in /tmoi/ml.
328 TABLE 2 MONITORING THE FUNCTIONALIZATION OF SOLID SUPPORTS FOR DNA SYNTHESIS Solid supports containing amino groups
Amino loading (/~mol/g)
Nucleoside loading a (/~mol/g)
1. Aminopropyl Fractosil 200 2. Aminopropyl-CPG 3. LCAA-CPG
66.50 49.24 34.02
64.90 49.10 33.30
a The nucleoside loadings are the mean from two separate experiments. for the reaction with D T D S and yet can still be titrated by acid and (ii) a part of the 4,4'-dimethoxytrityl cation released from the solid support m a y n o t be released in the surrounding solution but remained adsorbed to the solid support. The p r o p o s e d m e t h o d measures only those a m i n o groups which are accessible to D T D S . It does not measure those a m i n o groups which are sterically hindered or otherwise burried within the p o l y m e r matrix. Therefore, the n u m b e r of amino groups determined by the proposed m e t h o d would either be equal or less than the n u m b e r determined by nitrogen estimation or acid-base titration. However, the n u m b e r of amino groups determined by the p r o p o s e d m e t h o d would be an i m p o r t a n t parameter for calculating the a m o u n t of ligand required for immobilization. This was further confirmed by monitoring the functionalization of polymer supports for D N A synthesis. Three different p o l y m e r supports, viz, aminopropyl-Fractosil 200, aminopropylC P G and L C A A - C P G were chosen to test the new D T D S monitoring procedure. The amino loadings of all three p o l y m e r supports were determined b y the procedure mentioned in m e t h o d A. Suitably protected deoxynucleoside derivatives were then a n c h o r e d on a m i n o functionalized polymer supports [25]. Nucleoside loadings of all three functionalized p o l y m e r supports were then determined by the m e t h o d reported earlier [25]. The values of the amino loadings of all three polymer supports were found to be in good agreement with the nucleoside loadings of the corresponding functionalized p o l y m e r supports (Table 2). Thus, the solid support b o u n d a m i n o group determination by the p r o p o s e d m e t h o d could be a useful guide for the preparation of solid supports for the synthesis of oligonucleotides, peptides and matrices for affinity c h r o m a t o g r a p h y .
Simplified description of the method and its advantages A simple, sensitive and convenient spectrophotometric method for the determination of solid support bound amino groups is described. The method involves the use of the 2,4-dinitrophenyl ester of the commercially available 5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-succinate (DTS) and perchloric acid for the subsequent release of a strong chromophore of the bound DTDS, 4,4'-dimethoxytrityl cation. This method can be used to monitor the functionalization of all kinds of polymer supports including soft gels, for various applications in biotechnology. The method does not require any sophisticated instrumentation and the reagent DTDS can be synthesized in any laboratory without any problem.
329
Acknowledgements Financial support from the Department of Biotechnology, Government of India, N e w D e l h i is g r a t e f u l l y a c k n o w l e d g e d . T h a n k s a r e a l s o d u e t o M r . R a h i m f o r h i s t e c h n i c a l a s s i s t a n c e . O n e o f t h e a u t h o r s ( R . K . G . ) is t h a n k f u l t o t h e C S I R , N e w Delhi, for a research fellowship.
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