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2-(Trifluoromethyl)acrylic acid: a novel functional monomer in non-covalent molecular imprinting
ANALYTIC4
CHIMICA ACTA ELSEVIER
Analytica
Chimica Acta 343 (1997) 14
Letter
2-(Trifluoromethyl)acrylic acid: a novel functional monomer in non-covalent molecular imprinting Jun Matsui, Otto Doblhoff-Dier’, Toshifumi Takeuchi* Laboratory of Synthetic Biochemistry, Faculty of Information Sciences, Hiroshima City University, 151-5 Ozuka, Numata-cho, Asaminami-ku, Hiroshima 731-31. Japan Received 25 November
1996; accepted 2 December
1996
Abstract Molecular imprinting of nicotine was performed using an acidic functional monomer 2-(trifluoromethyl)acrylic acid (TFMAA). Chromatographic studies using the resultant polymers as stationary phases displayed that nicotine-selective affinity induced by the use of TFMAA was greater than that induced by the use of a conventional functional monomer methacrylic acid. Keywords: Nicotine; 2-(Trifluoromethyl)acrylic acid; Molecular imprinting
1. Introduction Molecular imprinting has been recognized as a template polymerization technique to produce biomolecule mimics [l-6]. Currently, the potential of molecularly imprinted polymers has been successfully demonstrated in various applications, especially as antibody/receptor mimics, and high selectivity comparable to natural antibodies has been exhibited [7-lo]. Regarding affinity, however, further improvements are still necessary for molecularly imprinted polymers to be feasible as biomolecule mimics. In molecular imprinting, self-assembling of a target molecule (as a template molecule) and polymerizable
*Corresponding author. Fax: +81 82 830 1610; e-mail: [email protected]. ‘On leave from Institute of Applied Microbiology, State University of Forestry and Agriculture, Vienna, Austria. 0003-2670/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOOO3-2670(96)00596-X
template-interacting molecules (functional monomers) is followed by crosslinking to freeze the selfassembled structure within the polymer matrices, and a subsequent extraction of the template molecule results in the formation of complementary binding sites (Fig. 1). From these procedures of molecular imprinting, it is apparent that the use of functional monomers interacting strongly with a template molecule is appropriate for generating high affinity binding sites, because they are expected to yield stable, selfassembled complexes at the crosslinking stage, which is critical for sharply imprinting the template molecule. Moreover, such functional monomers are expected to result in high affinity residues in binding sites of the resultant polymer, even without the imprint effect. We have recently reported [ 111 on a polymer selective for nicotine prepared by molecular imprinting using methacrylic acid (MAA), that has been exten-
J. Matsui et al. /Analytica
Chimica Acta 343 (1997) 14
selective rebinding
Fig. 1. Schematic
illustration
of the nicotine-imprinting
performed
in this study. Thick lines represent crosslinked
sively studied as a functional monomer and demonstrated to be useful for various template molecules. In that study, it was implied that the number of basic functional groups in the two basic sites of nicotine plays a more significant role for developing high affinity. Here, a parallel argument could be made for a functional monomer: more acidic functional monomers could be preferable for imprinting a basic template molecule, e.g. nicotine. On this basis, the feasibility of 2-(trifluoromethyl)acrylic acid (TFMAA) [12] was examined as a functional monomer for imprinting nicotine; the molecule is more acidic than methacrylic acid owing to the electron-withdrawing effect of the trifluoromethyl group.
bisisobutyronitrile were purchased from Waco. Acetonitrile, methanol and chloroform were from Katayama. Nicotine was purified by distillation prior to use. Other chemicals were used without further purification. Chromatographic studies were performed with a Waters HPLC system, consisting of a pump (model 626), a UV absorbance detector (model 486) and an autosample injector (model 717 plus). 2.2. Preparation
2.1. Materials and instruments Nicotine, cotinine, 3-methylpyridine, ethylene glyco1 dimethacrylate, methacrylic acid and 2,2’-azo-
and blank polymers
prepared
a Total amount of the functional imprinted polymers.
monomers
polymers
in this study
Functional
Nicotine-imprinted Blank
of the nicotine-imprinted
Four sets of nicotine-imprinted and non-imprinted blank polymers were prepared (Table 1). For preparing nicotine-imprinted polymers, nicotine (264 mg, 1.67 mmol) and functional monomers(s) (10.0 mmol) were dissolved in chloroform (25 ml). Into the mixture, a crosslinker (ethylene glycol dimethacrylate, 9.35 g) and an initiator (2,2’-azobisisobutyronitrile, 120 mg) were added. After nitrogen gas was sparged, the mixture was placed under UV irradiation at 3°C for 12 h. The resultant bulk rigid polymers were ground (<45 urn), and packed in stainless steel columns
2. Experimental
Table 1 Nicotine-imprinted
polymer network.
monomers
(MAA
: TFMAA) ’
6:0
4:2
214
0:6
PO(Nico) PO(Blk)
PZ(Nico) PZ(Blk)
P4fNico) P4(Blk)
P6(Nico) P6(Blk)
used was 10.0 mm01 for each polymer, that is six times that of the template species in the case of
.I. Matsui et al. /Analytica
50
(150 mmx4.6 mm i.d). Non-imprinted blank polymers were prepared in an identical fashion, without the addition of nicotine. 2.3. Chromatographic
40 ‘0 5 2
experiments
Columns filled with the polymer particles were attached to the HPLC pump and were washed with methanol/acetic acid (8 : 2, v/v). The eluent was acetonitrile/acetic acid (95 : 5, v/v) at a flow rate of 1.O ml min-‘, an eluted compounds were detected at 262 nm. The sample volume and concentration injected were 10 pl and 1.0 mM, respectively. Capacity factors were calculated by the equation, k’ = (tR - &)/to, where tR is the retention time of an analyte and to is the time to elute acetone. Separation factors were calculated as K(nicotine)lK (sample), where K(nicotine) is the capacity factor for nicotine.
3. Results and discussion The affinity for nicotine (Fig. 2) was evaluated by liquid chromatography [ 13-171. As shown in Fig. 3, the polymers prepared with increased amount of TFMAA showed longer retention, and consequently, P6(Nico) exhibited the largest capacity factor (K) among all the nicotine-imprinted polymers investigated, that is as 12 times large as the K of PO(Nico). By calculating ratios of K values of the imprinted polymer to those of the blank polymer, the degree of affinity induction by nicotine-imprinted, i.e. the imprint effect, could be evaluated. Although all the nicotine-imprinting polymers retained nicotine longer than the corresponding blank polymers, the greatest imprint effect was observed between P6(Nico) and P6(Blk), where K(P6(Nico))lK(P6(Blk)) is 11, while K(PO(Nico))/K(PO(Blk)) is 7.5, suggesting that
3
Chimica Acta 343 (1997) 14
.E 8 2 0
30 20 10 0 PO
P2
Fig. 3. The retention of nicotine in the imprinted non-imprinted blank (white) polymers.
(black) and the
TFMAA worked better in the imprinting process. Among the blank polymers, P6(Blk) exhibited the longest retention. This suggests that functional residues derived from TFh4AA are more reactive, and that TFMAA is again advantageous for high affinity. We also examined selectivity by testing cotinine and 3-methylpyridine (Fig. 2), a nicotine metabolite and a mimic of a part of nicotine structure, respectively. Separation factors on P6(Nico), P6(Blk) and PO(Nico) are listed in Table 2. As shown, P6(Nico) was more selective for nicotine than the non-imprinted P6(Blk), showing that the affinity was induced by the template-directed selectivity. Furthermore, the selectivity was not worse than that of PO(Nico) prepared by the conventional imprinting method. The results show that the use of TFMAA is favorable for both high affinity and selectivity in nicotine-imprinting.
Polymer
of the polymers
for nicotine
Separation Cotinine
1, nicotine; 2,
P6
Polymer
Table 2 Selectivity
Fig. 2. Structure of nicotine and tested compounds: cotinine; 3, 3-methylpyridine.
P4
P6(Nico) P6(Blk) PO(Nico)
5.8 2.6 4.0
factor 3-Methylpyridine 8.7 1.9 6.5
J. Matsui et al. /Analytica
4
4. Conclusions In this study, TFMAA was used for imprinting nicotine and resulted in high affinity nicotine-selective polymer. The results could allow a general conclusion to be drawn that high affinity can be achieved by careful selection of functional monomers. Further studies on the structures of the self-assembled complexes would be significantly helpful for making a better selection or for designing and synthesizing [ 181 appropriate functional monomers, if necessary, to perform molecular imprinting effectively.
Acknowledgements The authors thank the financial support of Kowa Life Science Foundation. This work was also supported by the Japan Securities Scholarship Foundation.
References [l] K. Mosbach 163.
and 0. Ramstrom,
Biotechnology,
14 (1996)
Chimica Acta 343 (1997) I-4 [2] R.J. Ansell, D. Kriz and K. Mosbach, Cum Opin. Biotechnol., 7 (1996) 89. [3] G. Wuff, Angew. Chem., Int. Ed. Engl., 34 (1995) 1812. [4] S. Vidyasankar and F.H. Arnold, Cum Opin. Biotechnol., 6 (1995) 218. [5] K.J. Shea, Trends Polym. Sci., 2 (1994) 166. [6] T. Takeuchi and J. Matsui, Acta. Polym., 47 (1996) 471. [7] G. Vlatakis, L.I. Andersson, R. Miller and K. Mosbach, Nature, 361 (1993) 645. [S] I. Matsui, Y. Miyoshi, 0. Doblhoff-Dier and T. Takeuchi, Anal. Chem., 67 (1995) 4404. [9] M.T. Muldoon and L.H. Stanker, J. Agric. Food Chem., 43 (1995) 1424. [lo] M. Siemann, L.I. Andersson and K. Mosbach, J. Agric. Food Chem., 44 (1996) 141. [ 111 J. Matsui, A. Kaneko, Y. Miyoshi, K. Yokoyama, E. Tamiya and T. Takeuchi, Anal. Lett., 29 (1996) 2071. [12] J. Matsui, Y. Miyoshi and T. Takeuchi, Chem. Lett., (1995) 1007. [13] I. Matsui, T. Kato, T. Takeuchi, S. Suzuki, K. Yokoyama, E. Tamiya and I. Karube, Anal. Chem., 65 (1993) 2223. [14] .I. Matsui, Y. Miyoshi, R. Matsui and T. Takeuchi, Anal. Sci., 11 (1995) 1017. [15] M. Kempe and K. Mosbach, J. Chromatogr. A, 694 (1995) 3. [16] B. Sellergren, in G. Subramanian (Ed.), Practical Approach Chiral Separations by Liquid Chromatography, VCH, Weinheim, 1994, p. 69. [17] LA. Nicholls, L.I. Andersson, K. Mosbach and B. Ekberg, Trends Biotechnol., 13 (1995) 47. [IS] K. Tanabe, T. Takeuchi, J. Matsui, K. Yano, K. Ikebukuro and I. Karube, J. Chem. Sot. Chem. Commun., (1995) 2303.
CHIMICA ACTA ELSEVIER
Analytica
Chimica Acta 343 (1997) 14
Letter
2-(Trifluoromethyl)acrylic acid: a novel functional monomer in non-covalent molecular imprinting Jun Matsui, Otto Doblhoff-Dier’, Toshifumi Takeuchi* Laboratory of Synthetic Biochemistry, Faculty of Information Sciences, Hiroshima City University, 151-5 Ozuka, Numata-cho, Asaminami-ku, Hiroshima 731-31. Japan Received 25 November
1996; accepted 2 December
1996
Abstract Molecular imprinting of nicotine was performed using an acidic functional monomer 2-(trifluoromethyl)acrylic acid (TFMAA). Chromatographic studies using the resultant polymers as stationary phases displayed that nicotine-selective affinity induced by the use of TFMAA was greater than that induced by the use of a conventional functional monomer methacrylic acid. Keywords: Nicotine; 2-(Trifluoromethyl)acrylic acid; Molecular imprinting
1. Introduction Molecular imprinting has been recognized as a template polymerization technique to produce biomolecule mimics [l-6]. Currently, the potential of molecularly imprinted polymers has been successfully demonstrated in various applications, especially as antibody/receptor mimics, and high selectivity comparable to natural antibodies has been exhibited [7-lo]. Regarding affinity, however, further improvements are still necessary for molecularly imprinted polymers to be feasible as biomolecule mimics. In molecular imprinting, self-assembling of a target molecule (as a template molecule) and polymerizable
*Corresponding author. Fax: +81 82 830 1610; e-mail: [email protected]. ‘On leave from Institute of Applied Microbiology, State University of Forestry and Agriculture, Vienna, Austria. 0003-2670/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOOO3-2670(96)00596-X
template-interacting molecules (functional monomers) is followed by crosslinking to freeze the selfassembled structure within the polymer matrices, and a subsequent extraction of the template molecule results in the formation of complementary binding sites (Fig. 1). From these procedures of molecular imprinting, it is apparent that the use of functional monomers interacting strongly with a template molecule is appropriate for generating high affinity binding sites, because they are expected to yield stable, selfassembled complexes at the crosslinking stage, which is critical for sharply imprinting the template molecule. Moreover, such functional monomers are expected to result in high affinity residues in binding sites of the resultant polymer, even without the imprint effect. We have recently reported [ 111 on a polymer selective for nicotine prepared by molecular imprinting using methacrylic acid (MAA), that has been exten-
J. Matsui et al. /Analytica
Chimica Acta 343 (1997) 14
selective rebinding
Fig. 1. Schematic
illustration
of the nicotine-imprinting
performed
in this study. Thick lines represent crosslinked
sively studied as a functional monomer and demonstrated to be useful for various template molecules. In that study, it was implied that the number of basic functional groups in the two basic sites of nicotine plays a more significant role for developing high affinity. Here, a parallel argument could be made for a functional monomer: more acidic functional monomers could be preferable for imprinting a basic template molecule, e.g. nicotine. On this basis, the feasibility of 2-(trifluoromethyl)acrylic acid (TFMAA) [12] was examined as a functional monomer for imprinting nicotine; the molecule is more acidic than methacrylic acid owing to the electron-withdrawing effect of the trifluoromethyl group.
bisisobutyronitrile were purchased from Waco. Acetonitrile, methanol and chloroform were from Katayama. Nicotine was purified by distillation prior to use. Other chemicals were used without further purification. Chromatographic studies were performed with a Waters HPLC system, consisting of a pump (model 626), a UV absorbance detector (model 486) and an autosample injector (model 717 plus). 2.2. Preparation
2.1. Materials and instruments Nicotine, cotinine, 3-methylpyridine, ethylene glyco1 dimethacrylate, methacrylic acid and 2,2’-azo-
and blank polymers
prepared
a Total amount of the functional imprinted polymers.
monomers
polymers
in this study
Functional
Nicotine-imprinted Blank
of the nicotine-imprinted
Four sets of nicotine-imprinted and non-imprinted blank polymers were prepared (Table 1). For preparing nicotine-imprinted polymers, nicotine (264 mg, 1.67 mmol) and functional monomers(s) (10.0 mmol) were dissolved in chloroform (25 ml). Into the mixture, a crosslinker (ethylene glycol dimethacrylate, 9.35 g) and an initiator (2,2’-azobisisobutyronitrile, 120 mg) were added. After nitrogen gas was sparged, the mixture was placed under UV irradiation at 3°C for 12 h. The resultant bulk rigid polymers were ground (<45 urn), and packed in stainless steel columns
2. Experimental
Table 1 Nicotine-imprinted
polymer network.
monomers
(MAA
: TFMAA) ’
6:0
4:2
214
0:6
PO(Nico) PO(Blk)
PZ(Nico) PZ(Blk)
P4fNico) P4(Blk)
P6(Nico) P6(Blk)
used was 10.0 mm01 for each polymer, that is six times that of the template species in the case of
.I. Matsui et al. /Analytica
50
(150 mmx4.6 mm i.d). Non-imprinted blank polymers were prepared in an identical fashion, without the addition of nicotine. 2.3. Chromatographic
40 ‘0 5 2
experiments
Columns filled with the polymer particles were attached to the HPLC pump and were washed with methanol/acetic acid (8 : 2, v/v). The eluent was acetonitrile/acetic acid (95 : 5, v/v) at a flow rate of 1.O ml min-‘, an eluted compounds were detected at 262 nm. The sample volume and concentration injected were 10 pl and 1.0 mM, respectively. Capacity factors were calculated by the equation, k’ = (tR - &)/to, where tR is the retention time of an analyte and to is the time to elute acetone. Separation factors were calculated as K(nicotine)lK (sample), where K(nicotine) is the capacity factor for nicotine.
3. Results and discussion The affinity for nicotine (Fig. 2) was evaluated by liquid chromatography [ 13-171. As shown in Fig. 3, the polymers prepared with increased amount of TFMAA showed longer retention, and consequently, P6(Nico) exhibited the largest capacity factor (K) among all the nicotine-imprinted polymers investigated, that is as 12 times large as the K of PO(Nico). By calculating ratios of K values of the imprinted polymer to those of the blank polymer, the degree of affinity induction by nicotine-imprinted, i.e. the imprint effect, could be evaluated. Although all the nicotine-imprinting polymers retained nicotine longer than the corresponding blank polymers, the greatest imprint effect was observed between P6(Nico) and P6(Blk), where K(P6(Nico))lK(P6(Blk)) is 11, while K(PO(Nico))/K(PO(Blk)) is 7.5, suggesting that
3
Chimica Acta 343 (1997) 14
.E 8 2 0
30 20 10 0 PO
P2
Fig. 3. The retention of nicotine in the imprinted non-imprinted blank (white) polymers.
(black) and the
TFMAA worked better in the imprinting process. Among the blank polymers, P6(Blk) exhibited the longest retention. This suggests that functional residues derived from TFh4AA are more reactive, and that TFMAA is again advantageous for high affinity. We also examined selectivity by testing cotinine and 3-methylpyridine (Fig. 2), a nicotine metabolite and a mimic of a part of nicotine structure, respectively. Separation factors on P6(Nico), P6(Blk) and PO(Nico) are listed in Table 2. As shown, P6(Nico) was more selective for nicotine than the non-imprinted P6(Blk), showing that the affinity was induced by the template-directed selectivity. Furthermore, the selectivity was not worse than that of PO(Nico) prepared by the conventional imprinting method. The results show that the use of TFMAA is favorable for both high affinity and selectivity in nicotine-imprinting.
Polymer
of the polymers
for nicotine
Separation Cotinine
1, nicotine; 2,
P6
Polymer
Table 2 Selectivity
Fig. 2. Structure of nicotine and tested compounds: cotinine; 3, 3-methylpyridine.
P4
P6(Nico) P6(Blk) PO(Nico)
5.8 2.6 4.0
factor 3-Methylpyridine 8.7 1.9 6.5
J. Matsui et al. /Analytica
4
4. Conclusions In this study, TFMAA was used for imprinting nicotine and resulted in high affinity nicotine-selective polymer. The results could allow a general conclusion to be drawn that high affinity can be achieved by careful selection of functional monomers. Further studies on the structures of the self-assembled complexes would be significantly helpful for making a better selection or for designing and synthesizing [ 181 appropriate functional monomers, if necessary, to perform molecular imprinting effectively.
Acknowledgements The authors thank the financial support of Kowa Life Science Foundation. This work was also supported by the Japan Securities Scholarship Foundation.
References [l] K. Mosbach 163.
and 0. Ramstrom,
Biotechnology,
14 (1996)
Chimica Acta 343 (1997) I-4 [2] R.J. Ansell, D. Kriz and K. Mosbach, Cum Opin. Biotechnol., 7 (1996) 89. [3] G. Wuff, Angew. Chem., Int. Ed. Engl., 34 (1995) 1812. [4] S. Vidyasankar and F.H. Arnold, Cum Opin. Biotechnol., 6 (1995) 218. [5] K.J. Shea, Trends Polym. Sci., 2 (1994) 166. [6] T. Takeuchi and J. Matsui, Acta. Polym., 47 (1996) 471. [7] G. Vlatakis, L.I. Andersson, R. Miller and K. Mosbach, Nature, 361 (1993) 645. [S] I. Matsui, Y. Miyoshi, 0. Doblhoff-Dier and T. Takeuchi, Anal. Chem., 67 (1995) 4404. [9] M.T. Muldoon and L.H. Stanker, J. Agric. Food Chem., 43 (1995) 1424. [lo] M. Siemann, L.I. Andersson and K. Mosbach, J. Agric. Food Chem., 44 (1996) 141. [ 111 J. Matsui, A. Kaneko, Y. Miyoshi, K. Yokoyama, E. Tamiya and T. Takeuchi, Anal. Lett., 29 (1996) 2071. [12] J. Matsui, Y. Miyoshi and T. Takeuchi, Chem. Lett., (1995) 1007. [13] I. Matsui, T. Kato, T. Takeuchi, S. Suzuki, K. Yokoyama, E. Tamiya and I. Karube, Anal. Chem., 65 (1993) 2223. [14] .I. Matsui, Y. Miyoshi, R. Matsui and T. Takeuchi, Anal. Sci., 11 (1995) 1017. [15] M. Kempe and K. Mosbach, J. Chromatogr. A, 694 (1995) 3. [16] B. Sellergren, in G. Subramanian (Ed.), Practical Approach Chiral Separations by Liquid Chromatography, VCH, Weinheim, 1994, p. 69. [17] LA. Nicholls, L.I. Andersson, K. Mosbach and B. Ekberg, Trends Biotechnol., 13 (1995) 47. [IS] K. Tanabe, T. Takeuchi, J. Matsui, K. Yano, K. Ikebukuro and I. Karube, J. Chem. Sot. Chem. Commun., (1995) 2303.