Abstract We prepared a crosslinked polymer as a sepa-ration and/or adsorption medium for CYN, as shown inFig. 1. The polymers were evaluated by high-performanceliquid chromatography (HPLC) and adsorption was exam-ined under batch conditions. Results from detailed HPLCevaluation and measurement of the difference between thebinding affinity for CYN and for other compounds showedthe prepared polymer had specific recognition ability forCYN.

Keywords Ion-exchange media · Highly hydrophiliccompounds · Interval immobilization technique · Cylindrospermopsin (CYN) · Cyanobacterium hepatotoxin

Introduction

Ion-exchange has been often used for separation and/oradsorption of ionic compounds, for example hydrophilicpolypeptides, DNA, or other hydrophilic ionic compounds.Because of the growing need for separation of such com-pounds, ion-exchange separation and/or adsorption mediawith selectivity for target compounds will be required.

Molecular imprinting (MI) is a successful means ofpreparing selective ion-exchange media for a particular tar-get compound. In this method we can easily obtain spe-cific molecular recognition of the molecule used as the tem-plate molecule. Many examples and useful applications ofMI have been reported [1, 2, 3, 4].

However, MI can be rarely be applied for highly hydro-philic compounds, rare naturally occurring compounds, andhighly toxic compounds, because it is necessary to use thereal target molecule as the template molecule in MI [5].

We have reported several applications of MI in which thereal target molecule is not used [6, 7, 8, 9, 10].

Another problem of MI using non-covalent-type inter-action between the template molecule and the functionalmonomers is the formation of rather heterogeneous recog-nition sites. The formation of heterogeneous recognitionsites in MI has been studied [11, 12, 13].

In MI an appropriate organic solvent is usually used aspolymerization solvent (porogenic solvent) to dissolve themonomers and the template molecule. In a relatively hy-drophobic organic solvent, hydrogen bonding works effec-tively between the template molecule and the functionalmonomers, resulting in formation of more accurate recog-nition sites. MI can, therefore, hardly be applied for highlyhydrophilic template molecules such as amino acids, hy-drophilic polypeptides, and organic ionic material, becausethose compounds dissolve only in water, in which typicalmonomers do not dissolve.

For example, many natural toxic compounds occur inenvironmental water. Cyanobacterium toxins, shellfish tox-ins, and fish toxins are examples. These toxic compoundsare highly hydrophilic because of their ionic properties,and so conventional adsorption methods based on hy-drophobic interaction are not effective at trapping them.

The occurrence of cyanobacterium toxins in freshwatercould be a serious danger to humans and domestic ani-mals if the water of lakes or ponds is used as drinking wa-ter [14, 15, 16, 17]. To avoid this danger quantitative analy-sis and effective removal of each toxin will be required.Environmental water is contaminated by many species,however, so direct analysis and selective removal of thetarget toxins is very difficult and novel separation mediawith selectivity for the highly hydrophilic toxins will benecessary.

In this report, we propose a novel separation mediumfor cylindrospermopsin (CYN), a powerful cyanobac-terium hepatotoxin (Fig. 1), as an example [18, 19, 20], byuse of a novel technique, called the “interval immobiliza-tion technique”. By use of this technique we believe thationic functional monomers can be immobilized at regulardistances and that these immobilized groups specifically

Takuya Kubo · Nobuo Tanaka · Ken Hosoya

Target-selective ion-exchange media for highly hydrophilic compounds: a possible solution by use of the “interval immobilization technique”

Anal Bioanal Chem (2004) 378 : 84–88DOI 10.1007/s00216-003-2320-4

Received: 27 August 2003 / Revised: 17 September 2003 / Accepted: 30 September 2003 / Published online: 13 November 2003

PAPER IN FOREFRONT

T. Kubo · N. Tanaka · K. Hosoya (✉)Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japane-mail: kenpc@ipc.kit.ac.jp

© Springer-Verlag 2003

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recognize target molecules with ionic groups separated bythe same distance as the immobilized ionic groups.

Experimental

For preparation of adsorbents, ethylene glycol dimethacrylate(EDMA) and 2-(diethylamino)ethyl methacrylate (DAEMA) werepurified by vacuum distillation to remove polymerization inhibi-tor. 2,2′-Azobis-(2,4-dimethylvaleronitrile) (ADVN), methyl (bro-momethyl)benzoate, 4-(bromomethyl)phenyl acetic acid, trimeth-ylsilyldiazomethane, and tetrabutylammonium chloride were pur-chased from Tokyo Kasei (Tokyo Japan). Methyl iodide, p-styrenesulfonic acid sodium salt (Ssa), methanol (MeOH), acetonitrile(AN), ethanol (EtOH), chloroform, dimethyl sulfoxide (DMSO),and NaCl were purchased from Wako Chemicals (Kyoto Japan)and used as received.

Synthesis of tributyl-(4-carboxybenzyl)ammonium (Tcba) as possible pseudo-template for CYN

Methyl (4-bromomethyl)benzoate (6.9 mmol, 1.5 g) and K2CO3(1.0 g) were dissolved in 50 mL AN. Tributylamine (4.2 mmol, 1.0 mL) was added slowly to the reaction mixture with stirring.The mixture was then stirred at 100 °C, under reflux, for 24 h un-der a nitrogen atmosphere. Generation of the target compound wasconfirmed by TLC with a detection reagent. When the reactionwas complete the mixture was separated by silica gel column chro-matography and tributyl-(4-methoxycarbonylbenzyl)-ammoniumchloride was obtained.

The tributyl-(4-methoxycarbonylbenzyl)ammonium chloridewas dissolved in hydrochloric acid (1.0 mol L–1, 50 mL) and heatedat 120 °C under reflux for 24 h under a nitrogen atmosphere. Afterconfirmation, by TLC, of loss of the methyl ester the resulting tribu-tyl-(4-carboxybenzyl)ammonium (Tcba), was extracted with chloro-form and purified by silica gel column chromatography.

Yield 90.2%. 1H NMR (in CD3OD) δ (ppm): 0.97 (m, 12H),1.36 (m, 8H), 1.76 (m, 8H), 3.13 (m, 8H), 4.78 (s, 2H), 7.42 (d,2H), 7.97 (d, 2H).

Synthesis of tributyl-(4-carboxymethylbenzyl)ammonium(Tcmba) as possible looser pseudo-template for CYN

4-(Bromomethyl)phenylacetic acid (2.0 g) and trimethylsilyldia-zomethane (2.0 mol L–1 solution in hexane, 10 mL) were dissolvedin 50 mL MeOH–Benzene, 15:35. The mixture was stirred at roomtemperature for 2 h. The solvent was then evaporated and theresidue was treated with tributylamine at 100 °C for 24 h. Tributyl-(4-methoxycarbonylmethylbenzyl)ammonium was isolated by sil-ica gel column chromatography and the resulting compound washydrolyzed with 1.0 mol L–1 aqueous NH3 solution.

Finally, the target compound, tributyl-(4-carboxymethylben-zyl)ammonium, was isolated by silica gel column chromatogra-phy.

Yield 64.3%. 1H NMR (in CD3OD) δ (ppm): 1.03 (m, 12H),1.41 (m, 8H), 1.82 (m, 8H), 3.18 (m, 8H), 3.60 (s, 2H), 4.75 (s,2H), 7.45 (d, 4H).

Isolation of CYN

A toxic strain of Cylindrospermopsis raciborskii (CRJ-1=AWT 205)was obtained from the Microbial Culture Collection (MCC-NIES)and grown in CT medium. Cells were separated from the mediumby centrifugation and lyophilized. CYN was extracted according toa method reported elsewhere [18]. The extracted CYN was puri-fied by HPLC on an Amide-80 column (10 mm×250 mm, TosohCorporation, Japan) with a 100 to 60% aqueous AN linear gradientin 20 min at 4.0 mL min–1. The isolated CYN was identified by NMRand MS. The spectrometric data were in good agreement withthose of CYN [18].

Preparation of polymer adsorbents

The complexes of p-styrene sulfonic acid sodium salt (Ssa) and theammonium compound were prepared as follows. Ssa was dis-

Fig. 1 Concept of interval immobilization technique for CYN

solved in water and extracted with chloroform containing one halfthe mole ratio of tetrabutylammonium chloride (or Tcba or Tcmba)relative to Ssa, by the phase transfer effect. The solvent was thenremoved to give the complexes. The polymer adsorbents were pre-pared with 1.0% w/w ADVN as radical initiator, at 50 °C, for 24 h.The polymers were ground, washed with MeOH, then treated withCH3I to generate the alkylammonium groups separate from base

polymer (P-base). The reaction was carried out in DMSO at 70 °Cfor 24 h as shown in Fig. 1. The symbols used for the polymers,and the polymer compositions, are also summarized in Table 1.

HPLC evaluation of polymer adsorbents

Each polymer was isolated in the size range 25–45 µm. The result-ing polymer particles were packed into stainless steel columns bya slurry method and evaluated by HPLC using a 90% methanolaqueous solution containing NaCl as mobile phase, because reten-tion of the ionic solutes was too large to be detected under salt-freeconditions. Chromatographic data were acquired with a Shimadzu(Japan) HPLC system consisting of an LC-6A pump, an SPD-M10A photodiode-array detector, and a CTO-10AC column oven.

Scatchard plot

To construct a Scatchard plot CYN solutions were prepared at con-centrations of 1.0–10–4 mmol L–1 in 90% MeOH aqueous solution.Each CYN solution (1.0 mL) was added to vials containing 10 mgP-BL or P-Tcba. After 12 h, during which the vials were shaken atregular time intervals, the amount of free CYN in the supernatantwas quantified by HPLC–MS with external standard calibration.Quantitative determination of CYN was performed by HPLC withan Amide-80 column (100 mm×2.0 mm, Tosoh, Japan) with 100 to60% AN linear gradient, in 20 min, at 0.2 mL min–1. CYN was de-tected by MS in SIM mode at m/z 414.

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Table 1 Composition of polymers

Symbol Composition

P-Base EDMA (5 mL, 26.5 mmol), 1:1 AN–EtOH (5 mL)

P-BL EDMA (5 mL, 26.5 mmol), (Non-MIP, blank) 1:1 AN–EtOH (5 mL), DAEMA (2 mmol),

Complex of Ssa and Tba (2 mmol)P-Tcba (M1P 1) EDMA (5 mL, 26.5 mmol),

1:1 AN–EtOH (5 mL), DAEMA (2 mmol), Complex of Ssa and Tcba (2 mmol)

P-Tcmba (M1P 2) EDMA (5 mL, 26.5 mmol), 1:1 AN–EtOH (5 mL), DAEMA (2 mmol), Complex of Ssa and Tcmba (2 mmol)

EDMA, ethylene glycol dimethacrylate; DAEMA, 2-(diethylami-no)ethyl methacrylate; Ssa, p-styrene sulfonic acid sodium salt;Tba, tetrabutylammonium; Tcba, tributyl-(4-carboxybenzyl)am-monium; Tcmba, tributyl-(4-carboxymethylbenzyl)ammonium

Fig. 2 Structures of thepseudo-template molecules anddistance between ionic groups

Results and discussion

To immobilize ionic functional groups on to the polymeradsorbents, two possible pseudo-template molecules weredesigned by computer modeling taking into considerationthe distance (interval) between the two ionic groups ofCYN. The structures of the functional groups and the dis-tance between them are shown in Fig. 2.

A possible pseudo-template molecule (Tcba) and twofunctional monomers in polymerization solvent were ex-amined by FTIR. Comparison of the spectra of Tcba andthe complexes with Ssa and Tcba revealed the specific ad-sorption band attributed to the sulfonyl group at approxi-mately 1200 cm–1. Moreover, although Ssa was not solu-

ble in the solvent the complex of Ssa and Tcba was read-ily soluble.

Therefore it seemed that the complex was stable in thissolvent. Comparison of the spectra of DAEMA and com-plex revealed the specific adsorption band attributed tohydrogen bonding between the carboxylic acid and amineat approximately 1600 cm–1. On the basis of these resultsit was therefore believed that the pseudo-print moleculeand each functional monomer could interact in each otherin the polymerization mixture.

Columns packed with the polymer adsorbents wereprepared and retention factors, k′, and separation factors,α, were determined for CYN and some ionic solutes. Theresults obtained from these examinations revealed thatboth ionic functional groups had been immobilized on

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Fig. 3 Comparison of separa-tion factor for CYN relative toother ionic solutes. HPLC con-ditions: mobile phase 9:1MeOH–1.0 mol L–1 NaCl aq;column size 100 mm×4.6 mm(i.d.); flow rate 0.5 mL min–1;detection photo-diode array;temperature 30 °C

Fig. 4 Scatchard plots forCYN on each polymer. The as-sociation constant (Ka) andnumber of binding sites weredetermined from the slope andy intercept, respectively, of thefitted line (n/Cf=–Kan+KaN)obtained by least-squares re-gression

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each polymer (P-BL, P-Tcba, and P-Tcmba) – this wasshown by comparison of retention factors for ionic soluteson P-BL, P-Tcba, and P-Tcmba with those on P-Base withionic functional groups attached.

Moreover, as is shown in Fig. 3, high selectivity wasobserved for CYN on P-Tcba, prepared with Tcba as thepossible pseudo-template molecule, for which the dis-tance between the functional groups was closer than thoseof CYN. This selectivity suggests that the distance be-tween the immobilized ionic groups obtained by use ofthe possible pseudo-template enabled selective recogni-tion of the CYN molecule.

The dependence of the retention factor for CYN on theconcentration of NaCl in the mobile phase was also ex-amined. It was shown that the slope of k′ for CYN on P-Tcba was much less steep than that for CYN on P-BL.This suggests that recognition sites on P-Tcba recognizeCYN more strongly. Thus retention of CYN was lowereven for higher NaCl concentrations.

Scatchard plot

To obtain more detailed information about the mechanismof recognition of CYN, the Scatchard plot was constructedunder batch conditions, because this evaluation is oftenperformed to examine the association constant in molecu-lar imprinting [11, 12, 13]. On the basis of the results fromthis analysis, the association constant Ka and the numberof binding sites N for the polymers were calculated ac-cording to the Langmuir model based on the Scatchardplot. The Scatchard plots are shown in Fig. 4 and the re-sults are summarized briefly in Table 2.

Table 2 shows that the association constants and num-ber of binding sites were similar on both polymers, andthe distribution of the plots was much broader at higherconcentrations. On the other hand, for P-Tcba the associa-tion constant was higher and the distribution of the plots wasnarrower than for P-BL at lower concentrations. Thesenotable differences suggest that the polymer prepared bythe interval immobilization technique, P-Tcba, has uni-

form high-affinity binding sites for CYN, as illustrated inFig. 1.

According to the results from the Scatchard plot it seemsthat a specific distance between binding sites for CYNwas achieved on P-Tcba by the interval immobilizationtechnique for ionic groups, resulting in a high associationconstant then the polymer P-Tcba was used.

Although the N value was larger on P-BL than that onP-Tcba, the Scatchard plot based on batch adsorptionstrongly suggests that specific recognition sites for CYNwere formed within the polymer prepared by the intervalimmobilization technique, for example P-Tcba, whereason P-BL non-specific adsorption sites are dominant.

We suggest that the technique proposed in this papercan be applied to other highly hydrophilic compounds, forexample as natural toxins. Moreover, the technique canalso be used for analysis of other biological compounds.For example, recognition of part of a protein by ionicgroups as a result of the interval immobilization techniquemight lead to analysis of specific groups of proteins.

References

1. Nilsson K, Lindell J, Norlow O, Sellergren B (1994) J Chro-matogr A 680:57

2. Kempe M, Mosbach K (1995) J Chromatogr A 691:3173. Kempe M, Fischer L, Mosbach K (1993) J Mol Recognit 6:254. Spivak D, Shea KJ (1999) J Org Chem 64:46275. Haginaka J, Sanbe H (2000) Anal Chem 72:52066. Yoshizako K, Hosoya K, Iwakoshi Y, Kimata K, Tanaka N

(1998) Anal Chem 70:3867. Hosoya K, Yoshizako K, Sasaki H, Kimata K, Tanaka N

(1998) J Chromatogr A 828:918. Hosoya K, Iwakoshi Y, Yoshizako K, Kimata K, Tanaka N,

Takehira H, Haginaka J (1999) J High Resolut Chromatogr 22:256

9. Hosoya K, Yoshizako K, Kubo T, Ikegami T, Tanaka N, Hagi-naka J (2002) Anal Sci 18:55

10. Kubo T, Hosoya K, Watabe Y, Ikegami T, Tanaka N, Sano T,Kaya K (2003) J Chromatogr A 987:389

11. Sajionz P, Kele M, Zhong G, Sellergren B, Guiochon G (1998)J Chromatogr A 810:1

12. Quaglis M, Chenon K, Hall AJ, Lorenzi ED, Sellergren B(2001) J Am Chem Soc 123:2146

13. Takeuchi T, Mukawa T, Matsui J, Higashi M, Shimizu KD(2001) Anal Chem 73:3869

14. Kaya K, Watanabe MM (1994) Microbiol Cult Coll June, Re-view

15. Codd GA, Metcalf JS, Kaya K (2001) J AOAC Int 84:162616. Hunter PR (1992) J Med Microbiol 36:30117. Elder GH, Hunter PR, Codd GA (1993) Lancet 341:151918. Harada K, Ohtani I, Terao K (1994) Toxicon 32:7319. Hawkins PR, Chandrasena NR, Fralconer IR (1997) Toxicon

35:34120. Li R, Carmichael WW, Brittain S, Kaya K, Watanabe MM

(2001) Toxicon 39:973

Table 2 Binding data determined by Scatchard analysis

Ka1 Ka2 N1 N2(mol–1 L) (mol–1 L) (µmol g–1) (µmol g–1)

P-BL (non-MIP, blank) 2.7×104 2.3×103 1.4 8.1P-Tcba (MIP 1) 8.9×104 3.2×103 0.82 7.6

The value of Ka and N were determined from the Scatchard plots ofeach polymer