Effect of Displacer Chemistry on Displacer Efficacy for a Sugar-Based

Survey of the year 2006 commercial optical biosensor literature. Rebecca L. Rich , David G. Myszka. Journal of Molecular Recognition 2007 20 (5), 300-...
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Ind. Eng. Chem. Res. 2006, 45, 9107-9114

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Effect of Displacer Chemistry on Displacer Efficacy for a Sugar-Based Anion Exchange Displacer Library Jia Liu,† Sun K. Park,‡ J. A. Moore,‡ and Steven M. Cramer*,† Department of Chemical and Biological Engineering and Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180

A homologous series of sugar-based anion exchange displacers were synthesized and evaluated by a parallel batch displacement assay as potential displacers for protein purification in anion exchange systems. The structural variation of these homologous molecules enabled a detailed study of the relationship between structure and efficacy. The percent protein displaced was evaluated in these batch systems for two model proteins, BSA and trypsin inhibitor, as a function of displacer concentration and chemistry on a Source 15Q anion exchange resin. The results indicated that the addition of aromatic rings at the periphery of the molecule close to the charged groups increased displacer affinity. It was observed that displacers with more structural flexibility had higher efficacy up to a certain value, beyond which flexibility did not appear to affect displacer efficacy for the molecules evaluated. It was also demonstrated that long hydrophobic side chains which are not associated with the charge may lower displacer efficacy because of undesired interactions with protein molecules or the formation of micelles. Stereochemistry was also not observed to play an important role in displacer efficacy for the molecules evaluated. Finally, low molecular mass displacers with affinities higher than those previously observed were identified as part of this study. Introduction Ion exchange displacement chromatography has attracted significant attention as a powerful technique for the purification of therapeutic biomolecules because of the high product concentration, purity, and yield that this technique can produce at high column loadings.1-3 While conventional high molecular weight displacers (MW > 2000) such as polyelectrolytes were used in ion exchange systems,4 it has also been demonstrated that low molecular weight displacers (MW < 2000) can be effective displacers for bioproduct purification.5 Low MW displacers have significant operational advantages compared to large polyelectrolyte displacers. If there is any overlap between the displacer and the protein of interest, these low MW molecules can be readily separated from the purified protein during postdisplacement processing using standard sizebased purification methods (e.g., size-exclusion chromatography, ultrafiltration). The salt-dependent adsorption behavior of these low MW displacers greatly facilitates column regeneration. Finally, the use of low MW displacers can result in separations utilizing the selective displacement mode, which results in elution of the weakly retained proteins in the induced salt gradient, displacement of the bioproduct of interest and closely associated impurities, and desorption of the more strongly retained impurities after the breakthrough of the displacer front. A variety of low molecular mass displacers have been identified including protected amino acids,6 dendrimers,7 antibiotics,8 pentaerythritol- and phloroglucinol-based salts,9,10 and aminoglycoside-polyamines.11 Despite these advances, the design of low molecular mass high-affinity displacers for the purification of highly retained biomolecules remains a challenge. The identification of appropriate displacer candidates has relied on the determination of their dynamic affinity based on the steric mass action (SMA) * To whom correspondence should be addressed. Tel.: (518) 2766198. Fax: (518) 276-4030. E-mail: [email protected]. † Isermann Department of Chemical and Biological Engineering. ‡ Department of Chemistry and Chemical Biology.

parameters of potential displacers.12 Shukla et al.9,13 synthesized pentaerythritol-based displacers and assessed their efficacy on a variety of cation exchange resins with different backbone chemistries to study the relationship between the displacers’ molecular structure and their efficacy. It has been shown that those displacers, consisting of pentaerythritol-bearing trimethylammonium groups, benzene rings, and heptyl or cyclohexyl groups, had different affinities and selectivities on cation exchange stationary phases. Tugcu et al.10 synthesized a homologous series of displacers based on either triazine or phloroglucinol. It was demonstrated that the balance between aromaticity and hydrophobicity is very important in increasing the affinity of displacers for anion exchange resins, regardless of their backbone chemistry. The results also indicated that a benzene ring is superior to a triazine ring in increasing the affinity of these anionic displacers. In addition, the data indicate that the location of an aromatic ring in the core enables the molecule to approach the stationary phase in a flat geometry, thereby increasing the number of charges interacting with the stationary phase. Although dynamic affinity determination is able to rank displacer efficacies successfully, it is time-consuming. In addition, this technique requires displacer retention data that might not be easily obtained for molecules not bearing UV chromophores. Therefore, a parallel batch screening technique was recently developed as a more generic and high-throughput screening (HTS) assay for displacer structure-efficacy relationships. Mazza et al.14 employed a large number of commercially available potential displacers with various affinities to displace proteins in batch ion exchange experiments and rapidly ranked their efficacies. In that work, displacer efficacy was measured indirectly by “percentage of protein displaced” at 10 mM displacer concentration. Tugcu et al.10 successfully employed parallel batch screening data in concert with structure-efficacy relationship modeling for designing displacers for the purification of antisense oligonucleotides. Rege et al.11 applied a modified version of this assay to a homologous cationic displacer library and successfully identified high-affinity displacers by

10.1021/ie060495m CCC: $33.50 © 2006 American Chemical Society Published on Web 11/16/2006

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Scheme 1. Synthetic Pathways to Displacers Based on a Disaccharide Core

searching for displacers with small “DC-50” values, the displacer concentration at which 50% of bound protein was displaced. These HTS assays can quickly rank displacer efficacy at certain operating concentrations and have been extensively employed for displacer screening work since they were developed.15 In previous work, it was shown that molecules containing aromatic rings have very high affinities for anion exchange stationary phases.10 Additionally, the sugar-based displacer sucrose octasulfate (SOS) also showed very high affinity although there is no aromatic/hydrophobic moiety in the structure.16 Therefore it is reasonable to expect that higher affinity displacers can be obtained by functionalizing SOS-type sugar-based molecules bearing affinity-improving moieties. In the present work, the HTS technique was employed to examine the percentage of protein displaced data over a range of displacer concentrations for a homologous sugar-based series of anionic displacers. All the displacer molecules were evaluated by their ability to displace the proteins bovine serum albumin (BSA) and trypsin inhibitor. These two proteins were selected because they exhibit comparable affinities in anion exchange but different affinities in hydrophobic interaction chromatography (HIC); thus they represent proteins with similar ion exchange retention but different hydrophobicities. The results

presented here demonstrate that the parallel batch screening approach combined with the chemical synthesis of a homologous displacer library can be a useful approach for providing insight into the design of low molecular weight displacers for ion exchange systems. Experimental Section Materials. Source 15Q (a hydrophilized poly(styrenedivinylbenzene) based quaternary ammonium strong anion exchange resin) was donated by GE Healthcare (Uppsala, Sweden). Bovine serum albumin (BSA), trypsin inhibitor, tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride, sodium phosphate (monobasic), sodium phosphate (dibasic), and sodium chloride were purchased from Sigma-Aldrich (St. Louis, MO). A TSK-Gel G3000SWXL sizeexclusion column (300 m × 7.8 mm i.d.) and the TSK-Gel SWXL (40 mm × 6 mm i.d.) guard column were gifts from TOSOH BIOSEP (Montgomeryville, PA). The displacers studied in this work, TH(PSNa)8, TH(SO3Na)8, TH(SO3Na)7, DBuTH(SO3Na)6, MMyTH(SO3Na)7, DMyTH(SO3Na)6, MtGlu(PSNa)4, MtGlu(BuSNa)4, OctGlu(PSNa)4, OctGlu(BuSNa)4, NpBrGlu(PSNa)4, NpBrGlu(BuSNa)4, PhGlu(BuSNa)4, MBzTH-

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(SO3Na)7, DBzTH(SO3Na)6, DBTH(PSNa)4, PhGal(PSNa)4, PhGlu(PSNa)4, MBuTH(SO3Na)7, PhGal(SO3Na)4, and DodGlu(PSNa)4, were synthesized as described in Schemes 1 and 2. Detailed procedure and characterization data are contained in the Supporting Information. Novozym-435 (lipase B from Canadida antarctica immobilized on an acrylic resin) was obtained from Novo Nordisk Bioindustries (Bagsvaerd, Denmark) and used without further treatment. Protease CL-15 (preparation from Bacillus sp.) was provided by Nagase Chemtex Corporation. Trehalose (anhydrous grade) was purchased from Acros Organic Chemicals. Methyl-R-D-glucopyranoside (MtGlu), octyl-β-D-glucopyranoside (OctGlu), n-dodecylβ-D-glucopyranoside (DodGlu), phenyl-β-D-glucopyranoside (PhGlu), phenyl-β-D-galactopyranoside (PhGal), and 6-bromo2-naphthyl-β-D-glucopyranoside (NpBrGlu) were purchased from Sigma-Aldrich. Vinyl butyrate and vinyl myristate were purchased from TCI America. Vinyl benzoate, 1,3-propansultone, sodium hydride, anhydrous dimethyl sulfoxide (DMSO), and anhydrous dimethylformamide (DMF) were obtained from Sigma-Aldrich. All reagents and solvents for synthesis of displacers were used as received unless otherwise noted. Instrumentations and Apparatus. Parallel batch screening experiments were carried out in 96-well Multiscreen-HV Durapore membrane-bottomed plates (Millipore, Bedford, MA). The supernatant solution from each well after equilibration with the displacer was recovered using a vacuum manifold (Millipore). The stationary phase was distributed using an Eppendorf Repeater Plus pipet. An Eppendorf eight-channel Finpipette (50-300 µL) was used to distribute equal amounts of supernatant solution into the analysis plates. Analysis of samples containing displacers without UV chromophores was accomplished in a 96-well quartz plate with a Perkin-Elmer HTS 7000 Plus plate reader and HTSoft 2 software. Supernatant analysis for samples containing displacers with UV chromophores was carried out by size-exclusion chromatography (SEC) using a Waters 600 multisolvent delivery system, a Waters 712 WISP autosampler, and a Waters 484 UV-vis absorbance detector controlled by a Millennium chromatography software manager (Waters). Surface plasmon resonance experiments were carried out in a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). 1H and 13C NMR spectral data were collected with a Varian Unity 500 NMR spectrometer. The sample solution prepared in DMSO-d6 and D2O was referenced

Figure 1. Reactions used for synthesis.

in parts per million relative to 2.49 and 4.8 ppm each for 1H NMR. In the case of 13C NMR using DMSO-d6, the reference value was 39.5 ppm. Electrospray ionization mass spectra (ESIMS) were obtained on an Agilent 1100 Series LC/MSD-SL ion trap system. Samples were introduced into the ion source using a syringe pump at a flow rate of ∼7 mL/min. The data were collected in negatively or positively charged ion modes. Instrument control, data acquisition, and data processing were performed using ChemStation 10.01 and IonTrap 5.2 softwares. (Note: NMR and MS data are given in the Supporting Information.) Displacer Synthesis. A sugar-based homologous displacer library was synthesized to determine the factors necessary for the design of high-affinity displacers and to provide insight into the effect of various chemical structural changes on displacer efficacy. The displacers based on trehalose or monosaccharides were prepared by the sulfonation or alkylsulfonation of the trehalose derivatives or monosaccharide derivatives with sulfur trioxide, propane sultone, or butane sultone. The reactions are shown in Figure 1, and the synthetic pathways to the homologous displacer library are shown in Schemes 1 and 2. The nonreducing R-glycoside trehalose contains two identical glucose residues bearing a total of eight hydroxyl groups that can be converted to charged functionalities with varying levels of selectivity. Using the enzyme Novozym-435 (step a), it is possible to introduce uncharged ester groups selectively at the hydroxyl groups at position 6 of each glucose residue. Similarly, using Protease CL-15, it is possible to react only one of the hydroxyl groups at the 6-positions (step c). In this effort, the esters were derived from butyric (saturated four-carbon), myris-

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tic (saturated 14-carbon), or benzoic (aromatic) acid. Subsequent introduction of charged functionalities enabled the construction of a stereochemically homogeneous family of displacers which varied in number of charged sites as well as in the nature of hydrocarbon side chains. Detailed procedure and characterization data are contained in the Supporting Information. Parallel Batch Screening. Twenty-one displacers with different structures were chosen to investigate the relationship between displacer structure and efficacy in anion exchange systems using the HTS technique.11 The bulk stationary phase (Source 15Q) was washed twice with deionized water and then three times with buffer (20 mM Tris with 30 mM NaCl, pH 7.5), and was allowed to equilibrate for 2 h. After gravity settling of the stationary phase, the supernatant solution was removed and 3.0 mL of the remaining stationary phase slurry was equilibrated with 36 mL containing 5 mg/mL protein (bovine serum albumin or trypsin inhibitor) in carrier buffer (20 mM Tris with 30 mM NaCl, pH 7.5), at 20 °C. The protein was kept in contact with the resin for 5 h to ensure equilibration, after which the stationary phase was allowed to settle. Upon settling, the supernatant solution was removed and the protein concentration was determined by measuring the UV absorbance at 280 nm as described below. The mass of the protein adsorbed on the stationary phase was then determined by mass balance. For the screening experiments, 120 µL of different initial concentrations of a displacer solution were added to 10 µL aliquots of the stationary phase slurry with bound protein. (Note that each well in the 96-well plate contained a different displacer molecule and/or concentration to enable parallel screening. Resin was distributed in the form of a 1:1 (v/v) slurry, which helped to minimize error to (5%.) Seven initial displacer concentrations varying from 0.25 to 10 mM were employed. After the system was equilibrated for 5 h with the displacer solution, the supernatant concentration became constant and equilibrium was achieved. The supernatant was then removed and tested for the determination of “percentage of protein displaced” for each initial displacer concentration. Supernatant Analysis. For the supernatant solution obtained from the batch displacement process using displacers without UV chromophores, 75 µL of the supernatant samples was transferred to 96-well quartz analysis plates using an eightchannel parallel pipet and UV absorbance at 280 nm was measured with a plate reader. For the samples containing displacers that absorb UV light, determination of protein concentration in the supernatant solution was performed by sizeexclusion chromatography (SEC). Samples (40 µL) were injected at a flow rate of 1 mL/min, using 50 mM phosphate with 100 mM NaCl, pH 7.5, as the carrier buffer. Both plate reader and SEC analyses were done in duplicate. The percentages of protein displaced were calculated for each aliquot based on protein mass balance, and the data were plotted as a function of the initial displacer concentration. The characteristic charges for PhGal(SO3Na)4, PhGal(PSNa)4, and PhGlu(BuSNa)4 were obtained at pH 7.5 using linear gradient experiments9 that were carried out with different slopes between 20 mM Tris with 30 mM NaCl, pH 7.5, and 20 mM Tris with 1980 mM NaCl, pH 7.5. The retentions were measured at 280 nm. Results and Discussion Previous work in our laboratory has demonstrated that sucrose octasulfate (SOS) has significant efficacy as a nontoxic, commercially available displacer for anion exchange systems. Accordingly, in the present work, a sugar-based homologous

Figure 2. Batch screening data of TH(PSNa)8, TH(SO3Na)8, TH(SO3Na)7, and SOS on Source 15Q with BSA and trypsin inhibitor as test proteins. (a) Displacement of BSA; (b) displacement of trypsin inhibitor.

Figure 3. Stereochemical difference between PhGal(PSNa)4 and PhGlu(PSNa)4.

displacer library was synthesized to determine the factors necessary for the design of high-affinity displacers and to provide insight into the effect of various chemical structural changes on displacer efficacy. Detailed procedure and characterization data are contained in the Supporting Information. The displacer efficacies were investigated by parallel batch screening assay as described in the Experimental Section. Identification of High-Affinity Displacers. The results for the displacers TH(PSNa)8, TH(SO3Na)8, and TH(SO3Na)7 are compared to those for SOS in Figure 2. As seen in the figure, TH(PSNa)8 and TH(SO3Na)8, which each have eight charges, exhibited affinity higher than or similar to that of SOS, which also has eight charges. In fact, these three molecules exhibited the highest affinities of all the displacers in the tested library. The other molecules in the library contained fewer charges and exhibited lower affinities. For example, TH(SO3Na)7 with only seven charges displaced significantly less protein (for

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Figure 4. Batch screening data of PhGal(PSNa)4 and PhGlu(PSNa)4 on Source 15Q with BSA and trypsin inhibitor as test proteins.

both proteins) even though it is structurally very similar to TH(SO3Na)8, with the eighth position occupied by an -OH group (Figure 2). The results in Figure 2 indicate that, for this particular displacer library, the number of charges is an important factor in determining displacer efficacy. While TH(PSNa)8 had a higher affinity (i.e., displaced more protein) than SOS at all concentrations, TH(SO3Na)8 had efficacy similar to that of SOS at low concentrations and only exhibited higher efficacy at displacer concentrations greater than 2 mM. Interestingly, the only difference between these two molecules is the length of the carbon linker between their structural cores and the charges. TH(PSNa)8 has each of its charges connected to the sugar residue by a three-carbon alkyl chain and TH(SO3Na)8 has no carbon chain at the corresponding position, which gives TH(PSNa)8 much more flexibility than TH(SO3Na)8. This confirms results from one of our previous studies10 that indicated that structural flexibility can be a contributing factor to displacer affinity. It is hypothesized that not all the charges of a displacer can access the resin surface simultaneously due to the displacer’s structure and that the longer alkyl chains give the charges more conformational freedom and a greater possibility of reaching binding sites on the resin surface. Investigation of the Effect of Stereochemistry on Displacer Efficacy. In this work, PhGal(PSNa)4 and PhGlu(PSNa)4 (Figure 3) are based on galactose and glucose cores. The displacers were then examined for their relative efficacy in displacing both BSA and trypsin inhibitor, and the results are shown in Figure 4. According to the batch displacement data shown in Figure 4, PhGal(PSNa)4 and PhGlu(PSNa)4 show no obvious difference in their efficacies on Source 15Q. This result indicates that PhGal(PSNa)4 and PhGlu(PSNa)4 have the same efficacies on Source 15Q resin whether the displaced protein is relatively hydrophobic (BSA) or hydrophilic (trypsin inhibitor). This result indicates that, for these displacers, chirality variation at one center plays no role in modulating the interaction of the displacers with the resin used. As a practical result, a considerable savings of labor and cost of synthesis can be realized by using simpler (achiral) polyfunctional starting materials, although at a loss of stereochemical specificity. Investigation of the Effect of Structural Flexibility on Efficacy. As described above, previous work has indicated that flexibility can play an important role in displacer efficacy.10 This effect may arise because the number of actual charges interacting with the resin surface may increase as the structural flexibility increases. In the present work, several displacers were

Figure 5. Effect of charge flexibility on displacer efficacy. (a) BSA displacement; (b) trypsin inhibitor displacement.

synthesized to investigate further the effect of structural flexibility. PhGal(SO3Na)4, PhGal(PSNa)4, and PhGlu(BuSNa)4 have similar structures, and they represent a displacer series in which the major difference is the length and flexibility of the spacer. From the data shown in Figure 5, it can be seen that flexibility improved displacer efficacy. For example, PhGal(PSNa)4, which has a three-carbon spacer, was a better displacer than PhGal(SO3Na)4, which had no spacer. Similar results were also observed when comparing the behavior of OctGlu(PSNa)4 and OctGlu(BuSNa)4 and of MtGlu(PSNa)4 and MtGlu(BuSNa)4. Interestingly, there was no further improvement in the efficacy by increasing the carbon spacer from three to four, as evidenced by the results with PhGal(PSNa)4 and PhGlu(BuSNa)4 in Figure 5. While this behavior was observed for both BSA and trypsin inhibitor, the results are more compelling for displacement of BSA. Since the number of effective charges involved in the ion exchange binding process (i.e., characteristic charge) can be less

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Figure 6. Batch screening data of MBzTH(SO3Na)7 and DBzTH(SO3Na)6 on Source 15Q with BSA and trypsin inhibitor as test proteins.

than the total number of formal charges, it is possible that the differences observed in Figure 5a are due to differences in the displacer characteristic charges. Linear gradient experiments were carried out to determine the characteristic charges of these displacers, and the results indicated that the characteristic charges of PhGal(SO3Na)4, PhGal(PSNa)4, and PhGlu(BuSNa)4 were 2.2, 2.9, and 2.8, respectively. Clearly, the displacers with more flexibility in the linker between the charged moiety and the displacer core (e.g., PhGal(PSNa)4 and PhGlu(BuSNa)4) had a higher characteristic charge resulting in higher efficacy. Investigation of the Effect of Number of Aromatic Groups on Efficacy. The displacers MBzTH(SO3Na)7 and DBzTH(SO3Na)6 were evaluated to study the impact of the number of

aromatic groups on displacer efficacy for this homologous series. While MBzTH(SO3Na)7 has one phenyl group and seven charges, DBzTH(SO3Na)6 has two phenyl groups located symmetrically on the opposite sides of the molecule along with six charges. As can be seen in Figure 6, a significant difference in displacer efficacy was observed between these two displacers for both proteins, even though there is only one charge difference between the two molecules. It is interesting to note that the molecule with one less charge, DBzTH(SO3Na)6, showed much higher efficacy as a displacer. Clearly, if charge was the only criterion, this result would not be the case. This result indicates that multiple aromatic groups in a given displacer can have a significant effect on displacer efficacy. The fact that this trend is true for both proteins indicates that this result may be applicable for a wide range of proteins. Since the importance of hydrophobic/aromatic interactions in ion exchange systems has been reported by many investigators17-19 and this result also corroborates previous results with a different anion exchange displacer library,10 it may be a generic result that aromaticity can significantly improve a displacer’s efficacy in anion exchange displacement systems. Investigation of the Effect of Alkyl Side Chain Length on Displacer Efficacy. The effect of alkyl side chain length on displacer efficacy was evaluated using three homologous molecules, MtGlu(PSNa)4, OctGlu(PSNa)4, and DodGlu(PSNa)4. The results from the parallel batch displacement experiments with both BSA and trypsin inhibitor are shown in Figure 7.

Figure 7. Batch screening data of MtGlu(PSNa)4, OctGlu(PSNa)4, and DodGlu(PSNa)4 on Source 15Q with BSA and trypsin inhibitor as test proteins. (a) BSA displacement; (b) trypsin inhibitor displacement.

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Figure 8. SPR data of protein-displacer interaction. (a) DodGlu(PSNa)4 on trypsin inhibitor chip; (b) MtGlu(PSNa)4 on trypsin inhibitor chip; (c) DodGlu(PSNa)4 on BSA chip; (d) MtGlu(PSNa)4 on BSA chip.

The displacer with the shortest side chain length, MtGlu(PSNa)4, exhibited the highest efficacy for BSA displacement. In addition, the other two molecules OctGlu(PSNa)4 and DodGlu(PSNa)4 behaved essentially the same at all concentrations for BSA displacement. In contrast, all three molecules exhibited comparable efficacy for displacing trypsin inhibitor. These results may be due to undesired secondary interactions between the displacer side chain and hydrophobic moieties on the BSA surface. Such binding could, in turn, result in a protein with many charged displacers reversibly bound to its surface, making it more difficult to displace the protein from the resin. Surface plasmon resonance (SPR) has been widely applied to study protein-protein interactions and was recently introduced to test the interactions between proteins and small molecules.20 Accordingly, SPR experiments were carried out to evaluate interactions between the displacers MtGlu(PSNa)4 and DodGlu(PSNa)4 with the proteins BSA and trypsin inhibitor. As can be seen in Figure 8, neither of these displacers bound to trypsin inhibitor. On the other hand, DodGlu(PSNa)4 clearly exhibited a binding response with BSA while MtGlu(PSNa)4 did not seem to interact. These results indicate that displacers with long side chains can bind to the surface of hydrophobic proteins, reducing their efficacy. Conclusion In this paper, a homologous series of sugar-based anion exchange displacers were synthesized and evaluated by a parallel batch displacement assay as potential displacers for protein

purification in anion exchange systems. This work extended the previous research from this laboratory and investigated the impacts of aromatic/hydrophobic moieties and charge flexibility on displacer binding affinity under various conditions. The results indicated that the addition of aromatic rings at the periphery of the molecule close to the charged groups increased displacer affinity. It was observed that displacers with more structural flexibility had higher efficacy up to a certain value, beyond which flexibility did not appear to affect displacer efficacy for the molecules evaluated. Future work will evaluate displacers with a wider range of flexibility along with various proteins and resins to determine if this “critical flexibility” is a generic phenomenon. It was also demonstrated by SPR experiments that long hydrophobic side chains that are not associated with the charge may lower displacer efficacy because of undesired interactions with protein molecules. Stereochemistry was also not observed to play an important role in displacer efficacy for the molecules evaluated. Importantly, low molecular mass displacers with affinities higher than those previously observed were identified as part of this study. This may have important implications for future applications of displacement chromatography for proteomic applications. Acknowledgment The work was supported by NIH Grant 5R01 GM047372. The authors thank Zachary A. Hilton for his assistance in the displacement screening experiments. Supporting Information Available: Discussion of displacer synthesis including the following sections: propylsulfonation

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of trehalose derivatives (Scheme 1); enzyme-catalyzed synthesis of disubstituted trehalose esters (Scheme 1); enzyme-catalyzed synthesis of monosubstituted trehalose esters (Scheme 1); sulfonation of trehalose derivatives (Scheme 1);21 sulfonation and alkylsufonation of monosaccharides (Scheme 2). This material is available free of charge via the Internet at http:// pubs.acs.org. Literature Cited (1) Horvath, C.; Nahum, A.; Frenz, J. H. High-Performance Displacement Chromatography. J. Chromatogr. 1981, 218, 365. (2) Kalasz, H.; Horvath, C. Preparative-Scale Separation of Polymyxins with An Analytical High-Performance Liquid Chromatography System by Using Displacement Chromatography. J. Chromatogr. 1981, 215, 295. (3) Vigh, G.; Varga-Puchony, Z.; Szepesi, G.; Gazdag, M. SemiPreparative High-Performance Reversed-Phase Displacement Chromatography of Insulins. J. Chromatogr. 1987, 386, 353. (4) Torres, A. R.; Peterson, E. A. Ion-Exchange Displacement Chromatography of Proteins, Using Narrow-Range Carboxymethyldextrans and A New Index of Affinity. Anal. Biochem. 1983, 130, 271. (5) Tugcu, N.; Deshmukh, R. R.; Sanghvi, Y. S.; Moore, J. A.; Cramer, S. M. Purification of An Oligonucleotide At High Column Loading by High Affinity, Low-Molecular-Mass Displacers. J. Chromatogr., A 2001, 923, 65. (6) Kundu, A.; Vunnum, S.; Jayaraman, G.; Cramer, S. M. Protected Amino Acids As Novel Low-Molecular-Weight Displacers In CationExchange Displacement Chromatography. Biotechnol. Bioeng. 1995, 48, 452. (7) Jayaraman, G.; Li, Y.; Moore, J. A.; Cramer, S. M. Ion-Exchange Displacement Chromatography of Proteins Dendritic Polymers as Novel Displacers. J. Chromatogr., A 1995, 702, 143. (8) Kundu, A.; Vunnum, S.; Cramer, S. M. Antibiotics As LowMolecular-Mass Displacers In Ion-Exchange Displacement Chromatography. J. Chromatogr., A 1995, 707, 57. (9) Shukla, A. A.; Barnthouse, K. A.; Bae, S. S.; Moore, J. A.; Cramer, S. M. Synthesis and Characterization of High-Affinity, Low Molecular Weight Dispalcers for Cation-Exchange Chromatography. Ind. Eng. Chem. Res. 1998, 37, 4090. (10) Tugcu, N.; Park, S. K.; Moore, J. A.; Cramer, S. M. Synthesis and Characterization of High-Affinity, Low-Molecular-Mass Displacers for Anion-Exchange Chromatography. Ind. Eng. Chem. Res. 2002, 41, 6482.

(11) Rege, K.; Hu, S.; Moore, J. A.; Dordick, J. A.; Cramer, S. M. Chemoenzymatic Synthesis and High-Throughput Screening of an Aminoglycoside-Polyamine Library: Identification of High-Affinity Displacers and DNA-Binding Ligands. J. Am. Chem. Soc. 2004, 126, 12306. (12) Brooks, C. A.; Cramer, S. M. Steric Mass-Action Ion Exchange: Displacement Profiles and Induced Salt Gradients. AIChE J. 1992, 38, 1969. (13) Shukla, A. A.; Barnthouse, K. A.; Bae, S. S.; Moore, J. A.; Cramer, S. M. Structural Characteristics of Low-Molecular-Mass Displacers for Cation-Exchange Chromatography. J. Chromatogr., A 1998, 814, 83. (14) Mazza, C. B.; Rege, K.; Breneman, C. M.; Sukumar, N.; Dordick, J. S.; Cramer, S. M. High Throughput Screening and Quantitative StructureEfficacy Relationship Models of Potential Displacer Molecules for IonExchange Systems. Biotechnol. Bioeng. 2002, 80, 60. (15) Liu, J.; Yang, T.; Ladiwala, A.; Breneman, C. M.; Cramer, S. M. High throughput determination and QSER modeling of displacer DC-50 values for ion exchange systems. Sep. Sci. Technol. 2006, 41, 3079. (16) Tugcu, N.; Ladiwala, A.; Breneman, C. M.; Cramer, S. M. Identification of Chemically Selective Displacers Using Parallel Batch Screening Experiments and Quantitative Structure Efficacy Relationship Models. Anal. Chem. 2003, 75, 5806. (17) Roth, C. M.; Unger, K. K.; Lenhoff, A. M. Mechanistic Model of Retention in Protein Ion-Exchange Chromatography. J. Chromatogr., A 1996, 726, 45. (18) Roth, C. M.; Lenhoff, A. M. Electrostatic and Van der Waals Contributions to Protein Adsorptions Computation of Equilibrium Constants. Langmuir 1993, 9, 962. (19) Stahlberg, J.; Jonsson, B.; Horvath, C. Combined Effect of Coulombic and Van der Waals Interactions in the Chromatography of Proteins. Anal. Chem. 1992, 64, 3118. (20) Myszka, D. G. Analysis of small-molecule interactions using Biacore S51 technology. Anal. Biochem. 2004, 329, 316. (21) Sun, X. L.; Grande, D.; Baskaran, S.; Hanson, S. R.; Chaikof, E. L. Glycosaminoglycan Mimetic Biomaterials. 4. Synthesis of Sulfated Lactose-Based Glycopolymers That Exhibit Anticoagulant Activity. Biomacromolecules 2002, 3, 1065.

ReceiVed for reView April 20, 2006 ReVised manuscript receiVed October 3, 2006 Accepted October 5, 2006 IE060495M