Novel Tertiary Amine Oxide Surfaces That Resist Nonspecific Protein

Aug 9, 2006 - Novel surfaces derivatized with tertiary amine oxides have been prepared and tested for their ability to resist nonspecific protein adso...
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Langmuir 2006, 22, 8144-8150

Novel Tertiary Amine Oxide Surfaces That Resist Nonspecific Protein Adsorption Suzanne J. Dilly,† Matthew P. Beecham,† Steven P. Brown,§ John M. Griffin,§ Andrew J. Clark,† Craig D. Griffin,‡ Jacqueline Marshall,‡ Richard M. Napier,‡ Paul C. Taylor,† and Andrew Marsh*,† Department of Chemistry, UniVersity of Warwick, CoVentry, CV4 7AL, UK, Warwick HRI, Wellesbourne, Warwick, CV35 9EF, UK, and Department of Physics, UniVersity of Warwick, CoVentry, CV4 7AL, UK ReceiVed March 20, 2006. In Final Form: June 9, 2006 Novel surfaces derivatized with tertiary amine oxides have been prepared and tested for their ability to resist nonspecific protein adsorption. The oxidation of tertiary amines supported on triazine units was carried out using mCPBA to give a format allowing conjugation of biologically active ligands alongside them. Adsorption to these surfaces was tested and compared to adsorption to a set of commercial and custom oligo-/poly(ethylene glycol) (OEG/PEG) supports by challenging them with a protein display library presented on bacteriophage λ. The new class of amine oxide surfaces is found to compare favorably with the performance of the OEG/PEG supports in the prevention of nonspecific binding.

Introduction Surfaces that resist adsorption of protein1 and cells2 have applications both in vivo and in the chemical biology laboratory, and there is a need for new, improved systems.3 For example, the coating of medical devices, contact lenses, and the microencapsulation of drugs4 have been studied to decrease biofouling of implants in vivo. Reducing adsorbed protein has been shown to minimize the immune response and improve biocompatibility.5 Likewise, in the laboratory many techniques can be improved by avoiding nonspecific protein adsorption. These include increased specificity in immunological studies,6 improved chromatography for protein purification,7 and, as reported in this paper, more efficient affinity screening of protein libraries.8 Although there are biological molecules and surfaces in use to confer degrees of protein resistance, the molecular mechanism(s) that underpin these effects continue to be discussed,9 and the construction of efficient synthetic surfaces for biomedical and biotechnological applications remains challenging. It has been noted that naturally occurring small molecule osmolytes that are able to order surrounding water molecules (kosmotropes) should display inherent protein resistance.1 One such molecule that has received considerable attention for its ability to counteract the * To whom correspondence should be addressed. E-mail: a.marsh@ warwick.ac.uk. † Department of Chemistry, University of Warwick. ‡ Warwick HRI. § Department of Physics, University of Warwick. (1) Kane, R. S.; Deschatelets, P.; Whitesides, G. M. Langmuir 2003, 19, 23882391. (2) Mrksich, M.; Chen, C. S.; Xia, Y. N.; Dike, L. E.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10775-10778. (3) Statz, A. R.; Meagher, R. J.; Barron, A. E.; Messersmith, P. B. J. Am. Chem. Soc. 2005, 127, 7972-7973. (4) Heuberger, R.; Sukhorukov, G.; Voros, J.; Textor, M.; Mohwald, H. AdV. Funct. Mater. 2005, 15, 357-366. (5) Lan, S.; Veiseh, M.; Zhang, M. Q. Biosens. Bioelectron. 2005, 20, 16971708. (6) Herrwerth, S.; Rosendahl, T.; Feng, C.; Fick, J.; Eck, W.; Himmelhaus, M.; Dahint, R.; Grunze, M. Langmuir 2003, 19, 1880-1887. (7) Barrett, D. A.; Hartshorne, M. S.; Hussain, M. A.; Shaw, P. N.; Davies, M. C. Anal. Chem. 2001, 73, 5232-5239. (8) Sche, P. P.; McKenzie, K. M.; White, J. D.; Austin, D. J. Chem. Biol. 1999, 6, 707-716. (9) Shimizu, S.; Smith, D. J. J. Chem. Phys. 2004, 121, 1148-1154.

protein denaturant urea10-14 and prevent misfolding15 is trimethylamine N-oxide (TMAO), although to the best of our knowledge amine oxide derivatives have not previously been examined for their ability to prevent protein adhesion to surfaces. Oligo-/poly(ethylene glycol) (OEG/PEG) is the established benchmark for protein resistance,2 and many studies have examined how it acts to achieve this.16,17 It is believed that both hydration and steric effects are crucial to impart protein resistance to a surface.18 The presence of water molecules within the PEG layer is essential for resistance,19 with a minimum of 2-3 molecules of water per ethyleneglycol unit, up to a maximum of 10 required for hydration.20 In fact, it has been shown that the organization of water molecules originating from a PEGylated surface can extend tens of nanometers from the surface, resulting in increased local viscosity.21 For a protein to approach the surface through both hydration and PEG layers, unfavorable conformational changes of the PEG chain and/or protein must occur, resulting in dehydration.22 The favorability of poly(ethylene glycol) over polypropyleneglycol and polytrimethyleneglycol has been shown to be due to enhanced disturbance of the water lattice by the poor matching of propylene- and methyleneglycol repeat units,16 resulting in better hydration for ethyleneglycol systems. Numerous examples of surfaces resistant to both cell and protein adhesion have been reported, including the use of (10) Baskakov, I.; Bolen, D. W. J. Biol. Chem. 1998, 273, 4831-4834. (11) Yancey, P. H.; Clark, M. E.; Hand, S. C.; Bowlus, R. D.; Somero, G. N. Science 1982, 217, 1214-1222. (12) Bolen, D. W.; Baskakov, I. J. Mol. Biol. 2001, 310, 955-963. (13) Auton, M.; Bolen, D. W. Biochemistry 2004, 43, 1329-1342. (14) Bolen, D. W. Methods 2004, 34, 312-322. (15) Bennion, B. J.; DeMarco, M. L.; Daggett, V. Biochemistry 2004, 43, 12955-12963. (16) Lee, J. H.; Lee, H. B.; Andrade, J. D. Prog. Polym. Sci. 1995, 20, 10431079. (17) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55-78. (18) Unsworth, L. D.; Sheardown, H.; Brash, J. L. Langmuir 2005, 21, 10361041. (19) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359-9366. (20) Heuberger, M.; Drobek, T.; Voros, J. Langmuir 2004, 20, 9445-9448. (21) Kim, H. I.; Kushmerick, J. G.; Houston, J. E.; Bunker, B. C. Langmuir 2003, 19, 9271-9275. (22) Li, L. Y.; Chen, S. F.; Zheng, J.; Ratner, B. D.; Jiang, S. Y. J. Phys. Chem. B 2005, 109, 2934-2941.

10.1021/la060743j CCC: $33.50 © 2006 American Chemical Society Published on Web 08/09/2006

NoVel Tertiary Amine Oxide Surfaces

Langmuir, Vol. 22, No. 19, 2006 8145 Scheme 1 a

a

Reagents and conditions: (i) cyanuric chloride, DIPEA, THF, room temperature; (ii) 2-(2-[2-aminoethoxy]ethoxy)ethanol, DIPEA, CHCl3, reflux; (iii) TFA, DCM.

differing polymeric molecules, arrangements, and PEG oligomer length displayed on a range of planar surfaces including gold, silver,19 graphite,23 silica,24,25 titanium,26 mica,27 and planar polystyrene.7 In summary, if linear PEG chains are present at sufficient density, as few as two ethylene glycol units are sufficient to impart protein resistance to a surface.28 However, six ethylene glycol units are required for similar levels of protein resistance if the layer is imperfect.29 Higher chain density can be achieved using dendritic PEG structures, but a comparison of linear and highly branched star PEG structures suggested that linear PEG chains confer greater protein resistance. Uneven covering of the surface was seen in the areas where the edges of two stars overlapped.25,30 In contrast, dendritic structures with other proteinresistant moieties have been shown to increase resistance. For example, highly branched dendritic poly(glycerol) has been shown to have protein-resistant properties similar to those of linear PEGs, while the linear glycerol equivalent does not.31 A highly branched linear carbohydrate has likewise been shown to demonstrate protein-resistant effects comparable to those of triethylene glycol.32 Tentagel is a polymer support commonly employed for affinity techniques requiring biocompatibility. It consists of a polystyrene core with pendant PEG chains of 2000-3000 MW. The highly cross-linked core ensures the matrix remains insoluble, while the hydrophilic chains form a conformationally stable layer in aqueous solution, imparting the desired protein resistance to the matrix. A major disadvantage of Tentagel is the low loading level (typically 0.2-0.3 mmol/g) caused by the high molecular weight PEG chains. As discussed above, this length of PEG chain may be unnecessary for many applications. The matrix developed is to be used in affinity screening with a protein library displayed on bacteriophage λ. The library to be screened consists of millions of independent bacteriophage clones, each with a different polypeptide displayed on its surface through fusion to a viral coat protein. In this case, the polypeptides are domain-sized fragments randomly derived from the transcriptome of the model plant Arabidopsis thaliana.33 An effective affinity matrix must therefore be resistant to nonspecific adsorption by both the protein fragments displayed on the surface of the bacteriophage and the protein coat of the bacteriophage itself. It must also be possible to display small molecule ligands on the exterior of the surface to allow specific binding between the molecule and its protein partner in the library. Matrixes typically employed in affinity screening, such as agarose, are problematic for synthetic chemistry and are often inappropriate for wider application. This report investigates polymer supports suitable for biological applications. These supports have the favorable properties of a

hydrophilic link and protein-resistant layer with higher loading (∼0.9 mmol/g) achieved by limiting PEG chain length, increasing PEG chain density, and using a dendrimeric core. Most importantly, we introduce amine oxides as a new class of proteinresistant moiety.

Synthesis Initial syntheses were carried out on Wang resin (0.9 mmol/ g), because this resin allowed on-bead analysis to be carried out by infrared spectroscopy and elemental analysis and by subsequent cleavage of the dendritic molecule from the support to confirm the structure as previously described.34,35 Following the successful synthesis on Wang resin, analogous syntheses on SynPhase Lanterns (rigid polymeric structures grafted with polyamide, with pendant amino functionality) and aminopropyl silica were performed. To create Generation 0 PEGylated resins (Scheme 1), the support was first treated with cyanuric chloride in the presence of diisopropylethylamine, giving dichloride 1. Symmetrical substitution of the remaining two [1,3,5]triazine sites was then achieved at elevated temperature to provide 2-fold 2-(2-[2-aminoethoxy]ethoxy)ethanol substituted product 2. Cleavage from Wang resin using 5% trifluoroacetic acid solution was done to yield the triazinone 3 for characterization. The sequence was repeated on both aminopropyl silica and SynPhase Lanterns via intermediates reported previously, to give supported triazines 5 (Scheme 2). First generation [1,3,5]triazine dendritic structures were created in an analogous fashion (Figure 1) using 2-(2-[2-aminoethoxy]ethoxy)ethanol, to give 8 as reported previously. (23) Ruegsegger, M. A.; Marchant, R. E. J. Biomed. Mater. Res. 2001, 56, 159-167. (24) Jon, S. Y.; Seong, J. H.; Khademhosseini, A.; Tran, T. N. T.; Laibinis, P. E.; Langer, R. Langmuir 2003, 19, 9989-9993. (25) Irvine, D. J.; Mayes, A. M.; Satija, S. K.; Barker, J. G.; Sofia-Allgor, S. J.; Griffith, L. G. J. Biomed. Mater. Res. 1998, 40, 498-509. (26) Dalsin, J. L.; Lin, L. J.; Tosatti, S.; Voros, J.; Textor, M.; Messersmith, P. B. Langmuir 2005, 21, 640-646. (27) Drobek, T.; Spencer, N. D.; Heuberger, M. Macromolecules 2005, 38, 5254-5259. (28) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 1071410721. (29) Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M. J. Am. Chem. Soc. 2000, 122, 8303-8304. (30) Fukai, R.; Dakwa, P. H. R.; Chen, W. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 5389-5400. (31) Siegers, C.; Biesalski, M.; Haag, R. Chem.-Eur. J. 2004, 10, 2831-2838. (32) Metzke, M.; Bai, J. Z.; Guan, Z. B. J. Am. Chem. Soc. 2003, 125, 77607761. (33) Bell, M. Ph.D. Thesis, Horticulture Research Institute, Wellesbourne, 2002. (34) Marsh, A.; Carlisle, S. J.; Smith, S. C. Tetrahedron Lett. 2001, 42, 493496. (35) Dilly, S. J.; Carlisle, S. J.; Clark, A. J.; Shepherd, A. R.; Smith, S. C.; Taylor, P. C.; Marsh, A. J. Polym. Sci., Part A: Polym. Chem. 2005, 44, 22482259.

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Dilly et al. Scheme 2 a

a

Reagents and conditions: (i) 2-(2-[2-aminoethoxy]ethoxy)ethanol, DIPEA, CHCl3, reflux.

Figure 1. Supported triazine dendrimers decorated with OEG moieties.

at -78 °C using mCPBA to yield the novel silica-supported protein-resistant coating (Scheme 3). The success of this second step is confirmed by the 13C CP-MAS spectrum of 10 in Figure 3, with the peak at 67 ppm corresponding to the two CH2 moieties adjacent to the amine oxide nitrogen. The chemical shifts of these signals correlate well with those obtained for a model compound prepared in solution (see Supporting Information). Differential substitution on the [1,3,5]triazine core is enabled by the established temperature requirements of the cyanuric chloride system.36 To ensure confidence in the distribution of our [1,3,5]triazine substituents (Scheme 4), the first substitution was carried out in solution phase at -78 °C to yield 11, and the resultant monsubstituted triazine was added to aminopropyl silica at ambient temperature to give 12. The final substitution with 2-(2-[2-aminoethoxy]ethoxy)ethanol was carried out at elevated temperature in the presence of diisopropylethylamine to ensure complete saturation of the [1,3,5]triazine sites, 13. Oxidation with mCPBA at -78 °C again provided the novel protein-resistant silica surface, 14, now bearing a versatile linker.

Testing for Resistance to Bacteriophage-Displayed Proteins

Figure 2. 13C CP-MAS NMR spectrum of silica-supported dendritic triazine 8b. Assignment: 10.1 ppm (SiCH2); 22.1 ppm (SiCH2CH2); 41.3 ppm (SiCH2CH2CH2 and NHCH2CH2NH); 61.2 ppm (NHCH2CH2O); 70.2 and 72.9 ppm (PEG CH2); 166.2 ppm (triazine).

Solid-state 13C cross-polarization (CP) magic-angle-spinning (MAS) NMR showed the desired derivatization of the final product 8b by the ethylene glycol chains (Figure 2). It should be noted that 13C CP-MAS NMR is not quantitative with peak intensities depending in a complex manner on the 13C-1H dipolar couplings, such that the relatively low intensity of the PEG CH2 resonances (70-73 ppm) is likely to be due to an increase in mobility (and hence reduction of the 13C-1H dipolar couplings) at greater distances from the silica support. [A reviewer has suggested that cross-linked byproducts may lead to lower peripheral display of ethylene glycol entities.] Amine oxide derivatives were synthesized in two formats. The first contains two amine oxide moieties, demonstrating the optimal coverage of our novel protein-resistant coating, and the second contains one amine oxide moiety and one PEG linker, potentially compromising the protein-resistant quality of the material, but allowing attachment of an appropriate ligand to the support through the terminal PEG hydroxyl group. The tertiary amine of the morpholino group in 9 was then selectively oxidized

The supports prepared above and a range of commercial supports were exposed to a library of proteins expressed on the surface of bacteriophage λ particles (Figure 4). The library has a diversity of 5 × 106 primary clones and, consequently, presents most nuclear-encoded proteins from the plant Arabidopsis. Bacteriophage input numbers were 109 for each experiment, and so around 1000 copies of each clone are presented during biopanning, exposing the supports to the widest of protein populations. After a series of washes, the number of phage particles adsorbed to each support was estimated by addition of E. coli cells, and results are presented as phage bound per 20 mg of beads processed. At least two replicates of each experiment were performed, with multiple counts of phage plaques to provide further estimation of errors, which are represented in Figure 4 as the standard deviation of the data (see Supporting Information for details). The commercial supports had primary amine functionality, with the exception of Tentagel, which was used with both amine and hydroxyl functionality. PEGylated supports were expected to exhibit low adsorption levels. This was the case for Tentagel, although PEGA, a poly(acrylamide) core with pendant PEG chains, retained around 10-fold more bacteriophage. Aminopropyl silica and SynPhase Lanterns were all found to perform poorly as compared to Tentagel, and Tentagel is clearly the benchmark product in the assay. The SynPhase Lantern dendritic series gave a 10-fold decrease in adsorbed phage for the Generation 0 PEG 5a lantern as compared to the plain Lantern, thereby achieving protein (36) Thurston, J. T.; Dudley, J. R.; Kaiser, D. W.; Hechenbleikner, I.; Schaefer, F. C.; Holm-Hansen, D. J. Am. Chem. Soc. 1951, 73, 2981-2983.

NoVel Tertiary Amine Oxide Surfaces

Langmuir, Vol. 22, No. 19, 2006 8147 Scheme 3 a

a

Reagents and conditions: (i) 4-(3-aminopropyl)morpholine, DIPEA, DMF, reflux; (ii) mCPBA, K2CO3, DCM, -78 °C.

proteins. In the presence of the amine oxide function, the system is at a thermodynamic minimum such that both the surface and the polypeptides attached to the phage remain hydrated, although the origin of this observation at the molecular level37 cannot be further elucidated from the work presented herein. This effect is currently under further investigation with the more classical proteins used in studying surface adhesion properties, such as fibrinogen and lysozyme, deposited on self-assembled monolayers probed with surface plasmon resonance, quartz crystal microbalance, and contact angle measurements. Figure 3. 13C CP-MAS NMR spectrum of silica-supported amine oxide 10. Assignment: 11.1 ppm (SiCH2); 23.4 ppm (SiCH2CH2 and NHCH2CH2CH2N+O-); 36.7 ppm (NHCH2CH2CH2N+O-); 43.7 ppm (SiCH2CH2CH2); 62.0 ppm (OCH2CH2 N+O-); 67.4 ppm (NHCH2CH2CH2N+O- and OCH2CH2N+O-); 166.3 ppm (triazine).

resistance properties close to those of Tentagel. No further decrease in adsorption was gained with the 1 PEG 8a lantern, which may be due to lower than expected levels of derivatization caused by partial cross-linking of dendrimeric triazines. In the aminopropyl silica series, a 10-fold decrease in protein adsorption was observed in going from the parent silica to the oxidized dimorpholino 10, representing levels of improvement similar to that seen in the Lantern-based materials. The resistance conferred by the amine oxide moiety is demonstrated to be due to the oxidation of the amine by comparison with 9, the same support prior to oxidation. Before oxidation, 9 shows a level of resistance similar to that of the parent aminopropyl silica. Furthermore, support 10 shows resistance to nonspecific adsorption of the phage-displayed proteins approaching that of Tentagel, the best of the commercial supports investigated, that bears much longer PEG chains. Significantly, the levels of protein adsorption are of the same order as those observed for similar molecular weight OEG derivatized 5a and 8a. Biopanning requires a support to be amenable to easy coupling to immobilize and present small molecule ligands for affinity selections. For this reason, silica support 14, which further demonstrated improved biofouling resistance by the amine oxide moiety (compare with unoxidized 13), is likely to be particularly useful for the display of ligands for biological screening purposes. This support and other amine oxides such as 10 may also be of use in vivo for biomedicine. The molecular mechanism for the observed protein resistance of the novel amine oxide supports is speculative, but we hypothesize that the dipolar amine oxide function, which is known to form a strong hydrogen bond with water molecules, is playing its role as a kosmotrope and ordering water molecules at the surface of the support. As such, the surface may be considered “preferentially hydrated” and will exclude proteins from the vicinity, consistent with the hypothesis1 that kosmotropic moieties at surfaces are able to prevent nonspecific interactions with

Conclusions This evaluation of immobilized supports for use in affinity screening against a protein library shows that Tentagel is a good commercial support with which to restrict nonspecific adsorption of phage and displayed protein. The dendritic PEG supports synthesized here show a level of protein resistance approaching that of Tentagel with the benefit that the chemistry used to produce them is straightforward and compatible with a wide range of surfaces. A novel protein-resistant tertiary amine oxide moiety has been introduced, and it shows enhanced resistance to “biofouling” as compared to both Tentagel and dendritic PEG supports when tested by presentation of a diverse protein library. Tertiary amine oxides offer a valuable addition to the protein resistance and biocompatibility toolset. Experimental Section General. Wang resin and polystyrene plugs were obtained from Polymer Laboratories, SynPhase Lanterns were obtained from Mimotopes, and other chemicals, solvents, and reagents were obtained from Acros, Aldrich, and Lancaster Synthesis and used without further purification. 1H NMR spectra were measured on a Bruker DPX300 spectrometer. Chemical shifts are reported in ppm for chloroform-d solution using TMS as an internal standard unless stated otherwise. Infrared spectra were recorded using an Avatar 320 FT-IR fitted with a “Golden Gate” attenuated total reflection attachment. Mass spectra were acquired on a Micromass Autospec (positive mode LSIMS). Elemental analysis was performed by Warwick Analytical Services using a Leeman Labs CE44U elemental analyzer. Theoretical loading is compared to observed loading on the supports by both %N ((0.3% ) 0.21 mmol/g) and %Cl ((0.3% ) 0.08 mmol/g) from this elemental analysis data. Assay for the Quantification of Nonspecific Adsorption of Phage-Displayed Proteins to Supports. The phage-displayed protein library33 used in this assay presents a huge diversity of protein domains from the Arabidopsis thaliana proteome displayed on bacteriophage lambda particles. The Arabidopsis proteins are carried as fusions to the capsid headgroup protein gpD.38 The library contains 5 × 106 (37) Timasheff, S. N. In AdVances in Protein Chemistry; Di Cera, E., Ed.; Academic: London, 1998; Vol. 51, pp 355-432. (38) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning - A Laboratory Manual, 2nd ed.; Cold Spring Harbor Press: Plainview, NY, 1989.

8148 Langmuir, Vol. 22, No. 19, 2006

Dilly et al. Scheme 4 a

a Reagents and conditions: (i) 4-(3-aminopropyl)morpholine, CHCl , -78 °C (27%); (ii) aminopropyl silica, DIPEA, CHCl , room temperature; 3 3 (iii) 2-(2-[2-aminoethoxy]ethoxy)ethanol, DIPEA, dioxane, 80 °C; (iv) mCPBA, K2CO3, DCM, -78 °C.

Figure 4. Nonspecific adsorption of bacteriophage to supports. The total number of plaque-forming units retained on each support after extensive washing is given for various supports, before and after derivatization. The data for Tentagel are shown for comparison. primary phage clones. One in three of these will represent inserts in frame with the fusion protein, giving the library a complexity of approximately 1.7 × 106 protein domains. Arabidopsis mRNA was prepared from whole, 7-day old seedlings grown aseptically. Conversion to cDNA used oligonucleotides designed to prime randomly. Size selection ensured inserts were of sufficient size to represent functional protein domains, and the inserts were ligated into a display vector based on λfooDc.33,38 The average insert size was approximately 600 base pairs, which, after deletion of framework sequence, gave an average fusion protein size of 130 residues. Sequencing 20 random clones confirmed them all to be different and all to be in the correct orientation. Screening the phage library with antibodies confirmed that proteins were actively displayed and cognate proteins could be selected efficiently within 2 or 3 rounds of biopanning.33 For biopanning, the library was prepared by propagating the phage particles in a host that promotes maximum valency of the displayed domains.38 Escherichia coli host TG1 was grown at 37 °C with shaking at 220 rpm to OD600 0.5, in 50 mL of NZCYM (1% N-ZAmine A, 86 mM NaCl, 0.5% yeast extract, 0.1% Hy-CaseAmino, 8.1 mM MgSO4). Next, 1010 phage particles were added to the culture, which was then shaken at 220 rpm, 37 °C, for 4 h. Cells were then

lysed and nucleic acids digested by the addition of 2% chloroform, 600 U DNase I, and 38 U RNase A, followed by shaking at 220 rpm, 37 °C, for 10 min. Cell debris was removed by centrifuging at 16 000g for 10 min at 4 °C. Phage particles were precipitated by adding 6.5% PEG 8000 and 650 mM NaCl, allowing 1 h on ice, and collected by centrifuging at 16 000g for 15 min at 4 °C. Phage particles were resuspended in SM (50 mM Tris-Cl pH 7.5, 10 mM NaCl, 8 mM MgCl2, 0.1% gelatin) ready for biopanning. The number of phage particles in the stock was determined in accordance with standard techniques.38 Each support was transferred to a 1.5 mL Eppendorf tube. For beaded supports, approximately 20 mg was used in each assay, and the exact mass was recorded. A single, weighed plug or lantern was used. To remove any residual reagents from the supports, they were washed twice with 1 mL of TBS (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 10 mM MgSO4), tumbled at a moderate rate for 1 min, and spun briefly in a microfuge (13 000 rpm, 30 s), and the buffer was removed. Each support was then washed in 1 mL of binding buffer (TBS 0.1% Tween 20, 1% γ-Globulins [Sigma G-5009]), allowing 5 min tumbling at a moderate rate, followed by three similar washes for 1 min. Phage particles 109 in 1 mL of binding buffer were added to each support and incubated for 60 min with tumbling at a moderate rate. Each support was washed repeatedly in 1 mL of wash buffer (TBS + 0.5% Tween 20), allowing 1 min tumbling at a moderate rate for each. After addition of 200 µL of SM to each sample, 400 µL of E. coli stock (strain Q526 in 10 mM MgCl2) was added, and the supports were shaken at 220 rpm for 20 min at 37 °C. After brief centrifugation to pellet the supports, supernatants were used to determine phage numbers as above. Chemical Synthesis. Wang-Supported Intermediate 1. A slurry of Wang resin (1.0 g, 0.9 mmol/g) in THF (30 mL) was treated with a solution of cyanuric chloride (0.8 g, 4.5 mmol). The mixture was treated with diisopropylethylamine (0.78 mL, 4.5 mmol) and shaken overnight. The resin was removed and washed successively with THF, 1:1 THF/DCM, DCM, 1:1 DCM/methanol, and DCM, then dried at 50 °C under vacuum; νmax, 3025, 2920, 1536, 1506, 1492, 1301, 1251, 1028, 696 cm-1. Anal. Calcd: C, 80.21; H, 6.41; N, 3.97; Cl, 6.20. Maximum theoretical loading: 0.79 mmol/g. Loading implied from percentage N, 0.9 mmol/g; from Cl, 0.9 mmol/g. Wang-Supported Dendrimer (Generation 0) 2. Wang-supported intermediate 1 (1.0 g, 0.79 mmol) was added to a solution of 2-(2-

NoVel Tertiary Amine Oxide Surfaces [2-aminoethoxy]ethoxy)ethanol (0.56 g, 2.3 mmol) in chloroform (20 mL), treated with diisopropylethylamine (0.4 mL, 2.3 mmol), and heated to reflux overnight. The resin was removed and washed successively with chloroform, DCM, 1:1 DCM/methanol, and DCM, then dried at 50 °C under vacuum; νmax, 3383, 3024, 2919, 1568, 1512, 1451, 1337, 1217, 1066, 1028, 749, 697 cm-1. Anal. Calcd: C, 74.54; H, 6.93; N, 4.26. Maximum theoretical loading: 0.67 mmol/g. Loading implied from percentage N: 0.6 mmol/g. CleaVed Product 3. Trifluoroacetic acid (0.15 mL, 2 mmol) was added dropwise to a slurry of Wang-supported dendrimer (Generation 0) 2 (0.2 g, 0.13 mmol) in DCM (10 mL), and the reaction was shaken overnight. The resin was removed and washed with DCM and 1:1 DCM/methanol. The organic fractions were combined and evaporated under reduced pressure to give the title product as a viscous oil; νmax, 3266, 1674, 1608, 1434, 1344, 1136 cm-1; δH (300 MHz, CDCl3) 3.10-3.79 (m, 20H); 4.47-4.49 (m, 4H); 8.93-9.02 (m, NH); 9.46-9.52 (m, NH) ppm; δC (75 MHz, CDCl3) 61.1, 63.7, 67.9, 70.0, 70.4, 72.0 153.7, 156.0, 160.0 ppm; m/z 392 (33%). Lantern-Supported Dendrimer (Generation 0) 5a. Lanternsupported intermediate 4a (5, 85 µmol) was suspended in a solution of 2-(2-[2-aminoethoxy]ethoxy)ethanol (0.053 g, 0.2 mmol) in chloroform (10 mL) and treated with diisopropylethylamine (0.04 mL, 0.2 mmol), and then heated to reflux overnight. The Lanterns were removed from the reaction and washed successively with DCM, 1:1 DCM/methanol, water, acetone, and DCM and dried at 50 °C under vacuum. Total weight change: 20.1 mg. Expected weight change: 20.3 mg. Silica-Supported Dendrimer (Generation 0) 5b. Silica-supported intermediate 4b (0.2 g, 0.23 mmol) was suspended in a solution of 2-(2-[2-aminoethoxy]ethoxy)ethanol (0.18 g, 1.2 mmol) in DMF (10 mL), treated with diisopropylethylamine (0.2 mL, 1.2 mmol), and heated to 100 °C overnight. The solid was removed from the reaction and washed successively with DCM, 1:1 DCM/methanol, and DCM, and then dried at 50 °C under vacuum; νmax, 3358, 2930, 1527, 1396, 1047, 807 cm-1. Anal. Calcd: C, 19.46; H, 3.17; N, 7.56. Found: C, 13.94; H, 2.67; N, 6.99. Maximum theoretical loading: 0.9 mmol/g. Loading implied from percentage N present: 0.8 mmol/g. Silica-Supported Dendrimer (Generation 1) 8b. This was prepared as described previously.35 The 13C CP-MAS spectrum in Figure 2 was recorded on a Varian/ Chemagnetics Infinity+ spectrometer operating at a 1H and 13C Larmor frequency of 300.1 and 75.5 MHz using a Bruker 4 mm double-resonance probe at a MAS frequency of 8 kHz. 20,200 transients were co-added with a recycle delay of 3 s (total experimental time ) 17 h). Ramped cross polarization39,40 from 1H to 13C with a CP contact time of 1.0 ms was used. Heteronuclear 1H decoupling in t2 was achieved using TPPM41 decoupling (ν1 ) 100 kHz) with a pulse duration of 5 µs and a phase increment of 15°. The 1H pulse length was 2.5 µs. For data and assignments, see Figure 2. Silica-Supported Dendrimer (Generation 0) 9. To a slurry of silica-supported intermediate 4b (0.5 g, 0.6 mmol) in DMF (20 mL) were added aminopropylmorpholine (0.5 mL, 3.6 mmol) and diisopropylethylamine (0.6 mL, 3.6 mmol), and then the reaction heated to 100 °C overnight. The solid was collected by filtration, washed successively with DMF, DCM, 1:1 DCM/methanol, DCM, and then dried under vacuum at 50 °C for 24 h; νmax, 3358, 2934, 1556, 1523, 1404, 1051, 808 cm-1. Anal. Calcd: C, 22.82; H, 3.54; N, 10.65. Found: C, 16.61; H, 2.81; N, 8.53. Calculated Maximum theoretical loading: 0.95 mmol/g. Loading implied from percentage N: 0.76 mmol/g. Silica-Supported Oxidized Dendrimer (Generation 0) 10. To a slurry of silica-supported dendrimer (Generation 0) 9 (0.6 g, 0.57 mmol) and potassium carbonate (0.2 g, 1.14 mmol) in DCM (20 mL) at -78 °C was added mCPBA (0.4 g, 1.14 mmol) in DCM (39) Hediger, S.; Meier, B. H.; Kurur, N. D.; Bodenhausen, G.; Ernst, R. R. Chem. Phys. Lett. 1994, 223, 283-288. (40) Metz, G.; Wu, X. L.; Smith, S. O. J. Magn. Reson., Ser. A 1994, 110, 219-227. (41) Bennett, A. E.; Rienstra, C. M.; Auger, M.; Lakshmi, K. V.; Griffin, R. G. J. Chem. Phys. 1995, 103, 6951-6958.

Langmuir, Vol. 22, No. 19, 2006 8149 (5 mL), and the reaction was maintained at -78 °C for 2 h. The reaction was allowed to warm to room temperature, and the solvent was removed by filtration and the solid washed successively with DMF, DCM, 1:1 DCM/methanol, water, acetone, and DCM, and then dried under vacuum at 50 °C for 24 h; νmax, 3043, 2948, 2789, 1571, 1524, 408, 1061, 1032, 808 cm-1. Anal. Calcd: C, 22.10; H, 3.43; N, 10.31. Found: C, 15.87; H, 2.69; N, 8.13. Maximum theoretical loading: 0.92 mmol/g. Loading implied from percentage N: 0.73 mmol/g. The 13C CP-MAS spectrum in Figure 3 was recorded on a Varian/ Chemagnetics Infinity spectrometer operating at a 1H and 13C Larmor frequency of 360.1 and 90.6 MHz, respectively, using a Bruker 4 mm double-resonance probe at a MAS frequency of 8 kHz. 16,384 transients were co-added with a recycle delay of 3.7 s (total experimental time ) 17 h). Ramped cross polarization39,40 from 1H to 13C with a CP contact time of 1.0 ms was used. Heteronuclear 1H decoupling in t was achieved using TPPM41 decoupling (ν ) 2 1 94 kHz) with a pulse duration of 5.2 µs and a phase increment of 25°. The 1H pulse length was 2.7 µs. For data and assignments, see Figure 3. Aminopropylmorpholine 3,5-Dichlorotriazine 11. To a solution of cyanuric chloride (2.0 g, 11 mmol) in chloroform (50 mL) at -78 °C was added aminopropylmorpholine (3.2 mL, 22 mmol). The reaction was quenched with brine after 10 min and extracted into chloroform (50 mL). The organic phase was separated and washed with water (50 mL) and brine (50 mL), and then dried (magnesium sulfate). The solvent was removed under reduced pressure to give a solid residue, which was purified by flash column chromatography (silica, DCM then 10% methanol/DCM) to yield the hydrochloride salt of aminopropylmorpholine 3,5-dichlorotriazine 11 as a white solid (1.13 g, 36%); mp 209-212 °C. νmax, 2923, 2772, 2682, 2607, 1741, 1591, 1560, 1533, 1431, 1259, 1110, 859, 804, 785 cm-1; δH (300 MHz, CDCl3) 1.76-1.89 (m, 2H); 2.40-2.66 (m, 6H); 3.523.67 (m, 2H); 3.74-3.89 (m, 4H); 7.80 (s, NH) ppm; m/z (LSIMS) 256 (35%) (M - Cl - H)+. Silica-Supported Intermediate 12. Aminopropyl silica (0.25 g, 0.35 mmol) was added to a solution of 11 (0.5 g, 1.8 mmol) in chloroform (10 mL), and the reaction was shaken for 3 days. The solvent was removed by filtration, and the solid was washed successively with DMF, DCM, 1:1 DCM/methanol, water, acetone, and DCM, then dried under vacuum at 50 °C for 24 h; νmax, 3295, 2964, 1578, 1529, 1044, 802 cm-1. Anal. Calcd: C, 15.61; H, 2.22; N, 8.40; Cl, 3.55. Found: C, 13.71; H, 2.33; N, 6.99; Cl, 3.26. Maximum theoretical loading: 1.0 mmol/g. Loading implied from percentage N, 0.83 mmol/g; Cl, 0.92 mmol/g. Silica-Supported Dendrimer (Generation 0) 13. To a suspension of silica-supported intermediate 12 (0.5 g, 1.03 mmol/g) in DMF (10 mL) were added 2-(2-[2-aminoethoxy]ethoxy)ethanol (0.65 g, 2.6 mmol) and diisopropylethylamine (0.36 g, 2.6 mmol), and the reaction was heated to 100 °C for 20 h. The solvent was removed by filtration and the solid washed successively with DMF, DCM, 1:1 DCM/methanol, water, acetone, and DCM, then dried under vacuum at 50 °C for 24 h; νmax, 3242, 1579, 1529, 1043, 785 cm-1. Anal. Calcd: C, 20.31; H, 3.23; N, 8.73. Found: C, 11.51; H, 2.23; N, 6.36. Maximum theoretical loading: 0.89 mmol/g. Loading implied from percentage N: 0.65 mmol/g. Silica-Supported Oxidized Dendrimer (Generation 0) 14. To a slurry of silica-supported dendrimer (Generation 0) 13 (0.2 g, 0.18 mmol) and potassium carbonate (0.02 g, 0.18 mmol) in DCM (10 mL) at -78 °C was added mCPBA (0.06 g, 0.18 mmol) in DCM (5 mL), and the reaction was maintained at -78 °C for 2 h. The reaction was then allowed to warm to room temperature, and the solvent was removed by filtration and the solid washed successively with DMF, DCM, 1:1 DCM/methanol, water, acetone, and DCM, then dried under vacuum at 50 °C for 24 h; νmax, 3043, 2973, 1653, 1583, 1536, 1046, 1034, 792 cm-1. Anal. Calcd: C, 20.08; H, 3.19; N, 8.63. Found: C, 12.73; H, 2.03; N, 5.62. Maximum theoretical loading: 0.88 mmol/g. Loading implied from percentage N: 0.57 mmol/g.

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Acknowledgment. We are grateful to the BBSRC (S.J.D., C.D.G. (GR 88/EGM17690)), EPSRC (S.P.B., J.M.G.), and Royal Society (S.P.B.) for funding and Dr. Andrew Thompson (Warwick HRI) for helpful discussions. We thank the reviewers for helpful suggestions.

Dilly et al.

Supporting Information Available: Tabulated data for Figure 4: Nonspecific adsorption of bacteriophage on supports. Solution synthesis of example amine oxides for spectroscopic comparison. This material is available free of charge via the Internet at http://pubs.acs.org. LA060743J