2.6.1 Materials
All commercial reagents were purchased from Aldrich and used as received unless stated otherwise. All solvents were purchased from Biosolve and used without further purification unless stated otherwise. Water was purified on an EMD Milipore Mili-Q Integral Water Purification System. Cy3- and Cy5-NHS esters were obtained from Lumiprobe.
Reactions were followed by thin-layer chromatography (precoated 0.25 mm, 60-F254 silica gel plates from Merck).
2.6.2 Instrumentation
Infrared spectra were recorded on a Perkin-Elmer Spectrum One 1600 FT-IR spectrometer.1H-NMR spectra were recorded on a Varian Mercury Vx 400MHz NMR spectrometer. Ultraviolet-visible absorbance spectra were recorded on a Jasco V-650 UV-vis spectrometer with a Jasco ETCT-762 temperature controller. Flash chromatography was performed on a Biotage flash chromatography system using 200-425 mesh silica gel (Type 60A Grade 633). Preparative reversed-phase high-pressure liquid chromatography (prep-HPLC) was performed on a system consisting of the following components: Shimadzu LC-8A preparative liquid chromatography pumps (with an Alltima C185 u (125x20 mm) preparative reversed-phase column and gradients of water-acetonitrile, supplemented with 0.1% trifluoroacetic acid), a Shimadzu CBM-20A prominence communications bus module and Shimadzu DGU 20A3 prominence degasser, Thermo Finnigan Surveyor PDA detector, Finigan LCQ Deca XP and Thermo Finnigan surveyor auto sampler.
Reversed-phase high-pressure liquid chromatography-mass spectrometry (RP-HPLC-MS) was performed on a system consisting of the following components: Shimadzu SCL-10A VP system controller with Shimadzu LC-10AD VP liquid chromatography pumps (with an Alltima C18 3 u (50x2.1 mm) reversed-phase column and gradients of water-acetonitrile supplemented with 0.1% formic acid, a Shimadzu DGU 20A3 prominence degasser, a Thermo Finnigan surveyor auto sampler, a Thermo Finnigan surveyor PDA detector and a Thermo Scientific LCQ Fleet.
2.6.3 Reduction of BTA-triazide to BTA-triamine
For the reduction, 102 mg (74 µmol) of the BTA-triazide and 106 mg (401 µmol) of triphenyl phosphine were dissolved in 13 ml of THF and 3 ml of H2O. The reaction was stirred overnight. The reaction was monitored using TLC (5%
MeOH/CHCl3). Another 107 mg (404 µmol) of triphenyl phosphine was added to the reaction mixture to promote complete reduction. After stirring at room temperature for another night, and completion of hydrolysis, analysis using IR-spectroscopy showed complete reduction of the starting material. The reaction mixture was separated into the different components using a Biotage silica column. Triphenyl phospine and its oxide could be removed by eluting with 5% MeOH/CHCl3. The product was obtained after addition of 2% i-PrNH2, yielding 70 mg (55 µmol) of the BTA-triamine as a colorless solid, resulting in a 74% yield. Analysis using1H-NMR and IR-spectroscopy showed complete reduction of the starting material.
2.6.4 Coupling of BTA-triamine with Cy3/Cy5 dyes
For dye labeling with Cy3, 25 mg (20 µmol) of BTA-triamine together with 9 mg (15 µmol) of the Cy3-NHS ester were dissolved in 1 ml of dry dimethylformamide (DMF). After adding a drop of TEA, the reaction mixture was allowed to stir overnight. After evaporation of the solvent, the mixture was purified via preparative-HPLC to isolate only the monosubstituted product, using a gradient from 40-50% MeCN. After purification and lyophilization, 1.6 mg (by weighing) of the desired product was isolated as a red powder-like solid. LC-MS analysis of the product revealed a single peak corresponding to the expected mass. The same procedure was followed for labelling with Cy5, which was isolated as a blue powder-like solid, where a yield of 1.2 mg (by weighing) was obtained.
2.6. MATERIALS AND METHODS
2.6.5 Stack-assembly
The assembly of supramolecular polymer samples was achieved through a dilution protocol. Stock solutions of BTA-3OH (10 mM in DMSO), and single labeled BTA-Cy3 (0.69 mM in DMSO) and BTA-Cy5 (0.43 mM in DMSO) were prepared. The stock solutions of dye labeled BTAs were standardized based on the absorbance of free dye in DMSO.
The stock solutions were combined and mixed to provide the correct concentration and dye ratio for the desired sample, and finally diluted with filtered Milli-Q water. The preparation involved mixing 10 µl of BTA-3OH solution with 2.9 µl of BTA-Cy3 solution and a separate batch that involved mixing 10 µl of BTA-3OH solution with 4.7 µl of BTA-Cy5 solution. Both were followed by dilution with 4 ml of water, producing 25µM BTA-solution concentrations, each with 2% dye labeling. The samples were then allowed to equilibrate for 24 hours before experiments, crucial for reliable stack formation.
C
HAPTER3
A
NTI-
FOULING SURFACEI
n order to overcome physisorption and the corresponding freezing of kinetics in BTA-fibers, we focus on the realization of an anti-fouling surface. In this approach we test a selection of different functional molecules that can react with the glass surface and change its physical properties, thus aiming to create a surface with anti-fouling properties.[29][30]3.1 Current method
In previous experiments involving the imaging of BTA-fibers, fibers were physisorbed to a glass coverslip surface used for microscopy. For sample preparation, a solution of BTA-fibers was flushed over such a glass slide. During a period of incubation, the polymers collide with the glass surface and a small proportion then remains adsorbed. This immobilization on the one hand allows us to image these fibers, but on the other hand it freezes the kinetics and prevents us from looking at the highly dynamic properties these BTA-fibers possess when they are in solution.
Imaging dynamic exchange of monomers along the chain of a single fiber is impossible with this method. Additionally, physisorption can be used as a general procedure for fiber sample preparation, the adsorption protocol needs to be optimized for different supramolecular polymers.
In the current protocol, samples with physisorbed BTA-fibers are measured to provide snap-shot information about their structure and composition. In order to achieve coverslips with excellent properties for BTA-imaging, the fiber-surface interaction is of great importance. Un-treated commercial slides suffer from surface contaminations and don’t always have a smooth surface. Intact BTA-fibers will not physisorb to these slides. Therefore, the glass coverslips are thoroughly cleaned using a selection of chemicals and solvents to remove contaminations.[25]
With this procedure, a smooth glass surface is obtained with active hydroxyl groups on the
surface. After incubation with a BTA-solution, these rinsed slides become covered BTA-fibers. An example of a surface obtained using this method can be seen in figure 3.1. This is an image of the surface of a cleaned glass coverslip and the physisorbed fiber-like aggregates are clearly visible.
To prevent fibers from adsorbing to the surface, the glass surface needs to be modified to avert physisorption.
FIGURE3.1. BTA-fibers physisorbed to the glass surface after cleaning using piranha-etch.