Materials and methods: organic synthesis

Solvents

All solvents used for synthesis were purchased in the highest quality available (puriss. p.a., absolute). Technical grade solvents were distilled prior to use and were stored over molecular sieves. Analytic and HPLC grade solvents were supplied from Acros Organics (Geel, Belgium), Sigma-Aldrich (Taufkirchen, Germany) and Fisher Scientific (Nidderau, Germany). Demineralized water was further purified on the water purification system Simplicity from Merck Millipore (Billerica, USA) prior to use.

Reagents

All reagents were purchased in the highest grade available and were used without further purification. Amino acids, coupling reagents and resins used in SPPS were purchased from NovaBiochem (Darmstadt, Germany), GL Biochem (Shanghai, China), ABCR (Karlsruhe, Germany), Bachem (Bubendorf, Switzerland), and IRIS Biotech (Marktredwitz, Germany).

Other reagents were purchased from Merck (Darmstadt, Germany), Carl Roth GmbH (Karlsruhe, Germany), Fisher Scientific GmbH (Nidderau, Germany), Alfa Aesar (Karlsruhe, Germany), Bachem (Bubendorf, Switzerland), TCI (Eschborn, Germany), VWR (Darmstadt, Germany), NovaBiochem (Darmstadt, Germany), Sigma Aldrich (Taufkirchen, Germany) and Acros Organics (Geel, Belgium). DNA oligomers were purchased in a HPLC-purified state from Biomers (Ulm, Germany). Biochemical reagents were purchased from Fermentas (St.

Leon-Rot, Germany), Biorad (Munich, Germany) and GERBU (Heidelberg, Germany).

Reactions

Air and moisture sensitive reactions were performed under an argon or nitrogen atmosphere.

Laboratory glassware was flame dried under reduced pressure and purged with dried nitrogen or argon. Solvents used in air sensitive reactions were degassed and flushed with argon prior to use. Reactants and solvents were added through a septum with the help of a syringe equipped with a cannula.

Lyophilization

Products were lyophilized from an aqueous solution, which was allowed to contain minimal amounts of acetonitrile, methanol or dioxane on a Christ Alpha-2-4 attached to a high vacuum pump. Freeze drying of larger samples was performed in a single-neck round bottom flasks. Smaller samples (<2 mL) were lyophilized in an Eppendorf safe-lock microcentrifuge tube in an evacuable Christ RCV-2-18 centrifuge, which was connected to the lyophilizer. All solvents were completely frozen in liquid nitrogen prior to lyophilization.

Thin layer chromatography (TLC)

Aluminium-backed plates coated with silica gel 60 F254 (layer thickness: 0.20 mm) from Merck were used for TLC. Substances were visualized by dipping the plate into a ninhydrin-staining solution (1.5 g ninhydrin, 3 mL acetic acid, 100 mL n-butanol) followed by spot pressure of 0.1 – 1 bar and fractions were collected in glass test tubes.

High performance liquid chromatography (HPLC)

Reverse phase HPLC (RP-HPLC) was performed on an Amersham Pharmacia Biotech system (Äkta basic, Pump-type P-900, UV detector 900). Time-dependent chromatograms were recorded by the detection of the compounds by means of UV absorption at 215 nm, 254 nm and 280 nm.

The following solvent systems were used:

A [99.9% H2O, 0.1%] to B [79.9% MeCN, 20% H2O, 0.1% TFA], A [99.9% H2O, 0.1%] to C [99.9% MeOH, 0.1% TFA]

The samples were dissolved in MilliQ water and either MeCN or MeOH was added in order to increase their solubility. Prior to injection, the samples were filtered using CHROMAFIL^® RC-45/15 MS (Macherey-Nagel) disposable syringe filters. The flow rates for analytical,

semi-preparative and preparative RP-HPLC runs were 1 mL/min, 3 mL/min and 10 mL/min.

The used columns were as indicated in the analytical section of the particular substances.

Nuclear magnetic resonance spectroscopy (NMR)

NMR spectroscopy was performed on a Varian instrument (Mercury 300, Unity 300, INOVA 500, INOVA 600). Chemical shifts are indicated in parts per million (ppm) downfield of the internal standard (TMS = 0 ppm). CDCl3, [D6]-DMSO, CD3OD and D2O were used as deuterated solvents. Signal multiplicities were abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Coupling constants ⁿJXX are stated in hertz (Hz), where n is the order of coupling and X stands for the nuclei.

Mass spectrometry

The compounds were characterized by electron spray ionization mass spectrometry (ESI-MS) on a Finnigan instruments (LCQ or TSQ 7000) or Bruker spectrometer (Apex-Q IV 7T and micrOTOF API). High resolution mass spectra were recorded on a Bruker Apex-Q IV 7T or on a Bruker micrOTOF.

UV spectroscopy

UV-vis spectra were recorded either on a Nanodrop ND-2000c (d = 0.1 cm) or on a JASCO V-550 UV-vis spectrophotometer (d = 1 cm). Hellma absorption cells with a path-length of 10 mm and a chamber volume of 1300 L or 700 L were used. Prior to the measurements, a blank spectrum was recorded, which was subtracted from the measured spectra. In cases UV-vis spectroscopy was used to determine the concentration of a compound, Lambert-Beer law was used for the calculations.

CD spectroscopy

Circular dichroism spectroscopy was either performed on a Jasco-810 or on a Jasco-1500 spectropolarimeter, which were equipped with a Jasco-PTC432S or a Julabo-F250 temperature controller. Hellma quartz precision cells with a thickness of either 1.0 mm or 10 mm were used for the measurements. The spectra were recorded in the range of 190–

280 nm at a constant temperature of 25 °C. The following parameters were applied for secondary structure measurements with Zf3 peptides: band width: 0.1 nm, response time:

0.1 s, sensitivity: high, data pitch: 0.1 nm and scanning speed: 50 nm/min. The final spectra were background corrected and were averaged from 12 accumulations. The means movement method was used to smooth the obtained curves.

Fluorescence spectroscopy

Fluorescence measurements were performed on a Jasco FP-6500 spectrofluorometer at 25 °C. The excitation and emission bandwidth was set to 5 nm, the data pitch was 1 nm, the response time was 0.1 s and the sensitivity was high. Hellma fluorescence cuvettes (semi micro Suprasil^® quartz, limit 200–2.500 nm spectral range, path-length 10 x 4 mm, chamber volume 1400 L) were used for measurements. All spectra were background subtracted and averaged from five measurements.

Microscale thermophoresis

MST measurements were performed on a Monolith NT.115 from NanoTemper Technologies (Munich, Germany). Fluorescence detection of the FITC labeled compounds was performed using the blue channel of the device. The LED power was set between 50–95% and the MST-power was set between 20–80%. The laser-on time was 30 s and the laser-off time was 5 s. The labeled small molecules were used in a final concentration of 60 nM whereas the labeled peptides were used in a final concentration of 120 nM, respectively. Ligand titrations were performed by preparing 1:1 dilution series starting with the highest concentration of the titrant and subsequent dilution with the buffer solution. Prior to capillary loading, all samples were centrifuged at 9.000 rpm for 10 min. All experiments were performed with premium capillaries, which were immediately sealed after loading.