Similarly, no striking enrichment was observed for selections on unmodified resin. enrichment of certain classes of structurally related compounds (Fig. 2). In addition to desthiobiotin, a biotin analogue with nanomolar affinity to streptavidin, which had been spiked into the library as positive control before selection, Cevipabulin fumarate we observed an enrichment of derivatives of the thioester moiety 78, of the ester moiety 49 as well as of other pharmacophores (e.g., 175). Fluorescent amide derivatives of compounds 49 and 78 had previously been found to Cevipabulin fumarate bind to streptavidin with dissociation constants in the millimolar range, as assessed by fluorescence polarization assays (7), whereas others (e.g., 175) had not previously been reported as streptavidin binders. Open in a separate window Fig. 2. Plots representing the frequency (i.e., sequence counts) of the 4,000 library members before selection, after selection on empty resin, and after selection on streptavidin resin, as revealed by high-throughput 454 sequencing. The chemical structures of some of the most relevant straptavidin binders are indicated. The building blocks used in the 2 2 synthetic steps are indicated Cevipabulin fumarate in green and red, respectively, together with the respective identification number. A known streptavidin binder (desthiobiotin) had been mixed with the library at low concentration before the selections serving as positive control. To evaluate whether the extensions of the pharmacophore 49 and 78 moieties within the 4,000-member chemical library (02, 07, 11, 15, 16, and 17 depicted in green in Fig. 2) contribute to an increased affinity toward streptavidin, we measured the dissociation constants of the most enriched compounds by fluorescence polarization at 25 C, after conjugation to fluorescein (Fig. 3; see also and and reveals that, after selection, compound 02-40 was identified 96 times of a total 39,092 identified sequence tags, whereas 50% of library members were detected between 1 and 10 counts, and 10% RGS7 of the compounds were identified 20 counts. By Cevipabulin fumarate using the diamino linker shows that IgG labeled with both the fluorophore Cy5 and with biotin could be completely and selectively captured from the supernatant and could be eluted by using 100 mM aqueous triethylamine solution. Open in a separate window Fig. 4. DEL4000 library selection with polyclonal human IgG. (+ 0.5 (((+ const + em c /em ) ? math xmlns:mml=”http://www.w3.org/1998/Math/MathML” id=”i1″ overflow=”scroll” mrow msqrt mrow mo stretchy=”false” ( /mo mo stretchy=”false” ( /mo mi x /mi mo + /mo mtext const /mtext mo + /mo mi c /mi mo stretchy=”false” ) /mo mover mn 2 /mn mo /mo /mover mo ? /mo mn 4 /mn mo /mo mi x /mi mo /mo mtext const /mtext mo stretchy=”false” ) /mo mo stretchy=”false” ) /mo mo stretchy=”false” ) /mo /mrow /msqrt /mrow /math ; const = 500 nM.] Synthesis of IgG Affinity Chromatography Resins (Containing the Compounds 02-40 Cevipabulin fumarate or 16-40). Details of the synthesis and analytics can be found in em SI Text /em . Polyclonal Human IgG Cy5 Labeling and Biotinilated Polyclonal Human IgG. Details of the preparations can be found in em SI Text /em . Affinity Chromatography of CHO Cells Supernatant Spiked with Human IgG Cy5-Labeled or Biotinylated Human IgG on IgG Binding. The resin containing compound 02-40 was loaded on a chromatography cartridge and washed 3 times with PBS before loading a CHO cell supernatant spiked with human IgG Cy5-labeled or biotinilated human IgG. The flow-through, the wash fractions (washing 1 time with PBS; 1 time with 500 mM NaCl, 0.5 mM EDTA; 1 time with 100 mM NaCl, 0.1% Tween 20, 0.5 mM EDTA) and the elutate (elution 3 times with 100 mM aqueous triethylamine solution) were collected and eventually concentrated back to the initial volume by centrifugation in a Vivaspin 500 tube (cut-off 10,000 MW). The samples were then analyzed by gel electrophoresis on a NuPAGE 4C12% Bis-Tris gel by using Mops SDS as running.