Easy parallel screening of reagent stability, quality control, and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

Abstract

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1-{[1-(Cyano-2-ethoxy-2-oxo-ethylidenaminooxy)dimethylamino-morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma-Aldrich, St. Louis, MO, USA) by SPPS of Leu-Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3-aminopropyl)triethyoxysilane-modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of the YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays.

Synthesis of the Novel Covalent Cysteine Proteases Inhibitor with Iodoacetic Functional Group

“…Application of bromo-, chloro-, and iodoacetic acids in the peptide solid phase synthesis requires changes in the standard synthesis protocol [14]. The results demonstrate a comparison of standard synthesis involving DIC (N,N -diisopropylcarbodiimide) with HOBt (N-hydroxybenzotriazole), with the newer activation reagent like COMU (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylaminomorpholino-carbenium hexafluorophosphate) [15][16][17]. Synthesis yield and product purity were checked by mass spectrometry [18].…”