Simple Preparation of Pacific Cod Trypsin for Enzymatic Peptide Synthesis

Trypsin from the pyloric caeca of Pacific cod (Gadus macrocephalus) was easily prepared by affinity chromatography on Benzamidine Sepharose 6B and gel filtration on Superdex 75. Pacific cod trypsin was composed of three isozymes, and their molecular masses were estimated 23,756.34 Da, 23,939.62 Da, and 24,114.81 Da by desorption/ionization time-of-flight mass spectroscopy (MALDI/TOF-MS) and their isoelectric points (pIs) were approximately 5.1, 6.0, and 6.2, respectively. The isolated Pacific cod trypsin showed high similarity to other frigid-zone fish trypsins. The kinetic behavior of tryptic hydrolysis toward N-p-tosyl-L-arginine methyl ester hydrochloride (TAME), N-benzoyl-L-arginine p-nitroanilide hydrochloride (BAPA), and p-amidinophenyl ester were also analyzed. In addition, the cod trypsin-catalyzed dipeptide synthesis was investigated using twelve series of “inverse subdtrates” that is p- and m-isomer of amidinophenyl, guanidinophenyl, (amidinomethyl)phenyl, (guanidinomethyl)phenyl, and four position isomers of guanidinonaphtyl esters derived from N-(tert-butoxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of the trypsin. All inverse substrates tested in this study undergo less enantioselective coupling reaction. The p-guanidinophenyl ester was most practical substrate in twelve series tested. The enzymatic hydrolysis of the resulting products was negligible

Synthesis of α‑, ω‑, and α,ω-End-Functionalized Poly(<i>n</i>‑butyl acrylate)s by Organocatalytic Group Transfer Polymerization Using Functional Initiator and Terminator

The α-functionalized (hydroxyl, ethynyl, vinyl, and norbornenyl), ω-functionalized (ethynyl, vinyl, hydroxyl, and bromo), and α,ω-functionalized polyacrylates were precisely synthesized by the <i>N</i>-(trimethylsilyl)­bis­(trifluoroethanesulfonyl)­imide (Me₃SiNTf₂)-catalyzed group transfer polymerization (GTP) of <i>n</i>-butyl acrylate (<i>n</i>BA). The α-functionalization and ω-functionalization were carried out using the functional triisopropylsilyl ketene acetal as the initiator (initiation approach) and 2-phenyl acrylate derivatives as the terminator (termination approach) for the organocatalytic GTP, respectively. All the polymerizations precisely occurred and produced well-defined end-functionalized poly­(<i>n</i>-butyl acrylate)­s which had predictable molecular weights and narrow molecular weight distributions. High-molecular-weight polyacrylates were easily synthesized using both approaches. In addition, the α,ω-functionalized (hetero)­telechelic polyacrylates were synthesized by the combination of the initiation and termination approaches. The structure of the obtained polyacrylates and degree of functionalization were confirmed by the ¹H NMR and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) measurements. The spectra of the ¹H NMR and MALDI-TOF MS showed that the end-functionalization quantitatively proceeded without any side reactions

A Photolithographic Approach to Spatially Resolved Cross-Linked Nanolayers

(Figure Presented). The preparation of cross-linked nanosheets with 1-2 nm thickness and predefined shape was achieved by lithographic immobilization of trimethacryloyl thioalkanoates onto the surface of Si wafers, which were functionalized with 2-(phenacylthio)acetamido groups via a photoinduced reaction. Subsequent cross-linking via free radical polymerization as well as a phototriggered Diels-Alder reaction under mild conditions on the surface led to the desired nanosheets. Electrospray ionization mass spectrometry (ESI-MS), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), as well as infrared reflection-absorption spectroscopy (IRRAS) confirmed the success of individual surface-modification and cross-linking reactions. The thickness and lateral size of the cross-linked structures were determined by atomic force microscopy (AFM) for samples prepared on Si wafers functionalized with a self-assembled monolayer of 1H,1H,2H,2H-perfluorodecyl groups bearing circular pores obtained via a polymer blend lithographic approach, which led to the cross-linking reactions occurring in circular nanoareas (diameter of 50-640 nm) yielding an average thickness of 1.2 nm (radical cross-linking), 1.8 nm (radical cross-linking in the presence of 2,2,2-trifluoroethyl methacrylate as a comonomer), and 1.1 nm (photochemical cross-linking) of the nanosheets. © 2015 American Chemical Society