Design, Synthesis and Catalytic Activity of Protein Containing Thiotyrosine as an Active Site Residue.

Native chemical ligation is a key reaction in the toolbox of chemical methods for the synthesis of native and modified proteins. The catalysis of ligation is commonly performed by using small aryl-thiol molecules added at high concentrations. In this work, we incorporated thiotyrosine, a non-canonical amino acid containing an aryl-thiol moiety, into a designed cyclic protein « sans queue ni tête ¼. Importantly, the protein environment reduced the pKa of the thiol group to 5.8-5.9, which is significantly lower than the previously reported value for thiotyrosine in a short peptide (pKa 6.4). Furthermore, we demonstrated the catalytic activity of this protein both as hydrolase and in native chemical ligation of peptides. These results will be useful for the development of efficient protein catalysts (enzymes) for protein synthesis and modification.

Dipeptides Containing Pyrene and Modified Photochemically Reactive Tyrosine: Noncovalent and Covalent Binding to Polynucleotides.

Dipeptides 1 and 2 were synthesized from unnatural amino acids containing pyrene as a fluorescent label and polynucleotide binding unit, and modified tyrosine as a photochemically reactive unit. Photophysical properties of the peptides were investigated by steady-state and time-resolved fluorescence. Both peptides are fluorescent (Φf = 0.3-0.4) and do not show a tendency to form pyrene excimers in the concentration range < 10-5 M, which is important for their application in the fluorescent labeling of polynucleotides. Furthermore, both peptides are photochemically reactive and undergo deamination delivering quinone methides (QMs) (ΦR = 0.01-0.02), as indicated from the preparative photomethanolysis study of the corresponding N-Boc protected derivatives 7 and 8. Both peptides form stable complexes with polynucleotides (log Ka > 6) by noncovalent interactions and similar affinities, binding to minor grooves, preferably to the AT reach regions. Peptide 2 with a longer spacer between the fluorophore and the photo-activable unit undergoes a more efficient deamination reaction, based on the comparison with the N-Boc protected derivatives. Upon light excitation of the complex 2·oligoAT10, the photo-generation of QM initiates the alkylation, which results in the fluorescent labeling of the oligonucleotide. This study demonstrated, as a proof of principle, that small molecules can combine dual forms of fluorescent labeling of polynucleotides, whereby initial addition of the dye rapidly forms a reversible high-affinity noncovalent complex with ds-DNA/RNA, which can be, upon irradiation by light, converted to the irreversible (covalent) form. Such a dual labeling ability of a dye could have many applications in biomedicinal sciences.

Amyloid engineering - how terminal capping modifies morphology and secondary structure of supramolecular peptide aggregates.

The effects of peptide N- and C-termini on aggregation behavior have been scarcely studied. Herein, we examine (105-115) peptide fragments of transthyretin (TTR) containing various functional groups at both termini and study their impact on the morphology and the secondary structure. We synthesized TTR(105-115) peptides functionalized with α-amino (H-), N-acetyl-α-amino (Ac-) or N,N-dimethyl-α-amino (DiMe-) groups at the N-terminus, and with amide (-NH2) or carboxyl (-OH) functions at the C-terminus. We also investigated quasi-racemic mixtures by mixing the L-enantiomers with the D-enantiomer capped by H- and -NH2 groups. We observed that fibril formation is promoted by the sufficient number of hydrogen bonds at peptides' termini. Moreover, the final morphology of the aggregates can be controlled by the functional groups at the N-terminus. Remarkably, all quasi-racemic mixtures resulted in the robust formation of fibrils. Overall, this work illustrates how modifications of peptide termini may help to engineer supramolecular aggregates with a predicted morphology.

Chemical grafting of cholesterol on monomer and PDMMLA polymers, a step towards the development of new polymers for biomedical applications.

Racemic α,α,ß-trisubstituted ß-lactones are the monomer units of poly((R,S)-3,3-dimethylmalic acid) (PDMMLA) derivatives, new biopolyesters showing great potential for biomedical applications. Using different groups during the synthesis of these ß-lactones allows a tailored synthesis of PDMMLA copolymers with adjustable hydrophilic/phobic ratio. The degradation kinetics of the employed material is one of the most important criteria in the development of bioresorbable implants. The degradation time of PDMMLA derivatives can be controlled using different ß-lactones of different hydrophilicity levels during the polymerization stage. Furthermore, PDMMLA has chemically available groups on its side chain allowing to graft functional groups on the polymer via covalent bonds. In this work, following a Steglich esterification protocol, the chemical grafting of cholesterol was carried out on a PDMMLA monomer derived ß-lactone as well as on homopolymer PDMMLA-H, and copolymer PDMMLAH40-co-Hex60 (PDMMLA 40/60). Nuclear magnetic resonance (NMR) analyses of the products confirm and quantify the grafting ratio. 100% of cholesterol grafting has been realized on the homopolymer PDMMLA-H giving PDMMLA-Chol, and 10% on the copolymer PDMMLA 40/60, giving PDMMLAH30-ter-Chol10-ter-Hex60 (PDMMLA-Chol 30/10/60) as wished. Fourier-transform infrared (FT-IR) spectra, elemental analysis on the ß-lactones and thermogravimetric analyses on the polymers also confirm the chemical modification of the products.