N-Methyl-D-isoleucine

The serotype II Dengue (DENV 2) virus is the most prevalent of all four known serotypes. Herein, we present nearly complete 1H, 15N, and 13C backbone and 1H, 13C isoleucine, valine, and leucine methyl resonance assignment of the apo S135A catalytically inactive variant of the DENV 2 protease enzyme folded as a tandem formed between the serine protease domain NS3pro and the cofactor NS2B, as well as the secondary structure prediction of this complex based on the assigned chemical shifts using the TALOS-N software. Our results provide a solid ground for future elucidation of the structure and dynamic of the apo NS3pro/NS2B complex, key for adequate development of inhibitors, and a thorough molecular understanding of their function(s).

Nuclear magnetic resonance (NMR) spectroscopy is a primary structural biology method to characterize protein dynamics in solution. For large macromolecular systems, methyl-labeling in a perdeuterated background significantly improves the relaxation properties, while providing sensitive probes for structure and dynamics analysis. However, how to prepare methyl-labeled proteins, especially for functional eukaryotic proteins, remains to be a major bottleneck in this field. Due to its advantages in eukaryotic co-translational and post-translational modification, as well as high-density fermentation, Pichia pastoris has been a cost-effective platform strain for 13C, 15N-labeling and deuterium labeling since the early 2000's. Recently, some substantial progress has been made in methyl-labeling, such as the feasibility of 13C isoleucine δ1 methyl-labeling in perdeuterated background and the increased uptake of the Val/Leu precursor. Here, we systematically introduce the isotope-labeling strategies in P. pastoris, including strain engineering and detailed fermentation protocols in 13C, 15N-labeling and methyl-labeling, providing instructions and guidance for the future improvement of sample preparation for NMR spectroscopy.

Aims: The present investigation is targeted towards the synthesis of a novel analogue of a natural peptide of marine origin. Background: Marine sponges are enriched with bioactive secondary metabolites, especially circu-lar peptides. Heterocycles are established organic compounds with potential biological value. Tak-ing into consideration the bio-properties of heterocycles and marine sponge-derived natural pep-tides, an effort was made for the synthesis of a heterocyclic analogue of a natural cyclopeptide. Objective: A heterocyclic analogue of a sponge-derived proline-containing cyclic peptide, rolloam-ide A, was synthesized by interaction of Boc-protected L-histidinyl-L-prolyl-L-valine and L-prolyl-L-leucyl-L-prolyl-L-isoleucine methyl ester and compared with synthetic rolloamide A with bioac-tivity against bacteria, fungi, and earthworms. Methods: The synthesis of cycloheptapeptide was accomplished employing the liquid phase method. The larger peptide segment was prepared by interaction of Boc-protected L-prolyl-L-leu-cine with L-prolyl-L-isoleucine methyl ester. Similarly, the tripeptide unit was synthesized from Boc-protected L-histidinyl-L-proline with L-valine ester. The linear heptapeptide segment (7) was cyclized by utilizing pentafluorophenyl (pfp) ester, and the structure was elucidated by elemental and spectral (IR, 1H/13C NMR, MS) analysis. The peptide was also screened for diverse bioactivities such as antibacterial, antifungal, and potential against earthworms and cytotoxicity. Results: The novel cyclooligopeptide was synthesized with 84% yield by making use of car-bodiimides. The synthesized cyclopeptide exhibited significant cytotoxicity against two cell lines. In addition, promising antifungal and antihelmintic properties were observed for newly synthesized heterocyclic peptide derivative (8) against dermatophytes and three earthworm species at 6 μg/mL and 2 mg/mL, respectively. Conclusion: Solution-phase technique employing carbodiimide chemistry was established to be promising for synthesizing the cycloheptapeptide derivative (8), and C5H5N was proved to be a better base for heptapeptide circling when compared to N-methylmorpholine and triethylamine.