Identification of inhibitors of an 80 kDa protease from Trypanosoma cruzi through the screening of a combinatorial peptide library

Two orthogonal peptide combinatorial libraries were screened to discover inhibitors of Tc80 protease, a novel target from Trypanosoma cruzi involved in host cell invasion. These libraries were composed of 15,625 structurally diversified tripeptides, partitioned in 125 mixtures. The screening led to a low micromolar inhibitor which was actually an HF cleavage by-product H-Ipe-D-Tic-D-Glu(S-paratolyl)-OH. IC50 values of several analogous molecules of this hit were determined and are discussed. For the best compounds, conformational analysis revealed a high degree of similarity in shape with a potent prolylendopeptidase inhibitor, SUAM-1221.     

Analysis of an asymmetric addition with a 2:1 mixed lithium amide/n-butyllithium aggregate

A 2:1 lithium amide/ n-butyllithium aggregate 1 is investigated as an asymmetric addition template in hydrocarbon solvents. Several different chiral lithium amides were synthesized from l-valine and tested in the asymmetric addition of n-BuLi to various aldehydes. Enantiomeric excesses up to 83% were obtained in the case of the addition of n-BuLi to pivaldehyde at -116 degrees C in pentane. (1)H and (13)C INEPT DOSY were utilized to characterize a new trimeric complex 12 between 2 equiv of lithium amide and 1 equiv of lithium alkoxide. This mixed aggregate strongly indicates the possibility of product-induced chirality inhibition that is detrimental to the enantioselectivity of asymmetric addition reaction.     

13C INEPT diffusion-ordered NMR spectroscopy (DOSY) with internal references

13C INEPT Diffusion-ordered NMR spectroscopy (DOSY) with an internal reference system was developed to study the aggregation state of THF-solvated LDA dimeric complex. Six components are clearly identified in the diffusion dimension, and their DOSY-generated 13C INEPT spectrum slices agree extremely well with their respective INEPT spectra. The correlation between log D and log FW of the linear least-squares fit to reference points of all components is exceptionally high: (r = 0.9985).     

Characterization of dimeric chiral lithium amide structures derived from N-isopropyl-O- triisopropylsilyl valinol

The dimeric structure is characterized for a chiral amide base complex consisting of an (S)-N-isopropyl-O-triisopropylsilyl valinol ligand and lithium. The complex is characterized by a variety of NMR techniques, including multinuclear one- and two-dimensional NMR experiments and diffusion-ordered NMR spectroscopy (DOSY) as well as diffusion coefficient-formula weight (D-fw) correlation analyses. Spartan calculations are presented which support the structural assignment. This structural characterization leads to an explanation of the behavior and the reactivity of these complexes in solution.     

Investigation of enzymatic C–P bond formation using multiple quantum HCP nuclear magnetic resonance spectroscopy

The biochemical mechanism for the formation of the C–P–C bond sequence found in l ‐phosphinothricin, a natural product with antibiotic and herbicidal activity, remains unclear. To obtain further insight into the catalytic mechanism of PhpK, the P‐methyltransferase responsible for the formation of the second C–P bond in l ‐phosphinothricin, we utilized a combination of stable isotopes and two‐dimensional nuclear magnetic resonance spectroscopy. Exploiting the newly emerged Bruker QCI probe (Bruker Corp.), we specifically designed and ran a ¹³C‐³¹P multiple quantum ¹H‐¹³C‐³¹P (HCP) experiment in ¹H‐³¹P two‐dimensional mode directly on a PhpK‐catalyzed reaction mixture using ¹³CH₃‐labeled methylcobalamin as the methyl group donor. This method is particularly advantageous because minimal sample purification is needed to maximize product visualization. The observed 3:1:1:3 multiplet specifically and unequivocally illustrates direct bond formation between ¹³CH₃ and ³¹P. Related nuclear magnetic resonance experiments based upon these principles may be designed for the study of enzymatic and/or synthetic chemical reaction mechanisms. Copyright © 2015 John Wiley & Sons, Ltd.