Experimental and Theoretical Studies on the Rearrangement of 2‐Oxoazepane α,α‐Amino Acids into 2′‐Oxopiperidine β2,3,3‐Amino Acids: An Example of Intramolecular Catalysis

Enantiopure β‐amino acids represent interesting scaffolds for peptidomimetics, foldamers and bioactive compounds. However, the synthesis of highly substituted analogues is still a major challenge. Herein, we describe the spontaneous rearrangement of 4‐carboxy‐2‐oxoazepane α,α‐amino acids to lead to 2′‐oxopiperidine‐containing β^2,3,3‐amino acids, upon basic or acid hydrolysis of the 2‐oxoazepane α,α‐amino acid ester. Under acidic conditions, a totally stereoselective synthetic route has been developed. The reordering process involved the spontaneous breakdown of an amide bond, which typically requires strong conditions, and the formation of a new bond leading to the six‐membered heterocycle. A quantum mechanical study was carried out to obtain insight into the remarkable ease of this rearrangement, which occurs at room temperature, either in solution or upon storage of the 4‐carboxylic acid substituted 2‐oxoazepane derivatives. This theoretical study suggests that the rearrangement process occurs through a concerted mechanism, in which the energy of the transition states can be lowered by the participation of a catalytic water molecule. Interestingly, it also suggested a role for the carboxylic acid at position 4 of the 2‐oxoazepane ring, which facilitates this rearrangement, participating directly in the intramolecular catalysis.     

Latex Fractionation by Sedimentation and Colloidal Crystallization: The Case of Poly(styrene-co-acrylamide)

Poly(styrene-co-acrylamide) (PS-AAM) latex was prepared, fractionated by sedimentation under gravity, and characterized by PCS, infrared spectra, secondary and backscattered electron imaging in the scanning electron microscope, and electron spectroscopy imaging in an analytical transmission electron microscope. Three latex fractions were obtained. The lower fraction was opalescent and its particles were the more uniform, concerning size, chemical composition, and topochemical features. This lower fraction was still further fractionated by zonal centrifugation in a density gradient, yielding two fractions with similar macrocrystal-forming abilities but different sizes and chemical compositions. These results confirm those previously obtained for the PS-HEMA latex. Copyright 2000 Academic Press.     

Modulating Protein–Protein Interactions by Cyclic and Macrocyclic Peptides. Prominent Strategies and Examples

Cyclic and macrocyclic peptides constitute advanced molecules for modulating protein–protein interactions (PPIs). Although still peptide derivatives, they are metabolically more stable than linear counterparts, and should have a lower degree of flexibility, with more defined secondary structure conformations that can be adapted to imitate protein interfaces. In this review, we analyze recent progress on the main methods to access cyclic/macrocyclic peptide derivatives, with emphasis in a few selected examples designed to interfere within PPIs. These types of peptides can be from natural origin, or prepared by biochemical or synthetic methodologies, and their design could be aided by computational approaches. Some advances to facilitate the permeability of these quite big molecules by conjugation with cell penetrating peptides, and the incorporation of β-amino acid and peptoid structures to improve metabolic stability, are also commented. It is predicted that this field of research could have an important future mission, running in parallel to the discovery of new, relevant PPIs involved in pathological processes.     

Entry to new conformationally constrained amino acids. First synthesis of 3-unsubstituted 4-alkyl-4-carboxy-2-azetidinone derivatives via an intramolecular N(alpha)-C(alpha)-cyclization strategy

A systematic study on the base-assisted intramolecular alkylation of N-benzyl-N-chloroacetyl amino acid derivatives is described. This study resulted in the first concise and versatile route to the preparation of 3-unsubstituted 4-alkyl-4-carboxy-2-azetidinones, to be included into the scarce family of beta-lactams with quaternary centers at the C(4) position. Particularly noteworthy is that the intramolecular N(alpha)-C(alpha)-cyclization of Phe and Leu derivatives afforded the corresponding beta-lactam derivatives with moderate enantioselectivity (up to 56%). It is suggested that, in these particular cases, the cyclization reaction proceeds by way of planar enolate intermediates, which possess dynamic chirality. The described sequence of reactions, that is compatible with commonly used protecting moieties for the alpha-carboxy group, cannot be applied to dipeptides, since the cyclization to the six-membered 2,5-diketopiperazine ring occurs preferentially.     

Antisense-mediated isoform switching of steroid receptor coactivator-1 in the central nucleus of the amygdala of the mouse brain

Background

Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon skipping by local injection in the central nucleus of the amygdala (CeA) of the mouse brain. As proof of principle we targeted the splicing of steroid receptor coactivator-1 (SRC-1), a protein involved in nuclear receptor function. This nuclear receptor coregulator exists in two splice variants (SRC-1a and SRC-1e) which display differential distribution and opposing activities in the brain, and whose mRNAs differ in a single SRC-1e specific exon.

Methods

For proof of principle of feasibility, we used immunofluorescent stainings to study uptake by different cell types, translocation to the nucleus and potential immunostimulatory effects at different time points after a local injection in the CeA of the mouse brain of a control AON targeting human dystrophin with no targets in the murine brain. To evaluate efficacy we designed an AON targeting the SRC-1e-specific exon and with qPCR analysis we measured the expression ratio of the two splice variants.

Results

We found that AONs were taken up by corticotropin releasing hormone expressing neurons and other cells in the CeA, and translocated into the cell nucleus. Immune responses after AON injection were comparable to those after sterile saline injection. A successful shift of the naturally occurring SRC-1a:SRC-1e expression ratio in favor of SRC-1a was observed, without changes in total SRC-1 expression.

Conclusions

We provide a proof of concept for local neuropharmacological use of exon skipping by manipulating the expression ratio of the two splice variants of SRC-1, which may be used to study nuclear receptor function in specific brain circuits. We established that exon skipping after local injection in the brain is a versatile and useful tool for the manipulation of splice variants for numerous genes that are relevant for brain function.