Peptide-conjugated antimiRs improve myotonic dystrophy type 1 phenotypes by promoting endogenous MBNL1 expression.

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the DMPK gene that generates toxic RNA with a myriad of downstream alterations in RNA metabolism. A key consequence is the sequestration of alternative splicing regulatory proteins MBNL1/2 by expanded transcripts in the affected tissues. MBNL1/2 depletion interferes with a developmental alternative splicing switch that causes the expression of fetal isoforms in adults. Boosting the endogenous expression of MBNL proteins by inhibiting the natural translational repressors miR-23b and miR-218 has previously been shown to be a promising therapeutic approach. We designed antimiRs against both miRNAs with a phosphorodiamidate morpholino oligonucleotide (PMO) chemistry conjugated to cell-penetrating peptides (CPPs) to improve delivery to affected tissues. In DM1 cells, CPP-PMOs significantly increased MBNL1 levels. In some candidates, this was achieved using concentrations less than two orders of magnitude below the median toxic concentration, with up to 5.38-fold better therapeutic window than previous antagomiRs. In HSALR mice, intravenous injections of CPP-PMOs improve molecular, histopathological, and functional phenotypes, without signs of toxicity. Our findings place CPP-PMOs as promising antimiR candidates to overcome the treatment delivery challenge in DM1 therapy.

Peptide-Conjugated PMOs for the Treatment of Myotonic Dystrophy.

Antisense oligonucleotides (ASOs) have shown great therapeutic potential in the treatment of many neuromuscular diseases including myotonic dystrophy 1 (DM1). However, systemically delivered ASOs display poor biodistribution and display limited penetration into skeletal muscle. The conjugation of cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligonucleotides (PMOs), a class of ASOs with a modified backbone, can be used to enhance ASO skeletal muscle penetration. Peptide-PMOs (P-PMOs) have been shown to be highly effective in correcting the DM1 skeletal muscle phenotype in both murine and cellular models of DM1 and at a molecular and functional level. Here we describe the synthesis and conjugation of P-PMOs and methods for analyzing their biodistribution and toxicity in the HSA-LR DM1 mouse model and their efficacy both in vitro and in vivo using FISH and RT-PCR splicing analysis.

Proof of concept of peptide-linked blockmiR-induced MBNL functional rescue in myotonic dystrophy type 1 mouse model.

Myotonic dystrophy type 1 is a debilitating neuromuscular disease causing muscle weakness, myotonia, and cardiac dysfunction. The phenotypes are caused by muscleblind-like (MBNL) protein sequestration by toxic RNA in the DM1 protein kinase (DMPK) gene. DM1 patients exhibit a pathogenic number of repetitions in DMPK, which leads to downstream symptoms. Another disease characteristic is altered microRNA (miRNA) expression. It was previously shown that miR-23b regulates the translation of MBNL1 into protein. Antisense oligonucleotide (AON) treatment targeting this miRNA can improve disease symptoms. Here, we present a refinement of this strategy targeting a miR-23b binding site on the MBNL1 3' UTR in DM1 model cells and mice by using AONs called blockmiRs. BlockmiRs linked to novel cell-penetrating peptide chemistry showed an increase in MBNL1 protein in DM1 model cells and HSALR mice. They also showed an increase in muscle strength and significant rescue of downstream splicing and histological phenotypes in mice without disturbing the endogenous levels of other miR-23b target transcripts.

N-methylation in amino acids and peptides: Scope and limitations.

Active pharmaceutical ingredients (APIs) can be divided into two types, namely chemical and biological entities. Traditionally, the former has been associated with the so-called small molecules. The revival of peptides in pharmaceutical industry results from their importance in many biological roles. However, low metabolic stability and the lack of oral availability of most peptides is the main drawback for peptide to fulfill that paradigmatic situation. In this regard, efforts are being channeled into addressing this issue by introducing restrictions into the flexible peptide backbone, mainly through N-methyl amino acids (NMAAs) or development of small cyclic peptides. In many cases, both the above restrictions are combined with the aim to enhance oral availability. The synthesis of NMAAs is complex and their introduction into the peptide chain brings additional synthetic challenges and also sometimes leads to side-reactions. Here we discuss the most efficient methods for the synthesis of NMAAs (either in solution or in solid phase) and also their introduction into peptide sequences. Special attention is also given to the detection of side reactions and the most efficient way to prevent them.

Solid-Phase Synthesis of Pyrrole Derivatives through a Multicomponent Reaction Involving Lys-Containing Peptides.

The synthesis of pyrroles has received considerable attention because of their biological and pharmaceutical activities. Herein we describe a solid-phase multicomponent reaction that utilizes Lys as a N donor, ß-nitrostyrenes, 1,3-dicarbonyl compounds, and FeCl3 as an easily accessible catalyst under microwave irradiation to afford the subsequent pyrrole derivatives in high conversions. The strategy combines three of the most powerful tools in modern synthetic chemistry: the solid-phase mode, microwave activation, and a multicomponent reaction. The excellent results in terms of rapidity, versatility, and purity obtained herein support once again that this combined strategy is efficient for gaining chemical diversity.

Teixobactin as a scaffold for unlimited new antimicrobial peptides: SAR study.

It looks that a new era of antimicrobial peptides (AMPs) started with the discovery of teixobactin, which is a "head to side-chain" cyclodepsipeptide. It was isolated from a soil gram-negative b-proteobacteria by means of a revolutionary technique. Since there, several groups have developed synthetic strategies for efficient synthesis of this peptide and its analogues as well. Herein, all chemistries reported as well as the biological activity of the analogues are analyzed. Finally, some inputs regarding new trends for the next generation of analogues are discussed.

Re-evaluating the stability of COMU in different solvents.

COMU is uronium-type coupling reagent based on OxymaPure. It showed several advantages over classical benzotriazole-based coupling reagents such as higher solubility, water-soluble byproduct, and monitoring the reaction by changing of color. Although COMU is well known to perform excellent in solution, but its hydrolytic stability in DMF limits its use in automatic peptide synthesizer. Herein, we evaluated the hydrolytic stability of COMU in γ-valerolactone (GVL), acetonitrile (ACN) and N-formylmorpholine (NFM) and compared its stability against DMF. The stability of COMU after 24 h was found to be 88 and 89% in GVL and ACN, respectively, when compared in DMF (14%). Further, the demanding Aib-ACP decapeptide and JR decapeptide were successfully synthesized using COMU dissolved in GVL or ACN while Fmoc amino acids were dissolved in DMF. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

2-Methyltetrahydrofuran and cyclopentyl methyl ether for green solid-phase peptide synthesis.

2-MeTHF and CPME were evaluated as greener alternatives for the most employed solvents in peptide synthesis. The ability of these solvents to dissolve amino acid derivatives and a range of coupling reagents were evaluated as well as the swelling of polystyrene and polyethylene glycol resins. In addition, racemization and coupling efficiencies were also determined. We concluded that the use of 2-MeTHF with combination of DIC/OxymaPure gave the lowest racemization level during stepwise synthesis of Z-Phg-Pro-NH2 and the highest purity during SPPS of Aib-enkephalin pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-NH2).

Lysine Scanning of Arg10-Teixobactin: Deciphering the Role of Hydrophobic and Hydrophilic Residues.

Teixobactin is a recently discovered antimicrobial cyclodepsipeptide with good activity against Gram positive bacteria. Taking Arg10-teixobactin as a reference, where the nonproteinogenic residue l-allo-enduracididine was substituted by arginine, a lysine scan was performed to identify the importance of keeping the balance between hydrophilic and hydrophobic amino acids for the antimicrobial activities of this peptide family. Thus, the substitution of four isoleucine residues present in the natural sequence by lysine led to a total loss of activity. On the other hand, the substitution of the polar noncharged residues and alanine by lysine allowed us to keep and in some cases to improve the antimicrobial activity.

Synthesis and Biological Evaluation of a Teixobactin Analogue.

The first synthesis and biological activity of a teixobactin analogue is reported. Substitution of the unusual L-allo-enduracididine residue by the naturally occurring L-arginine was achieved, and the analogue gave an activity trend similar to that of teixobactin (against Gram-postive bacteria) and meropenem, which was approved by the FDA in 1996. The synthetic route used allows for the synthesis of the natural product as well as the development of a program of medicinal chemistry.

Peptide synthesis beyond DMF: THF and ACN as excellent and friendlier alternatives.

To date, DMF has been considered as the only solvent suitable for peptide synthesis. Here we demonstrate the capacity of THF and ACN, which are friendlier solvents than DMF, to yield the product in higher purity than DMF. Using various peptide models, both THF and ACN reduced racemization in solution-phase and solid-phase synthesis when compared with DMF. Moreover, the use of ACN and THF in the solid-phase peptide synthesis of hindered peptides, such as Aib-enkephalin pentapeptide and Aib-ACP decapeptide, in combination with a complete polyethylene glycol resin (ChemMatrix), gave a better coupling efficiency than DMF.

Production and physicochemical assessment of new stevia amino acid sweeteners from the natural stevioside.

New stevia amino acid sweeteners, stevia glycine ethyl ester (ST-GL) and stevia l-alanine methyl ester (ST-GL), were synthesised and characterised by IR, NMR ((1)H NMR and (13)C NMR) and elemental analysis. The purity of the new sweeteners was determined by HPLC and their sensory properties were evaluated relative to sucrose in an aqueous system. Furthermore, the stevia derivatives (ST-GL and ST-AL) were evaluated for their acute toxicity, melting point, solubility and heat stability. The novel sweeteners were stable in acidic, neutral or basic aqueous solutions maintained at 100 °C for 2 h. The sweetness intensity rate of the novel sweeteners was higher than sucrose. Stevia amino acid (ST-GL and ST-AL) solutions had a clean sweetness taste without bitterness when compared to stevioside. The novel sweeteners can be utilised as non-caloric sweeteners in the production of low-calorie food.

TOMBU and COMBU as Novel Uronium-type peptide coupling reagents derived from Oxyma-B.

Here we describe two novel uronium salts, TOMBU and COMBU, derived from the recently described Oxyma-B for use in peptide bond synthesis. These coupling reagents are more stable than COMU in DMF. Furthermore, using various peptide synthetic models in solution and solid-phase synthesis, we reveal that they show better performance than HBTU in terms of preserving chiral integrity and coupling yields, but slightly worse performance than COMU.

Oxyma-B, an excellent racemization suppressor for peptide synthesis.

Peptide-bond formation is a key process in the synthesis of peptide oligomers. Among the many coupling techniques reported, carbodiimides combine strong acylation potency and smooth reaction conditions and are commonly used in the presence of additives. Recently, ethyl 2-cyano-2-(hydroxyimino)acetate (OxymaPure) has emerged as a highly reactive alternative to the classic and explosion-prone benzotriazolic additives, namely 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt). Here we report on a new oxime additive 5-(hydroxyimino)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (Oxyma-B). This new additive showed satisfactory solubility in various solvents (DMF, ACN, and THF). It was also more effective in the control of optical purity during the synthesis of Z-Phg-Pro-NH2, Z-Phe-Val-Pro-NH2, H-Gly-Ser-Phe-NH2, H-Gly-Cys-Phe-NH2, H-Gly-Cys(Acm)-Phe-NH2 and H-Gly-His-Phe-NH2 than related Oxyma- and benzotriazole-based reagents. Oxyma-B also proved to be advantageous compared to the related HONM, because the latter cannot be used with the carbodiimide. Furthermore, Oxyma-B showed satisfactory performance in assembling demanding sequences such as the Aib-enkephalin pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-NH2).

Oxime-based carbonates as useful reagents for both N-protection and peptide coupling.

We have demonstrated that oxime-based mixed carbonates are very effective reagents for both N-protection and peptide coupling.