A backbone amide protecting group for overcoming difficult sequences and suppressing aspartimide formation.

A backbone amide bond protecting group, 2-hydroxy-4-methoxy-5-nitrobenzyl (Hmnb), improved the synthesis of aggregation and aspartimide-prone peptides. Introduction of Hmnb is automated and carried out during peptide assembly by addition of 4-methoxy-5-nitrosalicylaldehyde to the peptidyl-resin and on-resin reduction to the secondary amine. Acylation of the hindered secondary amine is aided by the formation of an internal nitrophenol ester that undergoes a favourable O,N intramolecular acyl transfer. This activated ester participates in the coupling and generally gives complete reaction with standard coupling conditions. Hmnb is easily available in a single preparative step from commercially available material. Different methods for removing the amide protecting group were explored. The protecting group is labile to acidolysis, following reduction of the nitro group to the aniline. The two main uses of backbone protection of preventing aspartimide formation and of overcoming difficult sequences are demonstrated, first with the synthesis of a challenging aspartimide-prone test sequence and then with the classic difficult sequence ACP (65-74) and a 23-mer homopolymer of polyalanine.

HF-Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization.

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post-translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.

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.

Evaluating Efficacy of Peptide-Delivered Oligonucleotides Using the Severe Taiwanese SMA Mouse Model.

Oligonucleotides (ONs) are therapeutic macromolecules with great potential for the treatment of neurological conditions, including spinal muscular atrophy (SMA), a neurodegenerative disease. However, the neurovascular unit severely limits their distribution to the neural parenchyma of the brain and the spinal cord. Cell-penetrating peptides (CPPs) can be conjugated to oligonucleotides to increase their delivery across biological barriers. In this chapter, we describe the synthesis and conjugation of CPPs to oligonucleotides, and the use of a severe SMA mouse model to test in vivo the efficacy of CPP-delivered oligonucleotides, using ELISA, western blot, and TaqMan™ RT-qPCR assays.

Enhancing the Therapeutic Potential of Extracellular Vesicles Using Peptide Technology.

Extracellular vesicles are lipid-bilayer-enclosed nanoparticles present in the majority of biological fluids that mediate intercellular communication. EVs are able to transfer their contents (including nucleic acids, proteins, lipids, and small molecules) to recipient cells, and thus hold great promise as drug delivery vehicles. However, their therapeutic application is limited by lack of efficient cargo loading strategies, a need to improve EV tissue-targeting capabilities and a requirement to improve escape from the endolysosomal system. These challenges can be effectively addressed by modifying EVs with peptides which confer specific advantageous properties, thus enhancing their therapeutic potential. Here we provide an overview of the applications of peptide technology with respect to EV therapeutics. We focus on the utility of EV-modifying peptides for the purposes of promoting cargo loading, tissue-targeting and endosomal escape, leading to enhanced delivery of the EV cargo to desired cells/tissues and subcellular target locations. Both endogenous and exogenous methods for modifying EVs with peptides are considered.

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.

A Shortcut to the Synthesis of Peptide Thioesters.

Peptide thioesters serve as fundamental building blocks for the synthesis of proteins and cyclic peptides. Classically, methods to synthesize thioesters have been based on acid-labile amino-protecting groups for which final side-chain deprotection required the use of hazardous hydrogen fluoride (HF). Alternative protection schemes based on base-labile amino-protecting groups have become preferred methods but are not suitable due to the lability of thioester bonds toward bases. In this method, we employ a trifluoracetic acid/trimethylsilyl bromide (TFA/TMSBr) protocol using a hydroxymethyl resin obviating the need for HF. TFA/TMSBr is volatile enough to be easily removed yet less hazardous than HF, making it more practical for general peptide chemists. We describe optimized cleavage procedures and appropriate protecting group schemes and discuss in situ neutralization protocols. The method is relatively simple, straightforward, and easily scalable, allowing the facile preparation of alkyl and aryl thioesters.

Synthesis of Amide Backbone-Modified Peptides.

Solubility is a key property of peptides and of central importance to the success of solid-phase peptide synthesis and subsequent peptide purification and handling. Substitution of the backbone amide bond can dramatically increase peptide solubility. Backbone amide bond protection works by preventing the formation of interchain association and can be used both to synthesize aggregation-prone peptide sequences on solid phase and to improve solubility of a peptide post synthesis. Improving peptide solubility by judicial use of backbone protection is of growing importance, particularly for chemical protein synthesis by chemical ligation.

Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP.

The ubiquitylation of NF-κB essential modulator (NEMO) is part of the intracellular immune signalling pathway. Monoubiquitylated NEMO is required for exploring the mechanism of NEMO linear ubiquitylation by LUBAC (linear ubiquitin chain assembly complex), but is not accessible by biological techniques. Here we perform the chemical ubiquitylation of NEMO using a ligation auxiliary, which only requires a two-step synthesis, and is easily installed onto the lysine side-chain. Chemical ligation occurs directly on the lysine ε amine and remains efficient below pH 7. We show that ubiquitylated NEMO has similar affinity to linear diubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesised NEMOCoZi-Ub is accepted as a substrate for linear extension by the (RING-Between-RING) RBR domain of HOIL-1-interacting protein (HOIP) alone. Our results indicate that NEMO linear ubiquitylation consists of two-steps, an initial priming event and a separate extension step requiring different LUBAC components.

Fmoc-based synthesis of peptide thioacids for azide ligations via 2-cyanoethyl thioesters.

Rapid and efficient preparation of peptide thioacids from 2-cyanoethyl peptide thioesters has been accomplished. S-2-Cyanoethyl peptide thioesters were obtained cleanly without the need for purification from resin-bound tert-butyl peptide thioesters using 3-mercaptopropionitrile as a nucleophile. Elimination of the 2-cyanoethyl group proceeded rapidly (t(1/2) < 8 min) under mild conditions and furnished peptide thioacids up to the size of a 16-mer. Peptide thioacids could be isolated or formed in situ and reacted smoothly with electron-deficient azides yielding an amide as the ligation product.

Fmoc-based synthesis of peptide thioesters for native chemical ligation employing a tert-butyl thiol linker.

tert-Butyl thioesters display an astonishing stability toward secondary amines in basic milieu, in contrast to other alkyl and aryl thioesters. Exploiting this enhanced stability, peptide thioesters were synthesized in a direct manner, applying a tert-butyl thiol linker for Fmoc-based solid-phase peptide synthesis.