Growing Peptide Chain Research Articles

Complementary Strategies for Installation of Thioimidates into Peptide Backbones.

Thioimidates are a precursor and synthetic branch point to access either thioamide or amidine isosteres of the native amide (peptide bond). Previous syntheses of thioimidate-containing peptides were prone to side reactivity and required slow, cumbersome steps that were difficult to monitor. We describe a more efficient approach to directly couple thioimidates onto the growing peptide chain. This work also outlines optimal conditions for thioimidate formation on solid support and identifies potential off-target sites for alkylation that impact the choice of protecting group.

Access to Diverse Glutamic Acid Dendrons and a Janus Peptide Dendrimer Using an Atypical Solid Phase Synthesis.

The negligible cytotoxicity of anion surface-linked dendrons makes glutamic acid-based dendrons a potential candidate for materials and biological applications. Despite the inherent drawbacks of the conventional solution phase synthesis of glutamic acid-based dendrons, there have been no advancements in these protocols. Herein, we demonstrate the first-ever convergent solid phase synthesis of dendrons, up to fourth generation, having glutamic acid branching points produced by preactivation of dicarboxylic acid groups with N-hydroxysuccinimide and simultaneous coupling with amine groups of two growing peptide chains, with excellent yields (30-70%). In addition to the general advantages, such as the easy workup, a final single purification step, and an overall short synthesis duration, the convergent solid phase synthesis allowed us to chemically synthesize glutamic acid branching-based dendrons that cannot be accessed by standard divergent solid phase synthesis. This method has also been validated for its application in synthesizing hard-to-achieve Janus peptide dendrimers in a single stretch on a solid support. Our work corroborates the efficacy of controlled -COOH activation to accomplish an atypical solid phase synthesis of diverse glutamic acid dendrons in a convergent fashion. This is the first example of a Janus peptide dendrimer being synthesized on a solid support, utilizing both convergent and divergent approaches simultaneously.

GNN Codon Adjacency Tunes Protein Translation.

The central dogma treats the ribosome as a molecular machine that reads one mRNA codon at a time as it adds each amino acid to its growing peptide chain. However, this and previous studies suggest that ribosomes actually perceive pairs of adjacent codons as they take three-nucleotide steps along the mRNA. We examined GNN codons, which we find are surprisingly overrepresented in eukaryote protein-coding open reading frames (ORFs), especially immediately after NNU codons. Ribosome profiling experiments in yeast revealed that ribosomes with NNU at their aminoacyl (A) site have particularly elevated densities when NNU is immediately followed (3') by a GNN codon, indicating slower mRNA threading of the NNU codon from the ribosome's A to peptidyl (P) sites. Moreover, if the assessment was limited to ribosomes that have only recently arrived at the next codon, by examining 21-nucleotide ribosome footprints (21-nt RFPs), elevated densities were observed for multiple codon classes when followed by GNN. This striking translation slowdown at adjacent 5'-NNN GNN codon pairs is likely mediated, in part, by the ribosome's CAR surface, which acts as an extension of the A-site tRNA anticodon during ribosome translocation and interacts through hydrogen bonding and pi stacking with the GNN codon. The functional consequences of 5'-NNN GNN codon adjacency are expected to influence the evolution of protein coding sequences.

Inverse Peptide Synthesis Using Transient Protected Amino Acids.

Peptide therapeutics have experienced a rapid resurgence over the past three decades. While a few peptide drugs are biologically produced, most are manufactured via chemical synthesis. The cycle of prior protection of the amino group of an α-amino acid, activation of its carboxyl group, aminolysis with the free amino group of a growing peptide chain, and deprotection of the N-terminus constitutes the principle of conventional C → N peptide chemical synthesis. The mandatory use of the Nα-protecting group invokes two additional operations for incorporating each amino acid, resulting in poor step- and atom-economy. The burgeoning demand in the peptide therapeutic market necessitates cost-effective and environmentally friendly peptide manufacturing strategies. Inverse peptide chemical synthesis using unprotected amino acids has been proposed as an ideal and appealing strategy. However, it has remained unsuccessful for over 60 years due to severe racemization/epimerization during N → C peptide chain elongation. Herein, this challenge has been successfully addressed by ynamide coupling reagent employing a transient protection strategy. The activation, transient protection, aminolysis, and in situ deprotection were performed in one pot, thus offering a practical peptide chemical synthesis strategy formally using unprotected amino acids as the starting material. Its robustness was exemplified by syntheses of peptide active pharmaceutical ingredients. It is also amenable to fragment condensation and inverse solid-phase peptide synthesis. The compatibility to green solvents further enhances its application potential in large-scale peptide production. This study offered a cost-effective, operational convenient, and environmentally benign approach to peptides.

Molecular Coping Mechanisms: Reprogramming tRNAs To Regulate Codon-Biased Translation of Stress Response Proteins.

As part of the classic central dogma of molecular biology, transfer RNAs (tRNAs) are integral to protein translation as the adaptor molecules that link the genetic code in messenger RNA (mRNA) to the amino acids in the growing peptide chain. tRNA function is complicated by the existence of 61 codons to specify 20 amino acids, with most amino acids coded by two or more synonymous codons. Further, there are often fewer tRNAs with unique anticodons than there are synonymous codons for an amino acid, with a single anticodon able to decode several codons by "wobbling" of the base pairs arising between the third base of the codon and the first position on the anticodon. The complications introduced by synonymous codons and wobble base pairing began to resolve in the 1960s with the discovery of dozens of chemical modifications of the ribonucleotides in tRNA, which, by analogy to the epigenome, are now collectively referred to as the epitranscriptome for not changing the genetic code inherent to all RNA sequences. tRNA modifications were found to stabilize codon-anticodon interactions, prevent misinitiation of translation, and promote translational fidelity, among other functions, with modification deficiencies causing pathological phenotypes. This led to hypotheses that modification-dependent tRNA decoding efficiencies might play regulatory roles in cells. However, it was only with the advent of systems biology and convergent "omic" technologies that the higher level function of synonymous codons and tRNA modifications began to emerge.Here, we describe our laboratories' discovery of tRNA reprogramming and codon-biased translation as a mechanism linking tRNA modifications and synonymous codon usage to regulation of gene expression at the level of translation. Taking a historical approach, we recount how we discovered that the 8-10 modifications in each tRNA molecule undergo unique reprogramming in response to cellular stresses to promote translation of mRNA transcripts with unique codon usage patterns. These modification tunable transcripts (MoTTs) are enriched with specific codons that are differentially decoded by modified tRNAs and that fall into functional families of genes encoding proteins necessary to survive the specific stress. By developing and applying systems-level technologies, we showed that cells lacking specific tRNA modifications are sensitized to certain cellular stresses by mistranslation of proteins, disruption of mitochondrial function, and failure to translate critical stress response proteins. In essence, tRNA reprogramming serves as a cellular coping strategy, enabling rapid translation of proteins required for stress-specific cell response programs. Notably, this phenomenon has now been characterized in all organisms from viruses to humans and in response to all types of environmental changes. We also elaborate on recent findings that cancer cells hijack this mechanism to promote their own growth, metastasis, and chemotherapeutic resistance. We close by discussing how understanding of codon-biased translation in various systems can be exploited to develop new therapeutics and biomanufacturing processes.

Efficient Solution Phase Synthesis of PPII Helix Mimicking Ena/VASP EVH1 Inhibitors from Proline‐Derived Modules (ProMs)

AbstractIn the search for efficient inhibitors for the enabled/vasodilator‐stimulated phosphoprotein homology 1 (EVH1) domain to reduce cell motility in metastatic cancer, we previously developed a toolkit of proline‐derived modules (ProMs), which mimic the PPII helix found in the natural −FPPPP− binding motif of EVH1. In this work, we describe the modular assembly of these ProM‐based pentapeptidic EVH1 ligands through liquid phase peptide synthesis. We initially used pentafluorophenyl (Pfp) active esters for amide bond formation and built up the growing peptide chain from the C‐ to the N‐terminus. Switching to HATU/DIPEA coupling conditions and changing the directionality of the synthesis from the N‐ to the C‐terminus afforded the target ligands with improved overall yields and purity. Employing a Fmoc‐protected (instead of the N‐acetylated) phenylalanine derivative as N‐terminal building block significantly reduced epimerization. In contrast to the originally used solid phase peptide synthesis (SPPS), the developed solution phase method allowed for a facile alteration of the C‐terminal ProM unit and the production of various pentapeptidic ligands in an efficient fashion even on a multigram scale.

Glycine Peptide Chain Formation in the Gas Phase via Unimolecular Reactions

Peptide chain formation from amino acids such as glycine is a key step in the emergence of life. Unlike their synthesis by living systems, how peptide chains grow under abiotic conditions is an open question given the variety of organic compounds discovered in various astrophysical environments, comets and meteorites. We propose a new abiotic route in the presence of protonated molecular dimers of glycine in a cold gaseous atmosphere without further need for a solid catalytic substrate. The results provide evidence for the preferential formation of mixed protonated dimers of glycine consisting of a dipeptide and a glycine molecule instead of pure protonated glycine dimers. Additional measurements mimicking a cosmic-ray impact in terms of internal excitation show that a single gas-phase collision induces polymerization via dehydration in both the mixed and pure dimer ions. Peptide chain growth is thus demonstrated to occur via a unimolecular gas-phase reaction in an excited cluster ion.

Protease-catalyzed synthesis of α-poly-L-Lysine and amphiphilic poly(L-lysine-co-L-phenylalanine) in a neat non-toxic organic solvent.

Subtilisin Carlsberg (alkaline protease from Bacillus licheniformis) catalyzes the syntheses of high molecular weights (ca. 20 KDa) cationic α-poly-L-lysine and amphiphilic poly(α-L-lysine-co-L-phenylalanine) in neat organic solvent. The synthesis is conducted in liquid 1,1,1,2-tetrafluoroethane solvent, which is a hydrophobic non-toxic gas that does not deplete the ozone layer and approved for pharmaceutical applications. Solubility of substrates and adequate protease activity in this system with low water environment limits the reaction of hydrolysis of the growing peptide chains. The pressurization of this organic compressed fluid to liquid has low-pressure requirements (25bar, 40 ºC), and its complete evaporation at atmospheric pressure after completing the reaction ensures solvent-free residues in products. The resulting polypeptides present null cytotoxicity according to MTT and NR analyses, as well as Calcein/EthD-1 assay in human cells.

Incorporation of pseudoproline monomer (Fmoc-Thr[ψMe,Mepro]–OH) facilitates efficient solid-phase synthesis of difficult peptides

Solid-Phase Peptide Synthesis (SPPS) is a convenient and efficient method by which to prepare peptides. However, the sequences of compounds classified as “difficult” show aggregation of growing peptide chains and their synthesis is extremely challenging by stepwise SPPS. In this context, the concourse of pseudoproline derivatives helps to break the aggregation and facilitates the synthesis. Although pseudoproline moieties are traditionally incorporated as dipeptides, the possibility of using only the monomer adds flexibility to the synthesis. Herein, the introduction of the most hindered β-branched Thr[ψMe,Mepro] derivative is studied in terms of how difficult is its acylation is by the following amino acid and the impact of its use on the final purity of two well-known ”difficult peptides”.

Identification and analysis of putative tRNA genes in baculovirus genomes

Transfer RNAs (tRNAs) genes are both coded for and arranged along some viral genomes representing the entire virosphere and seem to play different biological functions during infection, other than transferring the correct amino acid to a growing peptide chain. Baculovirus genome description and annotation has focused mostly on protein-coding genes, microRNA, and homologous regions. Here we carried out a large-scale in silico search for putative tRNA genes in baculovirus genomes. Ninety-six of 257 baculovirus genomes analyzed was found to contain at least one putative tRNA gene. We found great diversity in primary and secondary structure, in location within the genome, in intron presence and size, and in anti-codon identity. In some cases, genes of tRNA-containing genomes were found to have a bias for the codons specified by the tRNAs present in such genomes. Moreover, analysis revealed that most of the putative tRNA genes possessed conserved motifs for tRNA type 2 promoters, including the A-box and B-box motifs with few mismatches from the eukaryotic canonical motifs. From publicly available small RNA deep sequencing datasets of baculovirus-infected insect cells, we found evidence that a putative Autographa californica multiple nucleopolyhedrovirus Gln-tRNA gene was transcribed and modified with the addition of the non-templated 3’-CCA tail found at the end of all tRNAs. Further research is needed to determine the expression and functionality of these viral tRNAs.

Liquid-Phase Peptide Synthesis (LPPS): A Third Wave for the Preparation of Peptides.

Since the last century, peptides have gained wide acceptance as drugs, with almost 100 already in the market and a large number in the pipeline. In this context, peptide synthesis has grown massively as a stringent field for pharmaceuticals around the globe. Three methodologies, namely, classical solution peptide synthesis (CSPS), solid-phase peptide synthesis (SPPS), and liquid-phase peptide synthesis (LPPS), have made significant contributions to the field. This review provides a comprehensive and integrated vision of LPPS as the third wave for peptide synthesis. LPPS combines the advantages of CSPS and SPPS, where peptide elongation is carried out in solution and the growing peptide chain is supported on a soluble tag, which confers characteristic properties. LPPS protocols allow the large-scale production of peptides and reduce the use of excess reagents and solvents, thus meeting the principles of green chemistry. In this review, tags associated with LPPS are broadly discussed under the following headings: polydisperse polyethylene glycol (PEG), membrane-enhanced peptide synthesis (MEPS), fluorous technology, ionic liquids (ILs), PolyCarbon, hydrophobic polymers, and group-assisted purification (GAP). It also highlights the signature accomplishments of LPPS tags and the limitations of the same.

Suppression of Simultaneous Fmoc-His(Trt)-OH Racemization and Nα-DIC-Endcapping in Solid-Phase Peptide Synthesis through Design of Experiments and Its Implication for an Amino Acid Activation Strategy in Peptide Synthesis

Coupling Fmoc-His(Trt)-OH in solid-phase peptide synthesis is frequently accompanied by significant racemization. Histidine is among the most susceptible amino acid residues inclined to racemize in peptide syntheses. In this study, the His racemized impurity could not be effectively purged by the applied chromatographic purification. Consequently, a Taguchi design of experiment (DOE) was first applied to screen the critical process parameters affecting the histidine racemization. Optimization of the DOE is subsequently performed to search for the optimum. The derived DOE models reveal that the conditions of Fmoc-His(Trt)-OH carboxylate pre-activation prior to its coupling to growing peptide chains are critical for the subject histidine racemization. Intensive Fmoc-His(Trt)-OH pre-activation stimulates this side reaction. On the other hand, without the amino acid pre-activation, by adopting the in situ Fmoc-Xaa-OH activation, another side reaction, that is, peptide Nα endcapping by N,N-diisopropylcarbodiimide, is boosted. A conflicting relationship between histidine racemization and peptide Nα endcapping has been detected through the DOE investigation. Significant models are established for the histidine racemization and peptide Nα endcapping, and reconciliation to balance these two side reactions is accomplished on this basis.

A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S

Significance Transfer RNA (tRNA) transfer appropriately activated amino acids to the end of the growing peptide chain, playing significant roles in primary metabolism. Importantly, tRNA have also been shown to function outside of translation. In this report, we present the characterization of a new class of aminoacyl-tRNA (aa-tRNA)–dependent enzyme, BlsK, from Streptomyces griseochromogenes . BlsK catalyzes an aa-tRNA–dependent leucyl transfer reaction in blasticidin S biosynthesis. BlsK contains a [3Fe-4S] cluster that is necessary for its function and unprecedented in previously identified tRNA-dependent transferases. Because BlsK does not show similarity to any known functional protein, it may represent a type of aa-tRNA–dependent enzyme. As such, this work has broad implications for both tRNA-dependent and iron–sulfur–containing enzymes.

TsRNAsearch: a pipeline for the identification of tRNA and ncRNA fragments from small RNA-sequencing data.

tRNAs were originally considered uni-functional RNA molecules involved in the delivery of amino acids to growing peptide chains on the ribosome. More recently, the liberation of tRNA fragments from tRNAs via specific enzyme cleavage has been characterized. Detection of tRNA fragments in sequencing data is difficult due to tRNA sequence redundancy and the short length of both tRNAs and their fragments. Here, we introduce tsRNAsearch, a Nextflow pipeline for the identification of differentially abundant tRNA fragments and other non-coding RNAs from small RNA-sequencing data. tsRNAsearch is intended for use when comparing two groups of datasets, such as control and treatment groups. tsRNAsearch comparatively searches for tRNAs and ncRNAs with irregular read distribution profiles (a proxy for RNA cleavage) using a combined score made up of four novel methods and a differential expression analysis, and reports the top ranked results in simple PDF and TEXT files. In this study, we used publicly available small RNA-seq data to replicate the identification of tsRNAs from chronic hepatitis-infected liver tissue data. In addition, we applied tsRNAsearch to pancreatic ductal adenocarcinoma (PDAC) and matched healthy pancreatic tissue small RNA-sequencing data. Our results support the identification of miR135b from the original study as a potential biomarker of PDAC and identify other potentially stronger miRNA biomarkers of PDAC. https://github.com/GiantSpaceRobot/tsRNAsearch. Supplementary data are available at Bioinformatics online.

The Role of tRNA Derived Small RNAs in Gene Regulation in Normal Tissues and Cancer

The function of tRNAs is not merely to provide the specific amino acids for a growing peptide chain during protein synthesis. Precursor and mature tRNAs can be enzymatically cleaved into various tRNA derived small RNAs. While there are thousands of different RNA species that can be generated, the function of most are not known. I will provide a brief introduction into this field and describe the mechanism of how at least one specific tsRNA derived from the 3′end of a mature tRNA is involved in regulating ribosome biogenesis (Kim et al. Nature 2017). This 3′tsRNA is upregulated in many tumors. Reducing the tsRNA in patient derived hepatocellular carcinomas using antisense oligonucleotides induced apoptosis and tumor shrinkage in mice. More recently, we have discovered the 3′tsRNAs are aminoacylated suggesting they are generated in the cytoplasm and perhaps on the ribosome. I will provide an update on current approaches we are developing to help us identify, quantify, and define the targets of the myriad of various tsRNAs present in various cell types. This will further allow investigation as to their role in maintaining health and disease states.

Activity, Binding, and Modeling Studies of a Reprogrammed Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket.

The gatekeeping adenylation (A) domain of the non-ribosomal peptide synthetase (NRPS) selectively incorporates specific proteinogenic/non-proteinogenic amino acid into a growing peptide chain. The EntE of the enterobactin NRPS is a discrete aryl acid A-domain with 2,3-dihydroxybenzoic acid (DHB) substrate specificity. Reprogrammed EntE N235G variant possesses an enlarged substrate recognition site, and is capable of accepting non-native aryl acids. Biochemical characterization of this unique substrate recognition site should provide a better understanding of activi-site microenvironments. Here, we synthesized a non-hydrolysable adenylate analogue with 2-aminobenzoic acid (2-ABA), 3-aminobenzoic acid (3-ABA), and 4-aminobenzoic acid (4-ABA) and used them to calculate the apparent inhibition constants (Kiapp.). Dose-response experiments using 3-ABA-sulfamoyladenosine (AMS) provided Kiapp. values of 596 nM for wild-type EntE and 2.4 nM for the N235G variants. These results suggest that 3-amino group of benzoic acid plays an important role in substrate recognition by the N235G variant. These findings would help designing aryl acid substrates with substituents at the 2- and 3-positions.

Opportunities and challenges in the synthesis of thioamidated peptides.

Chemical modifications of peptides hold great promise for modulating their pharmacological properties. In the last few decades amide to thioamide substitution has been widely explored to modulate the conformation, non-covalent interactions, and proteolytic stability of peptides. Despite widespread utilization, there are some potential limitations including epimerization and degradation under basic and acidic conditions, respectively. In this chapter, we present the synthetic method to build thio-precursors, their site-specific incorporation onto a growing peptide chain, and troubleshooting during the elongation of thioamidated peptides. This highly efficient, rapid, and robust method can be used for positional scanning of the thioamide bond.

Biosynthesis of depsipeptides, or Depsi: The peptides with varied generations.

Depsipeptides are compounds that contain both ester bonds and amide bonds. Important natural product depsipeptides include the piscicide antimycin, the K+ ionophores cereulide and valinomycin, the anticancer agent cryptophycin, and the antimicrobial kutzneride. Furthermore, database searches return hundreds of uncharacterized systems likely to produce novel depsipeptides. These compounds are made by specialized nonribosomal peptide synthetases (NRPSs). NRPSs are biosynthetic megaenzymes that use a module architecture and multi-step catalytic cycle to assemble monomer substrates into peptides, or in the case of specialized depsipeptide synthetases, depsipeptides. Two NRPS domains, the condensation domain and the thioesterase domain, catalyze ester bond formation, and ester bonds are introduced into depsipeptides in several different ways. The two most common occur during cyclization, in a reaction between a hydroxy-containing side chain and the C-terminal amino acid residue in a peptide intermediate, and during incorporation into the growing peptide chain of an α-hydroxy acyl moiety, recruited either by direct selection of an α-hydroxy acid substrate or by selection of an α-keto acid substrate that is reduced in situ. In this article, we discuss how and when these esters are introduced during depsipeptide synthesis, survey notable depsipeptide synthetases, and review insight into bacterial depsipeptide synthetases recently gained from structural studies.

Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on the Escherichia coli translation machinery

Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.

The Role of tRNA Derived Small RNAs in Gene Regulation in Normal Tissues and Cancer

The function of tRNAs is not merely to provide the specific amino acids for a growing peptide chain during protein synthesis. Precursor and mature tRNAs can be enzymatically cleaved into various tRNA derived small RNAs. While there are thousands of different RNA species that can be generated, the function of most are not known. I will provide a brief introduction into this field and describe the mechanism of how at least one specific tsRNA derived from the 3′end of a mature tRNA is involved in regulating ribosome biogenesis (Kim et al. Nature 2017). This 3′tsRNA is upregulated in many tumors. Reducing the tsRNA in patient derived hepatocellular carcinomas using antisense oligonucleotides induced apoptosis and tumor shrinkage in mice. This as well as other 3′tsRNAs are being studied as potential targets for cancer therapeutics. I will provide an update on current approaches we are developing to help us identify, quantify, and define the targets of the myriad of various tsRNAs present in various cell types. This will further allow investigation as to their role in maintaining health and disease states.Support or Funding InformationNational Institutes of Health ‐ National Institute of Diabetes and Digestive and Kidney Diseases R01‐DK114483