Solid-phase Chemistry Research Articles

A Universal Support for the Solid-Phase Synthesis of Peptidyl-tRNA Mimics.

Hydrolysis-resistant RNA-peptide conjugates that mimic peptidyl-tRNAs are often required for structural and functional studies of protein synthesis at the ribosome. These conjugates can be synthesized by solid-phase chemical synthesis, which allows maximum flexibility in both the peptide and RNA sequence. The commonly used strategy is based on (3'-N-aminoacyl)-3'-amino-3'-deoxyadenosine solid supports, which already contain the first C-terminal amino acid of the target peptidyl chain. This is a limitation in the sense that different individual supports must be synthesized for different C-terminal amino acids. In this study, we demonstrate a solution to this problem by introducing a novel universal support. The key is a free ribose 3'-NH2 group that can be coupled to any amino acid. This is made possible by a photocleavable ether moiety that links the ribose 2'-O to the support, thus avoiding the typical O-to-N migration that occurs when using 2'-O-acyl linked solid supports. Once assembled, the conjugate is readily cleaved by UV irradiation. The structural integrity of the obtained peptidyl-RNA conjugates was verified by mass spectrometry analysis. In conclusion, the new photocleavable solid support makes the synthesis of 3'-peptidyl tRNA mimics of different peptidyl chains significantly more efficient compared to the commonly used approaches.

Progressive cancer targeting by programmable aptamer-tethered nanostructures.

Scientific research in recent decades has affirmed an increase in cancer incidence as a cause of death globally. Cancer can be considered a plurality of various diseases rather than a single disease, which can be a multifaceted problem. Hence, cancer therapy techniques acquired more accelerated and urgent approvals compared to other therapeutic approaches. Radiotherapy, chemotherapy, immunotherapy, and surgery have been widely adopted as routine cancer treatment strategies to suppress disease progression and metastasis. These therapeutic approaches have lengthened the longevity of countless cancer patients. Nonetheless, some inherent limitations have restricted their application, including insignificant therapeutic efficacy, toxicity, negligible targeting, non-specific distribution, and multidrug resistance. The development of therapeutic oligomer nanoconstructs with the advantages of chemical solid-phase synthesis, programmable design, and precise adjustment is crucial for advancing smart targeted drug nanocarriers. This review focuses on the significance of the different aptamer-assembled nanoconstructs as multifunctional nucleic acid oligomeric nanoskeletons in efficient drug delivery. We discuss recent advancements in the design and utilization of aptamer-tethered nanostructures to enhance the efficacy of cancer treatment. Valuably, this comprehensive review highlights self-assembled aptamers as the exceptionally intelligent nano-biomaterials for targeted drug delivery based on their superior stability, high specificity, excellent recoverability, inherent biocompatibility, and versatile functions.

A novel antimicrobial peptide MP-4: Insights into its antimicrobial properties and intestinal regulation on E. coli-infected mice

The quest for novel, potent antimicrobial agents with low resistance potential poses a significant challenge for the advancement of the food and medical sectors. This study aimed to elucidate the antibacterial potency of antimicrobial peptides derived from koumiss in a murine model. Leveraging an antimicrobial peptide database, six peptides (MP-1 to MP-6) were meticulously predicted and screened for their antibacterial properties. These peptides were subsequently synthesized using chemical solid-phase methods and their antibacterial activities were rigorously validated. Remarkably, among the six peptides, MP-4 demonstrated a profound antibacterial effect against E. coli, achieving rapid bacterial eradication within 240 min. Flow cytometry analysis further corroborated its significant bactericidal activity. In vivo experiments conducted on mice infected with E. coli revealed that oral administration of MP-4 significantly ameliorated symptoms such as lethargy, anorexia, and weight loss. Additionally, it effectively reduced the colonic E. coli burden, attenuated inflammatory responses, and favorably modulated the intestinal microbiota composition. This study not only validates the robust antibacterial activity of the koumiss-derived antimicrobial peptide MP-4, but also underscores its potential therapeutic application in mitigating E. coli infections and promoting intestinal health.

Chemical Synthesis and Structure-Activity Relationship Studies of the Coagulation Factor Xa Inhibitor Tick Anticoagulant Peptide from the Hematophagous Parasite Ornithodoros moubata.

Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure-activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen-Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles.

Synthesis of Dual-Responsive Amphiphilic Glycomacromolecules: Controlled Release of Glycan Ligands via pH and UV Stimuli.

This work presents a versatile strategy for the synthesis of dual stimuli-responsive amphiphilic glycomacromolecules with tailored release properties. Amphiphilic precision glycomacromolecules (APGs) derived from tailor-made building blocks using solid phase polymer synthesis form glycofunctionalized micelles, a versatile class of materials with applications in drug delivery, as antiinfection agents as well as simple cell mimetics. In this work, this concept is extended by integrating cleavable building blocks into APGs now allowing stimuli-responsive release of glycan ligands or destruction of the micelles. This study incorporates a newly designed acid-labile building block, 4-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-1,3-dioxolan-2-yl)benzoic acid (DBA), suitable also for other types of solid phase or amide chemistry, and an established UV-cleavable 2-nitrobenzyl linker (PL). The results demonstrate that both linkers can be cleaved independently and thus allow dual stimuli-responsive release from the APG micelles. By choosing the APG design e.g., placing the cleavable linkers between glycomacromolecular blocks presenting different types of carbohydrates, they can tune APG and micellar stability as well as the interaction and cluster formation with a carbohydrate-recognizing lectin. Such dual-responsive glycofunctionalized micelles have wide potential for use in drug delivery applications or for the development as anti-adhesion agents in antiviral and antibacterial treatments.

Characterization of natural organic matter in Wyoming-type bentonites irradiated at varied moisture levels

Wyoming-type bentonite clay (MX-80; Wyoming, USA) will be used in a deep geological repository (DGR) for the long-term storage of used nuclear fuel in Canada. The natural organic matter (NOM) found in bentonite may serve as a microbial nutrient source and potentially compromise the performance of the used fuel containers. Previous investigations indicate that NOM is present in low concentrations in MX-80 and has undergone extensive diagenetic alteration, though limited knowledge is available regarding NOM chemistry under simulated DGR conditions. Of particular concern is the possibility for gamma-radiation to alter NOM dissolution and reactivity due to the presence of reactive species generated by water radiolysis in Wyoming-type bentonites. In this study, NOM chemistry was investigated using complementary molecular-level techniques following exposure to a total gamma-radiation dose of 100 kGy (1.08 kGy/h for 93 h) at varied moisture levels (20%, 40%, 60% and 80%) and room temperature. Treated samples exhibited relatively low total organic carbon concentrations (0.074–0.232%), with no evidence of any major changes in total, organic, and inorganic carbon concentrations. Solid-state 13C NMR spectroscopy detected no changes in solid-phase NOM chemistry after irradiation. Solubilization of NOM increased significantly with radiation exposure at 80% moisture, suggesting higher levels of water saturation may enhance dissolved organic matter (DOM) production. Solution-state 1H nuclear magnetic resonance (NMR), and UV–Vis analyses did not identify any significant differences in DOM composition. More sensitive, targeted compound analysis revealed significant decreases in total n-alkanol concentration at lower moisture content levels (20% and 40%). Several individual compound concentrations also differed significantly at the nanogram-level, including n-octacosanol and several n-alkanoic acids at elevated moisture levels (60% and 80%). These findings suggest the majority of NOM in MX-80 remains chemically stable under the anticipated initial conditions of the proposed DGR.

Experimental investigations of diacetylene ice photochemistry in Titan’s atmospheric conditions

Context. A large fraction of the organic species produced photochemically in the atmosphere of Titan can condense to form ice particles in the stratosphere and in the troposphere. According to various studies, diacetylene (C4H2) condenses below 100 km where it can be exposed to ultraviolet radiation. Aims. We studied experimentally the photochemistry of diacetylene ice (C4H2) to evaluate its potential role in the lower altitude photochemistry of Titan’s atmospheric ices. Methods. C4H2 ice films were irradiated with near-ultraviolet (near-UV) photons (λ > 300 nm) with different UV sources to assess the impact of the wavelengths of photons on the photochemistry of C4H2. The evolution of the ice’s composition was monitored using spectroscopic techniques. Results. Our results reveal that diacetylene ice is reactive through singlet-triplet absorption, similar to the photochemistry of other organic ices of Titan (such as dicyanoacetylene C4N2 ice) that we investigated previously. Several chemical processes occurred during the photolysis: the hydrogenation of C4H2 to form other C4 hydrocarbons (vinylacetylene C4H4 to butane C4H10); the formation of larger and highly polymerizable hydrocarbons, such as triacetylene (C6H2); and the formation of an organic polymer that is stable at room temperature. Conclusions. The nondetection of diacetylene ice in Titan’s atmosphere or surface could be rationalized based on our experimental results that C4H2 is photochemically highly reactive in the solid phase when exposed to near-UV radiation that reaches Titan’s lower altitudes and surface. C4H2 may be one of the key molecules promoting the chemistry in the ices and aerosols of Titan’s haze layers, especially in the case of co-condensation with other organic volatiles, with which it could initiate more complex solid-phase chemistry.

Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR, or DNA cloning. The oligonucleotide design, however, is a challenge to nonexperts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the rational design of oligonucleotides, calculated with kinetic parameters for enhanced in vitro transcription (ROCKET). The Python tool uses thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.

Solid Phase Peptide Carrier Conjugation.

Conjugation to carrier proteins is necessary for peptides to be able to induce antibody formation when injected into animals together with a suitable adjuvant. This is usually performed by conjugation in solution followed by mixing with the adjuvant. Alternatively, the carrier may be adsorbed onto a solid support followed by activation and conjugation with the peptide by solid-phase chemistry. Different reagents can be used for conjugation through peptide functional groups (-SH, -NH2, -COOH), and various carrier proteins may be used depending on the peptides and the intended use of the antibodies. The solid phase may be an ion exchange matrix, from which the conjugate can subsequently be eluted and mixed with adjuvant. Alternatively, the adjuvant aluminum hydroxide may be used as the solid-phase matrix, whereupon the carrier is immobilized and conjugated with peptide. The resulting adjuvant-carrier-peptide complexes may then be used directly for immunization.

SLAM (CD150) receptor homologous peptides block the peste des petits ruminants virus entry into B95a cells.

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC50 ) value of each peptide was greater than 1000 μg mL-1 . The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 μg mL-1 concentration showed a reduction of more than log10 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log10 4.8 to log10 2.5 and log10 3.1 respectively. The concentration of SLAM homologous peptide (25 μg mL-1 ) to exert its anti-PPRV effect was much less than its CC50 level (>1000 μg mL-1 ). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.

Access to High-Purity 7m G-cap RNA in Substantial Quantities by a Convenient All-Chemical Solid-Phase Method.

Given the importance of mRNA with 5'-cap, easy access to RNA substrates with different 7m G caps, of high quality and in large quantities is essential to elucidate the roles of RNA and the regulation of underlying processes. In addition to existing synthetic routes to 5'-cap RNA based on enzymatic, chemical or chemo-enzymatic methods, we present here an all-chemical method for synthetic RNA capping. The novelty of this study lies in the fact that the capping reaction is performed on solid-support after automated RNA assembly using commercial 2'-O-propionyloxymethyl ribonucleoside phosphoramidites, which enable final RNA deprotection under mild conditions while preserving both 7m G-cap and RNA integrity. The capping reaction is efficiently carried out between a 5'-phosphoroimidazolide RNA anchored on the support and 7m GDP in DMF in the presence of zinc chloride. Substantial amounts of 7m G-cap RNA (from 1 to 28 nucleotides in length and of any sequence with or without internal methylations) containing various cap structures (7m GpppA, 7m GpppAm , 7m Gpppm6 A, 7m Gpppm6 Am , 7m GpppG, 7m GpppGm ) were obtained with high purity after IEX-HPLC purification. This capping method using solid-phase chemistry is convenient to perform and provides access to valuable RNA substrates as useful research tools to unravel specific issues regarding cap-related processes.

2'-Modified Thymidines with Bioorthogonal Cyclopropene or Sydnone as Building Blocks for Copper-Free Postsynthetic Functionalization of Chemically Synthesized Oligonucleotides.

The development of facile methods for conjugating relevant probes, ligands, or delivery agents onto oligonucleotides (ONs) is highly desirable both for fundamental studies in chemical biology and for improving the pharmacology of ONs in medicinal chemistry. Numerous efforts have been focused on the introduction of bioorthogonal groups onto phosphoramidite building blocks, allowing the controlled chemical synthesis of reactive ONs for postsynthetic modifications. Among these building blocks, alkyne, cyclooctynes, trans-cyclooctene, and norbornene have been proved to be compatible with automated solid-phase chemistry. Herein, we present the development of novel 2'-functionalized nucleoside phosphoramidite monomers comprising bioorthogonal methylcyclopropene or sydnone moieties and their introduction for the first time to ON solid-phase synthesis. Traceless ON postsynthetic modifications with reactive complementary probes were successfully achieved through either inverse electron-demand Diels-Alder (iEDDA) reactions or strain-promoted sydnone-alkyne cycloaddition (SPSAC). These results expand the set of bioorthogonal phosphoramidite building blocks to generate ONs for postsynthetic labeling.

Development of an in vitro aggregation assay for long synthetic polypeptide, amyloidogenic gelsolin fragment AGelD187N 173-242.

Aggregation of the gelsolin protein fragment is the hallmark of the hereditary systemic disease gelsolin amyloidosis. As with other protein misfolding diseases, there is an urgent need for efficient disease-modifying treatment for gelsolin amyloidosis. The formation of amyloids can be reproduced by incubating the disease-causing amyloidogenic 8 kDa polypeptide, 70-residue gelsolin protein fragment, AGelD187N 173-242, in vitro and monitoring the process by thioflavin T dye. However, for screening of potential aggregation inhibitors, the required protein amounts are large and the biotechnological production of amyloidogenic proteins has many challenges. Conversely, use of shorter synthetic regions of AGelD187N 173-242 does not mimic the in vivo aggregation kinetics of full-length fragment as they have different aggregation propensity. In this study, we present an in vitro aggregation assay for full-length AGelD187N 173-242 that has been produced by solid-phase chemical synthesis and after that monomerized carefully. Chemical synthesis allows us to produce high quantities of full-length fragment efficiently and at low cost. We demonstrate that the generated aggregates are fibrillar in nature and how the purity, terminal modification, initial aggregates and seeding affect the aggregation kinetics of a synthetic gelsolin fragment. We also present sufficient quality criteria for the initial monomerized synthetic polypeptide.

Novel Approach to Enriching Glycosylated RNAs: Specific Capture of GlycoRNAs via Solid-Phase Chemistry.

Ribonuclease (RNA) modifications can alter cellular function and lead to differential immune responses by acting as discriminators between RNAs from different phyla. RNA glycosylation has recently been observed at the cell surface, and its dysregulation in disease may change RNA functions. However, determining which RNA substrates can be glycosylated remains to be explored. Here, we develop a solid-phase chemoenzymatic method (SPCgRNA) for targeting glycosylated RNAs, by which glycosylated RNA substrates can be specifically recognized. We found the differential N-glycosylation of small RNAs in hTERT-HPNE and MIA PaCa-2 cancer cells using SPCgRNA. RNA-Seq showed that the changes in glyco-miRNAs prepared from SPCgRNA were consistent with those of traditional methods. The KEGG signaling pathway analysis revealed that differential miRNA glycosylation can affect tumor cell proliferation and survival. Further studies found that NGI-1 significantly inhibited the proliferation, migration, and circulation of MIA PaCa-2 and promoted cell apoptosis. In addition, β-1,4-galactosyltransferase 1 (B4GALT1) not only affected the expression level of glycosylated miRNAs hsa-miR-21-5p but also promoted cell apoptosis and inhibited the cell cycle possibly through the p53 signaling pathway, while B4GALT1 and p53 were also affected following the hsa-miR-21-5p increase. These results suggest that B4GALT1 may catalyze miRNAs glycosylation, which further promotes cancer cell progression.

Practical Synthesis of N-Formylmethionylated Peptidyl-tRNA Mimics.

Hydrolysis-resistant RNA-peptide conjugates that mimic peptidyl-tRNAs are frequently needed for structural and functional studies of protein synthesis in the ribosome. Such conjugates are accessible by chemical solid-phase synthesis, allowing for the utmost flexibility of both the peptide and the RNA sequence. Commonly used protection group strategies, however, have severe limitations with respect to generating the characteristic Nα-formylmethionyl terminus because the formyl group of the conjugate synthesized at the solid support is easily cleaved during the final basic deprotection/release step. In this study, we demonstrate a simple solution to the problem by coupling appropriately activated Nα-formyl methionine to the fully deprotected conjugate. The structural integrity of the obtained Nα-formylmethionyl conjugate─and hence the chemoselectivity of the reaction─were verified by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry sequence analysis. Additionally, we confirmed the applicability of our procedure for structural studies by obtaining two structures of the ribosome in complex with either fMAI-nh-ACCA or fMFI-nh-ACCA in the P site and ACC-PMN in the A site of the bacterial ribosome at 2.65 and 2.60 Å resolution, respectively. In summary, our approach for hydrolysis-resistant Nα-formylated RNA-peptide conjugates is synthetically straightforward and opens up new avenues to explore ribosomal translation with high-precision substrate mimics.

Development of Hemagglutinin-Neuraminidase Homologous Peptides as Novel Promising Therapeutic Agents Against Peste des Petits Ruminants Virus.

The lack of specific antiviral therapy and complications associated with the existing peste des petits ruminants (PPR) vaccines accentuates the search of novel antiviral blocking agents in order to curtail the PPR infection at initial level. The synthetic hemagglutinin-neuraminidase (HN) homologous peptides may compete with the natural HN protein of PPR virus for binding to signaling lymphocytic activation molecule (SLAM) receptor, consequently, may disrupt peste des petits ruminants virus (PPRV) at entry level. Therefore, insilico analysis, synthesis, purification and subsequent characterization of HN homologous peptides were conducted in this study. The HN homologous peptides were synthesized by means of solid phase chemistry and were purified by reversed-phase-high performance liquid chromatography. The mass as well as sequence of HN homologous peptides were assessed by mass spectroscopy while its secondary structure was elucidated by circular dichroism spectroscopy. The binding (interaction) efficacy of HN homologous peptides with PPRV antibodies was assessed via indirect enzyme linked immunosorbent assay, visual detection test (red wine to purple), bathochromic shift under UV-Vis spectrophotometry and lateral flow immunochromatographic strip test. The antiviral properties and cytotoxicity of these peptides were also assessed in B95a cell line with changes in cytopathic effect and titer of PPRV (Sungri/96). The presence of green fluorescein isothiocyanate over the B95a cell surface pointed towards the binding of HN homologous peptides with surface SLAM receptor. Moreover, the intact beta sheet configuration in water and lower cytotoxicity [cytotoxic concentration 50 (CC50) > 1000µg/ml] of these peptides signifies its in vivo use. Among HN homologous peptides, the binding efficacy and antiviral properties of pep A was relatively high in comparison to pep B and Pep ppr peptides. The prerequisite concentration of HN homologous peptides (pep A = 12.5µg/ml; pep B = 25µg/ml; pep ppr = 25µg/ml) to exemplify its antiviral effect was much lower than its CC50 level. Hence, this study signifies the therapeutic potential of synthetic HN homologous peptides.

Characterization of a New Hypotensive Peptide from the Venom of Snake Bothrops jararaca (Bj)

Background: Snake venom has become a key source of many bioactive peptides, enzymes, and toxins associated with blood coagulation and neuronal toxicity. In the past, a number of bradykinin potentiating peptides have been isolated from snake venom that display hypotensive activity due to their inhibitory action towards Angiotensin-Converting Enzyme (ACE). Significant interest has developed to isolate, characterize, and subsequently design peptide analogs as potent ACE-inhibitors which may find therapeutic applications for the treatment of hypertension and associated diseases. Aim: The aim of this study is to search for new bioactive peptide/s in the venom of the snake Bothrops Jararaca (Bj). Objective: The objective is to isolate and characterize new hypotensive peptides from BJ venom. Methods: We examined the venom of Bj which is known to host a range of bioactive peptides. We have isolated a new peptide (BJ-1) which displayed in vitro potent hypotensive activity. The peptide was purified via Sephadex G25 column chromatography and RP-HPLC. It was characterized by mass spectrometry, amino acid analysis, N-terminal sequencing, and chemical synthesis. Result: The peptide was identified as an octa-decapeptide with amino acid sequence as DCPSDWSSYEGHCYKPFS where the two Cys residues are likely present in free state, although they can form an internal S-S bond upon oxidation. It was fully confirmed by comparing with synthetic peptide prepared by solid phase chemistry. Both have the same molecular mass (2,108 Da) and identical bioactivity. Furthermore, we rationalize that BJ-1 may be derived from precursor protein “Coagulation factor IX/factor X binding protein (CF-IX/X-BP)” by proteolytic cleavage at the Nterminus of its B-chain within the sequence KPFS18↓E19PKN. This cleavage site contains the recognition motif of enzyme PCSK8 (Proprotein Convertase Subtilisin Kexin8) also known as Subtilisin Kexin Isozyme 1 (SKI-1) or Site 1 Protease (S1P). Despite this observation, using a synthetic peptide encompassing the proposed cleavage site and recombinant PCSK8 enzyme, we found that the enzyme responsible for generation of BJ-1 is not PCSK8. Further studies will be needed to identify the associated enzyme and fully characterize the pharmacological and biological properties of the peptide. Conclusion: Our study revealed the presence of a novel hypotensive octa-decapeptide in the venom of the snake Bothrops jararaca. It is likely derived from the A-chain of protein CF-IX/X-BP via proteolytic cleavage at the N-terminus by a protease yet to be characterized.

O1-conotoxin Tx6.7 cloned from the genomic DNA of Conus textile that inhibits calcium currents.

Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDWCPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 μM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.

(Digital Presentation) Modern State and Prospects of Electrochemical CO2 Conversion in Molten Salts

Over the last century, anthropogenic activity has led to a significant increase in the concentration of carbon dioxide in the Earth's atmosphere. Today, the annual increase in CO2 is 3200-3600 million tons. Carbon dioxide together with other greenhouse gases (water vapor H2O, methane CH4, nitrous oxide N2O, sulfur hexafluoride SF6, etc.) causes global warming, which leads to serious environmental degradation - increasing the acidity of sea water, melting glaciers, strengthening the water cycle, which in some regions leads to heavy rainfall and floods, and in others - to extreme droughts. Therefore, the effective carbon dioxide utilization is becoming an urgent scientific and environmental task.One of the ways of carbon dioxide utilization is the electrochemical method. It consists in the electrochemical reduction of CO2 at the cathode. Depending on the conditions of electrolysis (composition of the electrolytic bath, temperature, current density, voltage in the bath, electrode materials), the chemical composition of cathode products changes dramatically.Report is a review of the present state of research concerning the electrochemical conversion of carbon dioxide from conducting liquid and solid media of different chemical composition. They include solid, aqueous, non-aqueous (organic and ionic liquids), and molten salt electrolytes. It contains a comparative analysis of the effectiveness of using these electrolytes, as well as products obtained by the electrochemical splitting of CO2 and the prospects for their use. Special emphasis has been made on the electrochemical decomposition of carbon dioxide from molten salts, several variants of decomposition have been considered, the advantages and disadvantages of each variant have been analyzed. An analysis of literature data on this subject as well as research results of the author on the direct electrochemical conversion of CO2 into valuable solid-phase chemicals with added value is presented.A feature of the electrochemical reduction of CO2 dissolved in molten salts, in contrast to aqueous, organic, solid electrolytes and ionic liquids is the possibility of forming a solid carbon phase at the cathode. Knowledge of the laws of electrode processes involving CO2 is necessary to create a scientific basis and control for the processes of electrochemical synthesis of nanosized carbon-containing inorganic compounds - nanotubes, nanofibers, graphene, amorphous carbon, carbides of refractory metals and numerous composites based on them.The voltammetric study of carbon electrowinning mechanism from carbon dioxide under excessive pressure and lithium carbonate in different gas media (air, argon, carbon dioxide) dissolved in chloride melts at temperatures of 550–800 °С was conducted. The characterization of the chemical and phase compositions, morphological and structural features of nanosized carbon phases obtained by electrolysis in the studied melts has been carried out; a correlation between the structure of the products and the synthesis conditions is established.Thermodynamic analysis of possible ways of electroreduction of CO2 and carbonates of alkali and alkaline earth metals in the temperature range 700 - 1000 K showed that the cathode product can be: (1) - solid carbon phase; (2) - carbon monoxide; (3) - carbide phase. At temperatures below 1000 K, the first path will be thermodynamically advantageous.Electrochemical behavior of CO2 in molten salt mixtures NaCl–KCl (0.5: 0.5 mol%); NaCl–KCl–CsCl (0.3: 0.245: 0.455 mol%); NaCl–KCl–NaF (0.423: 0.423: 0.154 mol.%) under gas excess pressure (1-17 atm.) was studied by cyclic voltammetry at different potential scan rates (0.02-10 Vs-1) and reverse potentials at Pt, Au and GC electrodes. It was established that CO2 electroreduction takes place in two stages, the electrode process is controlled by the charge transfer rate and the rate of CO2 diffusion in the melts. The solubility of CO2 in the melts of Na,K|Cl and Na,K|Cl,F was determined and the implementation of Henry's law in the studied pressure range was shown. An ECE (electrochemical–chemical–electrochemical) mechanism of cathodic reaction has been proposed for the cathode discharge of CO2. Oxidation of oxide- and carbonate- anions takes place at the anode, with the release of oxygen.Electrochemical reduction of Li2CO3 in molten equimolar mixture of Na,K|Cl in various gaseous media (air, argon, CO2) was studied by CV. Reduction of Li2CO3 to carbon in air occurs through the stage of preliminary chemical reaction of acid-base type with the formation of two electrochemically active particles of CO2 and {LixCO3}2-x at potentials -0.8 and -1.7 V, respectively, relative to Pb|PbCl2 reference electrode . Both processes are irreversible, and the limiting stage of reduction {LixCO3}2-x is the diffusion of the depolarizer to the electrode surface. In the atmosphere of argon and CO2, only {LixCO3}2-x participates in the electroreduction process.Promising areas of application of electrolytic carbon are presented.

Rapid identification of short oligonucleotide impurities using lithium adduct consolidated MALDI-TOF mass spectrometry

Nucleic acids are an increasingly popular platform for the development of pharmaceuticals to treat and prevent multiple diseases including those where traditional small molecule and protein-based drug development efforts have failed. Short oligonucleotide therapeutics, which consist of antisense oligonucleotides (ASOs) and short interfering ribonucleic acids (siRNAs), are prepared by solid phase chemical synthesis, which can generate various impuriies that have the potential to lower drug safety and efficacy. Drug substance-related impurities can be especially difficult to identify and characterize. To address this short coming, here we apply lithium adduct consolidation with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to afford a simplified, quick, and facile method for identification of a known drug substance impurity, isobutyryl (iBu) groups arising from incomplete deprotection during solid phase synthesis. We further employ high-resolution nuclear magnetic resonance (NMR) spectroscopy to confirm assessment of the iBu impurity. This lithium adduction consolidation method should find general applicability for routine quality assessment of synthetic oligonucleotides in both academic and industrial laboratories.