Biochemical Research Articles

Advancements in transition metal-catalyzed 1,2,3-triazole synthesis via azide–alkyne cycloaddition

Abstract 1,2,3-triazoles have emerged as important structural motifs in chemical biology, attracting increasing focus in recent years because of their wide range of uses. These compounds can be efficiently synthesized using click reactions. Its versatility makes it valuable in drug discovery and materials science. Significant advancements have been made in recent years in the process of making 1,2,3-triazoles, reflecting the growing curiosity and investment in the area of research. The catalytic performance of transition metals Cu, Ir, Rh, Ru, Ni, Pd, Au, Ag, and Zn, which were used as ligands and salts in the azide–alkyne cycloaddition method of 1,2,3-triazole synthesis, has been covered in this review. Cu-complexes and salts were found to be more effective for selective synthesis among all transition metals. Furthermore, it is determined that some azide–alkyne reactions are entirely catalytic in nature and cannot be carried out as such by switching transition metals.

M. I. Ravich-Shcherbo, the founder of the scientific School of biological chemistry in Kursk

The article is devoted to scientific and teaching activities of the Soviet biochemist M. I. Ravich-Shcherbo whose scientific career was largely spent in Kursk. The results of his studies into the development of ways to control malaria, carried out jointly with I. A. Smorodintsev and A. N. Adova during their work at the Tropical Institute of the RSFSR Peopleʼs Commissariat of Public Health (Narkomzdrav) in the 1920s and 1930s, and his research aimed at reducing the incidence of syphilis, conducted at the V. M. Bronner State Institute for Venereal Diseases in the 1930s – 1950s, are analyzed. His contribution to the development of wound healing and radiopaque contrast agents during World War II is also shown. Particular attention is given to the results of the studies into the mechanisms of biosynthesis of antibodies, gamma-globulin metabolism, and the ways to regulate these processes, carried out by Ravich-Shcherbo’s postgraduate and postdoctoral students in the 1950s – 1970s. The article also sheds light on the history of the scientific school of biological chemistry that became established at the Kursk Medical Institute.

Bioorthogonal Janus microparticles for photothermal and chemo‐therapy

AbstractBioorthogonal chemistry, recognized as a highly efficient tool in chemical biology, has shown significant value in cancer treatment. The primary objective is to develop efficient delivery strategies to achieve enhanced bioorthogonal drug treatment for tumors. Here, Janus microparticles (JMs) loaded with cyclooctene‐modified doxorubicin prodrug (TCO‐DOX) and tetrazine‐modified indocyanine green (Tz‐ICG) triggers are reported. Besides activating TCO‐DOX, Tz‐ICG is also a photothermal agent used in photothermal therapy (PTT), enabling the simultaneous use of biorthogonal chemotherapy and PTT. Additionally, the DOX could be significantly reduced in systemic toxicity with the modification of cyclooctene. Thus, the developed drug‐carrying JMs system exhibits effective tumor cell killing in vitro and effectively inhibits tumor local progress and distant lung metastasis after postoperative treatment with good safety. These results demonstrate that the prepared JMs provide a paradigm for bioorthogonal prodrug activation and localized delivery, and hold great promise for cancer therapy as well as other related applications.

Progress in Radical Fluorosulfonyl Reagents

AbstractSulfur(VI) fluoride exchange (SuFEx) chemistry that relies on the unique reactivity–stability balance of high valent organosulfur has emerged as a promising topic for the next-generation click reaction. Sulfonyl fluorides are the most widely used connective hubs of the SuFEx click reaction with widespread applications in the fields of chemical biology, drug discovery, and materials science. Compared with current methods, direct fluorosulfonylation with fluorosulfonyl radicals has emerged as a concise and efficient approach for the production of sulfonyl fluorides. The highly active SO2F radical was an unstable and inaccessible precursor until it was observed in the decomposition of fluorosulfonyl azide, which inspired efforts towards the production of fluorosulfonyl radical precursors for direct radical fluorosulfonylation processes. This review presents and discusses current breakthroughs in the generation of fluorosulfonyl radicals from various radical precursors, as well as their application in the synthesis of diverse functionalized sulfonyl fluorides.1 Introduction2 FSO2Cl as a Radical Precursor3 Benzimidazolium Fluorosulfonates as Radical Precursors4 Alk-1-ynylsulfonyl Fluorides as Radical Precursors5 (Diarylmethylene)sulfamoyl Fluorides as Radical Precursors6 Vinyl Fluorosulfates as Radical Precursors7 Conclusion and Outlook

Preface: 7th International Conference on Chemical Engineering and Advanced Materials (CEAM 2024)

The 2024 7th International Conference on Chemical Engineering and Advanced Materials (CEAM 2024) was held in Philadelphia, USA during September 28-29, 2024. This conference aims to provide opportunities for all participants to exchange new innovative ideas and application experiences as well as to provide a platform where possible innovators can think of new ventures, generate contacts and networks for future collaboration. The CEAM 2024 topics include chemical engineering, biological chemistry, biological pharmacy, food science, chemical materials, nano materials, materials processing engineering, and advanced materials applications. This conference brought together leaders from industry and academia to exchange and share their experiences, present research results, explore collaborations, and to spark new ideas, with the aim of developing new projects and exploiting new technology. On behalf of the conference organizers, we would like to thank the reviewers for their time and effort in reviewing the papers. Their feedbacks to the authors have been excellent and useful to ensure high quality papers for the conference. Our appreciation to all keynote speakers for sharing their thoughts and experiences with respect to various topics. Our sincere thanks also go to all attendees from all around the world for participating in the conference. Finally, we would like to thank the organizing committee members for their hard work, cooperation, and insights in ensuring the success of this conference. Scientific Committee of CEAM 2024 Philadelphia, USA

Noncanonical Amino Acid Tools and Their Application to Membrane Protein Studies.

Methods rooted in chemical biology have contributed significantly to studies of integral membrane proteins. One recent key approach has been the application of genetic code expansion (GCE), which enables the site-specific incorporation of noncanonical amino acids (ncAAs) with defined chemical properties into proteins. Efficient GCE is challenging, especially for membrane proteins, which have specialized biogenesis and cell trafficking machinery and tend to be expressed at low levels in cell membranes. Many eukaryotic membrane proteins cannot be expressed functionally in E. coli and are most effectively studied in mammalian cell culture systems. Recent advances have facilitated broader applications of GCE for studies of membrane proteins. First, AARS/tRNA pairs have been engineered to function efficiently in mammalian cells. Second, bioorthogonal chemical reactions, including cell-friendly copper-free "click" chemistry, have enabled linkage of small-molecule probes such as fluorophores to membrane proteins in live cells. Finally, in concert with advances in GCE methodology, the variety of available ncAAs has increased dramatically, thus enabling the investigation of protein structure and dynamics by multidisciplinary biochemical and biophysical approaches. These developments are reviewed in the historical framework of the development of GCE technology with a focus on applications to studies of membrane proteins.

Structural Insights into Helix–Loop–Helix Peptides for “Ligand-Targeting” Intracellular Drug Delivery via VEGF Receptor-Mediated Endocytosis

As a new alternative to antibody-drug conjugates, we developed “ligand-targeting” peptide-drug conjugates (PDCs), in which conformationally constrained helix-loop-helix (HLH) peptide M49 targeting human vascular endothelial growth factor-A (VEGF) was used as a drug carrier. The biochemical study showed that HLH peptide M49 made a complex with VEGF in the extracellular environment, and then the M49/VEGF complex interacts with the receptor on the cell surface to induce cellular internalization via the endocytic pathway. Here, we present an X-ray crystal structure of the M49/VEGF complex at 1.5 Å resolution using a protein crystal grown in microgravity. The structure illustrated the “ligand-targeting” cellular uptake mechanism for intracellular drug delivery and the molecular basis on the peptide-VEGF binding mode with tight binding and high target specificity. In addition, mutational studies and thermodynamic analysis provided information on the driving forces of the complex formation. This work would contribute to the design of mid-size molecular-targeting peptides as well as HLH peptides, advancing the research in drug discovery and chemical biology.

Construction of Aryl Azide‐ and Triazole‐Based Unnatural Amino Acid Motifs via the Pd(II)‐Catalyzed C(sp3)−H Arylation

AbstractThis paper reports a method for the synthesis of examples of aryl azide‐ and triazole moiety‐based unnatural amino acid motifs using the Pd(II)‐catalyzed arylation of the prochiral C(sp3)−H bonds of carboxamides of amino acids as one of the key transformations. Synthesis of racemic and enantioenriched aryl azide‐based unnatural amino acid motifs including, norvaline, leucine, norleucine, phenylalanine, 2‐aminobutyric acid, 2‐aminooctanoic acid, and non‐α‐amino acid derivatives were shown. Additionally, we attempted the synthesis of triazole moiety‐installed norvaline, leucine, norleucine, phenylalanine, 2‐aminobutyric acid, 2‐aminooctanoic acid, and non‐α‐amino acid derivatives via the Cu‐catalyzed Click reaction. We also demonstrated the removal of the protecting groups and the synthesis of free amino group‐containing aryl azide‐ or triazole‐based unnatural amino acid motifs and peptides. Generally, azide moiety‐containing unnatural amino acid motifs are important molecular tools in chemical biology. Furthermore, aryl azide‐ and triazole moiety‐containing unnatural amino acid scaffolds are notable substrates in medicinal chemistry and drug discovery research. Accordingly, this work contributes to enriching the library of aryl azide‐ and triazole‐based unnatural amino acid derivatives.

Design, Exploitation, and Rational Improvements of Diazirine-Based Universal Polymer Crosslinkers.

ConspectusAddition of new covalent bonds between the chains of thermoplastic polymers (i.e., crosslinking) provides improved mechanical strength and enhanced high-temperature performance while also providing an effective strategy for photopatterning. Traditionally, however, crosslinking of each polymer substrate has required the use of a specific crosslinking technology (hydrosilylation for PDMS, vulcanization for rubber, etc.). The lack of a general solution to the challenge of polymer crosslinking means that there are many thermoplastics (e.g., polypropylene or polyhydroxyalkanoates) that have desirable properties, but which cannot be upgraded by traditional crosslinking technologies.Our lab developed the first universal crosslinkers for aliphatic polymers by leveraging trifluoromethyl aryl diazirine motifs, functional groups that have been widely used in chemical biology for >30 years, but which have seldom been exploited in materials science. These novel reagents work (via C-H insertion) on essentially any commodity polymer that contains aliphatic C-H bonds, including industrial plastics like polypropylene (the crosslinking of which has been an outstanding challenge in the field for >50 years), as well as commercially important elastomers (e.g., polydimethylsiloxane), biodegradable polymers (e.g., polycaprolactone), and green polymer materials derived from biomass (e.g., polyhydroxyalkanoates).Subsequent structure-function work from our group led to crosslinkers that were >10-fold more effective in undergoing C-H insertion with aliphatic substrates. We then developed an improved synthesis of our electronically optimized diazirines and incorporated them into a family of cleavable crosslinker reagents, which permit the on-demand generation of reprocessable thermosets. At the same time, other groups replaced the perfluoropropyl linker in our first-generation crosslinker with a series of dynamic linkages; these permit the ready generation of vitrimeric materials and can be used in the reactive compatibilization of immiscible plastic waste.Since the publication of our initial Science paper in 2019, this burgeoning field of diazirine-based polymer crosslinkers has experienced an explosion of interest. Publications from our lab and others have described the use of these reagents in covalent adhesion, photopatterning of low dielectric materials for microelectronics, and direct optical printing of quantum dots. Our crosslinkers have also been shown to heighten the robustness of ice-phobic coatings and improve the performance of woven ballistic fabric, while─perhaps most unexpectedly─substantially improving the stability of high-performance perovskite solar cells. Electronically optimized diazirines can also be used to covalently link proteins to polymer surfaces, suggesting a broad range of applications in the biocompatibilization of medical devices. This Account will summarize the development of trifluoromethyl aryl diazirine reagents for materials science over the past 5 years. A brief comparison will also be made, in the Summary and Outlook section at the end of the Account, to competing (and often complementary) reagents based upon azide and diazoalkyl motifs. Finally, we have compiled a Frequently Asked Questions list that covers many practical aspects of crosslinker design and application; this is appended as Supporting Information.

A Rapid and Sensitive MicroPlate Assay (MPSA) Using an Alkyne-Modified CMP-Sialic Acid Donor to Evaluate Human Sialyltransferase Specificity.

Human sialyltransferases primarily utilize CMP-Sias, especially transferring Neu5Ac from CMP-Neu5Ac to various acceptors. Advances in chemical biology have led to the synthesis of novel CMP-Sia donors suitable for bioorthogonal reactions in cell-based assays. However, the compatibility of these donors with all human enzymes remains uncertain. We synthesized a non-natural CMP-Sia donor with an alkyne modification on the N-acyl group of Neu5Ac, which was effectively used by human ST6Gal I and ST3Gal I. A sensitive MicroPlate Sialyltransferase Assay (MPSA) was developed and expanded to a panel of 13 human STs acting on glycoproteins. All assayed enzymes tolerated CMP-SiaNAl, allowing for the determination of kinetic parameters and turnover numbers. This study enhances the biochemical characterization of human sialyltransferases and opens new avenues for developing sialyltransferase inhibitors.

Late-Stage Fluorination of Tyrosine Residues in Antiviral Protein Cyanovirin-N.

The applications of fluorinated molecules in chemical biology are rapidly expanding driven by the unique properties of C-F bonds, leading to increased interest in methodologies for controlled introduction of this atom. In this study, we present the first method for late-stage fluorination of tyrosine residues in proteins. Our results demonstrate that electrophilic fluorination using Selectfluor, a stable and non-toxic reagent, offers a straightforward and cost-effective method for labeling Cyanovirin-N (CVN), a 101-amino-acid lectin with effective antiviral activity. Uni- and bidimensional 1H, 13C and 19F NMR analyses, along with mass spectrometry, revealed chemoselective fluorination of the three tyrosine residues in CVN without affecting its overall structure or mannose-binding affinity. Additionally, we observed that other aromatic amino acids, such as tryptophan, phenylalanine, and histidine, are not fluorinated using this method. These findings advance our understanding of protein fluorination and its applications in studying structure, dynamics, and interactions, as well as other biological utilities.

Unveiling the Singular Conformation of the 3‐Fluoropyrrolidinium Cation

AbstractThe pyrrolidine ring is a 5‐membered ring that holds significant importance in biological chemistry. The introduction of fluorine at position 3 of pyrrolidine results in pseudo‐axial and pseudo‐equatorial C−F bonds. However, both quantum‐chemical calculations and infrared spectroscopy reveal that the 3‐fluoropyrrolidinium cation only adopts one conformation, with the C−F bond in the pseudo‐axial orientation. Upon deprotonation of 3‐fluoropyrrolidinium, leading to the formation of 3‐fluoropyrrolidine, four conformations are predicted based on CCSD/DGTZVP quantum‐chemical calculations. Among these, the conformation favoring an N−H⋅⋅⋅F−C interaction emerges as the most stable, both in the gas phase and within an implicit DMSO medium. The heightened ring puckering observed in 3‐fluoropyrrolidinium compared to the most stable conformation of 3‐fluoropyrrolidine suggests that the singular conformation of the cation arises from a robust attractive electrostatic interaction resembling hydrogen bonding, rather than a charge‐dipole gauche effect as previously assumed.

Nucleotide-mediated modulation of chemoselective protein functionalization in a liquid-like condensed phase

Liquid-like protein condensates are ubiquitous in cellular system and are increasingly recognized for their roles in physiological processes. Condensed phase harbors distinctive chemical microenvironment, markedly different than dilute aqueous phase. Herein, we demonstrate chemoselective modification pattern of nucleophilic canonical amino acid sidechains (namely – cysteine, tyrosine and lysine) of the protein towards 4-chloro-7-nitrobenzofurazan in the dilute and condensed phase. We also delineate how the effect of nucleotides and their in situ enzymatic dissociation temporally modulate the protein condensate’s pH and the protein’s corresponding chemoselective modification. We have shown that the pH of the condensate decreases in the presence of nucleoside triphosphate, whereas it increases in the presence of nucleoside monophosphates or phosphate ion. For instance, we find lysine-specific modification gets inhibited in the presence of adenosine triphosphate (ATP), but significantly enhanced in the presence of monophosphates. This feature enables us to gain temporal control over dynamic change in protein functionalization via enzymatic ATP hydrolysis. Overall, this work substantiates the alteration in pH-responsiveness of Brønsted basicity of a protein’s ε-amine in the condensed phase. Furthermore, this environment sensitivity in chemoselective protein functionalization in condensed phase will be important in adaptable protein engineering to the chemical biology of protein phase separation.

Detouring NSAID into Mitochondria to Induce Apoptosis in Cancer Cells.

In recent years, impairing mitochondria in cancer cells gained attention as alternative cancer therapy. In this context, non-steroidal anti-inflammatory (NSAID) drugs are interesting candidates to damage mitochondria in cancer cells. However, routing NSAIDs specifically into the mitochondria remained a major challenge and less explored. Herein, we have synthesized a small library of Meclofenamic acid and Naproxen derivatives having ester and amide linkage with substituted triphenylphosphonium cations for mitochondria targeting. Screening in cervical cancer (HeLa), breast cancer (MCF7) and colon cancer (HCT-116) cells revealed a Meclofenamic acid derivative having ester linkage with tri (4-methoxyphenyl) phosphonium cation (8A3) which induced mitochondrial damage through mitochondrial outer membrane permeabilization (MOMP) followed by generation of reactive oxygen species (ROS) in the HCT-116 cells. This 8A3-mediated mitochondrial impairment triggered apoptosis by inhibiting Cox-2, reduction in Bcl-2/Bcl-xl expression and Caspase-3/9 cleavage leading to remarkable HCT-116 cell death. This novel mitochondrion targeted Meclofenamic acid derivative has the potential to be used as a chemical biology tool to understand the role of NSAIDs in mitochondria towards cancer therapy.

ECBD: European chemical biology database.

The European Chemical Biology Database (ECBD, https://ecbd.eu) serves as the central repository for data generated by the EU-OPENSCREEN research infrastructure consortium. It is developed according to FAIR principles, which emphasize findability, accessibility, interoperabilityand reusability of data. This data is made available to the scientific community following open access principles. The ECBD stores both positive and negative results from the entire chemical biology project pipeline, including data from primary or counter-screening assays. The assays utilize a defined and diverse library of over 107000 compounds, the annotations of which are continuously enriched by external user supported screening projects and by internal EU-OPENSCREEN bioprofiling efforts. These compounds were screened in 89 currently deposited datasets (assays), with 48 already being publicly accessible, while the remaining will be published after a publication embargo period of up to 3 years. Together these datasets encompass ∼4.3 million experimental data points. All public data within ECBD can be accessed through its user interface, API or by database dump under the CC-BY 4.0 license.

Extensively harvested hay contaminated with common windgrass induced an ulcerative stomatitis–gingivitis outbreak in horses

SummaryAfter starting feeding on a new batch of hay, 29 out of 33 horses showed clinical signs of oral ulcerative and bleeding papules and mucous membrane erythema of the gingiva of the mandible within 2–3 days. The results of the complete blood cell count and biochemistry analysis did not yield significant findings in relation to horses affected by ulcerative stomatitis. Swab analyses of the oral mucosa for vesicular stomatitis virus (VS, Type Indiana and Type New Jersey) were negative in all horses (n = 33). Common windgrass with long awns in the deeper stomal‐gingival ulcerations was confirmed through botanical analysis by botanical identification. The hay batch fed to the affected horses was assessed via botanical analysis, finding 15% common windgrass (Apera spica‐venti). After the removal of the contaminated hay batch, full recovery was seen in all horses within 2–6 weeks depending on the severity of the lesions. The grassland from which the hay was harvested belonged to a semi‐natural landscape restored to increase biodiversity and as a protection zone in case of overflow of the river Rhine. In conclusion, feeding forages harvested from these semi‐natural landscapes may be harmful to the health of horses.

Oxadiazolone-Based Aromatic Annulations: A Nitrenoid Precursor for Tricyclic Aminoheterocycles.

Nitrogen-containing heterocycles are present in most approved drugs, reflecting the significance of their synthetic strategies. By utilizing oxadiazolone as a nitrenoid (nitrene-like) precursor, we have developed a general strategy for the annulation with nucleophilic heterocycles to access various polycyclic aminoheterocycles. We have discovered that 2-pyrryl-substituted substrates undergo a rearrangement, which indicates a spirocyclization-migration pathway. Given their fluorescence and biological activity as kinase hinge binders, these fragment-like aminoheterocycles represent potential starting points for applications in chemical biology and drug discovery.

Chem(Pro)2: the atlas of chemoproteomic probes labelling human proteins.

Chemoproteomic probes (CPPs) have been widely considered as powerful molecular biological tools that enable the highly efficient discovery of both binding proteins and modes of action for the studied compounds. They have been successfully used to validate targets and identify binders. The design of CPP has been considered extremely challenging, which asks for the generalization using a large number of probe data. However, none of the existing databases gives such valuable data of CPPs. Herein, a database entitled 'Chem(Pro)2' was therefore developed to systematically describe the atlas of diverse types of CPPs labelling human protein in living cell/lysate. With the booming application of chemoproteomic technique and artificial intelligence in current chemical biology study, Chem(Pro)2 was expected to facilitate the AI-based learning of interacting pattern among molecules for discovering innovative targets and new drugs. Till now, Chem(Pro)2 has been open to all users without any login requirement at: https://idrblab.org/chemprosquare/.

Systemic effect of the oncological process on the dog’s body

The article presents the results of an analysis of a non-randomized study of the prevalence of paraneoplastic syndromes in primary spontaneous malignant neoplasms in dogs. The study was conducted in 2022–2024 on the basis of the Department of Diseases of Small Domestic, Laboratory and Exotic Animals and the Research Laboratory of Oncology, Ophthalmology and Animal Biochemistry of the Russian Biotechnological University “ROSBIOTECH”. The object of the study were 948 dogs with primary spontaneous malignant neoplasms confirmed by morphological methods.The aim of the work was to study the frequency of paraneoplastic syndromes in dogs with neoplasia.The diagnosis of paraneoplastic syndrome was made by excluding other possible etiological factors for the development of clinical and laboratory abnormalities identified during a comprehensive examination of dogs. As a result of the study, the incidence of paraneoplasia was 54.54% of cases. Hematological (44.41%) and gastrointestinal (37.13%) paraneoplastic syndromes were most often recorded, less often endocrine (3.27%) and ophthalmological (3.38%), extremely rarely renal (1.27%), osteoarticular (0.42%) and dermatological (0.42%). The main hematological manifestations of the systemic effect of the oncological process were: anemia (12.87%), neutrophilic leukocytosis (12.76%) and thrombocytopenia (8.86%). Gastrointestinal paraneoplastic syndrome in most cases was manifested by a decrease in body weight (33.65%) and hyporexia (18.04%), inflammatory and erosive ulcerative lesions of the gastrointestinal tract (7.59%). During the study, a case of a nonspecific systemic reaction to a malignant tumor in the form of fever in the absence of infectious and inflammatory processes (0.11%) was registered.

Enhanced Synthesis of Sulfonyl Fluorides and Sulfamoyl Fluorides Using Fluorosulfonyl-Containing Radical Reagents

Sulfonyl fluorides and sulfamoyl fluorides, as the most significant species among sulfur(VI) fluorides, have garnered considerable attention in various fields, including organic synthesis, materials science, chemical biology, and drug discovery. Fluorosulfonyl-containing radical reagents have unique reactivity and exhibit notable advantages in constructing complex organic molecules, achieving selective functional group transformations. This short review provides a detailed introduction to the diverse applications of fluorosulfonyl-containing radical reagents in organic synthesis, such as C-H bond functionalizations, difunctionalization of olefins, and tandem reactions, highlighting their crucial role in synthesizing complex organic molecules.