Bioconjugation methodologies have proven to play a central enabling role in the recent development of biotherapeutics and chemical biology approaches. Recent endeavours in these fields shed light on unprecedented chemical challenges to attain bioselectivity, biocompatibility, and biostability required by modern applications. In this review the current developments in various techniques of selective bond forming reactions of proteins and peptides were highlighted. The utility of each endogenous amino acid-selective conjugation methodology in the fields of biology and protein science has been surveyed with emphasis on the most relevant among reported transformations; selectivity and practical use have been discussed.

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Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation

The native chemical ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro- and nanotechnology research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and physical chemistry studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O, S-, N, S-, or N, Se-acyl shift systems as thioester or selenoester surrogates and discusses the special molecular features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N, S-acyl migrations and selective dechalcogenation reactions.

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

5.3. Aromatic Ring Formation 1576 5.3.1. One Substituent at C-2 1576 5.3.2. Two Substituents at C-2 and C-3 1577 5.3.3. Two Substituents at C-2 and C-5 1577 5.3.4. Two Substituents at C-3 and C-4 1577 5.3.5. Three Substituents at C-2, C-3, and C-4 1578 5.3.6. Four Substituents at Carbon Atoms 1579 5.3.7. Benzotriazolyl Substituent at the Ring 1579 6. Furan 1580 6.1. Nonaromatic Rings 1580 6.1.1. Tetrahydrofurans 1580 6.1.2. 2,5-Dihydrofurans 1580 6.2. Electrophilic Substitution of the Aromatic Ring 1581 6.3. Aromatic Ring Formation 1582 7. Thiophene 1583 7.1. Nonaromatic Rings 1583 7.2. Electrophilic Substitution of the Aromatic Ring 1583 7.3. Aromatic Ring Formation 1583 8. Five-Membered Rings with Two Heteroatoms 1584 8.1. Pyrazole 1584 8.2. Imidazole 1586 8.3. Isoxazole 1589 8.4. Oxazole 1590 8.5. Thiazole 1592 9. Five-Membered Rings with Three or Four Heteroatoms 1594

Synthesis of Heterocycles Mediated by Benzotriazole. 1. Monocyclic Systems

We report on the synthesis and operation of a three-barrier, rotaxane-based, artificial molecular machine capable of sequence-specific β-homo (β3) peptide synthesis. The machine utilizes nonproteinogenic β3-amino acids, a class of amino acids not generally accepted by the ribosome, particularly consecutively. Successful operation of the machine via native chemical ligation (NCL) demonstrates that even challenging 15- and 19-membered ligation transition states are suitable for information translation using this artificial molecular machine. The peptide-bond-forming catalyst region can be removed from the transcribed peptide by peptidases, artificial and biomachines working in concert to generate a product that cannot be made by either machine alone.

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Sequence-specific β-peptide synthesis by a rotaxane-based molecular machine.

Peptides are ubiquitous in nature where they play crucial roles as catalysts (enzymes), cell membrane ion transporters, and structural elements (proteins) within biological systems. In addition, both linear and cyclic peptides have found use as pharmaceuticals and components of various conjugate molecular systems. Small wonder then that chemists throughout the ages have sought to mimic nature by synthesis of the amide polymers known as peptides and proteins. The fundamental reaction in the formation of a peptide bond is condensation of an amine of one amino acid with the activated carbonyl group of another. This "fragment condensation" has been achieved in many ways both in solution and by solid-phase peptide synthesis (SPSS) on resin. The most successful method for in-solution coupling is known as native chemical ligation (NCL), and the technique dates back to the pioneering work of Wieland (1953) and subsequently Kent (1994) among many others. This Account builds on the established principles of NCL as applied specifically to S-, O-, and N-isopeptides, molecules that are generally more soluble and less prone to aggregation than native peptides. This Account also covers NCL of isopeptides containing terminal and nonterminal S-acylated cysteine units, reactions that enable the synthesis of native peptides from S-acyl peptides without the use of auxiliaries. With C-terminal S-acyl isopeptides, NCL was carried out under microwave irradiation in phosphate buffer (pH 7.3) at 50 °C. Intramolecular acyl migration was observed through 5-19-membered transition states with relative rates, as assessed by product analysis, in the order, 5 > 10 > 11 > 14, 16, or 17 > 12 > 13, 15, or 19 > 18 ≫ 9 > 8. The rate/pH profile for the 15-membered TS showed a maximum for ligated product versus transacylation at pH 7.0-7.3 presumably associated with the pKa of the N-nucleophile in the hydrogen-bonded TS. Cysteine occurs at low abundance (1.7%) in natural peptides and is rarely available in a terminal position thus limiting the utility of the method. This Account reports, however, NCL at nonterminal acyl cysteine through 5-, 8-, 11-, and 14-membered TSs with relative rates of ligation in the order, 5 ≫ 14 > 11 ≫ 8, thus paralleling the results with acylated terminal cysteine residues. In an obvious sequel to the work with acylated cysteine, we discuss intramolecular O- to N-acyl shift in O-acyl serine and O-acyl tyrosine isopeptides where the story becomes more complex in terms of viable conditions and optimum size of the cyclic TS. N- to N-acyl migration in acyl tryptophan isopeptides is described, and finally, chemical ligation is applied to the synthesis of cyclic peptides. Conformational analysis and quantum chemical calculations are used to rationalize ligation through a range of cyclic transition states. This Account highlights the fact that NCL of acyl isopeptides is an extremely useful strategy for the synthesis of a wide variety of native peptides in good yields and under mild conditions. Mechanistic aspects of the ligations are not fully resolved, but theoretical studies indicate that hydrogen bonding within the various cyclic transition states plays a major role.

Traceless Chemical Ligation from S-, O-, and N-Acyl Isopeptides

Optically pure conjugates of quinolone antibiotics with naturally occurring amino acids are synthesized in 40–98% yields.

Synthesis of amino acid derivatives of quinolone antibiotics

S-Acyl cysteine peptides containing alpha-, beta- or gamma-amino acid residues undergo long-range S- to N-acyl transfer to give analogs of native tripeptides and tetrapeptides containing additional carbon atoms in the chain. The ease of intramolecular S -> N-acyl transfer relative to intermolecular transacylation is favored increasingly for 9?<?12?<?13?similar to?10-membered cyclic transition states; the observed order is explained on conformational and intermolecular interaction considerations

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S‐ to N‐Acyl transfer in S‐acylcysteine isopeptides via 9‐, 10‐, 12‐, and 13‐membered cyclic transition states

Dye-labelled nucleosides were obtained in 30-79% (average 45%) yields by treating N-(4-arylazobenzoyl)-1H-benzotriazoles 3a-b with appropriate nucleosides. Similarly, 3a-b afforded dye-labelled threoninol conjugates in 55-89% (average 67%) yields. All novel products were characterized by NMR and elemental analysis.

Dye labelling of nucleosides.

A new class of geminally-substituted nitroso compounds, i.e. α-benzotriazoyl nitroso derivatives, is presented. These compounds display a rather different behavior than other related nitroso compounds bearing a geminal electron-withdrawing group. An unexpected and spontaneous oxidation to the nitro analog is observed in solution.

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En route towards α-benzotriazoyl nitroso derivatives

Abstract Relying on an interview-based research carried out in Szeklerland (Eastern Transylvania) between 2011 and 2020, the present study investigates the impact of the new rural development paradigm on the first level indicated by Van der Ploeg et al. (2000): changes in the relationship between agriculture and local society. The analysed region does not form an administrative unit, it is the eastern region of the former Austro-Hungarian monarchy. It consists of rural settlements and small towns, the population of which is mostly of Hungarian ethnicity. More than half of the households have a small amount of land and are engaged in farming as their main or supplementary activity. Following a brief overview of the regional context, our paper analyzes the essential changes that have been taking place in the relationship between agriculture and the regional society over the last one or two decades. The focus of the analysis is how changes in the relationship between agriculture and the regional society appear in the innovative agricultural entrepreneurs’ practice and interpretations.

Judit Kovacs

Papers

Synthesis of amino acid derivatives of quinolone antibiotics

S‐ to N‐Acyl transfer in S‐acylcysteine isopeptides via 9‐, 10‐, 12‐, and 13‐membered cyclic transition states

Dye labelling of nucleosides.

En route towards α-benzotriazoyl nitroso derivatives

Changes in the Role of Agriculture from the Perspective of Innovative Agricultural Entrepreneurs. The Case of Szeklerland, Eastern Transylvania