Vladimir Kubyshkin

Controlling biological activity with light: diarylethene-containing cyclic peptidomimetics.

Photobiological processes in nature are usually triggered by nonpeptidic chromophores or by modified side chains. A system is presented in which the polypeptide backbone itself can be conformationally switched by light. An amino acid analogue was designed and synthesized based on a reversibly photoisomerizable diarylethene scaffold. This analogue was incorporated into the cyclic backbone of the antimicrobial peptide gramicidin S at several sites. The biological activity of the resulting peptidomimetics could then be effectively controlled by ultraviolet/visible light within strictly defined spatial and temporal limits.

Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology

The goal of xenobiology is to design biological systems endowed with unusual biochemical functions, whereas enzymology concerns the study of enzymes, the workhorses of biocatalysis. Biocatalysis employs enzymes and organisms to perform useful biotransformations in synthetic chemistry and biotechnology. During the past few years, the effects of incorporating noncanonical amino acids (ncAAs) into enzymes with potential applications in biocatalysis have been increasingly investigated. In this Review, we provide an overview of the effects of new chemical functionalities that have been introduced into proteins to improve various facets of enzymatic catalysis. We also discuss future research avenues that will complement unnatural mutagenesis with standard protein engineering to produce novel and versatile biocatalysts with applications in synthetic organic chemistry and biotechnology.

Energetic contribution to both acidity and conformational stability in peptide models

The acidity of N-acyl amino acids is dependent upon the rotameric state of the amide bond. In this work we systematically investigated the acidity difference of the rotamers (ΔpKa) in the frames of various acetylated amino acids. Our results indicated a mutual interaction of two carbonyl groups of an attractive type. We observed conservative ΔpKas for acyclic amino acids (2.2–3.0 kJ mol−1), whereas in the case of alicyclic amino acids, the experimental values revealed a strong dependency on the structural context (1.5–4.4 kJ mol−1). In homologous amino acids (α-, β-, γ-, etc.), the strength of the attraction decays in an exponential fashion. Furthermore, the interaction can accumulate through a chain of amide bonds in a cascade fashion, as demonstrated by an Ac-Pro-Pro dipeptide. As a result, we demonstrate that ΔpKa is an experimental parameter to estimate increments in the carbonyl–carbonyl alignment, as determined by the amino acid or peptidyl context. This parameter is also important in understanding the roles of amino acids in both protein folding and translation in biological systems as well as their evolutionary appearance in the genetic code.

A base promoted multigram synthesis of aminoisoxazoles: valuable building blocks for drug discovery and peptidomimetics

A practical multigram metal free synthesis of isoxazole-containing building blocks from commercially available amino acids was elaborated. The key reaction was a regioselective [3 + 2]-cycloaddition of in situ generated nitrile oxides with alkynes/enamines. The obtained building blocks were used in the preparation of bioactive compounds and peptidomimetics.

Applying γ-Substituted Prolines in the Foldon Peptide: Polarity Contradicts Preorganization

Rational choice of chemical modifications to proline residues allows the preorganization principle to be exploited for more stable assembly of the foldon domain as a tag for trimerization. With systematic knowledge of how chemical and steric variations of the ring substituents affect the relative stabilities of exo and endo puckers, the preorganization principle should then be usable in biotechnologically synthesized foldon mutants and applicable for protein tagging elsewhere.

Peptidyl-Prolyl Model Study: How Does the Electronic Effect Influence the Amide Bond Conformation?

The triple-helical structure of collagen, the most abundant protein in animal bodies, owes its stability to post-translationally installed hydroxyl groups at position 4 of prolyl residues. To shed light on the nature of this phenomenon, we have examined the influence of the 4-substituent on the amide isomerism in peptidyl-prolyl analogues. The rigid bicyclic skeleton of 2,4-methanoprolines allowed us to follow the through-bond impact of the substituent group (electronic effect) without the side-chain conformation being affected by a stereoelectronic effect. These proline analogues were prepared by [2 + 2] photocycloaddition of (2-allylamino)acrylic acid derivatives. Subsequent pKa studies demonstrated a remarkable electronic effect of the 4-fluorine substitution, while the effect of the 4-methyl group was negligible. The trans/cis amide ratio was measured in model compounds under low temperature conditions. The observed prevalence for a trans-amide is extraordinary, and in this regard, 2,4-methanoproline is closer to primary α-amino acids than to proline. At the same time the amide rotation velocities were 3-4 orders of magnitude higher when compared to N-acetylprolyl. Finally, our results indicate that the electronic effect of the 4-substituent only affects the kinetics of the amide isomerization but not the thermodynamic prevalence for the trans-rotamer.

Fluorine-Rich Planetary Environments as Possible Habitats for Life

In polar aprotic organic solvents, fluorine might be an element of choice for life that uses selected fluorinated building blocks as monomers of choice for self-assembling of its catalytic polymers. Organofluorine compounds are extremely rare in the chemistry of life as we know it. Biomolecules, when fluorinated such as peptides or proteins, exhibit a “fluorous effect”, i.e., they are fluorophilic (neither hydrophilic nor lipophilic). Such polymers, capable of creating self-sorting assemblies, resist denaturation by organic solvents by exclusion of fluorocarbon side chains from the organic phase. Fluorous cores consist of a compact interior, which is shielded from the surrounding solvent. Thus, we can anticipate that fluorine-containing “teflon”-like or “non-sticking” building blocks might be monomers of choice for the synthesis of organized polymeric structures in fluorine-rich planetary environments. Although no fluorine-rich planetary environment is known, theoretical considerations might help us to define chemistries that might support life in such environments. For example, one scenario is that all molecular oxygen may be used up by oxidation reactions on a planetary surface and fluorine gas could be released from F-rich magma later in the history of a planetary body to result in a fluorine-rich planetary environment.

cis–trans-Amide isomerism of the 3,4-dehydroproline residue, the ‘unpuckered’ proline

Summary Proline (Pro) is an outstanding amino acid in various biochemical and physicochemical perspectives, especially when considering the cis–trans isomerism of the peptidyl-Pro amide bond. Elucidation of the roles of Pro in chemical or biological systems and engineering of its features can be addressed with various Pro analogues. Here we report an experimental work investigating the basic physicochemical properties of two Pro analogues which possess a 3,4-double bond: 3,4-dehydroproline and 4-trifluoromethyl-3,4-dehydroproline. Both indicate a flat pyrroline ring in their crystal structures, in agreement with previous theoretical calculations. In solution, the peptide mimics exhibit an almost unchanged equilibrium of the trans/cis ratios compared to that of Pro and 4-trifluoromethylproline derivatives. Finally we demonstrate that the 3,4-double bond in the investigated structures leads to an increase of the amide rotational barriers, presumably due to an interplay with the transition state.

Hydrolysis, polarity, and conformational impact of C-terminal partially fluorinated ethyl esters in peptide models

Fluorinated moieties are highly valuable to chemists due to the sensitive NMR detectability of the 19F nucleus. Fluorination of molecular scaffolds can also selectively influence a molecule’s polarity, conformational preferences and chemical reactivity, properties that can be exploited for various chemical applications. A powerful route for incorporating fluorine atoms in biomolecules is last-stage fluorination of peptide scaffolds. One of these methods involves esterification of the C-terminus of peptides using a diazomethane species. Here, we provide an investigation of the physicochemical consequences of peptide esterification with partially fluorinated ethyl groups. Derivatives of N-acetylproline are used to model the effects of fluorination on the lipophilicity, hydrolytic stability and on conformational properties. The conformational impact of the 2,2-difluoromethyl ester on several neutral and charged oligopeptides was also investigated. Our results demonstrate that partially fluorinated esters undergo variable hydrolysis in biologically relevant buffers. The hydrolytic stability can be tailored over a broad pH range by varying the number of fluorine atoms in the ester moiety or by introducing adjacent charges in the peptide sequence.

Transmembrane Polyproline Helix

The third most abundant polypeptide conformation in nature, the polyproline-II helix, is a polar, extended secondary structure with a local organization stabilized by intercarbonyl interactions within the peptide chain. Here we design a hydrophobic polyproline-II helical peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold and demonstrate its transmembrane alignment in model lipid bilayers by means of solid-state 19F NMR. As result, we provide a first example of a purely artificial transmembrane peptide with a structural organization that is not based on hydrogen-bonding.