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In this work, we have applied the concept of α-hydrazino acid insertion in a peptide sequence as a means of structurally organizing a potential protein-protein interactions (PPI) inhibitor. Hydrazino peptides characterized by the incorporation of an α-hydrazino acid at specific positions introduce an additional nitrogen atom into their backbone. This modification leads to a change in the electrostatic properties of the peptide and induces the restructuring of its hydrogen bonding network, resulting in conformational changes toward more stable structural motifs. Despite the successful use of synthetic hydrazino oligomers in binding to nucleic acids, the structural changes due to the incorporation of α-hydrazino acid into short natural peptides in solution are still poorly understood. Based on NMR data, we report structural models of p53-derived hydrazino peptides with elements of localized peptide structuring in the form of an α-, ß-, or γ-turn as a result of hydrazino modification in the peptide backbone. The modifications could potentially lead to the preorganization of a helical secondary peptide structure in a solution that is favorable for binding to a biological receptor. Spectroscopically, we observed that the ensemble averaged rapidly interconverting conformations, including isomerization of the E-Z hydrazide bond. This further increases the adaptability by expanding the conformational space of hydrazine peptides as potential protein-protein interaction antagonists.

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The aim of this study was to assess the gelling potential of chiral oxalamide derivatives in vegetable oils. Special emphasis was given to the potential applications of the examined oil gels as sustained delivery systems and as fat substitutes in food products. The applicability of oil gelators is envisaged in food, cosmetics, and the pharmaceutical industry. The regulations requiring the elimination of saturated fats and rising concerns among consumers health motivated us to investigate small organic molecules capable of efficiently transforming from liquid oil to a gel state. The oxalamide organogelators showed remarkable gelation efficiency in vegetable oils, thermal and mechanical stability, self-healing properties, and a long period of stability. The physical properties of the gels were analysed by TEM microscopy, DSC calorimetry, and oscillatory rheology. The controlled release properties of acetylsalicylic acid, ibuprofen, and hydrocortisone were analysed by the LC-MS method. The influence of the oil type (sunflower, soybean, and olive oil) on gelation efficiency of diverse oxalamide derivatives was examined by oscillatory rheology. The oxalamide gelators showed thermoreversible and thixotropic properties in vegetable oils with a minimum gelation concentration of just 0.025 wt%. The substitution of palm fats with gelled sunflower oil applied in cocoa and milk spreads at gelator concentrations lower than 0.2 wt% have shown promising viscoelastic properties compared to that of the original food products.

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Lectins, carbohydrate-binding proteins, play important functions in all forms of life from bacteria and viruses to plants, animals, and humans, participating in cell-cell communication and pathogen binding. In an attempt to modify lectin functions, artificial lectin ligands were made usually as big dendrimeric or cluster multivalent glycomimetic structures. Here we synthesized a novel set of glycomimetic ligands through protection/deprotection multicomponent reactions (MCR) approach. Multivalent di-and tri-carbohydrate glycomimetics containing D-fructose, D-galactose, and D-allose moieties were prepared in 63-96% yield. MCR glycomimetics demonstrated different binding abilities for plant lectins Con A and UEA I, and human galectin-3. Information gained about the influence of molecule structure, multivalency and optical purity on the lectin binding ability can be used in lectin detection and sensitivity measurements to further facilitate understanding of carbohydrate recognition process.

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We report the synthesis of a structurally diverse library of chiral Nß-substituted 1,2-diazetidin-3-ones by a 4-center 3-component Ugi reaction comprising unprotected α-hydrazino acids. Various isocyanides and carbonyl compounds and both aldehydes and ketones were utilized. In addition, postcondensation modifications at three different sites have been demonstrated.

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A concise and practical strategy towards a novel class of 14-membered macrocycles containing an enediyne (Z-3-ene-1,5-diyne) structural unit is described. A highly modular assembly of various precursors via sequential Ugi/Sonogashira reactions allowed the preparation of hybrid enediyne-peptide macrocycles in most cases as single diastereoisomers. Selected macrocyclic compounds showed moderate antiproliferative activity, and can be considered as templates suitable for further diversification in terms of ring size, shape, and stereochemistry.

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Because of its quantitative character and capability for high-throughput screening, 1H nuclear magnetic resonance (NMR) spectroscopy is used extensively in the profiling of biofluids such as urine and blood plasma. However, the narrow frequency bandwidth of 1H NMR spectroscopy leads to a severe overlap of the spectra of components present in the complex mixtures such as biofluids. Therefore, 1H NMR-based metabolomics analysis is focused on targeted studies related to concentrations of the small number of metabolites. Here, we propose a library-based approach to quantify proportions of overlapping metabolites from 1H NMR mixture spectra. The method boils down to the linear non-negative least squares (NNLS) problem, whereas proportions of the pure components contained in the library stand for the unknowns. The method is validated on an estimation of the proportions of (i) the 78 pure spectra, presumably related to type 2 diabetes mellitus (T2DM), from their synthetic linear mixture; (ii) metabolites present in 62 1H NMR spectra of urine of subjects with T2DM and 62 1H NMR spectra of urine of control subjects. In both cases, the in-house library of 210 pure component 1H NMR spectra represented the design matrix in the related NNLS problem. The proposed method pinpoints 63 metabolites that in a statistically significant way discriminate the T2DM group from the control group and 46 metabolites discriminating control from the T2DM group. For several T2DM-discriminative metabolites, we prove their presence by independent analytical determination or by pointing out the corresponding findings in the published literature.

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C-glycosides represent an important group of naturally occurring glycosylation derivatives but are also efficient mimetics of native O-glycosides. Here, a one-pot four-component methodology is described toward a library of N-alkylated C-glycosyl amino acid derivatives comprising seven different isopropylidene-protected carbohydrate units. The applied methodology tolerates different amines and isocyanides and provides access to Ugi products in yields up to 85 %. X-ray analysis of selected products bearing three different carbohydrate motifs and comparison of their crystal structures with similar ones deposited in Cambridge Crystallographic Database revealed that four structures adopt different conformations, mostly not typical for peptide structures. This property opens the possibility to exploit here described N-alkylated C-glycosyl amino acid derivatives as templates to access different biotic and abiotic secondary structures.

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We applied a multicomponent approach to access a library of densely functionalized homo- and hetero-multivalent glycomimetics comprising aldehyde, amine, and isocyanide components related to isopropylidene-protected d-fructose, l-sorbose, d-galactose, and d-allose. Passerini products were obtained in very good yields (up to 78%) and high diastereoselectivities (up to 98:2). Three types of products were obtained by the Ugi reaction; along with the "classical" four-component product, α-acylaminoamides, a three-component α-aminoamides, and a four- component α-aminoacylamides were isolated. The presence of multiple pathways is rationalized by the structure of the imidate intermediate, mainly influenced by the amine component.

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Study of seven new guanidiniocarbonylpyrrole (GCP)-fluorophore conjugates interactions with dipeptidyl peptidase III (DPP III) showed that all compounds bind strongly (Ks ≈ µM) to enzyme active site, but with very different fluorimetric response (varying from quenching to strong increase), dependent on the fluorophore type and intramolecular pre-organisation of molecule. Positively charged lysine side chain improved significantly compound solubility but diminished fluorescence increase upon DPP III binding and completely abolished inhibitory effect on DPP III activity, whereas linker-neutral analogues showed stronger emission increase and were efficient enzyme inhibitors. By far the best fluorimetric response and inhibitive properties showed cyanine-GCP analogue, thus being promising lead compound for both enzyme sensing and bio-activity inhibiting (theragnostic) studies of DPP III in the future.Communicated by Ramaswamy H. Sarma.

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Carbohydrates and their conjugates are the most abundant natural products, with diverse and highly important biological roles. Synthetic glycoconjugates are versatile tools used to probe biological systems and interfere with them. In an endeavor to provide an efficient route to glycomimetics comprising structurally diverse carbohydrate units, we describe herein a robust, stereoselective, multicomponent approach. Isopropylidene-protected carbohydrate-derived aldehydes and ketones were utilized in the Passerini reaction, giving different glycosylated structures in high yields and diastereoselectivities up to 90:10 diastereomeric ratio (d.r). Access to highly valuable building blocks based on α-hydroxy C-glycosyl acids or more complex systems was elaborated by simple post-condensation methodologies.

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Due to its capability for high-throughput screening 1H nuclear magnetic resonance (NMR) spectroscopy is commonly used for metabolite research. The key problem in 1H NMR spectroscopy of multicomponent mixtures is overlapping of component signals and that is increasing with the number of components, their complexity and structural similarity. It makes metabolic profiling, that is carried out through matching acquired spectra with metabolites from the library, a hard problem. Here, we propose a method for nonlinear blind separation of highly correlated components spectra from a single 1H NMR mixture spectra. The method transforms a single nonlinear mixture into multiple high-dimensional reproducible kernel Hilbert Spaces (mRKHSs). Therein, highly correlated components are separated by sparseness constrained nonnegative matrix factorization in each induced RKHS. Afterwards, metabolites are identified through comparison of separated components with the library comprised of 160 pure components. Thereby, a significant number of them are expected to be related with diabetes type 2. Conceptually similar methodology for nonlinear blind separation of correlated components from two or more mixtures is presented in the Supplementary material. Single-mixture blind source separation is exemplified on: (i) annotation of five components spectra separated from one 1H NMR model mixture spectra; (ii) annotation of fifty five metabolites separated from one 1H NMR mixture spectra of urine of subjects with and without diabetes type 2. Arguably, it is for the first time a method for blind separation of a large number of components from a single nonlinear mixture has been proposed. Moreover, the proposed method pinpoints urinary creatine, glutamic acid and 5-hydroxyindoleacetic acid as the most prominent metabolites in samples from subjects with diabetes type 2, when compared to healthy controls.

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The γ-lactam motif is often found in naturally occurring compounds with diverse biological activities. We prepared a 28-member library of N-substituted γ-lactams following a single-pot, three-component Ugi reaction comprising bifunctional building block, l-glutamic acid methyl ester. The reaction tolerates structurally diverse carbonyl and isocyanide components providing a robust access to functionalized γ-lactams. Antimicrobial susceptibility testing, including agar well diffusion assay, serial microdilution broth assay, and antibiofilm activity testing, identified a potent compound with antibiofilm activity against Staphylococcus aureus ATCC 6538.

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We describe the utilization of bis-isopropylidene-protected d-fructose-derived aldehyde in the Passerini reaction with various acids and isocyanides. A library of densely functionalized glycomimetics bearing up to 3 carbohydrate units was obtained in high yields and diastereoselectivities. The configuration of the newly formed stereocenter was determined and the diastereoselectivity was rationalized by DFT calculations.

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Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.

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Multicomponent reactions represent a highly efficient approach to a broad spectrum of structurally diverse compounds starting from simple and affordable compounds. A focused library of tweezers-like compounds is prepared by employing the multicomponent Passerini reaction comprising enediyne-derived amino aldehydes. The reaction proceeds under mild conditions yielding Passerini products in good to excellent yields. Postcondensation modifications of Passerini products are demonstrated through a simple deprotection/coupling approach comprising amino functionality, furnishing enediyne cores with highly decorated arms.

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A series of novel hydrazino-based peptidomimetics and analogues comprising N-terminal lysine and C-terminal phenanthridinyl-l-alanine were prepared. The presented results demonstrate the up to now unknown possibility to finely modulate peptide interactions with DNA/RNA by α-hydrazino group insertion and how the different positioning of two α-hydrazino groups in peptides controls binding to various double stranded and single stranded DNA and RNA. All peptidomimetics bind with 1-10 micromolar affinity to ds-DNA/RNA, whereby the binding mode is a combination of electrostatic interactions and hydrophobic interactions within DNA/RNA grooves. Insertion of the α-hydrazino group into the peptide systematically decreased its fluorimetric response to DNA/RNA binding in the order: mono-hydrazino < alternating-hydrazino < sequential-hydrazino group. Binding studies of ss-polynucleotides suggest intercalation of phenanthridine between polynucleotide bases, whereby affinity and fluorimetric response decrease with the number of α-hydrazino groups in the peptide sequence. Particularly interesting was the interaction of two sequential α-hydrazino acids-peptidomimetic with poly rG, characterised by a specific strong increase of CD bands, while all other peptide/ssRNA combinations gave only a CD-band decrease. All mentioned interactions could also be reversibly controlled by adjusting the pH, due to the protonation of the fluorophore.

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Peptidomimetics based on hydrazino derivatives of α-amino acids represent an important class of peptidic foldamers with promising biological activities, like protease inhibition and antimicrobial activity. However, the lack of straightforward method for the synthesis of optically pure hydrazino acids and efficient incorporation of hydrazino building blocks into peptide sequence hamper wider exploitation of hydrazino peptidomimetics. Here we described the utility of N (α)-benzyl protected and unprotected hydrazino derivatives of natural α-amino acids in synthesis of peptidomimetics. While incorporation of N (α)-benzyl-hydrazino acids into peptide chain and deprotection of benzyl moiety proceeded with difficulties, unprotected hydrazino acids allowed fast and simple construction of hybrid peptidomimetics.

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The importance of turn-like peptide conformation for the copper(II) binding has been revealed by the synthesis of simple amino acid-based tweezers and the study of their interaction with copper(II). Amino acids Phe, Leu, Val, Ala and Gly were bridged through their C-terminuses with conformationally constrained motif, cis enediyne moiety ((Z)-octa-4-en-2,6-diyne-1,8-diamine). The interaction of prepared diamine ligands with copper(II) was studied by means of potentiometric titrations, UV-visible and EPR spectroscopic and mass spectrometric techniques. All ligands interact efficiently with copper(II) and form complexes of 1:1 stoichiometry differing in the protonation state of the ligand. LCu(2+) species were found predominant at pH<6.5, with log K* ranging from -8.06 to -6.65, while at higher pH deprotonation occurred, giving rise to LH(-1)Cu(+) complexes or LH(-2)Cu complex for the phenylalanine-related ligand. An additional species, LH(-3)Cu(-) were found at pH>9 for the valine- and alanine-related ligands, respectively. Comparing stability of studied complexes with those reported in previous work revealed that ligands effectively emulate properties of copper(II) binding peptides. Based on the results obtained in this work it can be concluded that structural rigidity significantly enhances coordination properties of the ligand, thus conforming importance of the turn-like peptide conformation for the copper(II) binding.

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Enediyne-peptide conjugates were designed with the aim to inhibit aminopeptidase N, a widespread ectoenzyme with a variety of functions, like protein digestion, inactivation of cytokines in the immune system and endogenous opioid peptides in the central nervous system. Enediyne moiety was embedded within the 12-membered ring with hydrophobic amino acid alanine, valine, leucine or phenylalanine used as carriers. Aromatic part of the enediyne bridging unit and the amino acid side chains were considered as pharmacophores for the binding to the aminopeptidase N (APN) active site. Additionally, the fused enediyne-amino acid "heads" were bound through a flexible linker to the L-lysine, an amino group donor. The synthesis included building the aromatic enediyne core at the C-terminal of amino acids and subsequent intramolecular N-alkylation. APN inhibition test revealed that the alanine-based derivative 9a inhibits the APN with IC(50) of 34 ± 11 µM. Enediyne-alanine conjugate 12 missing the flexible linker was much less effective in the APN inhibition. These results show that enediyne-fused amino acids have potential as new pharmacophores in the design of APN inhibitors.

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