Influence of a 4'-substituent on the efficiency of flavonol-based fluorescent indicators of ß-glycosidase activity.

This article presents novel fluorescent probes, based on the excited-state intramolecular proton transfer (ESIPT) phenomenon and flavonols, sensitive to the action of specific glycosidases. 4'-Substituted flavonols were synthesized, using various approaches, and glycosylated with d-glucose, N-acetyl-d-glucosamine and d-glucuronic acid. Evaluation of the ß-glycosidase activities was performed in neutral and acidic pH. In all the cases examined, an acidic environment accelerated enzymatic hydrolysis. It was demonstrated that the 4'-chloroflavonyl glycosides of all sugars tested, both in neutral and acidic pH, are the ones most sensitive to the presence of hydrolase. In turn, 4'-dimethylaminoflavonyl glucoside is not sensitive to glucosidase action at all. Generally, the rate of enzymatic hydrolysis increases as the electron-withdrawing nature of the 4'-substituent increases. An exception is the trifluoromethyl group which, in spite of having the most favourable Hammett constant, does not contribute enough to increase the rate of hydrolysis of its glucoside. The presented experimental results are supported by the electrostatic potential (ESP) analysis and related to the mechanisms of glycoside bond enzymatic hydrolysis.

Design, synthesis and biological evaluation of betulin-3-yl 2-amino-2-deoxy-ß-d-glycopyranosides.

Betulin is a natural pentacyclic triterpenoid, possessing a lupane-structure, with a wide range of pharmacological activities. Its weak hydrosolubility hinders the biological activity of the compound and its derivatives. To circumvent this problem, we synthesized and tested in vitro three d-glycosaminosides of betulin. The structure of betulin was modified by incorporation of 2-amino-2-deoxy-d-gluco- and -d-galactopyranosyl moieties to its C-3 position. So far betulinyl glycosides containing these amino-sugars have not been reported in the literature. The structure of the studied derivatives was confirmed by 1H and 13C NMR spectroscopy as well as mass spectrometry. The 28-O-acetylbetulin-3-yl 2-amino-2-deoxy-ß-d-glucopyranoside and betulin-3-yl 2-amino-2-deoxy-ß-d-gluco- and ß-d-galactopyranoside were tested against the human pathogenic fungi and Gram-positive and Gram-negative bacteria. Moreover, the MTT assay of their cytotoxicity was performed on the MCF-7 breast cancer cell line and on the HDFa, human dermal fibroblasts. The Ames test on mutagenic properties completed our biological assays.

Synthesis, Modification and Biological Activity of Diosgenyl ß-d-Glycosaminosides: An Overview.

Saponins are a structurally diverse class of natural glycosides that possess a broad spectrum of biological activities. They are composed of hydrophilic carbohydrate moiety and hydrophobic triterpenoid or steroid aglycon. Naturally occurring diosgenyl glycosides are the most abundant steroid saponins, and many of them exhibit various pharmacological properties. Herein, we present an overview of semisynthetic saponins syntheses-diosgenyl ß-d-glycosaminosides (d-gluco and d-galacto). These glycosides possess a 2-amino group, which creates great possibilities for further modifications. A wide group of glycosyl donors, different N-protecting groups and various reaction conditions used for their synthesis are presented. In addition, this paper demonstrates the possibilities of chemical modifications of diosgenyl ß-d-glycosaminosides, associated with functionalisation of the amino group. These provide N-acyl, N-alkyl, N,N-dialkyl, N-cinnamoyl, 2-ureido and 2-thiosemicarbazonyl derivatives of diosgenyl ß-d-glycosaminosides, for which the results of biological activity tests (antifungal, antibacterial, anti-cancer and hemolytic) are presented.

N-Aminoacyl and N-hydroxyacyl derivatives of diosgenyl 2-amino-2-deoxy-ß-d-glucopyranoside: Synthesis, antimicrobial and hemolytic activities.

Diosgenyl 2-amino-2-deoxy-ß-d-glucopyranoside is a semisynthetic saponin with antimicrobial and antitumor activities. To search for more effective analogues, N-aminoacyl and N-hydroxyacyl derivatives of this saponin were synthesized conventionally and with microwave assistance, and tested against the human pathogenic fungi and Gram-positive and Gram-negative bacteria. None of the tested compounds exhibit activity against Gram-negative bacteria. Almost all of the synthesized N-aminoacyl saponins exhibit antifungal activity and act effectively against Gram-positive bacteria, some better than the parent compound. The best acting saponins are the same size and possess sarcosine or l- or d-alanine attached to the parent glucosaminoside. Shorter and longer aminoacyl residues are less advantageous. d-Alanine derivative is the most effective against Gram positive bacteria. Structure-activity relationship (SAR) analysis indicates that the free α-amino group in aminoacyl residue is necessary for antimicrobial activities of the tested saponins. (N-Acetyl)aminoacyl and N-hydroxyacyl analogs are inactive. Measurements of the hemolytic activities demonstrate that the best acting saponins are not toxic towards human red blood cells.

5-Fluorouracil-Complete Insight into Its Neutral and Ionised Forms.

5-Fluorouracil (5FU), a common anti-cancer drug, occurs in four tautomeric forms and possesses two potential sites of both protonation and deprotonation. Tautomeric and resonance structures of the ionized forms of 5FU create the systems of connected equilibriums. Since there are contradictory reports on the ionized forms of 5FU in the literature, complex theoretical studies on neutral, protonated and deprotonated forms of 5FU, based on the broad spectrum of DFT methods, are presented. These indicate that the O4 oxygen is more willingly protonated than the O2 oxygen and the N1 nitrogen is more willingly deprotonated than the N3 nitrogen in a gas phase. Such preferences are due to advantageous charge delocalization of the respective ions, which is demonstrated by the NBO and ESP analyses. In an aqueous phase, stability differences between respective protonated and deprotonated forms of 5FU are significantly diminished due to the competition between the mesomeric effect and solvation. The calculated pKa values of the protonated, neutral and singly deprotonated 5FU indicate that 5FU does not exist in the protonated and double-deprotonated forms in the pH range of 0-14. The neutral form dominates below pH 8 and the N1 deprotonated form dominates above pH 8.

Diosgenyl 2-amino-2-deoxy-ß-D-galactopyranoside: synthesis, derivatives and antimicrobial activity.

The synthesis of diosgenyl 2-amino-2-deoxy-ß-D-galactopyranoside is presented for the first time. This synthetic saponin was transformed into its hydrochloride as well as N-acyl, 2-ureido, N-alkyl, and N,N-dialkyl derivatives. Antifungal and antibacterial studies show that some of the obtained compounds are active against Gram-positive bacteria and Candida type fungi.

Theoretical studies on the reaction of mono- and ditriflate derivatives of 1,4:3,6-dianhydro-D-mannitol with trimethylamine--Can a quaternary ammonium salt be a source of the methyl group?

DFT studies on the mechanism of the formation of "gemini" quaternary ammonium salts in the reaction of 1,4:3,6-dianhydro-D-mannitol ditriflate derivative with trimethylamine and its subsequent conversion to tertiary amine through the methyl-transfer reaction are discussed. Two alternative reaction pathways are presented in the gas phase and in ethanol. Additionally, the transformation of the monotriflate derivative of 1,4:3,6-dianhydro-D-mannitol into the single quaternary ammonium salt is presented. Two functionals (B3LYP, M062X) and two basis sets (6-31+G** and 6-311++G**) were used for the calculations. The effect of the substituent attached to the five-membered rings at the C2 (and/or C5) carbon atom on the activation barrier is described. The trimethylammonium group bond to the five-membered ring greatly reduces the activation barrier height. The preferred reaction pathway for the conversions was established. Including the London dispersion in the calculations increases the stabilization of all the points on the potential energy surface in relation to individual reactants.

N-Alkyl derivatives of diosgenyl 2-amino-2-deoxy-ß-D-glucopyranoside; synthesis and antimicrobial activity.

Diosgenyl 2-amino-2-deoxy-ß-D-glucopyranoside is a synthetic saponin exhibiting attractive pharmacological properties. Different pathways tested by us to obtain this glycoside are summarized here. Moreover, the synthesis of N-alkyl and N,N-dialkyl derivatives of the glucopyranoside is presented. Evaluation of antibacterial and antifungal activities of these derivatives indicates that they have no inhibitory activity against Gram-negative bacteria, whereas many of the tested N-alkyl saponins were found to inhibit the growth of Gram-positive bacteria and human pathogenic fungi.

Conformational preferences of guanine-containing threose nucleic acid building blocks in B3LYP studies.

In this paper, detailed and systematic gas-phase B3LYP conformational studies of four monomers of threose nucleic acid (TNA) with guanine attached at the C1' atom and bearing different substituents (OH, OP(=O)OH2 and OCH3) in the C2' and C3' positions of the α-l-threofuranose moiety are presented. All exocyclic single-bond (χ, ε and γ) rotations, as well as the ν0-ν4 endocyclic torsion angles, were taken into consideration. Three (threoguanosines TG1-TG3) or two (TG4) energy minima were found for the rotation about the χ torsion angle. The syn orientation (the A rotamer family) is strongly privileged in geometries TG1 and TG2, whereas the anti orientation (the C rotamer family) and the syn orientation are observed to be in equilibrium (with populations of 56% and 44%, respectively) for TG3. In the case of TG4, the high-anti orientation (the B rotamer family) turned out to be by far the most favourable, with the contribution exceeding 90% in equilibrium. Such a preference can be attributed to the inability of H-bonding between sugar and nucleobase and possibly because of the steric strains. The low-energy conformers of TG1-TG4 occupy the northeastern (P âˆ¼ 40°) and/or southern (P âˆ¼ 210°) parts of the pseudorotational wheel, which fits the A- and B-type DNA helices quite well. Additionally, in the case of TG4, some relatively stable geometries have the furanoid ring in conformation lying on the northwestern part of the pseudorotational wheel (P âˆ¼ 288°).

Identification of the furanose ring conformations and the factors driving their adoption.

Three groups of furanoses with restricted freedom of rotation on the C3-C4, C2-C3, and C1-C2 bonds, respectively, are presented. Conformational analysis of these furanoses is conducted based on the proton nuclear magnetic resonance (1H NMR) spectroscopy, density functional theory (DFT) calculations, and X-ray analysis. It is shown that the particular group of the presented furanoses is locked in the specific conformation. These are the 1T2-like, the 0E-like, and the 3T4-like conformation, respectively. Characteristic 1H NMR spectra of these three conformations are presented and the factors influencing the conformational preferences of the analyzed furanoses are discussed.

Synthesis, Antimicrobial and Mutagenic Activity of a New Class of d-Xylopyranosides.

Eight N-[2-(2',3',4'-tri-O-acetyl-α/ß-d-xylopyranosyloxy)ethyl]ammonium bromides, a new class of d-xylopyranosides containing a quaternary ammonium aglycone, were obtained. Their complete structure was confirmed using NMR spectroscopy (1H, 13C, COSY and HSQC) and high-resolution mass spectrometry (HRMS). An antimicrobial activity against fungi (Candida albicans, Candida glabrata) and bacteria (Staphylococcus aureus, Escherichia coli) and a mutagenic Ames test with Salmonella typhimurium TA 98 strain were performed for the obtained compounds. The greatest activity against the tested microorganisms was shown by glycosides with the longest (octyl) hydrocarbon chain in ammonium salt. None of the tested compounds exhibited mutagenic activity in the Ames test.

Characteristic 1H NMR spectra of ß-d-ribofuranosides and ribonucleosides: factors driving furanose ring conformations.

A series of ß-d-ribofuranosides and ribonucleosides fused with 2,3-O-isopropylidene ring was synthesized and studied in terms of their conformational preferences. Based on the 1H NMR spectra, DFT calculations, and X-ray analysis the E 0-like and E 4-like conformations adopted by these furanosides are identified. The 3 E-like and 2 E-like conformations are assigned to ribonucleosides without the 2,3-O-isopropylidene group. The studies are supported by analysis of the structural data of ß-d-ribofuranosides and ribonucleosides deposited in the Cambridge Crystallographic Data Center (CCDC) database. Finally, the factors influencing the conformational preferences of the furanose ring with the ß-d-ribo configuration are indicated. These are the unfavorable ecliptic orientation of the 2-OH and 3-OH groups, the 1,3-pseudodiaxial interaction of the aglycone and terminal hydroxymethyl group and the endo-anomeric effect. It is also proved that the exo-anomeric effect acts in ß-d-ribofuranosides.

Vibrationally Assisted Direct Intersystem Crossing between the Same Charge-Transfer States for Thermally Activated Delayed Fluorescence: Analysis by Marcus-Hush Theory Including Reorganization Energy.

Thermally activated delayed fluorescence (TADF) has recently become an extensively investigated phenomenon due to its high potential for application in organic optoelectronics. Currently, there is still lack of a model describing correctly basic photophysical parameters of organic TADF emitters. This article presents such a photophysical model describing the rates of intersystem crossing (ISC), reverse ISC (rISC), and radiative deactivation in various media and emphasizing key importance of molecular vibrations on the example of a popular TADF dye 9,10-dihydro-9,9-dimethyl-10-(4-(4,6-diphenyl-1,3,5-triazin-2-yl)phenyl)-acridine (DMAC-TRZ). The presented experimental and theoretical investigations prove that ISC and rISC can occur efficiently between the singlet and triplet states of the same charge-transfer nature (1CT and 3CT, respectively). In emitters with the orthogonal donor and acceptor fragments, such spin-forbidden 1CT ↔ 3CT transitions are activated by molecular vibrations. Namely, the change of dihedral angle between the donor and the acceptor affords reasonable spin-orbit coupling, which together with a small energy gap and reorganization energy enable 1CT ↔ 3CT transition rates reaching 1 × 107 s-1. Evidence of direct 1CT ↔ 3CT spin-flip and negligible role of a second triplet state, widely believed as a key parameter in the design of (r)ISC materials, change significantly the current understanding of TADF mechanism. In authors' opinion, photophysics, and molecular design principles of TADF emitters should be revised considering the importance of vibrationally enhanced 1CT ↔ 3CT transitions.

Cyclophosphamide and isophosphamide - DFT conformational studies in the gas phase and solution.

Cyclophosphamide and isophosphamide have been subjected to comprehensive conformational studies in the vacuum and solution using the SMD solvation model. Vacuum calculations were conducted using the B3LYP, M05-2X, M06-2X and ωB97XD functionals. Natural bond orbital (NBO) analysis has been performed for selected geometries. A preference for a chair conformation with the axial P=O bond is shown (1C4). The 5S0 conformation is 1.25-2.31 kcal/mol and 1.72-2.92 kcal/mol higher in energy than the global minimum conformations of cyclophosphamide and isophosphamide, respectively. In the gas phase, the chair conformation with the equatorial P=O bond (4C1) is of comparable stability or less stable than the skew form, depending on the method used, while it is slightly more favored than the 5S0 conformation in solution. The stereoelectronic effects do not differentiate the ring conformer stability. The steric strains between N(EtCl)1-2 and the C4 and C6 carbon atoms mainly influence the stability of cyclophosphamide and isophosphamide conformers.

Methyl 3-amino-2,3,6,-trideoxy-L-hexopyranosides in DFT level theory conformational studies.

Geometry optimizations at the B3LYP level of density functional theory are reported for the (1)C4 and (4)C1 conformations of four theoretically possible alpha and beta methyl 3-amino-2,3,6-trideoxy-L-hexopyranosides. The Gibbs free energies, relative Gibbs free energies, and geometry parameters are presented for all the optimized structures. Conformational analysis of the pyranose ring is performed for each stereoisomer on the basis of calculated rotamer populations. It is demonstrated that the alpha/beta-L-arabino, alpha/beta-L-lyxo, and alpha-L-ribo stereoisomers adopt the (1)C4 conformation, whereas beta-L-ribo and alpha/beta-L-xylo stereoisomers remain in (1)C4 <==> (4)C1 conformational equilibrium. The preference of the alpha over the beta anomers is due to the endo-anomeric effect. The factors affecting the stability of pyranose ring conformations are discussed, as is the influence of hydrogen bonds on the orientation of the hydroxyl and amino groups. Figures of the most stable conformers are presented.

Synthesis and conformational analysis of methyl 3-amino-2,3-dideoxyhexopyranosiduronic acids, new sugar amino acids, and their diglycotides.

The synthesis of methyl 3-azido- and 3-amino-2,3-dideoxyhexopyranosiduronic acids and their methyl esters with the -alpha,beta-D-arabino-, -alpha,beta-D-ribo, and -alpha,beta-L-lyxo configurations is presented. The conformations of the synthesized sugar amino acids and their precursors are discussed on the basis of 1H NMR data. The influence of the 5-carboxyl group on the pyranose ring conformation is assessed, and the bonding of the monosugar amino acids into dimeric glycotides, using conventional solution-phase peptide syntheses, is reported.

Synthesis and geometry of methyl (methyl 4-O-acetyl-3-azido-2,3-dideoxy-alpha/beta-D-arabino- and -alpha/beta-D-ribo-hexopyranosid)uronates.

The synthesis of methyl (methyl 4-O-acetyl-3-azido-2,3-dideoxy-alpha/beta-D-arabino- and -alpha/beta-D-ribo-hexopyranosid)uronates is presented. High resolution (1)H and (13)C NMR spectral data for all diastereoisomers and single-crystal X-ray diffraction analysis for methyl (methyl 3-azido-2,3-dideoxy-beta-D-arabino-hexopyranosid)uronate are reported. The planarity of the 4-OAc and 5-COOMe groups as well as the orientations of the aglycone and azide groups in the crystal lattice is discussed. The influence of the 5-COOMe group on the pyranose ring conformation is considered.

Comparative conformational studies of 3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxyhex-1-enitols at the DFT level.

B3LYP and M06-2X optimization and MP2 single point calculations are reported for the 4H5 and 5H4 conformations of 3,4,6-tri-O-acetyl-D-allal, 3,4,6-tri-O-acetyl-D-galactal, 3,4,6-tri-O-acetyl-D-glucal, and 3,4,6-tri-O-acetyl-D-gulal. Significant discrepancies in predictions of relative energies and conformers' population for B3LYP and M06-2X optimized geometries are observed. Generally, B3LYP overestimates the conformers' energies with respect to MP2, whereas M06-2X slightly underestimates the conformers' energies. B3LYP failed to estimate the 4H5⇄5H4 conformational equilibrium for 3,4,6-tri-O-acetyl-D-galactal and 3,4,6-tri-O-acetyl-D-glucal. The M06-2X functional showed good agreement with experimental results for all glycals studied. The 4H5⇄5H4 conformational equilibrium for 3,4,6-tri-O-acetyl-D-allal and 3,4,6-tri-O-acetyl-D-gulal is governed by the vinylogous anomeric effect (VAE), whereas competition between the VAE and quasi 1,3-diaxial interactions influence this equilibrium for 3,4,6-tri-O-acetyl-D-galactal and 3,4,6-tri-O-acetyl-D-glucal. The orientation of the 4-OAc group influences the strength of the quasi 1,3-diaxial interactions between the 3-OAc and 5-CH2OAc groups. AIM analysis shows weak bonding interaction between the 3-OAc and 5-CH2OAc groups.

Activity of Diosgenyl 2-amino-2-deoxy-ß-D-glucopyranoside, its Hydrochloride, and N,N-dialkyl Derivatives Against Non-albicans Candida Isolates.

BACKGROUND: Candida albicans belongs to the most common fungal pathogens in humans, but recently an increased proliferation of strains called non-albicans Candida has been reported. Species belonging to this group are often characterised by a reduced susceptibility to antifungal agents. OBJECTIVE: In view of the emergence of non-albicans Candida and their resistance to available antifungals, an attempt has been made to develop novel effective agents. Biological activities of the N,N-dialkyl diosgenyl glycosides, which were previously synthesized, were determined. METHOD: Minimum inhibitory concentration (MIC) was determined for group of clinical nonalbicans Candida isolates by serial dilution method in Sabouraud liquid medium. In order to assess the toxicity towards human cells the minimum haemolytic concentration (MHC) was determined on human erythrocytes by serial dilution method in phosphoric buffer. RESULTS: The saponins exhibited a strong activity towards clinical isolates of C. glabrata and C. parapsilosis comparable or even stronger than that of conventional antimicrobials. A high rate of resistance to fluconazole was shown among C. glabrata isolates. Among clinical strains of C. krusei and C. tropicalis, isolates with a decreased susceptibility to saponins were identified. All the tested C. krusei isolates showed resistance to fluconazole, while among C. tropicalis numerous strains were resistant to all tested azoles. The saponins did not show haemolytic activities at their microbiologically active concentrations. CONCLUSION: Results of the present work encourage to continue the study on steroidal saponins and their potential application for the treatment of candidemia.

Threocytidines: Insight into the Conformational Preferences of Artificial Threose Nucleic Acid (TNA) Building Blocks in B3LYP Studies.

A systematic DFT conformational studies of four building blocks of TNA with cytosine attached to the C1' atom of the α-L-threofuranose moiety are presented. Structures bearing 2'-OR and 3'-OR substituents, where R represents H, CH3 and phosphate groups, were used in the studies using a B3LYP functional in the gas phase. The χ angle (C2-N1-C1'-O4'), the ν0-ν4 endocyclic torsion angles and the exocyclic torsion angles ε (X-O2'-C2'-C1') and γ (X-O3'-C3'-C2') geometry parameter variations were taken into consideration. Three energy minima, high-anti, anti and syn, were found for the rotation about the C1'-N1 bond. The high-anti orientation of the base with respect to the sugar moiety, turned out to be preferred, regardless of the substituents at the C2' and C3' positions. Other orientations are at least 1.65 kcal/mol higher in Gibbs free energy than the high-anti one. It has been shown that intramolecular H-bonds and the anomeric effect of phosphate groups strongly affect the conformational preferences of the studied compounds. Further, the structure of substituents attached to the sugar moiety influence the pucker of the furanoid ring. The furanoid ring in the global minima of the compound with two OH groups (TC1) in the 2' and 3' positions, and the compound having a 3'-phosphate group (TC2), adopt roughly the same conformation located at the southern range of the pseudorotation wheel, and thus are close to those found in the B type DNA helix. The low-energy high-anti rotamers of the geometry with the phosphate group attached to the sugar ring in the 2' position (TC3) and the geometry with two methoxyl groups (TC4) have their furanoid rings in conformations resembling those found in A DNA and RNA helices (the northern range of the pseudorotation wheel).