Schiff Base Intermediate Research Articles

Recyclable and Stable α-Methylproline-Derived Chiral Ligands for the Chemical Dynamic Kinetic Resolution of free C,N-Unprotected α-Amino Acids.

A novel special designed, stable, and recyclable chiral ligand bearing a quaternary carbon was developed for chemical dynamic kinetic resolution (DKR) of free C,N-unprotected racemic α-amino acids via Schiff base intermediates. This method furnishes high yields with excellent enantioselectivity, has a broad substrate scope, and uses operationally simple and convenient conditions. The present chemical DKR is a practical and useful method for the preparation of enantiopure α-amino acids.

Glycation of Plant Proteins: Regulatory Roles and Interplay with Sugar Signalling?

Glycation can be defined as an array of non-enzymatic post-translational modifications of proteins formed by their interaction with reducing carbohydrates and carbonyl products of their degradation. Initial steps of this process rely on reducing sugars and result in the formation of early glycation products—Amadori and Heyns compounds via Schiff base intermediates, whereas their oxidative degradation or reactions of proteins with α-dicarbonyl compounds yield a heterogeneous group of advanced glycation end products (AGEs). These compounds accompany thermal processing of protein-containing foods and are known to impact on ageing, pathogenesis of diabetes mellitus and Alzheimer’s disease in mammals. Surprisingly, despite high tissue carbohydrate contents, glycation of plant proteins was addressed only recently and its physiological role in plants is still not understood. Therefore, here we summarize and critically discuss the first steps done in the field of plant protein glycation during the last decade. We consider the main features of plant glycated proteome and discuss them in the context of characteristic metabolic background. Further, we address the possible role of protein glycation in plants and consider its probable contribution to protein degradation, methylglyoxal and sugar signalling, as well as interplay with antioxidant defense.

Synthesis, Characterization and Antimicrobial Activity of p-Vanillin and Vanillin Schiff Bases

Introduction: The unending interest in the study of Schiff base ligands arise from the ease of their preparation and their versatility in several fields of Chemistry and Biochemistry. Some of the major biochemical processes such as trans-amination and glycosylation involve the formation of Schiff base intermediates. In addition, compounds containing the azomethine group have been found to often possess different biological activity such as anti-microbial, anti-viral and anti-inflammatory activity. In most cases, the extent of the potency depends on the nature of the constituent amino and or aldehyde moiety. Aims: The aim of this study was to synthesize, characterize and evaluate the antimicrobial activity of Schiff base ligands derived from p-vanillin and vanillin. Materials and Methods: The Schiff base ligands were prepared by condensing p-vanillin and vanillin with substituted aniline, aminonaphthalene and 3-aminopyridine respectively. The ligands have been characterized by elemental analysis, 1H- & 13C-NMR, infrared, Raman and electronic spectral data. The antimicrobial study was carried out by screening the prepared ligands against Staphylococcus aureus subsp. aureus ATCC® 6538™, Bacillus substillis subsp. spizizenii ATCC® 6633™*, Escherichia coli ATCC® 8739™* and Candida albicans ATCC® 2091™* using agar diffusion technique. Results: The azomethine, HC=N, 1H- & 13C-NMR signals were observed at 8.66-8.30 ppm and 164.42-157.99 ppm respectively. The infrared band appeared at 1622-1607 cm-1. The p-vanillin ligands exhibited higher activity than the vanillin based ligands. The p-hydroxyl and 3-aminopyridine ligands possess significant antimicrobial activity especially, antifungal activity. Conclusion: The spectroscopic spectral data of the compounds confirmed the formation of the Schiff base ligands. The antimicrobial activity of the compounds depends largely on the nature of the aldehyde moiety.

Histone Tail Sequences Balance Their Role in Genetic Regulation and the Need To Protect DNA against Destruction in Nucleosome Core Particles Containing Abasic Sites

Abasic sites (AP) are produced 10 000 times per day in a single cell. Strand cleavage at AP is accelerated ≈100-fold within a nucleosome core particle (NCP) compared to free DNA. The lysine-rich N-terminal tails of histone proteins catalyze single-strand breaks through a mechanism used by base-excision-repair enzymes, despite the general dearth of glutamic acid, aspartic acid, and histidine-the amino acids that are typically responsible for deprotonation of Schiff base intermediates. Incorporating glutamic acid, aspartic acid, or histidine proximal to lysine residues in histone N-terminal tails increases AP reactivity as much as sixfold. The rate acceleration is due to more facile DNA cleavage of Schiff-base intermediates. These observations raise the possibility that histone proteins could have evolved to minimize the presence of histidine, glutamic acid, and aspartic acid in their lysine-rich N-terminal tails to guard against enhancing the toxic effects of DNA damage.

Study of the Lyase Activity of Human DNA Polymerase β Using Analogues of the Intermediate Schiff Base Complex.

DNA polymerase β (Pol β) participates in mammalian base excision repair. The enzyme has a two-domain architecture, reflecting its dual functionality. The polymerase activity, which replaces damaged nucleosides removed during an initial excision process, is within the C-terminal 31 kDa domain, while the N-terminal 8 kDa domain participates in a lyase function, working to remove a 5'-deoxyribose phosphate (5'-dRP) moiety from the damaged DNA substrate. The currently accepted mechanism of the lyase reaction involves a transient covalent enzyme-DNA intermediate in the form of a Schiff base connecting Lys72 of the enzyme with the 5'-dRP moiety. The Schiff base intermediate is resolved via a β-elimination reaction, initiated by abstraction of a C2'-H atom from the 5'-dRP moiety. Presently, we describe the preparation of three Pol β enzymes modified at position 72 with aminooxy or hydrazinyl analogues of lysine. These enzymes form transient covalent bonds with the 5'-dRP moiety of the damaged DNA, in the form of an oxime or hydrazone, respectively. Both types of enzyme DNA intermediates are ultimately resolved by the lyase activities of each of the modified enzymes. Unsurprisingly, the formation and resolution of these E-S complexes proceed with diminished kinetics, and with an altered pH profile. The performed experiments provide additional support for Schiff base formation as an obligatory intermediate on the pathway to DNA repair, as well as for the proposed participation of Lys72 in effecting opening of the 5'-dRP ring via protonation of the ring oxygen atom, and for complex resolution via a β-elimination reaction.

Optimization of Enzyme Mechanism along the Evolutionary Trajectory of a Computationally Designed (Retro-)Aldolase.

De novo biocatalysts have been successfully generated by computational design and subsequent experimental optimization. Here, we examined the evolutionary history of the computationally designed (retro-)aldolase RA95. The modest activity of the starting enzyme was previously improved 105-fold over many rounds of mutagenesis and screening to afford a proficient biocatalyst for enantioselective cleavage and synthesis of β-hydroxyketones. Using a set of representative RA95 variants, we probed individual steps in the multistep reaction pathway to determine which processes limit steady-state turnover and how mutations that accumulated along the evolutionary trajectory influenced the kinetic mechanism. We found that the overall rate-limiting step for aldol cleavage shifted from C-C bond scission (or an earlier step in the pathway) for the computational design to product release for the evolved enzymes. Specifically, interconversion of Schiff base and enamine intermediates, formed covalently between acetone and the catalytic lysine residue, was found to be the slowest step for the most active variants. A complex hydrogen bond network of four active site residues, which was installed in the late stages of laboratory evolution, apparently enhances lysine reactivity and facilitates efficient proton shuffling. This catalytic tetrad accounts for the tremendous rate acceleration observed for all steps of the mechanism, most notably Schiff base formation and hydrolysis. Comparison of our results with kinetic and structural studies on natural aldolases provides valuable feedback for computational enzyme design and laboratory evolution approaches alike.

Synthesis, Characterization & Screening for Anti-Inflammatory & Analgesic Activity of Quinoline Derivatives Bearing Azetidinones Scaffolds.

Non-steroidal anti-inflammatory drugs (NSAIDS) are clinically used as anti-inflammatory, analgesic and antipyretic agents but they have the drawbacks such as gastric irritation and gastric ulceration. Recently, quinoline derivatives have shown significant anti-inflammatory and less ulcerogenic activity. The present study deals with the synthesis and pharmacological assessment of a series of novel quinoline derivatives bearing azetidinones scaffolds as anti-inflammatory and analgesic agents. A series of newer 3-chloro-1-(substituted)-4-(tetrazolo [1,5-a]quinolin-4- yl)azetidin-2-one derivatives (6a-l) was synthesized starting with acetanilide (1). Initially, acetanilide (1) was allowed to react with Vilsmeier-Haack reagent (DMF + POCl3) to form 2- chloro-3-formyl quinoline (2). The 2-chloro-3-formyl quinoline (2) was further treated with p-toluenesulphonic acid and sodium azide which yielded Tetrazolo [1,5-1] quinoline-4- carbaldehyde (3). The reaction of formyl group with various substituted amines (4a-l) formed corresponding Schiff base intermediates (5a-l), which were further allowed to react with chloroacetyl chloride to produce 3-chloro-1-(substituted)-4-(tetrazolo [1,5-a]quinolin-4-yl) azetidin-2-one derivatives (6a-l). The structure of the final analogues (6a-l) has been confirmed on the basis of elemental analysis, IR, 1H NMR, 13C NMR and mass spectra. All the synthesized compounds were evaluated for their anti-inflammatory and analgesic activities by using carrageenan induced rat paw model and Eddy's hot plate method respectively. All the values of elemental analysis, IR, 1H NMR, 13C NMR and mass spectra were found to be prominent. The anti-inflammatory activity test revealed that 3-chloro-1-(4-methoxyphenyl)- 4-(tetrazolo[1,5-a] quinolin-4-yl)azetidin-2-one (6b), 3-chloro-1-(2-methoxyphenyl)- 4-(tetrazolo[1,5-a]quinolin-4-yl)azetidin-2-one (6a) exhibited significant anti-inflammatory and analgesic activity as compared to control group. The results of the current study indicate that substitution at quinoline derivatives bearing azetidinones scaffolds showed potent analgesic and anti-inflammatory activities.

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

A One‐Pot Copper(II)‐Catalyzed Tandem Synthesis of 2‐Substituted Pyrrolo[1,2‐b]pyridazin‐4(1H)‐ones

AbstractA one‐pot copper(II)‐catalyzed tandem synthesis of 2‐substituted pyrrolo[1,2‐b]pyridazin‐4(1H)‐ones from N‐aminopyrroles was developed. This tandem reaction involves a Conrad–Limpach‐type reaction, including the thermal condensation of N‐aminopyrroles with the carbonyl group of β‐oxo esters followed by the cyclization of Schiff base intermediates. Compared to the traditional Conrad–Limpach quinoline synthesis, we herein successfully applied copper(II) as a catalyst in this transformation to furnish 2‐substituted pyrrolo[1,2‐b]pyridazin‐4(1H)‐ones for the first time. Most of the substrates bearing electron‐donating (EDG) and electron‐withdrawing (EWG) groups worked well with this procedure. The corresponding products could be converted directly into diverse pyrrolo[1,2‐b]pyridazine for drug discovery and materials science.

Reactivity of damaged pyrimidines: formation of a Schiff base intermediate at the glycosidic bond of saturated dihydrouridine.

DNA glycosylases catalyze the first step of the base excision repair (BER) pathway. The chemistry used by these enzymes for deglycosylation has been largely considered as the chemistry of the oxocarbenium ion, e.g., direct rupture of the C1'-N1 bond resulting in an oxocarbenium ion intermediate. Here we present mechanistic studies revealing the 2'-deoxyribose isomerization and subsequent deglycosylation processes in two pyrimidine lesions: 5,6-dihydro-2'-deoxyuridine (dHdU) and 5,6-dihydrothymidine (dHT), formed via ionizing radiation damage to 2'-deoxycytidine and thymidine, respectively, under anoxic conditions. Acid or heat treatment of these two lesions leads to the production of two pairs of C1' epimers containing a pyranose and a furanose, respectively, indicating that both lesions favor the rupture of the C1'-O4' bond, resulting in a Schiff base intermediate at the N-glycosidic bond. Such a Schiff base intermediate was trapped and characterized by either Pd-catalyzed hydrogenation or thiol-mediated addition reaction. In contrast, in undamaged 2'-deoxyuridine and thymidine, reactions at elevated temperatures lead to the release of nucleobases most likely via the traditional oxocarbenium ion pathway. DFT calculations further support the experimental findings, suggesting that the oxocarbenium ion intermediate is responsible for the deglycosylation process if the integrity of the pyrimidine ring is maintained, while the Schiff base intermediate is preferred if the C5═C6 bond is saturated. Currently, the oxocarbenium ion pathway is indicated to be solely responsible for the deglycosylation in BER enzymes, however our results suggest an alternative Schiff base mechanism which may be responsible for the repair of saturated pyrimidine damages.

ChemInform Abstract: Mechanisms for Enzymatic Cleavage of the N‐Glycosidic Bond in DNA

AbstractReview: 101 refs.

Design, Synthesis and Cytotoxic evaluation of Novel 2-(4-N, N-Dimethyl) pyridine containing 1, 3, 4-oxadiazole moiety

Anew series of novel 1,3,4-oxadiazoles derivatives have been synthesized by linear synthetic method and screened these compounds for anticancer activity on HeLa, MCF7, Caco-2 cell lines. Final 1, 3, 4-oxadiazoles 6a-6f were characterized by LCMS,1H NMR,13C, and elemental analysis. Oxidative cyclisation reaction of the corresponding key intermediate Schiff base derivatives 5a-5f yielded the desired novel 1, 3, 4-oxadiazoles. Most of the compounds in this series showed moderate cytotoxicity on all the cell lines, but compounds 6d and 6f is more cytotoxic on Caco-2 and HeLa cell lines respectively. The standard used was 5-FU and cytotoxicity of the compounds were compared with the 5-FU. The IC50 values of compounds 6d and 6fis 2.4μM and 5.3μM on Caco-2 and HeLa cell lines respectively.

ChemInform Abstract: Synthesis and Functionalization of 3‐Azolylquinoxalin‐2(1H)‐ones.

AbstractThe formation of compound (IX) as a single isomer starting from an 1:1 cis/trans‐mixture of diamine (VIII) is explained by epimerization during formation of the intermediate Schiff base.

ChemInform Abstract: Synthesis of 2,5‐Disubstitued Benzimidazole Using SnCl2‐Catalyzed Reduction System at Room Temperature.

AbstractReaction of aniline (I) with (II) gives an intermediate Schiff base which is directly converted into benzimidazole products in high yields.

Discovery of the Highly Potent Fluoroquinolone‐Based Benzothiazolyl‐4‐thiazolidinone Hybrids as Antibacterials

A new series of fluoroquinolone-based benzothiazolyl-4-thiazolidinone hybrids has been yielded via sulfated tungstate-promoted highly accelerated N-formylation at a piperazine residue of ciprofloxacin and norfloxacin entities. The formylated fluoroquinolone moieties were then coupled with substituted 2-aminobenzothiazoles, which were generated from their respective para-substituted amines to form corresponding Schiff base intermediates. The Schiff bases were then treated with thioglycolic acid to equip a new class of 4-thiazolidinones to be analyzed for their antibacterial effects against two Gram-positive (Staphylococcus aureus and Bacillus subtilis) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains and were found highly potent with lowest Minimum inhibitory concentrations (MIC), 1-2 μg/mL, that is, more potent than control drugs ciprofloxacin (3.12-6.25 μg/mL). Initial outcomes provided for these novel molecular systems will aid researchers to design and develop new antibacterial drugs. The structural assignments of the new products were done on the basis of FT-IR, (1) H NMR and (13) C NMR spectroscopy, and elemental analysis.

Synthesis of highly potent piperazine-linked benzothiazolyl-4-thiazolidinones via catalytic N-formylation: antibacterial drug design

A new series of piperazine-based benzothiazolyl-4-thiazolidinones has been yielded efficiently via highly accelerated N-formylation of 1-(4-methoxyphenyl) piperazine using sulfated tungstate, a mild heterogeneous catalyst. The formylated piperazine moiety was then condensed with substituted 2-aminobenzothiazoles to form corresponding Schiff base intermediates which were then treated with thioglycolic acid to equip a new class of 4-thiazolidinones. New products were examined for their antibacterial effects against two Gram-positive (S. aureus and B. subtilis) and two Gram-negative (E. coli and P. aeruginosa) strains and were highly potent with lowest MIC values 1–4 μg mL−1, more potent than control drugs ciprofloxacin (3.12–6.25 μg mL−1). The structural assignments of the new products were done on the basis of FT-IR, 1H NMR, 13C NMR spectroscopy, and elemental analysis.

QM/MM studies of the mechanism of unusual bifunctional fructose-1,6-bisphosphate aldolase/phosphatase.

Archaeal fructose-1,6-bisphosphate aldolase/phosphatase (FBPA/P) is a newly identified unusual bifunctional enzyme (Nature, 2010, 464, 1077), which contains one single catalytic domain but catalyzes two chemically distinct reactions of gluconeogenesis. It is different from the ordinary enzymes whose active sites are responsible for a specific reaction. To explore the catalytic characteristic of FBPA/P, the aldol condensation mechanism of bifunctional FBPA/P has been investigated using quantum mechanics/molecular mechanics (QM/MM) method. The whole reaction process can be divided into two half-reactions involving seven elementary steps. A Schiff base intermediate is theoretically confirmed, agreeing well with the recently resolved crystal structures (Nature, 2011, 478, 538). The free energy barrier of the rate-limiting step is calculated to be 22.2 kcal mol(-1), which is a concerted process of a nucleophilic attack by the enolic carbon to the ketonic carbon and a proton transfer from Tyr229 to the ketonic oxygen. Lys232 plays an important role in forming a Schiff base intermediate with the substrate (DHAP). Tyr229 functions as a proton shuttle during the catalysis. This is the first theoretical study on the aldol condensation mechanism of FBPA/P, which may provide useful information for understanding bifunctional enzymes.

Synthesis of Some New Pyrimidine-Azitidinone Analogues and Their Antioxidant,In VitroAntimicrobial, and Antitubercular Activities

A series of 1-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-3-chloro-4-(2-mercaptoquinolin-3-yl)azetidin-2-one (7a-j) have been synthesized from the condensation of aromatic amines withN-phenylacetamide. The thione nucleus formed from 2-chloroquionoline-3-carbadehyde using sodium sulphide in dimethyl formamide (DMF) was followed by the reaction with pyrimidine amine to form the Schiff base intermediates. Attempt has been made to derive final azetidinone analogues from Schiff bases by using chloroacetyl chloride. The newly synthesized analogues were examined for the antimicrobial activity against some bacterial and fungal strains andin vitroantituberculosis activity against mycobacterium tuberculosis. These observations provide some predictions to design further antibacterial and antituberculosis active compounds prior to their synthesis according to molecular studies.

Structural Basis for Substrate Specificity and Mechanism of N-Acetyl-d-neuraminic Acid Lyase from Pasteurella multocida

N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac, the most common form of sialic acid) to form pyruvate and N-acetyl-d-mannosamine. Although equilibrium favors sialic acid cleavage, these enzymes can be used for high-yield chemoenzymatic synthesis of structurally diverse sialic acids in the presence of excess pyruvate. Engineering these enzymes to synthesize structurally modified natural sialic acids and their non-natural derivatives holds promise in creating novel therapeutic agents. Atomic-resolution structures of these enzymes will greatly assist in guiding mutagenic and modeling studies to engineer enzymes with altered substrate specificity. We report here the crystal structures of wild-type Pasteurella multocida N-acetylneuraminate lyase and its K164A mutant. Like other bacterial lyases, it assembles into a homotetramer with each monomer folding into a classic (β/α)₈ TIM barrel. Two wild-type structures were determined, in the absence of substrates, and trapped in a Schiff base intermediate between Lys164 and pyruvate, respectively. Three structures of the K164A variant were determined: one in the absence of substrates and two binary complexes with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Both sialic acids bind to the active site in the open-chain ketone form of the monosaccharide. The structures reveal that every hydroxyl group of the linear sugars makes hydrogen bond interactions with the enzyme, and the residues that determine specificity were identified. Additionally, the structures provide some clues for explaining the natural discrimination of sialic acid substrates between the P. multocida and Escherichia coli NALs.

One-Step Simultaneous Differential Scanning Calorimetry-FTIR Microspectroscopy to Quickly Detect Continuous Pathways in the Solid-State Glucose/Asparagine Maillard Reaction

The stepwise reaction pathway of the solid-state Maillard reaction between glucose (Glc) and asparagine (Asn) was investigated using simultaneous differential scanning calorimetry (DSC)-FTIR microspectroscopy. The color change and FTIR spectra of Glc-Asn physical mixtures (molar ratio = 1:1) preheated to different temperatures followed by cooling were also examined. The successive reaction products such as Schiff base intermediate, Amadori product, and decarboxylated Amadori product in the solid-state Glc-Asn Maillard reaction were first simultaneously evidenced by this unique DSC-FTIR microspectroscopy. The color changed from white to yellow-brown to dark brown, and appearance of new IR peaks confirmed the formation of Maillard reaction products. The present study clearly indicates that this unique DSC-FTIR technique not only accelerates but also detects precursors and products of the Maillard reaction in real time.