Carbonyldiimidazole (CDI) mediated synthesis of Nα-protected amino acid azides: application to the one-pot preparation of ureidopeptides

Synthesis of Nα-protected amino acyl azides starting from corresponding acids via the carbonyldiimidazole (CDI) activation is described. The protocol is extended for a one-pot preparation of ureido peptides that circumvents the isolation of acyl azide and isocyanate intermediates. The reaction was accomplished without using any additives and base. The protocol is simple, clean, high yielding and free from racemization.     

Azide promotion of alkane oxidations catalyzed by metal complexes in solution

The oxidation of light alkanes catalyzed by metal complexes in solution is promoted by Group 1 metal azides. Yields of oxygenated reaction products are greatly enhanced when catalytic amounts of azides are added to the reaction mixture. The addition of sodium azide to oxidations catalyzed by transition metal acetylacetonates, heteropolyacids, polyoxometallates, phthalocyanines, bis-(pyridylimino)isoindolines, porphyrins and Schiff bases significantly enhances rates of low-temperature catalytic oxidation reactions in the liquid phase. Earlier work showed that Cr(III), Mn(III), Fe(III) and Co(III) complexes of electron-deficient macrocyclic complexes exhibited remarkable catalytic activity for oxidizing light alkanes. Such complexes bearing axial azide ligands were far more active than their axial chloride or acelate counterparts.     

THE NEMATICIDAL PROPERTIES OF AZIDES

The use of sodium azide and organic acid azides for the control of potato root eelworm has been investigated: in suitable conditions they were effective both in greenhouse tests and in field trials. The biological activity of azides has been shown to arise from liberation of undissociated hydrazoic acid. The acid is only liberated in effective quantities in acid soils, but in such soils it is decomposed with formation of nitrogen. Organic acid azides are decomposed by a wide range of dust diluents and by soil, and there is some evidence that in diluents the main decomposition reaction is the formation of isocyanate by the Curtius rearrangement, whereas on wet soils extensive hydrolysis occurs giving hydrazoic acid, which is subsequently decomposed.     

The azidation of starch

Starch is an inexpensive commodity that has been used for non-food purposes for many years. Some of these uses include cross-linked starches that are synthesized with a variety of multifunctional reagents. One unexplored possibility is the use of azides for cross-linking. To this end, azide derivatives of different starches have been synthesized, including the first reported synthesis of 6-deoxy-6-azido-amylopectin. Lithium salts, which were found to not be essential for the dissolution of starch in the reaction, were replaced with sodium azide. The time for this derivatization reaction to reach completion was determined to be 1 h. N,N-dimethylacetamide was also found to be a suitable solvent. Initial experiments suggest that the azide derivative does cross-link starch when activated by heat.     

Effect of glutathione L-cystein and L-djenkolic acid in the synthesis and mutagenicity of azide metabolite in Bacillus subtilis ATCC 6633 strain.

The Bacillus subtilis ATCC 6633 strain synthesizes a mutagenic metabolite from sodium azide and O-acetylserine. Mutagenicity of azide was decreased in growth media containing 10(-4) M glutathione, L-cysteine or L-djenkolic acid whereas dithiothritol (DTT) added at the same concentration did not reduce the mutagenicity of azide. Likewise, glutathione, L-cysteine, L-djenkolic acid, and DTT were found to have no effect in reducing the mutagenicity of the in vitro produced metabolite using bacterial cell-free extract. These results suggest that O-acetyl-serine sulfhydrylase catalyzes the reaction of azide and O-acetylserine to form a mutagenic metabolite, which is ninhydrin positive and migrates in TLC to an Rf value similar to that of azidoalanine in both acidic and basic solvent systems.     

Studies on the origin of stereoselectivity in the synthesis of 1,2-trans glycofuranosyl azides

The stereoselectivity of the 1,2-trans directed, Lewis acid-catalysed azidation of peracylated furanoses was found to depend on the reactivity of the azide donor (azide nucleophilicity) and the configuration at the anomeric centre relative to the neighbouring 2-O-acyl group. Reactions of 1,2-trans glycosyl esters with highly nucleophilic azide donors, generated from SnCl4 and Me3SiN3, were stereospecific. The results are interpreted in terms of the rapid reaction of the azide species with bicyclic 1,2-acyloxonium (1,2-O-alkyliumdiyl-D-glycofuranose) ions, which were the primarily formed reactive intermediates. When using 1,2-cis glycosyl esters as starting materials the selectivity was reduced (90-94% de); the same is true with 1,2-trans counterparts if less nucleophilic Me3SiN3 in combination with Me3SiOTf catalyst was used. This occurred due to the appearance of the more reactive but less selective oxocarbenium (glycofuranoxonium) ions either as primarily formed reactive intermediates in the former case or after equilibration with acyloxonium ions in the latter case. Protected 1,2-trans beta-D-glycofuranosyl azides with ribo, xylo and 3-deoxy-erythro-pento configurations were best prepared from the corresponding glycosyl esters using 0.05 equivalents of SnCl4, i.e., under anomerization-free conditions. Azidation of methyl glycofuranosides proceeds with inferior (80-90% de) and less predictable selectivity irrespective of the starting anomeric configuration.     

Syntheses of amphiphilic glycosylamides from glycosyl azides without transient reduction to glycosylamines

Protected glycosyl azides react with acyl chlorides in the presence of triphenylphosphine to afford glycosylamides in high yields, at room temperature. Starting from the beta-glycosyl azides, the reaction is highly stereoselective and occurs with retention of configuration, whereas the alpha-azido anomers display a lower stereoselectivity giving rise to alpha/beta mixtures of glycosylamides. The reaction was applied to several monosaccharidic azides and to lactosyl azide with various acyl chlorides; it was shown to be of general use for preparing 1,2-trans beta-glycosylamides.     

A novel synthesis of β-d-mannopyranosyl azide by phase transfer catalysis

A simple stereoselective synthesis of per-O-benzoyl-β-d-mannopyranosyl azide from per-O-benzoyl-α-d-mannopyranosyl bromide using phase transfer catalysis was developed. The stereochemistry at C-1 of the anomeric O-benzoylated α- and β-d-mannopyranosyl azides was unambiguously established using 2D NOESY NMR spectroscopy. Pure deprotected β-d-mannopyranosyl azide was prepared by debenzoylation with sodium methoxide in methanol.     

Azide reduction during peptide cleavage from solid support—the choice of thioscavenger?

Peptide azides acquired growing impact because of application in bioconjugation via ‘click chemistry’ or Staudinger ligation. Furthermore, there are many methods established in organic synthesis addressing the reduction of azides to amines, but no observation of a reductive transformation of peptide azides during SPPS cleavage was yet reported. In the present study, the reduction of peptide azides during SPPS cleavage was investigated depending on the choice of thioscavenger, reacting as reductive species. First observed for short PNA/peptide conjugates the occurring extensive side reaction was also validated for one of the applied azide amino acid building blocks and was further investigated by applying different cleavage cocktails to a series of peptides varying in hydrophobicity and position of the azide moiety in the oligomer sequence. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of the reactive intermediates produced upon photolysis of p-azidoacetophenone and its tetrafluoro analogue in aqueous and organic solvents: implications for photoaffinity labeling

Photolysis of p-azidoacetophenone (1a) or 2,3,5,6-tetrafluoro-p-azidoacetophenone (1b) releases the corresponding singlet nitrenes 2a and 2b. In aqueous solutions singlet nitrenes relax (1.1 ps and 43 ns, respectively) to the lower energy triplet nitrenes 3a and 3b, intermediates which do not react to form cross-links or adducts with typical amino acids and nucleic acids. In a hydrophobic environment singlet nitrene 2a partitions between forming triplet nitrene 3a and an acyl-substituted didehydroazepine 4a, which can be detected by LFP and time-resolved IR spectroscopy. The absolute rate constant of reaction of didehydroazepine 4a with water, in acetonitrile, was determined (3.5 x 10(4) M-1 s-1) by laser flash photolysis (LFP) techniques with IR detection at ambient temperature. Photolysis of tetrafluoro azide 1b releases singlet nitrene 2b, which has a lifetime of 172 ns in benzene and can readily be intercepted by pyridine to form ylide 10b (lambdamax = 415 nm). Singlet nitrene 2b reacts with the unactivated CH bonds of cyclohexane to form adduct 8b in 46% yield. Absolute rate constants of reaction of 1b with N-methylimidazole, phenol, dibutyl sulfide, indole, methanol, and dimethyl sulfoxide were determined using the pyridine ylide probe method. It is concluded that photolysis of p-azidoacetophenone (1a) will not lead to cross-link formation but that tetrafluorinated azide 1b can form useful singlet nitrene derived adducts upon photolysis.     

Modification of nucleic acids using [3 + 2]-dipolar cycloaddition of azides and alkynes

The use of azide and alkyne cycloaddition reaction in the synthesis of conjugates of nucleic acids and oligodeoxyribonucleotides is reviewed. Data on the chemical and enzymatic methods for introducing azides and alkynes into DNA are summarized.     

Ring Closure Reactions of Heterocyclic Azides with the Assistance of DSC [1]

5-Azido-pyrido[2, 3-d]pyrimidine-2,4,7-triones or 6-azido-uracils with reactive ortho-substituents such as aryl-, acyl- or nitro groups were prepared from the corresponding hydroxy compounds by chlorination (or tosylation) and reaction with sodium azide. The azides cyclize thermically to the corresponding indoles, isoxazoles or furoxanes. The cyclization conditions depend upon the structural properties of the ortho-substituents and range between 50 and 150 °C. The determination of the reaction temperature and the suitable solvents was carried out with the aid of DSC (Differential Scanning Calorimetry), and also further reactions such as the desoxygenation of the obtained furoxanes could be investigated by DSC in order to find the suitable reaction conditions.     

Fourier-transform infrared studies on azide-binding to the binuclear center of the Escherichia coli bo-type ubiquinol oxidase.

Azide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier-transform infrared spectroscopy. Deconvolution analyses of infrared spectra of the azide (14N3)-inhibited air-oxidized form showed a major infrared azide antisymmetric stretching band at 2041 cm(-1). An additional band developed at 2062.5 cm(-1) during a longer incubation. Isotope substitutions with terminally 15N-labelled azides did not show a splitting of the major band, indicating that the geometry of the bound azide is mainly in a bridging configuration between high-spin heme o and CuB. The band at 2062.5 cm(-1) showed clear splittings upon substitution with the terminally 15N-labelled azides, indicating the Cu(2+)B-N=N=N structure. Partial reduction of the oxidase with beta-NADH in the presence of azide caused an appearance of new infrared bands at 2038.5 (major) and 2009 (minor) cm(-1). The former band also showed clear splittings in the presence of the terminally 15N-labelled azides, indicating that reduction of low-spin heme b alters the structure of the binuclear center leading to the Fe(3+)o-N=N=N configuration.     

Reduction of aryl azides by thiols: implications for the use of photoaffinity reagents.

Aryl azides are rapidly reduced by dithiothreitol at room temperature to the corresponding aryl amines. Glutathione and 2-mercaptoethanol react much more slowly. The relevance of this reaction to experiments involving aryl azide photoaffinity reagents is discussed.     

Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli

The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.     

Application of click chemistry towards an efficient synthesis of 1,2,3-1H-triazolyl glycohybrids as enzyme inhibitors

An efficient synthesis of novel 1,2,3-1H-triazolyl glycohybrids with two or more than two sugar units or a chromenone moiety via copper-catalysed azide–alkyne cycloaddition (CuAAC), a 1,3-dipolar cycloaddition of glycosyl azides to 2,3-unsaturated alkynyl glycosides or propargyloxy coumarins is described. The synthesised glycohybrids were screened for their α-glucosidase, glycogen phosphorylase and glucose-6-phosphatase inhibitory activities. A few of the glycohybrids showed promising inhibitory activities against these enzymes.     

"Clickable" agarose for affinity chromatography

Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.     

Subzero temperature quenching and electrophoretic methods for the isolation of protein reaction intermediates

A quenching technique for the study of rapid protein reactions is described which consists of injecting a small volume of aqueous solution of reactants into a large volume (× 10) of hydro-organic solvent cooled at subzero temperature and mechanically shaken. The protein reaction intermediates, stabilized at subzero temperature and brought into a hydro-organic solution, can then be separated by subzero temperature electrophoretic methods, such as isoelectric focusing, in the same solvent. The alkaline hydrolysis of 2,4-dinitrophenylacetate was studied by the use of this quenching technique in order to compare the quenching and the rate constants of the reaction with those obtained by normal rapid quenching methods. It was found that first-order reactions having rate constants up to about 5 s^?1 can be satisfactorily studied by this technique. The technique is not suitable for the study of faster reactions because of the high value of the quenching time (40–100 ms). The hybridization reaction of carboxyhemoglobins A and C in aqueous solution at 22°C was studied as an example of the application of this quenching technique and of the isoelectric focusing method at subzero temperature to the isolation of unstable intermediates in a protein reaction.     

Conjugation of chemical handles and functional moieties to DNA during solid phase synthesis with sulfonyl azides

Labelling of oligonucleotides with dyes, targeting ligands, and other moieties has become ever more essential in life-sciences. Conventionally, modifications are introduced to oligonucleotides during solid phase synthesis by special phosphoramidites functionalised with a chemical handle or the desired functional group. In this work, we present a facile and inexpensive method to introduce modifications to oligonucleotides without the need for special phosphoramidites. Sulfonyl azides are applied to react with one or more selected phosphite intermediates during solid phase synthesis. We have prepared 11 sulfonyl azides with different chemical handles such as amine, azide, alkyne, and thiol, and we have further introduced functionalities such as pyrene, other dyes, photo-switchable azobenzenes, and a steroid. The method is compatible with current phosphoramidite-based automated oligonucleotide synthesis and serves as a simple alternative to the unstable and expensive special phosphoramidites currently used for conjugation to oligonucleotides.     

Reductive alkylation of hyaluronic acid for the synthesis of biocompatible hydrogels by click chemistry

Hyaluronan (HA) based hydrogels have been synthesized combining chemical modification of the polysaccharide by partial oxidation, reductive amination and 'click chemistry'. HA was oxidized by 4-acetamido-TEMPO-mediated reaction, using sodium hypochlorite as primary oxidant and NaBr in buffered pH, so that the produced aldehyde moieties (hemiacetals) were trapped in situ by adding primary amines containing azide or alkyne-terminal groups. The structure of the reaction products, oxidized-HA and primary amines bonded to HA, was elucidated using 2D NMR spectroscopy. SEC-MALLS analysis of the modified substrates showed a negligible degradation of the polysaccharide using this procedure. Furthermore, azido- and alkynyl derivatives underwent cross-linking by click chemistry into hydrogels, which were characterized by NMR, FT-IR, swelling degree and mechanical properties. Possible application of the material as scaffold for tissue engineering was tested by seeding and proliferation of chondrocytes for up to 15 days.