The structure of monoamine oxidase from Aspergillus niger provides a molecular context for improvements in activity obtained by directed evolution

Monoamine oxidase from Aspergillus niger (MAO-N) is a flavoenzyme that catalyses the oxidative deamination of primary amines. MAO-N has been used as the starting model for a series of directed evolution experiments, resulting in mutants of improved activity and broader substrate specificity, suitable for application in the preparative deracemisation of primary, secondary and tertiary amines when used as part of a chemoenzymatic oxidation-reduction cycle. The structures of a three-point mutant (Asn336Ser/Met348Lys/Ile246Met or MAO-N-D3) and a five-point mutant (Asn336Ser/Met348Lys/Ile246Met/Thr384Asn/Asp385Ser or MAO-N-D5) have been obtained using a multiple-wavelength anomalous diffraction experiment on a selenomethionine derivative of the truncated MAO-N-D5 enzyme. MAO-N exists as a homotetramer with a large channel at its centre and shares some structural features with human MAO B (MAO-B). A hydrophobic cavity extends from the protein surface to the active site, where a non-covalently bound flavin adenine dinucleotide (FAD) sits at the base of an ‘aromatic cage,’ the sides of which are formed by Trp430 and Phe466. A molecule of l-proline was observed near the FAD, and this ligand superimposed well with isatin, a reversible inhibitor of MAO-B, when the structures of MAO-N proline and MAO-B-isatin were overlaid. Of the mutations that confer the ability to catalyse the oxidation of secondary amines in MAO-N-D3, Asn336Ser reduces steric bulk behind Trp430 of the aromatic cage and Ile246Met confers greater flexibility within the substrate binding site. The two additional mutations, Thr384Asn and Asp385Ser, that occur in the MAO-N-D5 variant, which is able to oxidise tertiary amines, appear to influence the active-site environment remotely through changes in tertiary structure that perturb the side chain of Phe382, again altering the steric and electronic character of the active site near FAD. The possible implications of the change in steric and electronic environment caused by relevant mutations are discussed with respect to the improved catalytic efficiency of the MAO-N variants described in the literature.     

Cloning and upscale production of monoamine oxidase N (MAO-N D5) by <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To clone monoamine oxidase N, that catalyses the selective oxidative deamination or deracemisation of amines into imines, in Pichia pastoris and prove the importance of choosing the proper expression system for its recombinant production.

Results

Monoamine oxidase, originating from Aspergillus niger and subjected to directed evolution (MAO-N D5), was cloned and expressed in Pichia pastoris CBS7435 Mut^S strain for the first time. Various transformants were screened at microscale level. The production of the clone expressing the most active enzyme was scaled-up to a 1.5 l fermenter and preparation of MAO-N D5 as a crude enzyme extract was optimised. The obstacles in the production of the enzyme in both expression systems, Escherichia coli and P. pastoris, are discussed and demonstrated.

Conclusions

There was an improvement in specific productivity, which was 83 times higher in P. pastoris, clearly proving the importance of choosing the right expression host system for the specific enzymes.

     

Substrate profiling of cyclohexylamine oxidase and its mutants reveals new biocatalytic potential in deracemization of racemic amines

A cyclohexylamine oxidase (CHAO) of bacterial origin was previously shown to be a potentially useful catalyst in the deracemization of racemic primary amines. To further explore the properties and application of this enzyme, five single-amino acid substitution mutants (L199A, M226A, Y321A, Y321F, and L353M) were created based on superimposition of the tertiary structure of CHAO and the monoamine oxidase (MAO) B homolog. The substrate specificity of the purified wild-type and five mutant enzymes were examined towards 38 structurally diverse amines. All the enzymes exhibited better activity for primary amines than secondary and tertiary amines and in general exhibited high stereoselectivity. Among the mutant enzymes, M226A displayed an enhanced activity (5–400 %) towards most substrates, and L353M showed 7–445 % higher activity towards primary aliphatic amines with cycloalkane or aromatic moieties. Kinetic parameters revealed that both Y321 mutants showed higher catalytic efficiency towards cyclooctanamine, whereas the wild-type CHAO (wt CHAO) was most efficient towards cyclohexylamine. The wt CHAO or variant L353M in combination with a borane–ammonia complex as reducing agent was applied to the deracemization of 1-aminotetraline to give the (R)-enantiomer, a precursor of an antidepressant drug Norsertraline, in good yield (73–76 %), demonstrating their application potential in chiral amine synthesis.     

Microbial deracemisation of aromatic β-hydroxy acid esters

Aromatic β-hydroxy acid esters were found to undergo deracemisation using whole cells of Candida parapsilosis. The conditions for the deracemisation reaction were optimised where 75% isolated yield and >95% enantiomeric excess of the product was achieved. The effect of electron donating as well as electron withdrawing groups present in the standard substrate, ethyl 3-hydroxy 3-phenyl propionate was studied to establish the generality of the reaction. The enantiomeric excess of the product remains high (>95%) irrespective of the different substituents in the para position but substitution at the ortho position obstructs the process. Similarly, ethyl and methyl esters of the standard substrate undergo deracemisation reaction giving high ee of the product, but the benzyl ester of the standard substrate did not undergo deracemisation.     

Cloning,sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.     

Convenient synthesis of bifunctional tetraaza macrocycles.

A convenient synthesis of 4-nitrobenzyl-substituted macrocyclic tetraamines and their conversion to bifunctional poly(amino carboxylate) chelating agents is described. Cyclization of (4-nitrobenzyl)-ethylenediamine with appropriate BOC-protected amino disuccinimido esters in dioxane at 90 degrees C resulted in the formation of 12- and 14-membered ring diamides in 40% and 44% yield, respectively. A 12-membered macrocyclic triamide was also prepared in 44% yield by cyclization of N-(2-aminoethyl)-4-nitrophenylalaninamide with disuccinimidyl N-(tert-butoxycarbonyl)iminodiacetate. Deprotection (HCl/dioxane) and reduction with borane gave the substituted macrocyclic amines which were then alkylated with either bromoacetic acid or tert-butyl bromoacetate. Preparation of the isothiocyanate derivatives and 14C labeled chelating agents are described. Attempts to prepare a 9-membered macrocyclic diamide using this cyclization technique resulted instead in a 20% yield of a 10:1 mixture of isomeric fused 5,6 ring acylamidines. Deprotection (HCl/dioxane) and reduction with borane gave a substituted piperazine derivative in 55% yield.     

Enzyme catalysed deracemisation and dynamic kinetic resolution reactions

New catalysts and reaction conditions have been developed for the dynamic kinetic resolution or deracemisation of racemic mixtures of chiral compounds. Specific functional groups that lend themselves particularly well to this approach include chiral secondary alcohols, alpha-amino acids, amines and carboxylic acids. A general theme of these processes is the combination of an enantioselective enzyme with a chemical reagent, the latter being used either to racemise the unreactive enantiomer or alternatively recycle an intermediate in the deracemisation process. In some examples of dynamic kinetic resolution, a second enzyme (racemase) is used to interconvert the enantiomers of the starting material.     

Candida parapsilosis ATCC 7330 can also deracemise 1-arylethanols

Abstract

Candida parapsilosis ATCC 7330 grown using different culture conditions (inoculum size 4% (v/v), inoculum age 12 h, and harvest time 14 h) from those previously reported (inoculum size 2% (v/v), inoculum age 24 h, and harvest time 44 h) successfully deracemised racemic 1-arylethanols and 4-phenyl-2-butanol to the (R)-enantiomer (ee up to >99%). The deracemisation of racemic 1-aryl ethanol proceeds via (i) enantioselective oxidation of (S)-enantiomer followed by (ii) reduction of the ketone formed to give the racemic alcohol which gets kinetically resolved thus enriching for the (R)-enantiomer from the racemate. This is the first report on the deracemisation of 1-arylethanols using Candida parapsilosis ATTC 7330 via dynamic kinetic resolution.     

Amine boranes as alternative reducing agents for reductive alkylation of proteins

The use of several commercially available amine-borane complexes was investigated for the reductive methylation of amino groups of several proteins. An earlier study in our laboratory, using turkey ovomucoid as the model protein, showed that dimethylamine borane is a slightly weaker reducing agent, but a good, less toxic substitute for sodium cyanoborohydride (K. F. Geoghegan, J. C. Cabacungan, H. B. F. Dixon, and R. E. Feeney, 1981, Int. J. Peptide Protein Res.17, 345–352). N-α-Acetyl-l-lysine, poly-l-lysine, turkey ovomucoid, bovine serum albumin, chicken ovalbumin, β-lactoglobulin, casein, and soybean protein were reductively methylated with dimethylamine borane and trimethylamine borane. The latter produced a consistently lower degree of modification even in the presence of sodium dodecyl sulfate. In a comparison that included the boranes triethylamine, t-butylamine, morpholine, and pyridine, pyridine borane was found to be slightly stronger than sodium cyanoborohydride. In a pH 7 solution containing 2 mmN-α-acetyl-l-lysine and 20 mm formaldehyde, complete dimethylation was achieved with about 10 mm pyridine borane after 2 h incubation at 22°C, while more than 15 mm was necessary with sodium cyanoborohydride. Like dimethylamine borane, both pyridine borane and triethylamine borane showed a reducing capacity at pH 7 which was as high as that at pH 9. Reductive alkylation under neutral to mild acid conditions allows modification of alkaline labile proteins and also limits the side reactions between proteins and formaldehyde.     

Activation studies with amines and amino acids of the β-carbonic anhydrase from the pathogenic protozoan Leishmania donovani chagasi

The activation of a β-class carbonic anhydrase (CAs, EC 4.2.1.1) from Leishmania donovani chagasi (LdcCA) was investigated using a panel of natural and non-natural amino acids and amines. The most effective activators belonged to the amine class, with histamine, dopamine, serotonin, 2-pyridyl-methylamine and 4-(2-aminoethyl)-morpholine with activation constants in the range of 0.23–0.94 µM. In addition, 2-(2-aminoethyl)pyridine and 1-(aminoethyl)-piperazine were even more effective activators (K_As of 9–12 nM). Amino acids such as L-/D-His, L-/D-Phe, L-/D-DOPA, L-/D-Trp and L-/D-Tyr were slightly less effective activators compared to the amines, but showed activation constants in the low micromolar range (1.27–9.16 µM). Many of the investigated activators are autacoids that are present in rather high concentrations in different tissues of the host mammals infected by these parasites. As CA activators have not yet been investigated for protozoan CAs, this study may be relevant for an improved understanding of the role of this enzyme in the life cycle of Leishmania.     

Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.     

Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis

The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were selected as targets for site-directed mutagenesis, and six mutants (E175N, K576D, K174D, G176D, S575D and N577K) were designed using the RosettaDesign server. The E175N and K576D mutants exhibited improved thermostability in preliminary experiments, so the double mutant E175N/K576D was constructed. These three mutants (E175N, K576D, E175N/K576D) were characterized in detail. The results indicate that the three mutants exhibit a slightly increased optimal temperature (35°C), compared with that of the wild-type enzyme (30°C). The mutants also share an identical pH optimum of 6.0, which is similar to that of the wild-type enzyme. The half-lives of the E175N, K576D and E175N/K576D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the K_m values for the E175N, K576D and E175N/K576D mutants decreased by 6.6%, 2.0% and 11.0%, respectively, and their k_cat/K_m values increased by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, we found that the E175N, K576D and E175N/K576D mutants displayed slightly improved isomaltulose yields, compared with the wild-type enzyme. Therefore, the mutants produced in this study would be more suitable for industrial biosynthesis of isomaltulose.     

Effects of the processing methods of corn grain and soybean meal on milk protein expression profiles in dairy cows

A proteomic approach was used to investigate the effects of the processing method of corn grain and soybean meal on the milk protein expression profile in lactating dairy cows. A total of 12 multiparous Holstein dairy cows were used in a 4×4 Latin square design with a 2×2 factorial arrangement. The primary factors examined were corn (finely ground (FGC) v. steam-flaked (SFC)) and soybean meal (solvent-extracted (SSBM) v. heat-treated (HSBM)), which were used to formulate four diets with the same basal ingredient: 27% FGC and 9% SSBM; 27% SFC and 9% SSBM; 27% FGC and 9% HSBM; and 27% SFC and 9% HSBM. Each period lasted for 21 days. Milk samples were collected on days 18, 19 and 20 of each period. Changes in the milk proteins were assessed by two-dimensional (2D) electrophoresis and ImageMaster 2D Platinum 6.0 software. A total of 13 spots displayed variations in protein spot abundance according to the statistical analysis. These spots were identified by a matrix-assisted laser desorption/ionization-time of flight/time of flight MS. According to the gels, the relative abundance of α_s2-casein (CN) fragments was higher in the cows fed the SFC-HSBM than that for SFC-SSBM, whereas β-CN, α-lactalbumin and zinc-alpha-2-glycoprotein fragments were down-regulated in HSBM-fed cows. The relative decrease of β-CN expression was validated by western blot and agreed with the MS data. These results suggested that the method used to process soybean meal modified the synthesis and secretion of milk proteins in lactating dairy cows’ mammary glands.     

The Efficacy and Safety of Co-Administration of Sitagliptin With Metformin in Patients With Type 2 Diabetes at Hospital Discharge

Objective: Few randomized controlled trials have focused on the optimal management of patients with type 2 diabetes (T2D) during the transition from the inpatient to outpatient setting. This multicenter open-label study explored a discharge strategy based on admission hemoglobin A1c (HbA1c) to guide therapy in general medicine and surgery patients with T2D.Methods: Patients with HbA1c ≤7% (53 mmol/mol) were discharged on sitagliptin and metformin; patients with HbA1c between 7 and 9% (53–75 mmol/mol) and those >9% (75 mmol/mol) were discharged on sitagliptinmetformin with glargine U-100 at 50% or 80% of the hospital daily dose. The primary outcome was change in HbA1c at 3 and 6 months after discharge.Results: Mean HbA1c on admission for the entire cohort (N = 253) was 8.70 ± 2.3% and decreased to 7.30 ± 1.5% and 7.30 ± 1.7% at 3 and 6 months (P<.001). Patients with HbA1c <7% went from 6.3 ± 0.5% to 6.3 ± 0.80% and 6.2 ± 1.0% at 3 and 6 months. Patients with HbA1c between 7 and 9% had a reduction from 8.0 ± 0.6% to 7.3 ± 1.1% and 7.3 ± 1.3%, and those with HbA1c >9% from 11.3 ± 1.7% to 8.0 ± 1.8% and 8.0 ± 2.0% at 3 and 6 months after discharge (both P<.001). Clinically significant hypoglycemia (<54 mg/dL) was observed in 4%, 4%, and 7% among patients with a HbA1c <7%, 7 to 9%, and >9%, while a glucose <40 mg/dL was reported in <1% in all groups.Conclusion: The proposed HbA1c-based hospital discharge algorithm using a combination of sitagliptin-metformin was safe and significantly improved glycemic control after hospital discharge in general medicine and surgery patients with T2D.Abbreviations: BG = blood glucose; DPP-4 = dipeptidyl peptidase-4; eGFR = estimated glomerular filtration rate; HbA1c = hemoglobin A1c; T2D = type 2 diabetes     

Uptake of glycine and release of primary amines by the polychaete Nereis virens (Sars) and the mud snail Hydrobia neglecta muus

Uptake of glycine and release of primary amines by the polychaete Nereis virens (Sars) and the mud snail Hydrobia neglecta Muus was investigated. In a flow system, Nereis virens had a net influx of glycine of 32 to 39 nmol·g^?1 fresh wt·h from a 5-μM solution of glycine. Simultaneously, an efflux of primary amines of 14 nmol·g^?1·h^?1 was measured. The experiments indicated that glycine uptake and release of primary amines are two independent processes. Glycine was absorved by Hydrobia neglecta from 50-μM solutions at an influx of 175 nmol·g^?1 organic wt·h^?1, and simultaneously, there was an independent efflux of 25 nmol primary amine·g^?1·h^?1. In the experiments, released primary amines from both species increased the ambient concentration of primary amines, but after various periods of time these concentrations apparently stabilized. Finally, the significance of micro-organisms in amino-acid uptake experiments is considered.     

Secondary structure models of D2-D3 expansion segments of 28S rRNA for Hoplolaiminae species

The D2-D3 expansion segments of the 28S ribosomal RNA (rRNA) were sequenced and compared to predict secondary structures for Hoplolaiminae species based on free energy minimization and comparative sequence analysis. The free energy based prediction method provides putative stem regions within primary structure and these base pairings in stems were confirmed manually by compensatory base changes among closely and distantly related species. Sequence differences ranged from identical between Hoplolaimus columbus and H. seinhorsti to 20.8% between Scutellonema brachyurum and H. concaudajuvencus. The comparative sequence analysis and energy minimization method yielded 9 stems in the D2 and 6 stems in the D3 which showed complete or partial compensatory base changes. At least 75% of nucleotides in the D2 and 68% of nucleotides in the D3 were related with formation of base pairings to maintain secondary structure. GC contents in stems ranged from 61 to 73% for the D2 and from 64 to 71% for the D3 region. These ranges are higher than G-C contents in loops which ranged from 37 to 48% in the D2 and 33-45% in the D3. In stems, G-C/C-G base pairings were the most common in the D2 and the D3 and also non-canonical base pairs including A•A and U•U, C•U/U•C, and G•A/A•G occurred in stems. The predicted secondary model and new sequence alignment based on predicted secondary structures for the D2 and D3 expansion segments provide useful information to assign positional nucleotide homology and reconstruction of more reliable phylogenetic trees.     

Histamine-producing bacteria in blue scad (Decapterus maruadsi) and their abilities to produce histamine and other biogenic amines

Using decarboxylation medium and 16S rDNA sequence analysis, histamine-producing bacteria (HPB) in blue scad (Decapterus maruadsi) were isolated and identified, and the histamine-producing abilities of the isolated HPB were determined. Nine mesophilic strains (H1–H9) isolated from the muscle of blue scad were identified as the genera of HPB, including Arthrobacter bergeri (H1), Pseudomonas sp. (H2, H5 and H6), Psychrobacter sp. (H3), Shewanella baltica (H4 and H7), and Aeromonas salmonicida (H8 and H9), respectively. Results showed that most of the HPB strains were weak on histamine formation (13.0–20.4 mg/l), except for the H8 strain with the ability of producing 115 mg of histamine/l in trypticase soy broth containing 1.0 % l-histidine. As the strongest HPB in blue scad, bacterial strain H8 also presented a strong ability to produce other biogenic amines, such as putrescine, cadaverine, spermidine, spermine, tyramine and tryptamine. Therefore, the H8 strain identified as the genus of A. salmonicida was the dominant mesophilic HPB strain for producing histamine and other biogenic amines in blue scad at room temperature.     

Poly-3-hydroxybutyrate synthase from the periplasm of Escherichia coli

This is the first report of a poly-3-hydroxybutyrate (PHB) synthase in Escherichiacoli. The enzyme was isolated from the periplasm using ammonium sulfate fractionation, hydrophobic, and size-exclusion chromatography and identified by LC/MS/MS as YdcS, a component of a putative ABC transporter. Green Fluorescent Protein-tagged ydcS, purified by 2D native gel electrophoresis, also exhibited PHB synthase activity. Optimal conditions for enzyme activity were 37 °C, pH 6.8-7.5, 100 mM KCl. K_m was 0.14 mM and V_max was 18.7 nmol/mg protein/min. The periplasms of deletion mutants displayed <25% of the activity of the parent strain. Deletion mutants exhibited ∼25% less growth in M9 medium, glucose, and contained ∼30% less PHB complexed to proteins (cPHB) in the outer membranes, but the same concentration of chloroform-extractable PHB as wild-type cells. The primary sequence of YdcS suggests it may belong to the α-/β-hydrolase superfamily which includes polyhydroxybutyrate (PHB) synthases, lipases, and esterases.     

An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[³H]vitamin D₃ (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1–20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).     

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.