Chemo-enzymatic synthesis and structure-activity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide

Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation.     

Site-dependent effect of O-glycosylation on the conformation and biological activity of calcitonin

We synthesized seven O-glycosylated calcitonin derivatives, each with a single GalNAc residue attached to either Ser or Thr, and studied their three-dimensional structure and biological activity to examine site-dependent effects of O-glycosylation. The CD spectra in an aqueous trifluoroethanol solution showed that the GalNAc attachment at Thr6 or Thr21 reduced the helical content of calcitonin, indicating that the O-glycosylated residue functions as a stronger helix breaker than the original amino acid residue. Only the GalNAc attachment at Ser2 or Thr21 retained the hypocalcemic activity of calcitonin. This result corresponded well to that of the calcitonin-receptor binding assay. The GalNAc attachment other than Ser2 or Thr21 perturbed the interaction with the receptor, resulting in the loss of the hypocalcemic activity. The biodistribution did not change much among the seven derivatives, but some site dependency could also be observed. Thus, we can conclude that the O-glycosylation affects both the conformation and biological activity in a site-dependent manner.     

Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.     

Glycosylation reduces the bioactivity of calcitonin

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.     

Studies on the synthesis and the antimicrobial and antioxidant activities of a novel class of fluorescein-based glycosides

Facile glycosylation of a fluorescein diol derivative with per-O-acetyl/benzoyl sugar derivatives using BF₃·Et₂O catalyst resulted in the formation of the expected glycosides in 54–66% yield. The biological screening of the glycosides against different microbes shows good inhibitory activity. The antioxidant activity of the fluorescein-based glycosides shows remarkable inhibition (IC₅₀ ∼80%).     

Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structures of eel calcitonin (CT) and two glycosylated CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man6-GlcNAc2)3]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-helix consisting of residues Ser5 through Leu19 followed by an unstructured region at the C-terminus. The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relative exchange rates of the Leu12 amide proton might suggest the possibility that fluctuations of the alpha-helix are reduced by glycosylation. The presence of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc and CT-M6 and the amide proton chemical shift data suggested that the carbohydrate interacted with the peptide, and this might account for the conformational stabilization of the alpha-helix. Both the unmodified CT and the glycosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.     

Conformational flexibility and biological activity of salmon calcitonin

We have assessed the biological activity of salmon calcitonin I (sCT) using an in vivo biological assay of hypocalcemic activity in rats. The changes in biological activity observed are explained on the basis of changes in the conformational properties of the hormone analogues. Helical content in the presence and absence of lipids and detergents was assessed by using circular dichroism, and the section of the molecule that folds into a helix was predicted on the basis of the helix-coil transition theory of Mattice and co-workers. In the amino acid sequence of sCT, residue 8 is valine and residue 16 is leucine. The synthetic calcitonin derivatives [Gly8]sCT and [Ala16]sCT have higher biological activity than the native hormone although they have a lower helical content. The increased biological activity of these derivatives is ascribed to an increase in their conformational flexibility resulting from the substitution of amino acid residues with less bulky side chains and less tendency to form helical structures. The derivative [Met8]sCT has less substitution than sCT on the beta-carbon at position 8, but it has increased helix-forming potential in the region of residues 8-12. These two factors affect conformational flexibility in opposite ways, resulting in the biological activity of [Met8]sCT being slightly higher than that of sCT. However, increased conformational flexibility does not always increase biological activity. Substitution of the L-arginine at residue 24 for a D-arginine has little effect on the conformational properties or biological activity of sCT. However, [Gly8, D-Arg24]sCT is less active than sCT, [Gly8]sCT, or [D-Arg24]sCT.(ABSTRACT TRUNCATED AT 250 WORDS)     

An NMR study of O-glycosylation induced structural changes in the alpha-helix of calcitonin

We previously reported that two out of seven artificially O-glycosylated calcitonin derivatives had an altered peptide backbone conformation as indicated by decreased helical contents, determined by CD measurement. In the present study, two of those derivatives, in which a GalNAc residue is attached to Thr6 or Thr21 of calcitonin, were analyzed by NMR in order to determine the structural changes induced by the O-glycosylation in more detail. Deviations in the chemical shifts suggest that the structural change is not global but only a local one and is located in the vicinity of each O-glycosylation site. The intensities of the NOE cross peaks, an indicator of -helical structure, also were decreased around the O-glycosylation site. The hydrogen/deuterium exchange rates of the main chain amide protons increased at the N- or C-terminal portion of the -helix corresponding to the respective O-glycosylation site and explains the results of the CD experiments. The inter-residual NOE cross peaks between the carbohydrate and the peptide portions, other than the O-glycosylated amino acid residue, showed that local structural contacts extended three or two residue distance for Thr6- or Thr21-glycosylated derivative, respectively. Thus, we conclude that the O-glycosylation induced a change in the local structure and that this structural perturbation modulated the original -helical structure of calcitonin, resulting in the apparent decrease in the helical content deduced from CD spectra.     

Glycan structures of the structural subunit (HtH1) of <Emphasis Type="Italic">Haliotis tuberculata</Emphasis> hemocyanin

The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin (HtH) were studied by mass spectral sequence analysis of the glycans. The proposed structures are based on MALDI-TOF-MS data before and after treatment with the specific exoglycosidases β1-3,4,6-galactosidase and α1-6(>2,3,4) fucosidase followed by sequence analysis via electrospray ionization MS/MS-spectra. In total, 15 glycans were identified as a highly heterogeneous group of structures. As in most molluscan hemocyanins, the glycans of HtH1 contain a terminal MeHex, but more interestingly, a novel structural motif was observed: MeHex[Fuc(α1-3)-]GlcNAc, including thus MeHex and (α1-3)-Fuc residues being linked to an internal GlcNAc residue. While the functional unit (FU) c (HtH1-c) is completely lacking any potential glycosylation site, FU-h possesses a second exposed sugar attachment site between beta-strands 8 and 9 within the beta sandwich domain compared to the other FUs. The glycosylation pattern/sites show a high degree of conservation. In FU-h two prominent potential glycosylation sites can be detected. The finding that HtH1 is not able to form multidecameric structures in vivo could be explained by the presence of the exposed glycan on the surface of FU-h.     

Biologically potent analogues of salmon calcitonin which do not contain an N-terminal disulfide-bridged ring structure

The disulfide bridge formed between the cysteine residues at positions 1 and 7 of salmon calcitonin (sCT) is not required for biological activity. The analogues [Ala1,7]sCT,[AcmCys1,7]sCT and [AmcCys1,Ala7]sCT (AcmC = S-acetamido-methylcysteine) are linear sequences which retain full hypocalcemic activity in the intact rat and ability to activate adenylate cyclase of rat renal membranes. The secondary structure of these peptides in aqueous solution in the presence or absence of lipid is not greatly perturbed by the opening of the disulfide ring. In contrast with salmon calcitonin, substitution of Cys by AcmCys in human calcitonin results in greatly reduced hypocalcemic activity but no loss in the ability of the peptide to activate renal adenylate cyclase. Thus in vitro activation of adenylate cyclase by human calcitonin analogues is not always correlated with in vivo hypocalcemic potency.     

Syntheses and Biological Activities of Danicalipin A Derivatives

Synthesis of three derivatives of danicalipin A, tetrachloride, trisulfate and a fluorescent probe was achieved through Wittig reaction strategy. Toxicity of the derivatives against brine shrimp (Artemia salina) as also investigated to provide useful information for the biological activity; i) less chloride derivative showed similar toxicity to danicalipin A, ii) the amphiphilic property, a characteristic feature of danicalipin A, was crucial because trisulfate considerably decreased the toxicity and iii) fluorescent derivative kept brine shrimp toxicity of danicalipin A.     

Expression of Soluble Bovine Pancreatic Ribonuclease A in Pichia pastoris and its Purification and Characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased k _cat, 60-70% of the activity of wild-type RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since T _m of the glycosylated RNase A was decreased by 6°C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

Tuning the conformational properties of the prion peptide

Previously, we disclosed that O‐linked glycosylation of Ser‐132 or Ser‐135 could dramatically change the amyloidogenic property of the hamster prion peptide (sequence 108–144). This peptide, which corresponds to the flexible loop and the first β‐strand in the structure of the prion protein, is a random coil when it is initially dissolved in buffer, but amyloid fibrils are formed with time. Thus, it offers a convenient model system to observe and compare how different chemical modifications and sequence mutations alter the amyloidogenic property of the peptide within a reasonable experimental time frame. In our earlier study, aside from uncovering a site‐specificity of the glycosylation on the fibrillogenesis, different effects of α‐GalNAc and β‐GlcNAc were observed. In this work, we explore further how different sugar configurations affect the conformational property of the polypeptide chain. We compare the effects of O‐linked glycosylation by the common sugars α‐GalNAc, β‐GlcNAc with their non‐native analogs β‐GalNAc, α‐GlcNAc in an effort to uncover the origin of the sugar‐specificity on the fibril formation. We find that the anomeric configuration of the sugar is the most important factor affecting the fibrillogenesis. Sugars with the glycosidic bond in the α‐configuration at Ser‐135 have a dramatic inhibitory effect on the structural conversion of the glycosylated peptide. Because O‐glycosylation of Ser‐135 with α‐linked sugars also promote the formation of three slowly converting conformations at the site of glycosylation, we surmise that the amyloidogenic property of the peptide is related to its conformational flexibility, and the proclivity of this region of the peptide to undergo the structural conversion from the random coil to form the β‐structure. Upon O‐glycosylation with an α‐linked sugar, this conversion is inhibited and the nucleation of fibril formation is largely retarded. Consistent with this scenario, Arg‐136 is the residue most affected in the TOCSY NMR spectra of the glycosylated peptides, other than the serine site modified. In addition, when Arg‐136 is substituted by Gly, a mutation that should provide higher structural flexibility in this part of the peptide, the amyloidogenic property of the peptide is greatly enhanced, and the inhibition effect of glycosylation is largely diminished. These results are consistent with Ser‐135 and Arg‐136 being part of the kink region involved in the structural conversion. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Design,Synthesis, and Biological Activity of Guaiazulene Derivatives

Guaiazulene and related derivatives were famous for diverse biological activities. In an effort to discover new highly efficient candidate drugs derived from guaiazulene, four series of guaiazulene derivatives were designed, synthesized, and evaluated for antiproliferation, antiviral, anti-inflammatory and peroxisome proliferators-activated receptor γ (PPARγ) signalling pathway agonist activities. Among them, two guaiazulene condensation derivatives showed selective cytotoxic activities towards K562 cell with IC₅₀ values 5.21 μM and 5.14 μM, respectively, accompanied by slight effects on normal cell viability. For the first time, one guaiazulene derivative from series I exhibited potent antiviral activity towards influenza A virus with IC₅₀ of 17.5 μM. A guaiazulene-based chalcone showed higher anti-inflammatory activity than positive drug indomethacin with an inhibitory rate of 34.29 % in zebrafish model in vivo. One guaiazulene-based flavonoid could strongly agitate PPARγ pathway at 20 μM, indicating the potential of guaiazulene derivatives to reduce obesity development and ameliorate hepatic steatosis. Preliminary in silico ADME studies predicted the excellent drug-likeness properties of bioactive guaiazulene derivatives.     

Bacteriophage predation promotes serovar diversification in Listeria monocytogenes

Listeria monocytogenes is a bacterial pathogen classified into distinct serovars (SVs) based on somatic and flagellar antigens. To correlate phenotype with genetic variation, we analyzed the wall teichoic acid (WTA) glycosylation genes of SV 1/2, 3 and 7 strains, which differ in decoration of the ribitol‐phosphate backbone with N‐acetylglucosamine (GlcNAc) and/or rhamnose. Inactivation of lmo1080 or the dTDP‐l ‐rhamnose biosynthesis genes rmlACBD (lmo1081–1084) resulted in loss of rhamnose, whereas disruption of lmo1079 led to GlcNAc deficiency. We found that all SV 3 and 7 strains actually originate from a SV 1/2 background, as a result of small mutations in WTA rhamnosylation and/or GlcNAcylation genes. Genetic complementation of different SV 3 and 7 isolates using intact alleles fully restored a characteristic SV 1/2 WTA carbohydrate pattern, including antisera reactions and phage adsorption. Intriguingly, phage‐resistant L. monocytogenes EGDe (SV 1/2a) isolates featured the same glycosylation gene mutations and were serotyped as SV 3 or 7 respectively. Again, genetic complementation restored both carbohydrate antigens and phage susceptibility. Taken together, our data demonstrate that L. monocytogenes SV 3 and 7 originate from point mutations in glycosylation genes, and we show that phage predation represents a major driving force for serovar diversification and evolution of L. monocytogenes.     

Synthesis of New Lathyrane Diterpenoid Derivatives from Euphorbia lathyris and Evaluation of Their Anti‐Inflammatory Activities

Euphorbia factor L₃, a lathyrane diterpenoid extracted from Euphorbia lathyris, was found to display good anti‐inflammatory activity with very low cytotoxicity. To find more potent anti‐inflammatory drugs, two series of Euphorbia factor L₃ derivatives with fatty and aromatic acids were designed and synthesized. Among them, lathyrane derivative 5n exhibited most potent inhibition on LPS‐induced NO production in RAW264.7 cells with no obvious cytotoxicity. To determine the key characteristics of Euphorbia factor L₃ derivatives that contribute to anti‐inflammatory activity, we conducted a structure‐activity relationship study of these compounds.     

In vitro antimicrobial activity and mechanism of action of novel carbohydrate fatty acid derivatives against Staphylococcus aureus and MRSA

Aims: This study investigates the antimicrobial activity and mode of action of novel carbohydrate fatty acid (CFA) derivatives against Staphylococcus aureus and methicillin‐resistant Staph. aureus (MRSA). Methods and Results: Minimum inhibitory concentrations (MICs) and the effect of CFA derivatives on lag phase were determined using a broth microdilution method. Lauric acid carbohydrate esters and corresponding ether analogues showed the greatest antimicrobial activity with MIC values between 0·04 and 0·16 mmol l^?1. Leakage studies at 260 nm following exposure to CFA derivatives at 4× MIC showed a significant increase in membrane permeability for all compounds, after c. 15 min exposure except for the lauric beta ether CFA derivative. Further assessment using both BacLight and luminescence ATP assays confirmed that an increase in membrane permeability and reduced metabolic activity was associated with CFA treatment. Conclusions: All strains were significantly inhibited by the novel compounds studied, and efficacy was related to specific structural features. Cell‐membrane permeabilization was associated with CFA treatment and may account for at least a component of the mode of action of these compounds. Significance and Impact of the Study: This study reports the antimicrobial action of CFA compounds against a range of Staph. aureus and MRSA strains, and provides insights into their mode of action.     

Design,synthesis and biological activity of pyrazinamide derivatives for anti-Mycobacterium tuberculosis

A total of 11 pyrazinamide derivatives were designed and synthesised using pyrazinamide as the lead compound, which was optimised by structural modification with alkyl chains, six-membered rings, and bioisosterism, respectively. The target compounds were synthesised using pyrazinecarboxylic acid as the starting material by acylation, amidation, and alkylation, respectively. Their structures were confirmed by ¹H NMR, ¹³C NMR, HRESIMS, and elemental analysis, respectively. The bioactivities of derivatives were assayed using bacteriostatic experiment and minimum inhibitory concentration experiment. It was showed that the derivatives had good inhibitory effect on Mycobacterium tuberculosis. The biological activity of derivative 1f was the best among all compounds, its antibacterial activity was 99.6%, and the minimum inhibitory concentration was 8.0?µg/mL.     

New Pyrano-4H-benzo[g]chromene-5,10-diones with Antiparasitic and Antioxidant Activities

New pyranonaphthoquinone derivatives were synthesized and investigated for their activity against Trypanosoma brucei, Leishmania major, and Toxoplasma gondii parasites. The pentafluorophenyl derivative was efficacious against T. brucei with single digit micromolar EC₅₀ values and against T. gondii with even sub-micromolar values. The 3-chloro-4,5-dimethoxyphenyl derivative showed an activity against amastigotes of Leishmania major parasites comparable to that of amphotericin B. In addition, antioxidant activities were observed for the bromophenyl derivatives, and their redox behavior was studied by cyclovoltammetry. Anti-parasitic and antioxidative activities of the new naphthoquinone derivatives appear uncorrelated.