Synthesis and applications of alkylated C-sugars as peptide bioconjugates

Summary Permethylated C-sugars affect the stability and solubility of their carbohydrate precursors and may represent an important group of bioconjugates. When properly functionalized, these units can be appended to the N- and C-termini or to the side chains of peptides or other therapeutic candidates. In this report, we describe the synthesis of an amine-functionalized alkylated mannose derivative and confirm the configuration by determining the X-ray crystal structure of its nitrile precursor. An acid functionalized counterpart, when attached to the N-terminus of a NR box peptide analog, improved binding to estrogen receptor β (ERβ) but not to ERα.     

Synthesis and evaluation of novel bioconjugates as antiviral agents.

Novel lipid (mannito-stearate) antiviral nucleoside and oligonucleotide conjugates were prepared with improved lipophilic and membrane associating properties of drugs. Potential advantages of these liponucleotide prodrugs are lower toxicity, increased cellular uptake, nuclease resistivity and antiviral activity. Oligonucleotide conjugate complementary to a unique segment of viral genome may selectively disrupt the processes dependent on the segment by hybridisation.     

Synthesis and characterization of bioconjugates of S-layer proteins

The self-assembling proteins that form crystalline surface layers (S-layers) on many microbial species have found numerous applications due to their nanostructured nature. To devise a new method to construct surface displays that exploit S-layer self-assembly activity and nanostructural properties, we have constructed polymer bioconjugates of S-layer proteins. The conjugates formed are similar in function to the monomer alkanethiols that form self-assembled monolayers (SAMs) on gold surfaces. However, the self-assembly is driven by the protein "headgroup" that positions polymer-tethered endgroups on a surface. This paper examines the integration of protein purification, conjugation, and surface assembly that has led to the development of this new method for the formation of nanostructured surfaces. Purified S-layer proteins from Lactobacillus brevis were conjugated with small molecule probes and polymers using amine-based reactions. To keep multiple labeling of protein amine groups to acceptable levels, the conjugations were performed at pH 6.5, allowing for limited yields (24-39%) as determined by mass spectrometry and SDS-polyacrylamide gel electrophoresis. As the presence of high levels of unlabeled S-layer proteins is undesired, we have developed a protocol for further purification that employs monomeric avidin affinity chromatography. The surface self-assembly of the polymer bioconjugates onto amine-terminated microspheres was studied using epi-fluorescence, confocal, and scanning electron microscopy. The surfaces obtained exhibited homogeneous distributions of tethered molecules. Also, in cases where the modular assembly of two distinct types of tethered endgroups was accomplished, there was no evidence for phase separation in the surfaces. The modular assembly method will provide a potential route to controlling surface display density as the starting assembly conditions guide displayed endgroup concentrations in mixed molecular monolayers.     

Synthesis, functionalization and photo-Bergman chemistry of enediyne bioconjugates

Methodology is outlined for the chemical synthesis of versatile photo-Bergman enediyne building blocks and their conjugates. Routes to both mono and bis conjugated enediyne templates are detailed together with representative examples of their bioconjugates, nanoconjugates, PEG derivatives and water soluble salts. The immunocompetence of antibody conjugates is retained, and application in the form of reagents for photodynamic therapy (PDT) advanced.     

Synthesis, characterization and immunogenicity of silk fibroin-L-asparaginase bioconjugates

L-asparaginase (ASNase) is one basic drug in the treatment of acute lymphoblastic leukemia (ALL). Because its half-life time is too short and it is easy to arouse allergic reaction, use in practical clinic is considerably limited. Silk fibroin (SF) with different molecular mass from 40 to 120 kDa is a natural biocompatible protein and could be used as a novel bioconjugate for enzyme modification to overcome its usual shortcomings mentioned above. When the enzyme was bioconjugated covalently with the water-soluble fibroin by glutaraldehyde, the enzyme kinetic properties and immune characteristics in vivo of the resulting silk fibroin-L-asparaginase (SF-ASNase) bioconjugates were investigated in detail. The results show that the modified ASNase was characterized by its higher residual activity (nearly 80%), increased heat and storage stability and resistance to trypsin digestion, and its longer half-life (63 h) than that of intact ASNase (33 h). The abilities of intact and modified ASNases to arouse allergic reaction are 2(4) and 2(1) antibody titers, respectively. Bioconjugation of silk fibroin significantly helps to reduce the immunogenicity and antigenicity of the enzyme. The apparent Michaelis constants of the modified ASNase (K(m(app))=0.844 x 10(-3)mol L(-1)) was approximately six times lower than that of enzyme alone, which suggests that the affinity of the enzyme to substrate l-asparagine elevated when bioconjugated covalently with silk fibroin. SF-ASNase bioconjugates could overcome the common shortcomings of the native form. Therefore, the modified ASNase coupled with silk fibroin has the potential values of being studied and developed as a new bioconjugate drug.     

Design,solid phase synthesis and evaluation of cationic ferrocenoyl peptide bioconjugates as potential antioxidant enzyme mimics

Synthetic C-terminal amidated cationic ferrocenoyl peptide bioconjugates Fc-Orn-Orn-Orn (1) and Fc-Tyr-Orn-Orn-Orn (2) were rationally designed as superoxide dismutase (SOD) mimics based on the structure of the iron SOD from Escherichia coli. Ferrocenoyl peptide bioconjugates 1, 2 and ferrrocenecarboxylic acid (4) were subsequently evaluated as SOD mimics and as inhibitors of peroxynitrite-mediated tyrosine nitration. Due to their cationic character, ferrocenoyl peptide bioconjugates 1 and 2 exerted an acceptable SOD activity (EC₅₀ = 575 μM and 310 μM, respectively) in comparison with 4 (EC₅₀ = 1.4 mM). The C-terminal amidated cationic peptide Ac-Tyr-Orn-Orn-Orn (3), designed as marker of peroxynitrite, was used to evaluate the inhibitory activity of 1 and 4 towards peroxynitrite-mediated tyrosine nitration. Both compounds proved to inhibit the nitration especially the cationic ferrocenoyl peptide bioconjugates 1. The ferrocene moiety of conjugate 2 displayed a strong inhibitory activity of peroxynitrite-mediated nitration of the neighboring tyrosine.     

Synthesis and anti-HIV activity of alkylated quinoline 2,4-diols

Naturally occurring quinolone alkaloids, buchapine (1) and compound 2 were synthesized as reported in literature and evaluated for anti-HIV potential in human CD4+ T cell line CEM-GFP, infected with HIV-1_NL4.3 virus by p24 antigen capture ELISA assay. The compounds 1 and 2 showed potent inhibitory activity with IC₅₀ value of 2.99 and 3.80 μM, respectively. Further, 45 alkylated derivatives of quinoline 2,4-diol were synthesized and tested for anti-HIV potential in human CD4+ T cell line CEM-GFP. Among these, 13 derivatives have shown more than 60% inhibition. We have identified three most potent inhibitors 6, 9 and 23; compound 6 was found to be more potent than lead molecule 1 with IC₅₀ value of 2.35 μM and had better therapeutic index (26.64) as compared to AZT (23.07). Five derivatives 7, 19a, 19d, 21 and 24 have displayed good noticeable anti-HIV activity. All active compounds showed higher CC₅₀ values which indicate that they have better therapeutic indices.     

Synthesis and properties of the first all-aza analogue of a biologically active peptide

The synthesis of the first all-aza-amino acid analogue ( 2 ) of a peptidic renin inhibitor is described. The X-ray structural analysis and molecular modelling investigations of this novel compound reveal interesting conformational features which have a significant impact on its biological activity. In addition, insight into conformational features of azapeptides in general in comparison with the corresponding purely peptidic compounds is given.     

Synthesis, photophysical effects, and DNA targeting properties of oxazole yellow-peptide bioconjugates

The design of a dsDNA-sensitive fluorescent bioconjugate capable of targeting a specific DNA sequence with high efficiency is described. The bioconjugate has the molecular recognition features of the polypeptide from a DNA-binding protein and the dsDNA-dependent fluorescence of an intercalating dye. The DNA sequence selectivity of the probe was characterized, as were the changes in photophysical properties of the dye upon covalent linkage to the peptide to assess whether such bioconjugates could function as molecular probes of gene sequences. The oxazole yellow-peptide bioconjugate exhibits DNA recognition and binding affinity comparable to the native Hin recombinase protein. Examination of photophysical effects to dye conjugation indicates a negligible affect on the fluorescence quantum yield. Fluorescence studies indicate this molecular probe is useful to determine the presence of a given DNA target sequence and gives negligible fluorescence in the absence of a given target site. Using the synthetic route described here, bioconjugates could be designed using different combinations of DNA recognition polypeptides and cyanine dyes to generate an array of sequence specific and wavelength specific probes.     

Synthesis of purine modified 2'-C-methyl nucleosides as potential anti-HCV agents

Based on the anti-hepatitis C activity of 2′-C-methyl-adenosine and 2′-C-methyl-guanosine, a series of new modified purine 2′-C-methyl nucleosides was prepared as potential anti-hepatitis C virus agents. Herein, we report the synthesis of both 6-modified and 2-modified purine 2′-C-methyl-nucleosides along with their anti-HCV replication activity and cytotoxicity in different cells.     

Synthesis and applications of chitosan mercaptanes as heavy metal retention agent

Chitosan, poly-beta(1-->4)-2 amino-2-deoxy-D-Glucopyranose is a biopolymer obtained by extensive deacetylation of chitin [poly-beta(1-->4)-2-acetamide-2-deoxy-D-Glucopyranose], main constituent of crustacean shells. The present study was carried out using crab shells from nylon shrimps (Heterocarpus reedi). Despite the abundance of raw material in our environment, little work has been published in this field using derivatives. The main goal of this work is to develop a good method to prepare chitosan mercaptanes derivatives using mercaptoacetic acid and 1-chloro-2,3-epoxy propane propionic acid. The evaluation of the retention capacities using several concentrations of copper and mercury solutions with concentrations ranging from 10 to 104 ppm at pH 2.5 and 4.5 are tested. A comparison of the absorption isotherms with Langmuir isotherms is also reported. Full characterization of the derivatives was carried out using FTIR, elemental analysis and TGA. The morphology of chitosan and derivatives is compared before and after treating polymers with mercury and copper ions.     

Synthesis and biological activity of novel peptide mimetics as melanocortin receptor agonists

A series of novel peptidomimetic analogs was prepared containing cyclohexyl, phenyl, or heterocyclic groups to ostensibly orient the guanidine or mimic of an arginine in a putative melanocortin receptor ligand pharmacophore. Some binding affinity at the melanocortin receptors MC(3) and MC(4) was noted. In silico docking also indicated that the relative positions of the hydrogen-bonding sites and hydrophobic regions of the compounds are reasonably well matched to the receptor-binding site. This may present a lead entry into a selective series of MC(4)R agonists.     

Synthesis of cyclic peptide homologs of glutathione as potential antitumor agents

Two cyclic tripeptide homologs, cyclo(Glu[Cys-beta-Ala-]-OH) 8a, and cyclo(Glu[Cys-Gaba-]-OH) 8b, were synthesized by the pentafluorophenyl ester method in solution. These cyclic peptides are cyclo homologs of glutathione and are designed as potential antitumor agents. The 1H- and 13C-n.m.r. spectral parameters of cyclo(Glu[Cys(Bzl)-beta-Ala-]-OH) 7a were measured in DMSO-d6 and a possible conformation has been proposed. The cyclic peptide 8a showed low cytotoxic activities against three human tumor cell lines: KB, HeLa, and Colo 205.     

Synthesis of RGD amphiphilic cyclic peptide as fibrinogen or fibronectin antagonist

Summary This paper investigates the effect of the incorporation of a diazaethylene glycol derivative (Deg,2) into a cyclic peptide containing the tripeptide sequence Arg-Gly-Asp (RGD). This motif is a common structural element of many integrin ligands. The synthesis of cyclo-(Arg-Gly-Asp-Deg) (7) has been accomplished in solution using standard peptide chemistry. The intent was to improve the bioavailability of this new RGD cyclic peptide, which is shown to interact with α_IIbβ₃ or α₅β₁ receptors. A preliminary step for the conformational study of peptide7 was done in DMSO-d ₆ using nuclear magnetic resonance spectroscopy techniques.     

Synthesis and analgesic activity of alkylated,reduced and constrained oligoheterocyclic peptidomimetic analogs of Leu-enkephalin

We report the design and the parallel solid phase synthesis of linear and oligoheterocyclic peptidomimetic analogs of Leu-enkephalin. The described peptidomimetics represent different unique scaffolds that distribute in the space the peptidyl side chains of amino acids essential for biological activity and mimic the bioactive conformation of the Leu-enkephalin peptide. All the compounds were screened in competitive radioligand binding assays to determine their affinities for μ-(MOR), and κ-(KOR) opioid receptors. A reduced analog of Leu-enkephalin TPI1879-26 with activity K_i = 60 nM for the mu receptor was identified.     

Synthesis and characterization of a peptide identified as a functional element in alphaA-crystallin

Eye lens alpha-crystallin is a member of the small heat shock protein (sHSP) family and forms large multimeric structures. Earlier studies have shown that it can act like a molecular chaperone and form a stable complex with partially unfolded proteins. We have observed that prior binding of the hydrophobic protein melittin to alpha-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence of mutually exclusive sites for these proteins in alpha-crystallin. To investigate the mechanism of the interaction between alpha-crystallin and substrate proteins, we determined the melittin-binding sites in alpha-crystallin by cross-linking studies. Localization of melittin-binding sites in alpha-crystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLTVK of alphaA-crystallin and FSVNLDVK of alphaB-crystallin as the chaperone sites. Of these sites, FVIFLDVKHFSPEDLTVK and FSVNLDVK were identified earlier as 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bis-ANS)-binding hydrophobic sites. Here we also report the synthesis and characterization of the peptide, KFVIFLDVKHFSPEDLTVK, having the melittin as well as bis-ANS-binding sequence of alphaA-crystallin. We show that this peptide has characteristics similar to that of alphaA-crystallin by in vitro thermal aggregation assay, gel filtration study, CD spectroscopy, and bis-ANS interaction studies. The peptide sequence corresponds to the beta3 and beta4 region present in the alpha-crystallin domain of sHSP 16.5. We hypothesize that the alpha-crystallin domain in other sHSPs may have a similar function and would likely possess the anti-aggregation property even when separated from the native protein.     

Synthesis, 18F-radiolabelling and biological evaluations of C-6 alkylated pyrimidine nucleoside analogues

Synthesis of pyrimidine derivatives with a side-chain attached to the C-6 of pyrimidine ring (6-14) is reported. Target compounds 8 and 12 were subjected to in vitro phosphorylation tests, determination of their binding affinities to herpes simplex virus (HSV-1) thymidine kinase (TK) and catalytic turnover constants. Fluorinated pyrimidine derivative 12 (40 microM) exhibited better binding affinity for HSV-1 TK than acyclovir (ACV, 170 microM) and ganciclovir (GCV, 48 microM). Catalytic turnover constant (k(cat)) of 12 (0.08 s(-1)) was close to the k(cat) values of ACV (0.10 s(-1)) and GCV (0.10 s(-1)). Furthermore, compounds 8 and 12 showed no cytotoxic effects in HSV-1 TK-transduced and non-transduced cell lines. Besides, compounds 8 and 12 did not exhibit antiviral or cytostatic activities against several viruses and malignant tumor cell lines that were evaluated. The new fluorinated pyrimidine derivative 16 that is phosphorylated by HSV-1 TK could be developed as non-toxic PET-tracer molecule. Thus, 18F labelling of the precursor 14 was performed by nucleophilic substitution using [18F] tetrabutylammonium fluoride as the fluorinating reagent.     

Synthesis and in vitro cytotoxicity of cis,cis,trans-diamminedichloridodisuccinatoplatinum(IV)-peptide bioconjugates

The synthesis and characterization of four Pt(IV)-peptide conjugates, containing one or two peptides in the axial position, designed for the purpose of targeted drug delivery to tumor cells, are described. The precursor cis,cis,trans-diamminedichloridodisuccinatoplatinum(IV) was coupled in the last step of standard solid-phase peptide synthesis (SSPS) with an analogue of neurotensin (pseudo-neurotensin = Lys-Lys-Pro-Tyr-Ile-Leu) and with octreotate (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH), an analogue of somatostatin, respectively. For all peptides, the SSPS reactions afforded both mono- and diconjugated Pt-peptide species, which were separated and purified by RP-HPLC. The two couples of conjugates, together with the precursor, were tested as cytotoxic agents towards different cancer cell lines. In general all conjugates are good inhibitors of cellular proliferation when compared to a nontargeting platinum(IV) parent compound, so that its relatively low cytotoxicity is greatly improved by addition of the peptides.