ABSTRACT
2-benzothiazoles and 2-(aminophenyl)benzothiazoles represent biologically interesting heterocycles with high pharmacological activity. The combination of these heterocycles with amino acids and peptides is of special interest, as such structures combine the advantages of amino acids and peptides with the advantages of the 2-benzothiazolyl and 2-(aminophenyl)benzothiazolyl pharmacophore group. In this work, we developed an easy and efficient method for the solid-phase synthesis of 2-benzothiazolyl (BTH) and 2-(aminophenyl)benzothiazolyl (AP-BTH) C-terminal modified amino acids and peptides with high chiral purity.
Subject(s)
Amino Acids , Solid-Phase Synthesis Techniques , Amino Acids/chemistry , Amines/chemistry , Benzothiazoles/chemistry , PeptidesABSTRACT
Thioether containing peptides were obtained following three synthetic routes. In route A, halo acids esterified on 2-chlorotrityl(Cltr) resin were reacted with N-fluorenylmethoxycarbonyl (Fmoc) aminothiols. These were either cleaved from the resin to the corresponding (Fmoc-aminothiol)carboxylic acids, which were used as key building blocks in solid phase peptide synthesis (SPPS), or the N-Fmoc group was deprotected and peptide chains were elongated by standard SPPS. The obtained N-Fmoc protected thioether containing peptides were then condensed either in solution, or on solid support, with the appropriate amino components of peptides. In route B, the thioether containing peptides were obtained by the reaction of N-Fmoc aminothiols with bromoacetylated peptides, which were synthesized on Cltr-resin, followed by removal of the N-Fmoc group and subsequent peptide elongation by standard SPPS. In route C, the thioether containing peptides were obtained by the condensation of a haloacylated peptide synthesized on Cltr-resin and a thiol-peptide synthesized either on 4-methoxytrityl(Mmt) or trityl(Trt) resin.
Subject(s)
Peptides/chemistry , Peptides/chemical synthesis , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/chemical synthesis , Solid-Phase Synthesis TechniquesABSTRACT
N-mercaptoalkylglycine residues were inserted into peptides by reacting N-free amino groups of peptides, which were initially synthesized on 2-chlorotrityl resin (Cltr) using the Fmoc/tBu method, with bromoacetic acid and subsequent nucleophilic replacement of the bromide by reacting with S-4-methoxytrityl- (Mmt)/S-trityl- (Trt) protected aminothiols. The synthesized thiols containing peptide-peptoid hybrids were cleaved from the resin, either protected by treatment with dichloromethane (DCM)/trifluoroethanol (TFE)/acetic acid (AcOH) (7:2:1), or deprotected (fully or partially) by treatment with trifluoroacetic acid (TFA) solution using triethylsilane (TES) as a scavenger.
Subject(s)
Glycine/chemistry , Peptides/chemistry , Molecular Structure , Peptoids/chemistry , Solid-Phase Synthesis Techniques , Sulfhydryl Compounds/chemical synthesis , Sulfhydryl Compounds/chemistryABSTRACT
Amyloid ß (Aß) species are considered as potential targets for the development of diagnostics/therapeutics towards Alzheimer's disease (AD). Nanoliposomes which are decorated with molecules having high affinity for Aß species may be considered as potential carriers for AD theragnostics. Herein, benzothiazolyl (BTH) decorated nanoliposomes were prepared for the first time, after synthesis of a lipidic BTH derivative (lipid-BTH). The synthetic pathway included acylation of bis(2-aminophenyl) disulfide with palmitic acid or palmitoyl chloride and subsequent reduction of the oxidized dithiol derivative. The liberated thiols were able to cyclize to the corresponding benzothiazolyl derivatives only after acidification of the reaction mixture. Each step of the procedure was monitored by HPLC analysis in order to identify all the important parameters for the formation of the BTH-group. Finally, the optimal methodology was identified, and was applied for the synthesis of the lipid-BTH derivative. BTH-decorated nanoliposomes were then prepared and characterized for physicochemical properties (size distribution, surface charge, physical stability, and membrane integrity during incubation in presence of buffer and plasma proteins). Pegylated BTH-nanoliposomes were demonstrated to have high integrity in the presence of proteins (in comparison to non-peglated ones) justifying their further exploitation as potential theragnostic systems for AD.
Subject(s)
Benzothiazoles/chemical synthesis , Nanoparticles/chemistry , Benzothiazoles/chemistry , Liposomes , Particle Size , Polyethylene Glycols/chemistryABSTRACT
Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.
Subject(s)
Chloramphenicol/pharmacology , Fluorenes/chemistry , Peptides/pharmacology , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Ribosomes/drug effects , Amino Acid Sequence , Binding Sites , Chloramphenicol/analogs & derivatives , Chloramphenicol/chemical synthesis , Escherichia coli K12/chemistry , Escherichia coli K12/genetics , Escherichia coli K12/metabolism , Green Fluorescent Proteins/antagonists & inhibitors , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Internal Ribosome Entry Sites/drug effects , Models, Molecular , Peptides/chemical synthesis , Peptidyl Transferases/antagonists & inhibitors , Peptidyl Transferases/genetics , Peptidyl Transferases/metabolism , Poly A/genetics , Poly A/metabolism , Protein Binding , Protein Synthesis Inhibitors/chemical synthesis , Puromycin/pharmacology , Ribosomes/genetics , Ribosomes/metabolismABSTRACT
A fast and efficient microwave (MW)-assisted solid-phase peptide synthesis protocol using the 2-chlorotrityl chloride resin and the Fmoc/tBu methodology, has been developed. The established protocol combines the advantages of MW irradiation and the acid labile 2-chlorotrityl chloride resin. The effect of temperature during the MW irradiation, the degree of resin substitution during the coupling of the first amino acids and the rate of racemization for each amino acid were evaluated. The suggested solid phase methodology is applicable for orthogonal peptide synthesis and for the synthesis of cyclic peptides.
Subject(s)
Chemistry Techniques, Analytical/methods , Peptides, Cyclic/chemical synthesis , Solid-Phase Synthesis Techniques , Trityl Compounds/chemistry , Microwaves , Peptides, Cyclic/chemistry , TemperatureABSTRACT
Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/drug therapy , Epitopes/chemistry , Myelin-Oligodendrocyte Glycoprotein/chemistry , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Amino Acid Sequence , Animals , Encephalomyelitis, Autoimmune, Experimental/metabolism , Epitopes/metabolism , Epitopes/pharmacology , Female , Humans , Ligands , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Multiple Sclerosis/drug therapy , Multiple Sclerosis/metabolism , Myelin Proteins/chemistry , Myelin Proteins/metabolism , Myelin-Oligodendrocyte Glycoprotein/metabolism , Peptide Fragments/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Trityl Compounds/chemistryABSTRACT
A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60-110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60-110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys(77)-Cys(109) bond, followed by iodine oxidation to form the Cys(67)-Cys(99) bond.
Subject(s)
Carrier Proteins/chemistry , Cytokines/chemistry , Microwaves , Peptides/chemical synthesis , Resins, Synthetic/chemistry , Trityl Compounds/chemistry , Humans , Molecular Structure , Peptides/chemistry , Peptides/isolation & purification , Resins, Synthetic/isolation & purificationABSTRACT
Human gene 2 relaxin (RLX) is a member of the insulin superfamily and is a multi-functional factor playing a vital role in pregnancy, aging, fibrosis, cardioprotection, vasodilation, inflammation, and angiogenesis. RLX is currently applied in clinical trials to cure among others acute heart failure, fibrosis, and preeclampsia. The synthesis of RLX by chemical methods is difficult because of the insolubility of its B-chain and the required laborious and low yielding site-directed combination of its A (RLXA) and B (RLXB) chains. We report here that oxidation of the Met(25) residue of RLXB improves its solubility, allowing its effective solid-phase synthesis and application in random interchain combination reactions with RLXA. Linear Met(O)(25)-RLX B-chain (RLXBO) reacts with a mixture of isomers of bicyclic A-chain (bcRLXA) giving exclusively the native interchain combination. Applying this method Met(O)(25)-RLX (RLXO) was obtained in 62% yield and was easily converted to RLX in 78% yield, by reduction with ammonium iodide.
Subject(s)
Relaxin/chemical synthesis , Amino Acid Sequence , Chromatography, Liquid , Humans , Molecular Sequence Data , Oxidation-Reduction , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, UltravioletABSTRACT
The RING finger domain of the Mdm2, located at the C-terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48-residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid-phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C-terminus as the amino component. Best results were achieved using solution condensation where the N-component was applied with the C-terminal carboxyl group left unprotected. The developed method is well suited for large-scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid-phase and solution synthesis.
Subject(s)
Cysteine/chemistry , Proto-Oncogene Proteins c-mdm2 , RING Finger Domains , Amino Acid Sequence , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/genetics , Proto-Oncogene Proteins c-mdm2/chemical synthesis , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/genetics , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/geneticsABSTRACT
Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system, and it has been established that autoreactive T helper (Th) cells play a crucial role in its pathogenesis. Myelin basic protein (MBP) epitopes are major autoantigens in MS, and the sequence MBP87-99 is an immunodominant epitope. We have previously reported that MBP87-99 peptides with modifications at principal T-cell receptor (TCR) contact sites suppressed the induction of EAE symptoms in rats and SJL/J mice, diverted the immune response from Th1 to Th2 and generated antibodies that did not cross react with the native MBP protein. In this study, the linear and cyclic analogs of the MBP87-99 epitope, namely linear (Ala91,Ala96)MBP87-99 (P2) and cyclo(87-99)(Ala91,Ala96)MBP87-99 (P3), were evaluated for their binding to HLA-DR4, stability to lysosomal enzymes, their effect on cytokine secretion by peripheral blood mononuclear cells (PBMC) derived from MS patients or healthy subjects (controls), and their effect in rat EAE. P1 peptide (wild-type, MBP87-99) was used as control. P2 and P3 did not alter significantly the cytokine secretion by control PBMC, in contrast to P1 that induced moderate IL-10 production. In MS PBMC, P2 and P3 induced the production of IL-2 and IFN-γ, with a simultaneous decrease of IL-10, whereas P1 caused a reduction of IL-10 secretion only. The cellular response to P3 indicated that cyclization did not affect the critical TCR contact sites in MS PBMC. Interestingly, the cyclic P3 analog was found to be a stronger binder to HLA-DR4 compared to linear P2. Moreover, cyclic P3 was more stable to proteolysis compared to linear P2. Finally, both P2 and P3 suppressed EAE induced by an encephalitogenic guinea pig MBP74-85 epitope in Lewis rats whereas P1 failed to do so. In conclusion, cyclization of myelin altered peptide ligand (Ala91,Ala96)MBP87-99 improved binding affinity to HLA-DR4, resistance to proteolysis and antigen-specific immunomodulation, rendering cyclo(87-99)(Ala91,Ala96)MBP87-99 an important candidate drug for MS immunotherapy.
Subject(s)
Immunotherapy , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Myelin Basic Protein/pharmacology , Peptide Fragments/pharmacology , Adolescent , Adult , Aged , Animals , Cell Proliferation/drug effects , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Humans , Leukocytes, Mononuclear/drug effects , Ligands , Male , Middle Aged , Molecular Structure , Multiple Sclerosis/pathology , Myelin Basic Protein/chemical synthesis , Myelin Basic Protein/chemistry , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Rats , Rats, Inbred Lew , Young AdultABSTRACT
Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide.
Subject(s)
Disulfides/chemistry , Peptide Fragments/chemistry , Serpins/chemistry , Serpins/chemical synthesis , Amino Acid Sequence , Molecular Sequence Data , Oxidation-Reduction , Peptide Fragments/genetics , Protein Structure, Tertiary , Serpins/geneticsABSTRACT
Hirudin variant 1 (HV1), a small protein consisting of 65 amino acids and three disulfide bonds, was synthesized by using Fmoc-based convergent methods on 2-chlorotrityl resin (CLTR). The linear sequence was assembled by the sequential condensation of 7 protected fragments, on the resin-bound 55-65 fragment. The conditions of fragment assembly were carefully studied to determine the most efficient synthetic protocol. Crude reduced [Cys(16, 28)(Acm)]-HV1 thus obtained was easily purified to homogeneity by RP-HPLC. Disulfide bridges were successfully formed by a two-step procedure, involving an oxidative folding step to form Cys(6)-Cys(14) and Cys(22)-Cys(39) linkages, followed by iodine oxidation to form the Cys(16)-Cys(28) bond. The correct disulfide bond alignment was established by peptide mapping using Staphylococcus aureus V8 protease at pH 4.5.
Subject(s)
Hirudins/chemical synthesis , Amino Acid Sequence , Chromatography, High Pressure Liquid , Molecular Sequence Data , Oxidation-Reduction , Peptide Fragments/chemistryABSTRACT
Monophthaloyl diamines derived from naturally occurring amino acids were attached through their free amino functions to resins of the trityl type. The phthaloyl groups were removed by hydrazinolysis, and peptide chains were assembled using Fmoc/tBu-amino acids on the liberated amino functions. The peptidyl aminoalkyl amides obtained were cleaved from the resins by mild acidolysis, with the tBu-side chain protection remaining intact.