Applications of biaryl cyclization in the synthesis of cyclic enkephalin analogs with a highly restricted flexibility.

A series of 10 cyclic, biaryl analogs of enkephalin, with Tyr or Phe residues at positions 1 and 4, were synthesized according to the Miyaura borylation and Suzuki coupling methodology. Biaryl bridges formed by side chains of the two aromatic amino acid residues are of the meta-meta, meta-para, para-meta, and para-para configuration. Conformational properties of the peptides were studied by CD and NMR. CD studies allowed only to compare conformations of individual peptides while NMR investigations followed by XPLOR calculations provided detailed information on their conformation. Reliability of the XPLOR calculations was confirmed by quantum chemical ones performed for one of the analogs. No intramolecular hydrogen bonds were found in all the peptides. They are folded and adopt the type IV ß-turn conformation. Due to a large steric strain, the aromatic carbon atoms forming the biaryl bond are distinctly pyramidalized. Seven of the peptides were tested in vitro for their affinity for the µ-opioid receptor.

Alkyl Thiocyanurates as Thioester Mimetics. Transthioesterification and Ligation Reactions with High Potential in Dynamic Covalent Chemistry.

Alkyl thiocyanurates, the compounds formed in the SN reaction of thiocyanuric acid and alkyl halides, are susceptible to transthioesterification and ligation with molecules containing cysteamine, analogous to native chemical ligation of thioesters with peptides with an N-terminal cysteine moiety. The ligation is irreversible and results in the formation of mono- and disubstituted products dominantly. Transthioesterification, in contrast, is fully reversible and may be used in constructing dynamic systems. The application of this reactivity in dynamic covalent chemistry has been exemplified by the preparation of a library of mixed thiocyanurates of glutathione and thioglycolic acid with self-assembly abilities and metathesis between thiocyanurates of tris(carboxymethyl) and tris(carboxamidomethyl) catalyzed by MESNa (sodium 2-mercaptoethylsulphonate) or MPAA (4-mercaptophenylacetic acid). Differences in reactivity of thiocyanurates toward cysteamines and thiols has been explained based on conceptual DFT.

Self-Synthesizing Models of Helical Proteins Based on Aromatic Disulfide Chemistry.

A new method of synthesis of peptide conjugates with aromatic moieties substituted with two sulfhydryl groups at 1,3-positions is proposed. Amphiphilic peptides derivatized in such a way under oxidative conditions spontaneously form cyclic, covalent trimers and tetramers dominated by α-helical conformations. The tendency to form tri- or tetrahelical bundles depends on sequences of the peptides and on the oxidation conditions, pH, and additives.

A general method for preparation of N-Boc-protected or N-Fmoc-protected α,ß-didehydropeptide building blocks and their use in the solid-phase peptide synthesis.

N-(tert-butyloxycarbonyl) or N-(9-fluorenylmethoxycarbonyl) dipeptides with C-terminal (Z)-α,ß-didehydrophenylalanine (∆Z Phe), (Z)-α,ß-didehydrotyrosine (∆Z Tyr), (Z)-α,ß-didehydrotryptophan (∆Z Trp), (Z)-α,ß-didehydromethionine (∆Z Met), (Z)-α,ß-didehydroleucine (∆Z Leu), and (Z/E)-α,ß-didehydroisoleucine (∆Z/E Ile) were synthesised from their saturated analogues via oxidation of intermediate 2,5-disubstituted-oxazol-5-(4H)-ones (also known as azlactones) with pyridinium tribromide followed by opening of the produced unsaturated oxazol-5-(4H)-one derivatives in organic-aqueous solution with a catalytic amount of trifluoroacetic acid or by a basic hydrolysis. In all cases, a very strong preference for Z isomers of α,ß-didehydro-α-amino acid residues was observed except of the ΔIle, which was obtained as the equimolar mixture of Z and E isomers. Reasons for the (Z)-stereoselectivity and the increased stability of the aromatic α,ß-didehydro-α-amino acid residue oxazol-5-(4H)-ones over the corresponding aliphatic ones are also discussed. It is the first use of such a procedure to synthesise peptides with the C-terminal unsaturated residues and a peptide with 2 consecutive ΔPhe residues. This approach is very effective especially in the synthesis of peptides with aliphatic α,ß-didehydro-α-amino acid residues that are difficult to obtain by other methods. It allowed the first synthesis of the ∆Met residue. It is also more cost-effective and less laborious than other synthesis protocols. The dipeptide building blocks obtained were used in the solid-phase synthesis of model peptides on a polystyrene-based solid support. Peptides containing aromatic α,ß-didehydro-α-amino acid residues were obtained with PyBOP or TBTU as a coupling agent with good yields and purities. In the case of aliphatic α,ß-didehydro-α-amino acid residues, a good efficiency was achieved only with DPPA as a coupling agent.

Synthesis, receptor binding studies, optical spectroscopic and in silico structural characterization of morphiceptin analogs with cis-4-amino-L-proline residues.

Three novel morphiceptin analogs, in which Pro in position 2 and/or 4 was replaced by cis-4-aminoproline connected with the preceding amino acid through the primary amino group, were synthesized. The opioid receptor affinities, functional assay results, enzymatic degradation studies and experimental and in silico structural analysis of such analogs are presented. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Novel analogs of alloferon: Synthesis, conformational studies, pro-apoptotic and antiviral activity.

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala(9)]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys(9)]-, [Phg(9)]-, [Lys(12)]-, and [Phe(12)]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.

Stapling of leu-enkephalin analogs with bifunctional reagents for prolonged analgesic activity.

The design and synthesis of leu-enkephalin analogs by replacing the glycine residues with N-(2-thioethyl)glycines and opening the cyclisation potential is presented. The cyclization (stapling) was achieved using bifunctional reagents (hexafluorobenzene and trithiocyanuric acid derivatives). The CD conformational studies of the stapled analogs suggest that the peptides adopt the type I ß-turn conformation, which is in agreement with the theoretical analysis. The analog containing a trithiocyanuric acid derivative with a benzyl substituent shows potent analgesic activity.

Enhanced ß-turn conformational stability of tripeptides containing ΔPhe in cis over trans configuration.

Conformations of three pairs of dehydropeptides with the opposite configuration of the ΔPhe residue, Boc-Gly-Δ(Z/E)Phe-Phe-p-NA (Z- p -NA and E- p -NA), Boc-Gly-Δ(Z/E)Phe-Phe-OMe (Z-OMe and E-OMe), and Boc-Gly-Δ(Z/E)Phe-Phe-OH (Z-OH and E-OH) were compared on the basis of CD and NMR studies in MeOH, TFE, and DMSO. The CD results were used as the additional input data for the NMR-based calculations of the detailed solution conformations of the peptides. It was found that Z- p -NA, E- p -NA, Z-OMe, and Z-OH adopt the ß-turn conformations and E-OMe and E-OH are unordered. There are two overlapping type III ß-turns in Z- p -NA, type II' ß-turn in E- p -NA, and type II ß-turn in Z-OMe and Z-OH. The results obtained indicate that in the case of methyl esters and peptides with a free carboxyl group, Δ(Z)Phe is a much stronger inducer of ordered conformations than Δ(E)Phe. It was also found that temperature coefficients of the amide protons are not reliable indicators of intramolecular hydrogen bonds donors in small peptides.

Two phosphonodehydrotripeptides: Boc0-Gly1-Δ(Z)Phe2-α-Abu3PO3Me2 and Boc0-Gly1-Δ(Z)Phe2-α-Nva3PO3Et2.

The present paper reports the crystal structures of two short phosphonotripeptides (one in two crystal forms) containing one ΔPhe (dehydrophenylalanine) residue, namely dimethyl (3-{[tert-butoxycarbonylglycyl-α,ß-(Z)-dehydrophenylalanyl]amino}propyl)phosphonate, Boc(0)-Gly(1)-Δ(Z)Phe(2)-α-Abu(3)PO3Me2, C21H32N3O7P, (I), and diethyl (4-{[tert-butoxycarbonylglycyl-α,ß-(Z)-dehydrophenylalanyl]amino}butyl)phosphonate, Boc(0)-Gly(1)-Δ(Z)Phe(2)-α-Nva(3)PO3Et2, as the propan-2-ol monosolvate 0.122-hydrate, C24H38N3O7P·C3H8O·0.122H2O, (II), and the ethanol monosolvate 0.076-hydrate, C24H38N3O7P·C2H6O·0.076H2O, (III). The crystals of (II) and (III) are isomorphous but differ in the type of solvent. The phosphono group is linked directly to the last Cα atom in the main chain for all three peptides. All the amino acids are trans linked in the main chains. The crystal structures exhibit no intramolecular hydrogen bonds and are stabilized by intermolecular hydrogen bonds only.

Intramolecular cross-linking in the native JHBP molecule.

Juvenile hormone binding protein (JHBP) acts as a shuttle, carrying one of the most crucial hormones for insect development to target tissues. We have found that although the JHBP molecule does not contain tryptophan residues, it exhibits a weak fluorescence maximum near 420nm upon excitation at 315nm. Gel filtration experiments performed in denaturing conditions and ESI-MS analyses excluded the possibility that some low molecular ligand was bound to the protein molecules. Further UV and CD spectroscopy studies, as well as immunoblotting, showed that the unusual JHBP optical properties were due to dityrosine intramolecular cross-linking. These bridges were detected both in native and recombinant protein molecules. We believe that in Galleria mellonella hemolymph the DT generation occurs via ROS-mediated oxidation leading to the formation of cross-linked JHBP monomers. MS analyses of peptides generated after JHBP proteolysis indicated, that the dityrosine bridge occurs between the Y128 and Y130 residues.

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes.

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-ß-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47 kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.

Two pentadehydropeptides with different configurations of the DeltaPhe residues.

Comparison of the crystal structures of two pentadehydropeptides containing DeltaPhe residues, namely (Z,Z)-N-(tert-butoxycarbonyl)glycyl-alpha,beta-phenylalanylglycyl-alpha,beta-phenylalanylglycine (or Boc(0)-Gly(1)-Delta(Z)Phe(2)-Gly(3)-Delta(Z)Phe(4)-Gly(5)-OH) methanol solvate, C(29)H(33)N(5)O(8) x CH(4)O, (I), and (E,E)-N-(tert-butoxycarbonyl)glycyl-alpha,beta-phenylalanylglycyl-alpha,beta-phenylalanylglycine (or Boc(0)-Gly(1)-Delta(E)Phe(2)-Gly(3)-Delta(E)Phe(4)-Gly(5)-OH), C(29)H(33)N(5)O(8), (II), indicates that the Delta(Z)Phe residue is a more effective inducer of folded structures than the Delta(E)Phe residue. The values of the torsion angles phi and psi show the presence of two type-III' beta-turns at the Delta(Z)Phe residues and one type-II beta-turn at the Delta(E)Phe residue. All amino acids are linked trans to each other in both peptides. Beta-turns present in the peptides are stabilized by intramolecular 4-->1 hydrogen bonds. Molecules in both structures form two-dimensional hydrogen-bond networks parallel to the (100) plane.

4-Methylpseudoproline analogues of cyclolinopeptide A: Synthesis, structural analysis and evaluation of their suppressive effects in selected immunological assays.

The synthesis of new analogues of cyclolinopeptide A (CLA) and their linear precursors modified with (R)- and (S)-4-methylpseudoproline in the Pro3-Pro4 fragment are presented. The peptides were tested in comparison with cyclosporine A (CsA) in concanavalin A (Con A) and pokeweed mitogen (PWM)-induced mouse splenocyte proliferation and in secondary humoral immune response in vitro to sheep erythrocytes (SRBC). Their effects on expression of selected signaling molecules in the Jurkat T cell line were also determined. In addition, the structural features of the peptides, applying nuclear magnetic resonance and circular dichroism, were analyzed. The results showed that only peptides 7 and 8 modified with (R)-4-methylpseudoproline residue (c(Leu1-Val2-(R)-(αMe)Ser(ΨPro)3-Pro4-Phe5-Phe6-Leu7-Ile8-Ile9) and c(Leu1-Val2-Pro3-(R)-(αMe)Ser(ΨPro)4-Phe5-Phe6-Leu7-Ile8-Ile9), respectively) strongly suppressed mitogen-induced splenocyte proliferation and the humoral immune response, with peptide 8 being more potent. Likewise, peptide 8 more strongly elevated expression of Fas, a proapoptotic signaling molecule in Jurkat cells. We postulate that the increased biological activity of peptide 8, compared to the parent molecule and other studied peptides, resulted from its more flexible structure, found on the basis of both CD and NMR studies. CD and NMR spectra showed that replacement of Pro3 by (R)-(αMe)Ser(¬Pro) caused much greater conformational changes than the same replacement of the Pro4 residue. Such a modification could lead to increased conformational freedom of peptide 8, resulting in a greater ability to adopt a more compact structure, better suited to its putative receptor. In conclusion, peptide 8 is a potent immune suppressor which may find application in controlling immune disorders.

Isoform-specific variation in the intrinsic disorder of the ecdysteroid receptor N-terminal domain.

The Drosophila melanogaster ecdysteroid receptor (EcR) is a member of the nuclear hormone receptor superfamily. EcR controls animal development and metamorphosis by activating or repressing the transcription of target genes. There are three EcR isoforms, EcRA, EcRB1, and EcRB2 that exhibit diverse spatial and temporal distributions within various tissues and reveal essential functional differences. These differences can be attributed to the isoform-specific N-terminal domains (NTDs), which differ in length and primary structure. To lay a foundation for understanding of the molecular mechanism underlying functional diversity of the isoforms, we have carried out a comprehensive biochemical and biophysical analysis of purified hexahistidine-tagged EcRA and EcRB1 NTDs (EcRA-NTD and EcRB1-NTD). The results, along with in silico examinations of the primary structures indicate that the EcR NTDs exhibit properties of premolten globule-like intrinsically disordered proteins. Furthermore, we demonstrate for the first time that NTDs of isoforms of a particular nuclear hormone receptor exhibit distinct structural properties. In silico analysis revealed that the EcRA-NTD sequence has a bigger tendency for disorder than the EcRB1-NTD sequence. Accordingly, the circular dichroism experiments demonstrated that EcRA-NTD has lower regular secondary structure content than EcRB1-NTD and the size-exclusion chromatography showed that EcRA-NTD is less compact than EcRB1-NTD. Furthermore, the limited proteolysis analysis revealed that the C-terminal region common to both NTDs is more susceptible to the enzymatic cleavage in EcRA-NTD than in EcRB1-NTD. We postulate that unique conformational states of EcRA-NTD and EcRB1-NTD might act as the starting points for the functional diversity of EcRA and EcRB1 isoforms.

Starmaker exhibits properties of an intrinsically disordered protein.

Fish otoliths composed of calcium carbonate and an organic matrix play a primary role in gravity sensing and the perception of sound. Starmaker (Stm) was the first protein found to be capable of influencing the process of biomineralization of otoliths. Stm dictates the shape, size, and selection of calcium carbonate polymorphs in a concentration-dependent manner. To facilitate exploration of the molecular basis of Stm function, we have developed and optimized a protocol for efficient expression and purification of the homogeneous nontagged Stm. The homogeneous nontagged Stm corresponds to its functional form, which is devoid of a signal peptide. A comprehensive biochemical and biophysical analysis of recombinant Stm, along with in silico examinations, indicate for the first time that Stm exhibits the properties of intrinsically disordered proteins. The functional significance of Stm having intrinsically disordered protein properties and its possible role in controlling the formation of otoliths is discussed.

Solid-phase synthesis of peptides containing aminoadipic semialdehyde moiety and their cyclisations.

Pathological levels of oxidative stress (OS) have been implicated in many diseases including diabetes mellitus, neurodegenerative diseases, inflammatory diseases, atherosclerosis, and cancer. Studies of oxidative stress are however complicated by the low concentration of oxidation products. To resolve this problem, we tested a new derivative of aminoadipic semialdehyde (Fmoc-Aea-OH) in the solid-phase synthesis of carbonylated peptides. We prepared a series of peptides with free and acetylated N-terminal amino groups using the Fmoc-Aea-OH reagent. LC-MS, ESI-MS, and MS/MS spectra confirmed the sequences of the modified peptides, although the LC-MS and ESI-MS spectra were dominated by signals corresponding to dehydration products. NMR studies of acetylated products revealed that the dominant product formed in this reaction contains a 1,2,3,4-tetrahydropyridine-2-carboxylic acid residue. Another side reaction in this system was the cleavage of the amide bond between the Aea residue and the amino acid moiety preceding it resulting in the formation of a side product with a six-membered ring at the N-terminus (2,3,4,5-tetrahydropyridine-2-carboxylic acid residue). We found that, depending on the peptide sequence, one of those side products is predominant. Our work suggests new methods for the solid-state synthesis of peptides containing unnatural amino acids.

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH.

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.

Albumin binds self-assembling dyes as specific polymolecular ligands.

Self-assembling dyes with a structure related to Congo red (e.g. Evans blue) form polymolecular complexes with albumin. The dyes, which are lacking a self-assembling property (Trypan blue, ANS) bind as single molecules. The supramolecular character of dye ligands bound to albumin was demonstrated by indicating the complexation of dye molecules outnumbering the binding sites in albumin and by measuring the hydrodynamic radius of albumin which is growing upon complexation of self-assembling dye in contrast to dyes lacking this property. The self-assembled character of Congo red was also proved using it as a carrier introducing to albumin the intercalated nonbonding foreign compounds. Supramolecular, ordered character of the dye in the complex with albumin was also revealed by finding that self-assembling dyes become chiral upon complexation. Congo red complexation makes albumin less resistant to low pH as concluded from the facilitated N-F transition, observed in studies based on the measurement of hydrodynamic radius. This particular interference with protein stability and the specific changes in digestion resulted from binding of Congo red suggest that the self-assembled dye penetrates the central crevice of albumin.

Mapping of an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine mono- and bi-layer binding site in erythroid beta-spectrin.

It has been shown previously that binding of vesicles and monolayers containing PE (phosphatidylethanolamine) by either erythroid or non-erythroid spectrin proved sensitive to inhibition by purified erythrocyte ankyrin. We tested the lipid-binding affinities of the purified ankyrin-binding domain of beta-spectrin and of its truncated mutants in four ways, by analysing: (1) penetration of 'loose' PE/PC (phosphatidylcholine) monolayers; (2) binding to liposomes in suspension; (3) competition with spectrin for liposomes; and (4) binding of a PE/PC monolayer in a surface plasmon resonance system. The results obtained indicated that the full-length ankyrin-binding domain bound PE/PC mono- and bi-layers with moderate affinity, penetrated monolayers and competed with spectrin for liposomes. Moreover, its truncated mutants that retained the N-terminal part, in contrast with those lacking eight or 38 N-terminal residues (which bound lipid mono- and bi-layers with lower affinity), bound PE/PC mono- and bi-layers with an affinity and capacity comparable with those of the full-length ankyrin-binding domain, and this activity was inhibited by purified erythrocyte ankyrin. The full-length domain, in contrast with the mutant lacking 38 N-terminal residues, induced a small increase in the fluidity of PE/PC membranes when probed with 5'-doxyl stearate, similar to the effect of purified spectrin. Therefore we conclude that the binding site for PE-rich lipids, which is sensitive to ankyrin inhibition, is located in a 38-residue N-terminal fragment of the beta-spectrin ankyrin-binding domain, and that the first eight residues play a key role in this activity.

Metallacrowns as products of the aqueous medium self-assembly of histidinehydroxamic acid-containing polypeptides.

Self-assembly is a widely studied, spontaneous, and reversible phenomenon leading to the formation of the ordered structures by non-covalent specific interactions among starting molecules. In this work, a new template for the self-assembly of polypeptides based on peptides containing the C-terminal histidinehydroxamic acid moiety and Cu(2+) ions is characterized. Two peptide (tripeptide and pentadecapeptide) hydroxamic acid systems were synthesized and their interactions with Cu(2+) ions were investigated, revealing a high stability of the supramolecular assemblies formed. The supramolecular metallacrown-based L4Cu5 complexes exist at physiological pH in the presence of Cu(2+) ions as is evidenced from the spectroscopic methods, ESI mass spectrometry, and physicochemical techniques.