Synthetic, site-specific biotinylated analogs of human MCP-1.

Human monocyte chemoattractant protein 1 (MCP-1, CCL2) is a 8.6-kDa protein that has been implicated in a number of diseases including atherosclerosis, rheumatoid arthritis, chronic obstructive pulmonary disease and cancer. As part of a program to identify antibodies against MCP-1, we synthesized site-specific, biotinylated human MCP-1 analogs to be used for panning of an antibody phage display library. In contrast to material obtained from random biotinylation, the site-specific biotinylated analogs were homogeneous and retained full activity.     

Peptide Fragments and Structural Analogues of Chemokine MCP-1: Synthesis and Effect on the MCP-1-Induced Migration of Mononuclear Cells

Fourteen fragments and structural analogues of chemokine MCP-1 were synthesized using the Fmoc-strategy of solid phase peptide synthesis. The effect of synthesized peptides on the MCP-1-stimulated migration of mononuclear cells was examined. Both in vitro stimulants and inhibitors of the monocyte migration were found among the peptides. A possible participation of the C-terminal part of the MCP-1 molecule in the inhibition of the MCP-1-stimulated cell migration was found for the first time.     

Synthesis of 5(6)-dihydro-OSW-1 analogs bearing three kinds of disaccharides linking at 15-hydroxy and their antitumor activities

In order to study the SAR of 5(6)-dihydro-OSW-1, eight 15(α)β-O-glycosyl analogs (26-33) carrying three kinds of disaccharides including [β-d-Xylp-(1-3)-α-l-Arap], [β-d-Xylp-(1-4)-α-l-Arap] and [α-l-Rhap-(1-2)-(α)β-d-Glcp] were designed and synthesized. Their in vitro antitumor activities were tested by the standard MTT assay which disclosed that compound 33 (IC₅₀ = 0.28-0.52 μM) showed potential antitumor activities.     

Synthesis and DNA-binding ability of Spl protein zinc finger domain and its peptidomimetics

The second zinc finger fragment of Sp1 (Spl-ZF2), its mutant (Spl-ZF2/HT. E20→H, R23→T), and two mimic analogues (ZF20 and ZF15) were synthesized by stepwise solid phase technique. The CD spectra and UV-visible spectrum with CoCl2 indicated that the formation of zinc finger structure was affected not only by the hy-drophobic amino acids but also by the change of the distance between Cys and His. Gel-retardation electrophoresis as-says indicated that the Grlu and Arg residues are very important for recognition. A single zinc finger like Spl-ZF2 isable to bind DNA sequence specifically.     

The Total Chemical Synthesis of Monocyte Chemotactic Protein-1 (MCP-1)

The affinity-based N^α-amino protecting group tetrabenzo [a,c,g,i]fluorenyl-17-methoxycarbonyl (Tbfmoc) has been utilized as a hydrophobic probe to allow the simple, quick and highly effective isolation of a 76 residue cysteine-containing protein (MCP-1). The base-labile Tbfmoc group can be removed under very mild conditions, which preserve the thiol-con taining protein in the reduced state. Oxidative folding was then used to furnish the biologically active β-chemokine MCP-1.     

Cross‐reactivity of a human IgG1 anticitrullinated fibrinogen monoclonal antibody to a citrullinated profilaggrin peptide

Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease. It is characterized by persistent joint inflammation, resulting in loss of joint function, morbidity and premature mortality. The presence of antibodies against citrullinated proteins is a characteristic feature of RA and up to 70% of RA patients are anticitrullinated protein antibody (ACPA) positive. ACPA responses have been widely studied and are suggested to be heterogeneous, favoring antibody cross‐reactivity to citrullinated proteins. In this study, we examined factors that may influence cross‐reactivity between a commercial human anticitrullinated fibrinogen monoclonal antibody and a citrullinated peptide. Using a citrullinated profilaggrin sequence (HQCHQEST‐ Cit‐GRSRGRCGRSGS) as template, cyclic and linear truncated peptide versions were tested for reactivity to the monoclonal antibody. Factors such as structure, peptide length and flanking amino acids were found to have a notable impact on antibody cross‐reactivity. The results achieved contribute to the understanding of the interactions between citrullinated peptides and ACPA, which may aid in the development of improved diagnostics of ACPA.     

Synthesis, conformational analysis and CB1 binding affinity of hairpin-like anandamide pseudopeptide mimetics.

We have designed, synthesized and evaluated the CB(1) binding affinity of a number of new conformationally restricted lipopeptides (1-17). All of them present some of the AEA key structural elements incorporated in a hairpinlike peptide framework. Among them, compounds 1-3 and 8 showed CB(1) affinities in competitive binding assays with K(i) values in the micromolar range (K(i) of AEA = 0.8 microM in the same assay). The remaining pseudopeptides showed little binding to the CB(1) receptor (with K(i) values >or= 50 microM). Conformational analysis on two representative compounds, performed by a combination of NMR studies, restrained molecular dynamics and QM calculations, allowed us to shed light on the structure-activity relationships (SAR), pointing to a correlation between the predominance of the hairpin-like structural motif and the CB(1) binding affinity. In a more general context, the present study may also prove useful in gaining additional insight into the biological relevance of the various AEA conformations.     

Synthesis,characterization, and biological activity of poly(arginine)‐derived cancer‐targeting peptides in HepG2 liver cancer cells

The solid‐phase synthesis, structural characterization, and biological evaluation of a small library of cancer‐targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose‐regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3–12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14–46%) and crude purities >95% as analyzed by liquid chromatography–mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure–activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose‐regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose‐response (0–1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer‐targeting delivery agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1)

Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 μM. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.     

Synthesis of multiple antigenic peptides (MAPs)—strategies and limitations

Dendrimeric platforms such as MAPs can be synthesized either entirely by solid‐phase methods (SPPS, direct approach) or by conjugation in solution of preformed, SPPS‐made building blocks (indirect approach). Although MAPs and MAP‐like constructs have been extensively and successfully used for various biological (mainly immunological) applications, experimental reports are most often lacking in chemical detail about their preparation and characterization. Here, we provide complete accounts of the synthesis and analytical documentation of MAPs and similar dendrimers by either all‐SPPS (direct) or chemoselective thioether ligation (indirect) methods. We have chosen as model epitopes a 24‐residue sequence of the ectodomain of protein M2 from influenza virus (M2e), which is found to be a rather challenging peptide epitope, and a far more manageable, shortened (12‐residue) version of the same peptide. The advantages and shortcomings of both direct and indirect methods are discussed. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Tryptic hydrolysis of hGH-RH(1-29)-NH2 analogues containing Lys or Orn in positions 12 and 21.

Two analogues of the 29 amino acid sequence of human growth hormone-releasing hormone, namely [Nle27]hGH-RH(1-29)-NH2 and [Orn(12,21),Nle27]hGH-RH(1-29)-NH2, have been synthesized and subjected to digestion by trypsin. The course of degradation was followed using RP-HPLC and ESI-MS. Several intermediates and final products of degradation were identified and conclusions regarding the rate of cleavages at different positions occupied by Lys and Arg residues were drawn. The analogue containing ornithine was found to be less susceptible to hydrolysis by trypsin: the 12-13 and 21-22 peptide bonds were completely resistant to the cleavage. The results show that by replacing Lys with Orn, a possibility exists to design new peptides, which could be more stable in biological fluids.     

The expression of monocyte chemotactic protein (MCP-1) in human vascular endotheliumin vitro andin vivo

Total 6-phosphofructo-1-kinase (PFK) activity, amounts of each type of PFK subunit, and levels of fructose-2,6-P₂ in the cerebral cortex, midbrain, pons-medulla, and cerebellum of 3, 12, and 25 month rats were measured. Further, the role of fructose-2,6-P₂ in the regulation of brain PFK activity was examined. A positive correlation was found to exist between the reported losses of glucose utilization as measured by 2-deoxy-D-glucose uptake and PFK activity in each region. That is, both parameters decreased to their lowest level by 12 months of age and remained decreased and fairly constant thereafter. Fructose-2,6-P₂ levels did not appear to directly correlate with regional changes in glucose utilization. Also, region-specific and age-related alterations of the PFK subunits were found although these changes apparently did not correlate with decreased glucose utilization. Brain PFK is apparently saturated with fructose-2,6-P₂ due to the high endogenous levels, and it contains a large proportion of the C-type subunit which dampens catalytic efficiency. Consequently, brain PFK could exist in a conformational state such that it can readily consume fructose-6-P rather than in an inhibited state requiring activation. This may explain, in part, the ability of brain to efficiently but conservatively utilize available glucose in energy production.Abbreviations fructose-2,6-P₂ D-fructose 2,6-bisphosphate - fructose-6-P D-fructose 6-phosphate - PAGE Polyacrylamide Gel Electrophoresis - PFK 6-phosphofructo-1-kinase - PP_i-PFK Pyrophosphate-dependent Phosphofructokinase, ribose-1,5-P₂, ribose-1,5-bisphosphate - SDS Sodium Dodecyl Sulfate     

Preparation and characterization of two LysB29 specifically labelled fluorescent derivatives of human insulin.

The preparation and characterization of two novel LysB29 selectively labelled fluorescent derivatives of human insulin are described. Two probes were chosen: 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD) and 7-methoxycoumarin-4-acetic acid (MCA), which have a relatively small, compact structure and are able to react with amino groups to form highly fluorescent derivatives. The combination of solid phase peptide synthesis and enzymatic semisynthesis was chosen for preparation of these fluorescent derivatives. Using two different protocols of solid-phase peptide synthesis, two fluorescent octapeptides were prepared corresponding to the position B23-B30 of human insulin, each with a different fluorescent label, NBD or MCA, on the epsilon-amino group of lysine. Then, the fluorescent octapeptides were coupled to desoctapeptide-(B23-B30)-insulin by a trypsin catalysed reaction. The receptor binding affinities of two novel fluorescent derivatives of human insulin with NBD and MCA (HI-NBD and HI-MCA) were determined on rat adipose tissue plasma membranes. Both fluorescent insulins, HI-NBD and HI-MCA, had only slightly reduced binding affinity and will be used for studying the interaction of insulin with its receptor.     

Gel-Phase synthesis of hydrophobic (thr14) (thr19) galanin (1 -19) fragment on a high capacity flexible crosslinked polystyrene support.

A hydrophobic analogue of human galanin (1-19) fragment has been synthesized using Boc/Bzl tactics to demonstrate the synthetic utility of the flexible crosslinked polystyrene support prepared by the suspension polymerization of styrene and 1,4-butanediol dimethacrylate. The copolymer was chloromethylated to 2.36 mmol Cl/g. The functionalized resin was found to possess all the physicochemical properties similar to Merrifield resin. The free peptide was obtained in high yield and purity as judged by RP-HPLC and characterized by amino acid analysis and ESI-MS.     

Backbone amide linker (BAL) strategy for Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of peptide aldehydes.

A rapid and efficient strategy has been developed for the general synthesis of complex peptide aldehydes. N(alpha)-Benzyloxycarbonylamino acids were converted to protected aldehyde building blocks for solid-phase synthesis in four steps and moderate overall yields. The aldehydes were protected as 1,3-dioxolanes except for one case where a dimethyl acetal was used. These protected amino aldehyde monomers were then incorporated onto 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyryl-resin (BAL-PEG-PS) by reductive amination, following which the penultimate residue was introduced by HATU-mediated acylation. The resultant resin-bound dipeptide unit, anchored by a backbone amide linkage (BAL), was extended further by routine Fmoc chemistry procedures. Several model peptide aldehydes were prepared in good yields and purities. Some epimerization of the C-terminal residue occurred (10% to 25%), due to the intrinsic stereolability conferred by the aldehyde functional group, rather than any drawbacks to the synthesis procedure.     

Synthesis and spectroscopic properties of 4-ethoxymethylene-2-(1)-naphthyl-5(4H)-oxazolone-labeled fluorescent peptides

Strategies for the preparation of new fluorescent oligopeptide conjugates labeled with 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx-OEt) at the N-terminal on solid support or in solution have been devised. These procedures are simple and easy to carry out by reacting naOx-OEt or N(alpha)-naOx-amino acid with side chain protected peptide chains attached to resins. The integrity of the N-alkyl bond was maintained even after the trifluoracetic acid or HF based cleavages procedures. Our data show that the naOx fluorophore is compatible with both Fmoc/tBu and Boc/Bzl methods and also suggest that naOx-amino acid could be utilized as building blocks for solid phase peptide synthesis. Comparative analysis of fluorescence properties of naOx-conjugates indicated that the spectral properties of the fluorophore do not change after incorporating into peptides. The compact size, the definite chemical reaction for its introduction in combination with the appropriate spectral features (e.g., intense emission, pH independent fluorescent characteristics, and beneficial photobleaching dose constant and rates) and with chemical and spectral stability, naOx-based labeling could be attractive for novel cellular fluorescent techniques (e.g., in laser scanning confocal FRET) to study peptide-protein and protein-protein interactions even in biological matrices.     

Total synthesis,structural, and biological evaluation of stylissatin A and related analogs

The natural product cyclic peptide stylissatin A ( 1a ) was reported to inhibit nitric oxide production in LPS‐stimulated murine macrophage RAW 264.7 cells. In the current study, solid‐phase total synthesis of stylissatin A was performed by using a safety‐catch linker and yielded the peptide with a trans‐Phe⁷‐Pro⁶ linkage, whereas the natural product is the cis rotamer at this position as evidenced by a marked difference in NMR chemical shifts. In order to preclude the possibility of 1b being an epimer of the natural product, we repeated the synthesis using d ‐allo‐Ile in place of l ‐Ile and a different site for macrocyclization. The resulting product (d ‐allo‐Ile²)‐stylissatin A ( 1c ) was also found to have the trans‐Phe⁷‐Pro⁶ peptide conformations like rotamer 1b . Applying the second route to the synthesis of stylissatin A itself, we obtained stylissatin A natural rotamer 1a accompanied by rotamer 1b as the major product. Rotamers 1a , 1b , and the epimer 1c were separable by HPLC, and 1a was found to match the natural product in structure and biological activity. Six related analogs 2–7 of stylissatin A were synthesized on Wang resin and characterized by spectral analysis. The natural product ( 1a ), the rotamer ( 1b ), and (d ‐allo‐Ile²)‐stylissatin A ( 1c ) exhibited significant inhibition of NO^.. Further investigations were focused on 1b , which also inhibited proliferation of T‐cells and inflammatory cytokine IL‐2 production. The analogs 2–7 weakly inhibited NO^. production, but strongly inhibited IL‐2 cytokine production compared with synthetic peptide 1b . All analogs inhibited the proliferation of T‐cells, with analog 7 having the strongest effect. In the analogs, the Pro⁶ residue was replaced by Glu/Ala, and the SAR indicates that the nature of this residue plays a role in the biological function of these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Cu(OBt)2 and Cu(OAt)2, copper(II)-based racemization suppressors ready for use in fully automated solid-phase peptide synthesis.

The complexes Cu(OBt)2 and Cu(OAt)2, which are derived from copper(II) and HOBt and HOAt, respectively, are shown to be more effective in suppressing racemization during solid-phase peptide synthesis (SPPS) than are those compounds currently being used for this purpose. These compounds can readily be used in conjunction with the commonly applied coupling reagents in fully automated systems for solid-phase peptide chemistry.     

Polymer-supported biopolymer synthesis: 1. High load solid (gel) phase strategy for peptide synthesis with a phenolic poly(acryloylmorpholine)-based support

An efficient, low-cost, reaction strategy for the solid (gel) phase synthesis of peptides and protected peptide segments has been developed. The strategy involves the use of a new poly(acryloylmorpholine)-based phenolic support matrix, Koch-Light Peptide Resin A. Illustrative syntheses of N-terminal Boc- and Z-protected[Leu]- enkephalin derivatives, including C-terminal acid hydrazides and esters, are described. The strategy, which is effective for the synthesis of peptides at high matrix loadings, is adopted readily for large-scale application.     

Assessing the correlation of microscopy‐based and volumetry‐based measurements for resin swelling in a range of potential greener solvents for SPPS

The degree of resin swelling in a particular solvent system is one of the critical parameters for solid‐phase peptide synthesis (SPPS) and for solid‐phase synthesis in general. Methods used for measuring the degree of resin swelling include microscopy‐based and volumetry‐based methods. This study describes and compares the use of both methods for a number of commercially available resins commonly used in SPPS, with a range of solvents, which have been identified in the literature as ‘greener’ than DCM, DMF and NMP. The results were analysed by statistical methods, and a significant correlation between the two distinct methods has been demonstrated for the first time. The results will likely be used, in conjunction with other literature methods, to help in choosing both the resin and solvent system for greener SPPS, as well as for continuous flow SPPS, which is of growing importance.