An efficient gel-phase synthesis of peptides on a high capacity flexible crosslinked polystyrene support: comparison with Merrifield resin.

A highly solvating copolymer was prepared in high yield by introducing a flexible crosslinker, 1,4-butanedioldimethacrylate, into the polystyrene matrix by a free radical aqueous suspension polymerization. A 2 mol% crosslinked resin showed rigidity and mechanical characteristics comparable to those of divinylbenzene-crosslinked polystyrene (Merrifield resin, DVB-PS) support. Swelling and solvation characteristics of the new resin, BDDMA-PS, were much higher than DVB-PS support in all solvents used for solid phase peptide synthesis. The diacrylate crosslinks in the resin network were found to be highly stable even after 48 h treatment with neat TFA, 6 N HCl and 6 N KOH at 110 degrees C. To demonstrate the usefulness of the new resin in high capacity peptide synthesis, a typical difficult peptide, acyl carrier protein (ACP) fragment (65-74), was synthesized on commercially available 1 mol% crosslinked DVB-PS and 2 mol% crosslinked BDDMA-PS resins under identical conditions. A protocol using NMP/DMSO mediated coupling was employed for chain assembly. The yield and purity of the product from BDDMA-PS resin was higher than when the DVB-PS resin was used. The mechanistic reason behind the synthetic efficiency of the new resin was found to be its ability to induce random coil conformation to the growing peptide chains.     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Studies leading to optimization of butanedioldimethacrylate-crosslinked polystyrene supports (BDDMA–PS) forsolid phase peptide synthesis are delineated. BDDMA–PScopolymers with different crosslink densities were prepared andfunctionalised with chloromethyl groups. The reactivity of theLys(2-Cl-Z)-OH residue bound to these polymers through a benzylester linkage was investigated by following the kinetics ofacylation by the HOBt active ester of Boc-Alanine. From theresults it was observed that the rate of peptide bond formationwas maximum for a 2% BDDMA crosslinked resin. This resin wascompared with a 2% DVB-crosslinked polystyrene resin (DVB–PS). Synthesis of an extremely insoluble, hydrophobic,antiparallel -sheeted difficult sequencepeptide LMVGGVVIA ( 34–42), C-terminal fragment of -amyloid protein, (1–42), wascarried out on both 2% DVB–PS and 2% BDDMA-crosslinkedpolystyrene supports. The synthesis of the peptide was carriedout using Boc amino acid strategy. Greater extent of swellingof the resino peptide, increased coupling efficiency during theassembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA–PS polymer.     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

Summary The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

Solid phase synthesis of hydrophobic difficult sequence peptides on BDDMA-PS support.

This article illustrates the successful and efficient solid phase assembly of hydrophobic difficult sequence peptides following both t-Boc and Fmoc chemistry. The peptides were synthesized on an optimized 1,4-butanediol dimethacrylate-crosslinked polystyrene support (BDDMA-PS). Four difficult sequence test peptides, VAVAG, VIVIG, QVGQVELG and VQAAIDYING, were synthesized in relatively good yield and purity without any aggregation problems. The peptides were assembled on chloromethylated and 4-hydroxymethylphenoxymethyl (HMP) BDDMA-PS resins. The peptides were fabricated using Boc amino acid 1-hydroxybenzotriazolyl and Fmoc amino acid pentafluorophenyl active esters in coupling reactions. The peptides after synthesis were cleaved from the polymeric support by exposing the peptidyl resin to 90% trifluroacetic acid/5% thioanisole/5% EDT mixture. The HPLC and MALDI TOF MS studies of the peptides revealed the high homogeneity of the synthesized peptides. Chloromethylated resin having a functional group loading of 1.14 mmol Cl/g was used for the synthesis. The yield and homogeneity of these peptides synthesized using the new support were high when compared with the conventional DVB-PS resin.     

Polyethyleneglycol graft copoly (styrene-1,4-butanediol dimethacrylate) resin for gel-phase peptide synthesis

Summary Synthesis and characterization of a flexible crosslinked polystyrene grafted polyethyleneglycol (PEG) resin which allows for efficient synthesis of aggregating peptides in high yield and purity has been described. The resin showed rigidity, mechanical and chemical stability, and improved swelling and solvation characteristics essential for the successful synthesis of peptides. To demonstrate the usefulness of the new resin in polypeptide synthesis, a 4-(hydroxymethyl)phenoxyacetic acid (HMPA) handle was anchored to the free terminus of PEG and a typical hydrophobic peptide, Alzheimer’s β-amyloid plaque protein (33–42) fragment, was synthesized using Fmoc/tBu tactics. The new resin was compared with commercially available 1 mol% divinylbenzene (DVB)-crosslinked Tentagel resin under identical conditions. HPLC profiles and LC/MS analyses of the crude products revealed the high synthetic efficiency of the newly developed support. Efficiency of the resin was further illustrated by the gel-phase synthesis of a 15-residue peptide, (28–42) fragment of β-amyloid protein.     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

Synthesis by an improved solid-phase method of a highly acidic peptide from nucleolar nonhistone protein C23

The total synthesis of a 42-amino acid residue peptide corresponding to the proposed sequence of a highly acidic fragment of nucleolar nonhistone protein C23 was accomplished by an improved, mild solid-phase method, employing a p-alkoxybenzyl alcohol polystyrene resin. The symmetrical anhydride and active ester coupling were used exclusively. Coupling monitoring and amino acid analyses were carried out during the stepwise synthesis. The synthetic product was purified by gel filtration and ion-exchange chromatography, and found to be homogeneous by seven additional criteria. Phosphorylation of serine residues was attempted enzymatically, and the phosphopeptides obtained had electrophoretic mobilities comparable to that of the natural product.     

γ-Radiation Induced Polymerization of a Chiral Monomer: A New Way to Produce Chiral Amplification

The treatment of the terpene β(−)pinene with γ radiation (at dose level: 150, 300 and 600 kGy) causes its polymerization into a resin and into a dimer. The yield of the resin and of the dimer appears to be linearly dependent to the radiation dose. The structure of the products was studied by FT–IR spectroscopy also in comparison to a reference β(−)pinene resin prepared by cationic polymerization. A highly ordered structure was found in the case of the radiopolymer in comparison to the resin from cationic polymerization. Polarimetric measurements have shown astonishing enhancement in the optical activity of the radiopolymer and radiodimer in comparison to the starting optical activity of the β(−)pinene monomer. Also DSC (differential scanning calorimetry) data supports the unexpected highly ordered structure for the β(−)pinene radiopolymer in comparison to the resin prepared by cationic polymerization. The results have been discussed in terms of amplification of chirality caused by γ radiation and the implications of this fact on the mechanism of chiral amplification on prebiotic molecules. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Soft shell resins for solid-phase peptide synthesis.

Soft shell (SS) resins with a lightly or noncross-linked shell layer were prepared by reducing the amount of cross-linking agent, divinylbenzene (DVB), during seed suspension polymerization from polystyrene (PS) resin. These SS resins have a lower swelling volume than that produced by normal cross-linking. Despite its lower swelling, however, SS (10-00) resin, which consists of the 1% DVB-cross-linked core and the noncross-linked surface layer, showed higher efficiency in peptide synthesis compared with 1% DVB-PS resin and other SS resins.     

Exploration of the mechanism for LPFFD inhibiting the formation of β-sheet conformation of Aβ(1–42) in water

The main component of senile plaques found in AD brain is amyloid β-peptide (Aβ), and the neurotoxicity and aggregation of Aβ are associated with the formation of β-sheet structure. Experimentally, beta sheet breaker (BSB) peptide fragment Leu-Pro-Phe-Phe-Asp (LPFFD) can combine with Aβ, which can inhibit the aggregation of Aβ. In order to explore why LPFFD can inhibit the formation of β-sheet conformation of Aβ at atomic level, first, molecular docking is performed to obtain the binding sites of LPFFD on the Aβ(1–42) (LPFFD/Aβ(1–42)), which is taken as the initial conformation for MD simulations. Then, MD simulations on LPFFD/Aβ(1–42) in water are carried out. The results demonstrate that LPFFD can inhibit the conformational transition from α-helix to β-sheet structure for the C-terminus of Aβ(1–42), which may be attributed to the hydrophobicity decreasing of C-terminus residues of Aβ(1–42) and formation probability decreasing of the salt bridge Asp23-Lys28 in the presence of LPFFD.     

Conformational analysis of sea cucumber (Caudina arenicola) C globin 94-97 fragment,Pro-Glu-Leu-Leu

The tetrapeptide Pro-Glu-Leu-Leu forms the 94-97 fragment of C globin in sea cucumber. 2% Butanediol dimethacrylate-cross linked polystyrene (2% BDDMA-PS), which had been optimized, was used for the synthesis of the tetrapeptide Pro-Glu-Leu-Leu. The peptide was synthesized by using Boc-amino acid strategy. The peptide purity was checked by RP-HPLC and the peptide was characterized by (1)H NMR spectroscopy and amino acid analysis. Conformation of the peptide was studied by 1D- and 2D- homonuclear (1)H NMR, in DMSO-d6 at 300K. The conformation of the synthetic tetrapeptide (extended backbone conformation) is not in agreement with that in C globin.     

Metal effects on the membrane interactions of amyloid-β peptides

Aβ(1–42) peptide, found as aggregated species in Alzheimer’s disease brain, is linked to the onset of dementia. We detail results of ³¹P and ²H solid-state NMR studies of model membranes with Aβ peptides and the effect of metal ions (Cu²⁺ and Zn²⁺), which are found concentrated in amyloid plaques. The effects on the lipid bilayer and the peptide structure are different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induce formation of smaller vesicles, but not when Aβ(1–42) is associated with the bilayer membrane. Aβ(25–35), a fragment from the C-terminal end of Aβ(1–42), which lacks the metal coordinating sites found in the full length peptide, is neurotoxic to cortical cortex cell cultures. Addition of metal ions has little effect on membrane bilayers with Aβ(25–35) peptides. ³¹P magic angle spinning NMR data show that Aβ(1–42) and Aβ(1–42)-Cu²⁺ complexes interact at the surface of anionic phospholipid membranes. Incorporated peptides, however, appear to disrupt the membrane more severely than associated peptides. Solid-state ¹³C NMR was used to compare structural changes of Aβ(1–42) to those of Aβ(25–35) in model membrane systems of anionic phospholipids and cholesterol. The Aβ peptides appeared to have an increase in β-strand structure at the C-terminus when added to phospholipid liposomes. The inclusion of Cu²⁺ also influenced the observed chemical shift of residues from the C-terminal half, providing structural clues for the lipid-associated Aβ/metal complex. The results point to the complex pathway(s) for toxicity of the full-length peptide. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience.     

Synthesis of amyloid β-peptides in solution employing chloroform-phenol mixed solvent for facile segment condensation of sparingly soluble protected peptides

Summary Segment condensation reaction of sparingly soluble protected peptides proceeded smoothly in CHCl₃-phenol mixed solvent without danger of epimerization or of significant ester formation with the carboxyl component when 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was employed in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). The optimal conditions for enhancement of peptide coupling mediated by EDC/HOOBt in CHCl₃-phenol were determined and successfully applied to the synthesis of amyloid β-peptide (1–42), (1–43) and [Pyr³]-(3–42). These peptides of high homogeneity were used to examine the relation between structure and amyloidogenesis by means of CD spectra and fluorimetric assay.     

Polyethyleneglycol graft copoly (styrene-1,4-butanediol dimethacrylate) resin for gel-phase peptide synthesis

Synthesis and characterization of a flexible crosslinked polystyrene graftedpolyethyleneglycol (PEG) resin which allows for efficient synthesis of aggregating peptides in high yield and purity has been described. The resin showed rigidity, mechanical and chemical stability, and improved swelling and solvation characteristics essential for the successful synthesis of peptides. To demonstrate the usefulness of the new resin in polypeptide synthesis, a 4-(hydroxymethyl)phenoxyacetic acid (HMPA) handle was anchored to the free terminus of PEG and a typical hydrophobic peptide, Alzheimer's -amyloid plaque protein (33–42) fragment, was synthesized using Fmoc/t-Bu tactics. The new resin was compared with commercially available 1 mol% divinylbenzene (DVB)-crosslinked Tentagel resin under identical conditions. HPLC profiles and LC/MS analyses of the crude products revealed the high synthetic efficiency of the newly developed support. Efficiency of the resin was further illustrated by the gel-phase synthesis of a 15-residue peptide, (28–42) fragment of -amyloid protein.     

Amyloid beta–heme peroxidase promoted protein nitrotyrosination: relevance to widespread protein nitration in Alzheimer’s disease

Amyloid beta (Aβ) peptide accumulation has been demonstrated to play a central role in Alzheimer’s disease (AD). Substantial evidence indicates that protein nitrotyrosination contributes to Aβ-dependent neurotoxicity; however, the molecular mechanism is unknown. Recent research has shown that Aβ complexes with heme to form Aβ–heme, and increases the pseudo-peroxidase activity of heme. We found that Aβ–heme uses H₂O₂ and NO₂ ⁻ to cause nitration of enolase and synaptic proteins more effectively than heme. Thus, the increased peroxidase activity of Aβ–heme may be the molecular link between excess Aβ and the widespread protein nitration in AD. Interestingly, the site of enolase nitration that was catalyzed by Aβ–heme is different from that induced by heme. Moreover, the secondary structural perturbations of Aβ–heme-treated and heme-treated enolase are also different. These observations suggest that Aβ–heme targets specific amino acid sequences in enolase. Furthermore, our data show that Aβ–heme peroxidase activity is independent of the aggregation state of Aβ, suggesting an important role of soluble Aβ in addition to Aβ aggregates and oligomers in AD pathogenesis.     

Effects of beta-amyloid on proliferation and morphology of yeast Saccharomyces cerevisiae

Summary Deposition of beta-amyloid peptide (1–42) (βAP) in the brain is an early event linked with pathogenesis of cell injury and death in Alzheimer disease. Previous studies have demonstrated that βAP induces cytotoxicity in several types of human cells. Surprisingly, the peptide was found not only to be non toxic for yeast cells, but to stimulate growth of yeast culture. The results are consistent with βAP binding to yeast cell as illustrated by binding isotherms with the apparent dissociation constant of 8×10⁻⁷ M and B_max of 4.7×10⁴ molecules/cell.     

Efficient production of diltiazem chiral intermediate using immobilized lipase from <Emphasis Type="Italic">Serratia marcescens</Emphasis>

The lipase from Serratia marcescence ECU1010 (Sml) was capable of enantioselectively catalyzing the synthesis of many chiral drug precursors. This paper investigated the immobilization of Sml on appropriate supporting materials and its performance in bioreactor. Chitosan, Celite 545, and DEAE-cellulose were found to be the ideal supports among 8 carriers tested with respect to enzyme load and activity recovery of lipase. When Sml was immobilized, significant improvements of stability against pH, thermal, and operational deactivation were observed with all the 3 better supports, and the best stability was observed when the lipase was immobilized on glutaraldehyde activated chitosan. As for the effect of organic solvent in the biphasic reaction system, the hydrolytic activity of the immobilized lipase on trans-3-(4′-methoxyphenyl)glycidic acid methyl ester ((±)-MPGM) observed in isopropyl ether was 6 and 3 times higher than those in toluene and methyl tert-butyl ether. The lipasecatalyzed production of (−)-MPGM by enzymatic resolution of (±)-MPGM with chitosan-Sml in isopropyl etherwater biphasic system was carried out in a 2 L stirred-tank reactor. The batch operation was more efficient operation mode for the enantioselective hydrolysis of (±)-MPGM, affording enantiopure (−)-MPGM in 44.3% overall yield, in contrast to 29.3% in a continuous reactor.     

β-Amyloid enhances intracellular calcium rises mediated by repeated activation of intracellular calcium stores and nicotinic receptors in acutely dissociated rat basal forebrain neurons

β-Amyloid, a 39–43 amino acid peptide, may exert its biological effects via neuronal nicotinic acetylcholine receptors. Using the ratiometric dye, fura-2, we examined the effect of soluble β-amyloid_1–42 on the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in acutely dissociated rat basal forebrain neurons. Focal applications of nicotine (0.5–20 mM), evoked dose-dependent increases in intracellular [Ca²⁺]_i that were mediated by the entry of extracellular Ca²⁺ via nicotinic acetylcholine receptors, and the release of intracellular Ca²⁺ from stores. With repeated nicotine challenges, the nicotinic responses were potentiated by 98 ± 12% (P < 0.05) while β-amyloid_1–42 (100 nM) was present for ∼5 min. This potentiation became larger during the subsequent washout of β-amyloid_1–42, which was associated with a gradual rise in baseline [Ca²⁺]_i. Application of β-amyloid_1–42 by itself did not alter [Ca²⁺]_i, and β-amyloid_1–42 also had no significant effect on the response to repeated KCl challenges. Therefore, β-amyloid_1–42 caused neither gross disturbance of cellular Ca²⁺ homeostasis nor enhancement of voltage-gated Ca²⁺ channels. Interestingly, β-amyloid_1–42 transiently potentiated the response to repeated caffeine challenges, which was also associated with a transient rise in baseline [Ca²⁺]_i. β-amyloid_1–42 potentiation of nicotine-evoked rises in [Ca²⁺]_i was reversed by the SERCA pump inhibitor, thapsigargin, and the mitochondrial Na⁺/Ca²⁺ exchanger inhibitor, CGP-37157. These results suggest that the dysregulation of [Ca²⁺]_i by β-amyloid_1–42 during multiple challenges with nicotine or caffeine involved the sensitization or overfilling of intracellular stores that are maintained by SERCA pump and Ca²⁺ efflux from the mitochondria.     

To Rink or Not to Rink Amide Link, that is the Question to Address for More Economical and Environmentally Sound Solid-Phase Peptide Synthesis

A comparative study is presented on the solid-phase peptide synthesis (SPPS) of the acyl carrier protein (ACP 65–74) sequence on a series of Rink amide resins possessing different matrix structures: poly(vinyl alcohol)-graft-poly(ethylene glycol) (PVA-g-PEG, 4), Tentagel-S-RAM (TG, 5), NovaGel (NG, 6), ChemMatrix (CM, 7) and polystyrene-divinylbenzene (PS-DVB, 8). In this comparison, the PEG-containing resins proved significantly better suited for the synthesis of pure ACP target sequence than the conventional PS-DVB solid supports (75–90% versus 52% crude purity). Amongst themselves, the PEG resins 4-7 exhibited similar capacity for providing pure peptide. Selecting PVA-g-PEG resin for a comparison of Rink amide linker versus no linker, the ACP (65–74) sequence was synthesized directly on the PVA-g-PEG resin 1, under identical conditions as employed in the synthesis on resin 4 bearing the Fmoc Rink linker, except for the final cleavage step, which was performed under more environmentally sound conditions using ester displacement with aqueous ammonia. Relative to its Rink amide counterpart 4, PVA-g-PEG resin 1 was cheaper to produce and possessed twice as much loading capacity (0.48 vs. 0.81 mmol/g). Moreover, Rink-less resin 1 gave higher yields of isolated pure peptide (61 vs. 45%) relative to its Fmoc Rink linker counterpart 4. In light of these results, the importance of the linker has been brought into question. As the need for large scale solid-phase peptide synthesis grows with greater demand for peptide products, ideal resins should be inexpensive to produce and employable under environmentally sound conditions to provide pure products. In this light, PVA-g-PEG resin 1 has demonstrated significant promise for economic and “green” SPPS.     

Selective removal of anti-α-Gal antibodies from human serum by using synthetic α-Gal epitope on a core-shell type resin

A novel α-Gal resin was chemo-enzymatically synthesized for the efficient adsorption of anti-α-Gal antibodies in human serum for xenotransplantation. To covalently conjugate a hexanoate linker with lactose and N-acetylglucosamine, both acceptor sugars were acetylated and brominated. Then, α-and β-galactoses were sequentially added to the linker-containing saccharides at their non-reducing ends by using recombinant α-(1,3)-and β-(1,4)-galactosyltransferases from E. coli. Finally, the synthesized α-Gal derivatives were immobilized on HiCore, a core-shell type resin, that was functionalized with amino groups on the shell region, as a packing material on-column. Using this method we were able to demonstrate that the α-Gal HiCore resin had a reduced level of non-specific protein adsorption compared with the commercially available polystyrene supports, TentaGel, and agarose-based supports, when Lectin BS-I was used as the model binding protein. Furthermore, the α-Gal HiCore resin was more efficient at eliminating anti-α-Gal IgGs from the total human IgGs through immunoadsorption than the other two α-Gal resins, α-Gal TentaGel and α-Gal agarose. The α-Gal HiCore resin developed in this study can be utilized in a wide range of applications including ex vivo immunoadsorption and as a quantitative assay of anti-Gal antibody in human sera.