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.     

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

Summary Studies leading to optimization of butanediol dimethacrylate-crosslinked polystyrene supports (BDDMA-PS) for solid phase peptide synthesis are delineated. BDDMA-PS copolymers with different crosslink densities were prepared and functionalised with chloromethyl groups. The reactivity of the Lys(2-Cl−Z)−OH residue bound to these polymers through a benzyl ester linkage was investigated by following the kinetics of acylation by the HOBt active ester of Boc-Alanine. From the results it was observed that the rate of peptide bond formation was maximum for a 2% BDDMA crosslinked resin. This resin was compared with a 2% DVB-crosslinked polystyrene resin (DVB-PS). Synthesis of an extremely insoluble, hydrophobic, antiparallel β-sheeted difficult sequence peptide LMVGGVVIA (β 34–42), C-terminal fragment of β-amyloid protein, β (1–42), was carried out on both 2% DVB-PS and 2% BDDMA-crosslinked polystyrene supports. The synthesis of the peptide was carried out using Boc amino acid strategy. Greater extent of swelling of the resino peptide, increased coupling efficiency during the assembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA-PS polymer.     

Tri(propylene glycol) glycerolate diacrylate cross-linked polystyrene: a new resin support for solid-phase peptide synthesis.

A highly flexible, mechanically and chemically stable copolymer, tri(propylene glycol) glycerolate diacrylate cross-linked polystyrene (PS-TRPGGDA), was synthesized by the suspension polymerization and employed as a solid support for peptide synthesis. The beaded polymer support containing secondary hydroxyl functional groups in the cross-linker was used as the growth site for peptide synthesis. The procedure is unique and cost-effective in that it avoids the initial functionalization steps required for most of the styrene-based polymer supports. The resin was characterized by 13C-CP-MAS NMR spectroscopy and the morphologic features of the resin were investigated using scanning electron microscopy. Swelling studies conducted on the new support revealed that the PS-TRPGGDA resin undergoes more effective swelling and solvation than PS-DVB resin in all solvents used in peptide synthesis. The efficiency of the new support was demonstrated by synthesizing a 'difficult' sequence Ala-Arg-(Ala)6-Lys and comparing it with commercially available Merrifield and Sheppard resins. The synthetic efficiency was further demonstrated by the synthesis of a 24-residue NR 2A peptide substrate of calcium/calmodulin-binding peptide. The high yield and purity of the peptide synthesized on the novel support indicates the positive role of the flexible and hydrophilic cross-linking agent in the solid support.     

An improved synthesis of 2- and 4-bromoestradiols.

Bromination of estradiol-17beta by 2,4,4,6-tetrabromocyclohexa-2,5-dienone gives 2- and 4-bromoestradiols in good yields.     

An improved synthesis of the C-linked glucuronide of N-(4-hydroxyphenyl)retinamide

Retinoic acid analogues such as N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventatives and chemotherapeutics for numerous types of cancer. The C-linked analogue of the O-glucuronide of 4-HPR (4-HPRCG) has been shown to be a more effective agent. The synthetic route to this molecule has been significantly improved by access to a key C-benzyl-glucuronide intermediate through employment of a Suzuki coupling reaction between an exoanomeric methylene sugar and an aryl bromide. Preliminary evidence shows 4-HPRCG has chemotherapeutic activity.     

An improved CPG support for the synthesis of 3'-amine-tailed oligonucleotides.

A new controlled-pore glass (CPG) support is described that allows for the direct synthesis of oligonucleotides bearing a 3'-aminohexyl tail. This solid support (AH-CPG) exhibits superior performance as compared to a commercially available 3'-amine CPG. The AH-CPG is prepared from 6-aminohexan-1-ol with a unique protecting group for the amine that also functions as the site of attachment to the CPG. A 3'-amine-tailed oligodeoxynucleotide (ODN) was prepared from this support using standard phosphoramidite coupling and deprotection conditions. The 3'-amine-tailed ODN was subsequently modified with an acridinylpropionic acid tetrafluorophenyl ester. Facile synthesis of the AH-CPG and the stability of the deprotected product makes this functionalized solid support especially useful for preparation of oligonucleotides bearing 3'-amine tails and other modifications.     

An improved synthesis of potassium hexacyanoruthenate(II)

A new procedure for the synthesis of K₄Ru(CN)₆ is detailed. The synthesis involves bromine oxidation of an aqueous solution of ruthenium chloride, conversion of the perruthenate to ruthenate with base, and reaction of ruthenate with cyanide ion. Good yields (77%) of pure compound are obtained.     

3-Hydroxy-(4H)-benzopyran-4-ones as potential iron chelating agents in vivo

Increasing evidence suggests that iron plays an important role in tissue damage both during chronic iron overload diseases (i.e., hemochromatosis) and when, in the absence of actual tissue iron overload, iron is delocalised from specific carriers or intracellular sites (inflammation, neurodegenerative diseases, post-ischaemic reperfusion, etc.). In order to be used for therapeutical purposes in vivo, a reliable iron chelator should be capable of preventing the undesired effects that follow the electrochemical activation of iron (see below). Bearing in mind the molecular structure of some flavonols that are able to chelate iron, we synthesised a new oral iron-chelator, 2-methyl-3-hydroxy-4H-benzopyran-4-one (MCOH). We demonstrate that MCOH chelates iron in a 2:1 ratio showing a stability constant of approximately 10(10). MCOH is able to cross cell membranes (erythrocytes, ascite tumour cells) in both directions. Following intraperitoneal administration to rats, it is quickly taken up by the liver and excreted in the urine within 24h. A similar behaviour has been documented after oral administration. We propose that MCOH may represent the prototype of a new class of iron chelating agents to be developed for iron-removal therapy in vivo with the goal of preventing tissue damage caused by the iron redox cycle.     

Evaluation of new base-labile 2-(4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc)-amino acids for solid-phase peptide synthesis.

The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base-labile protecting group for solid-phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with Nalpha-Nsc-protected amino acids for the synthesis of some melanotropins important to our research, namely, gamma-melanocyte-stimulating hormone (gamma-MSH), its [Nle3]-analogue, and a cyclic alpha-MSH/beta-MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in >98% purity. The gamma-MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc-chemistry but was accompanied by side products. Finally, the Nle3-gamma-MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc-chemistry might not be the best methodology for certain sequences.     

Multiple peptide synthesis using a single support (MPS3)

An automated multiple peptide synthesis method to synthesize, cleave, and purify several peptides simultaneously in a single batch has been developed. The technique is based on the synthesis of multiple peptides on a single solid phase support and is easily adapted to manual or to automated methods. The approach relies on coupling of amino acid mixtures to the resin and it has been found that DCC/HOBt gives the best coupling performance. Fast Atom Bombardment Mass Spectrometry (FAB-MS) was used to rapidly and efficiently identify the peptides in each synthetic mixture which significantly assisted the purification process by HPLC. The method has been successfully applied to the synthesis of magainin 2 and angiotensinogen peptides.     

An improved large-scale synthesis of PEG-peptides for gene delivery.

Polyethylene glycol (PEG)-peptides are under development as components of nonviral gene delivery systems. Several earlier reports have demonstrated that covalent attachment of PEG to the surface of peptide condensed DNA particles blocks non-specific biodistribution during gene targeting. In this study, we report an improved large-scale synthesis and purification of a DNA condensing PEG-peptide used for gene delivery. The new method takes advantage of low-pressure cation-exchange chromatography to isolate dimeric Cys-Trp-Lys(18). The dimeric peptide was reduced and directly conjugated with PEG-maleimide resulting in PEG-Cys-Trp-Lys(18). The PEG-peptide was purified by low-pressure chromatography affording 50 mumol (400 mg) quantities of PEG-peptide in >95% purity. The approach offers the advantage of avoiding preparative high-performance liquid chromatography (HPLC) purifications of polylysine peptides to increase yield and capacity.     

4-Hydroxy-3-nitrophenylglyoxal. A chromophoric reagent for arginyl residues in proteins.

The chromophoric reagent, 4-hydroxy-3-nitrophenylglyoxal, is highly selective for the modification of arginine in aqueous solution at pH 7--9. The reagent also inactivates creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) in a manner analogous to that reported with phenylglyoxal.     

An imidazoline pseudodipeptide suitable for solid phase peptide synthesis.

This paper describes the synthesis of two diastereoisomers of an imidazoline dipeptide mimetic (a 4,5 dihydroimidazole-4-carboxylic acid), suitably protected for incorporation into solid phase peptide synthesis (SPPS) using the Fmoc protocol, from a phenylalanine-derived thioimidate and an alpha,beta-diaminopropanoic acid ester, followed by protecting group manipulation.     

An improved divergent synthesis of comb-type branched oligodeoxyribonucleotides (bDNA) containing multiple secondary sequences.

The divergent synthesis of branched DNA (bDNA) comb structures is described. This new type of bDNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branch network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb structures were assembled on a solid support and several synthesis parameters were investigated and optimized. The bDNA comb molecules were characterized by polyacrylamide gel electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The developed chemistry allows synthesis of bDNA comb molecules containing multiple secondary sequences. In the accompanying article we describe the synthesis and characterization of large bDNA combs containing all four deoxynucleotides for use as signal amplifiers in nucleic acid quantification assays.     

An improved synthesis of NHS-MAG3 for conjugation and radiolabeling of biomolecules with (99m)Tc at room temperature

The mercaptoacetyltriglycine (MAG3) chelator has been shown to stably complex technetium-99m (99mTc) for nuclear imaging and radiorhenium (186/188Re) for tumor radiation therapy studies. The bifunctional N-hydroxysuccinimidyl ester of MAG3 with S-acetyl protection (N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycinate (NHS-MAG3)) has been successfully used to covalently conjugate a MAG3 chelator to primary amine functionalized biomolecules. We describe herein a simplified synthesis of NHS-MAG3 that begins with the preparation of the N-hydroxysuccinimidyl ester of S-acetylmercaptoacetic acid (N-succinimidyl S-acetylmercaptoacetate (SATA)) from mercaptoacetic acid and is followed by the synthesis of S-acetylmercaptoacetyltriglycine from SATA, together requiring about 14 days. Finally, the synthesis of NHS-MAG3 from S-acetylmercaptoacetyltriglycine requires a further 5 days. We had earlier described a method for the preparation of MAG3-conjugated and 99mTc-radiolabeled biomolecules that required elevated temperatures during postconjugation purification. We now report a modified method for the preparation that is accomplished at room temperature and therefore applicable to temperature-sensitive biomolecules. The conjugation and radiolabeling of bovine serum albumin is used as an example. The conjugation and purification requires about 2-3 h and the radiolabeling and postlabeling purification requires about an additional 2 h.     

Use of the excluded protecting group (EPG) method for peptide synthesis.

The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin-1, [Ala(19), Asn(22)]magainin-1. In the approach reported, the C-terminal amino acid is esterified to the 2-position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl-steroid as the Fmoc-pentafluorophenyl-ester. The Fmoc group is removed with Et(2)NH/DMF ( approximately 15% v/v) and, after evaporation to approximately 10 mL, the solution chromatographed on Sephadex LH-20 in DMF. The dipeptidyl-steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc-amino acid-pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn degrees /AcOH to yield the peptide with intact alpha-amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C-terminal amino acids in other EPG syntheses.