Application of carboxylic-phosphinic mixed anhydrides in the fragment peptide synthesis of protected analogues of substance P.

Mixed carboxylic-phosphinic anhydrides derived from peptide acids and 1-oxo-1-chlorophospholane have been applied in the synthesis of the protected [Leu11]-SP by the fragment coupling strategy. The yields from fragment couplings were ca. 75%, the products were of high purity while the conditions of formation and coupling of the corresponding mixed phosphinic anhydrides, for optimum yields, have been evaluated.     

Efficient activation of nucleoside phosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis.

A new activator for the coupling of phosphoramidites to the 5'-hydroxyl group during oligonucleotide synthesis is introduced. The observed time to complete coupling is twice as fast with 4, 5-dicyanoimidazole (DCI) as the activator, compared with 1 H -tetrazole. The effectiveness of DCI is thought to be based on its nucleophilicity. DCI is soluble in acetonitrile up to 1.1 M at room temperature and can be used as the sole coupling activator during routine automated solid phase synthesis of oligonucleotides. The addition of 0.1 M N -methylimidazole to 0.45 M 1 H -tetrazole also results in higher product yields during oligonucleotide synthesis than observed with 1 H -tetrazole alone.     

Investigation of the automated solid-phase synthesis of a 38mer peptide with difficult sequence pattern under different synthesis strategies

Difficult peptides are a constant challenge in solid-phase peptide synthesis. In particular, hydroxyl amino acids such as serine can cause severe breakdowns in coupling yields even several amino acids after the insertion of the critical amino acid. This paper investigates several methods of improving synthesis yields of difficult peptides including the use of different resins, activators and the incorporation of a structure-breaking pseudoproline dipeptide building block both alone and in combination with each other.     

Versatile synthesis of oligodeoxyribonucleotide-oligospermine conjugates

A protocol for the rapid, automated synthesis of oligospermine-oligonucleotide sequences is described. To this end, a protected spermine phosphoramidite derivative was synthesized in six steps from spermine and used as the fifth synthon in an oligonucleotide synthesizer. Parameters were optimized to reach greater than 95% coupling yields. Cationic oligonucleotides show enhanced hybridization and strand invasion properties, and hence are an alternative to conventional oligonucleotides for molecular biology, diagnostic and potential therapeutic applications. A multi-gram-scale synthesis of the spermine phosphoramidite allowing several hundred coupling steps takes 2-3 weeks. Oligonucleotide synthesis and purification takes approximately 3 d.     

Extending the applicability of carboxyfluorescein in solid-phase synthesis

Optimized coupling protocols are presented for the efficient and automated generation of carboxyfluorescein-labeled peptides. Side products, generated when applying earlier protocols for the in situ activation of carboxyfluorescein, were eliminated by a simple procedure, yielding highly pure fluorescent peptides and minimizing postsynthesis workup. For the cost-efficient labeling of large compound collections, coupling protocols were developed reducing the amount of coupling reagent and fluorophore. To enable further chemical derivatization of carboxyfluorescein-labeled peptides in solid-phase synthesis, the on-resin introduction of the trityl group was devised as a protecting group strategy for carboxyfluorescein. This protecting group strategy was exploited for the synthesis of peptides labeled with two different fluorescent dyes, essential tools for bioanalytical applications based on fluorescence resonance energy transfer (FRET). Tritylation and optimized labeling conditions led to the development of a fluorescein-preloaded resin for the automated synthesis of fluorescein-labeled compound collections with uniform labeling yields.     

A study of the efficiency and the problem of sulfonation of several condensing reagents and their mechanisms for the chemical synthesis of deoxyoligoribonucleotides.

The efficiencies and problems of sulfonation of several condensing reagents for deoxyoligoribonucleotide synthesis have been studied. While 2,4,6-triisopropylbenzenesulfonyl chloride (TPSC1) gave very low yield and slow rate of coupling, the new 1:3 mixture of TPSC1 and tetrazole afforded excellent yield and extremely fast rate of reaction with the lowest rate of sulfonation. The difference and advantages of this mixture over triisopropylbenzenesulfonyl tetrazole (TPSTe) and mesitylenesulfonyl tetrazole (MSTe) are discussed. Mechanisms for the coupling reactions with these condensing reagents to account for the difference in their rates and yields of coupling are discussed.     

Solid-phase synthesis of polynucleotides. III. Synthesis of polynucleotides with defined sequences by the block coupling phosphotriester method.

Preparation of the three hexadecanucleotides, dGpTpApTpCpApCpGpApGpGpCpCpCpTpT, dCpGpApCpGpApGpCpGpTpGpApCpApCpC and cTpGpCpCpGpGpCpCpApCpGpApTpGpCpG, is described by a rapid and simple solid-phase method on polyacrylamide supports. The synthesis were performed by the extension of the method described in the previous paper using di and trinucleotides of defined sequences as an incoming 3'-phosphodiester unit. Although the coupling yields to form phosphotriester bonds are slightly lower than those for the homothymidylic acid series, pure polydeoxyribonucleotides of defined sequences can be synthesized without any major difficulty.     

Solid phase synthesis of apamin, the principal neurotoxin in bee venom. Isolation and characterization of acetamidomethyl apamin

The synthesis of apamin, the principal neurotoxin in bee venom, has been accomplished by the solid phase method on a benzhydrylamine resin, 2-Phenylisopropyloxycarbonyl amino acids were used throughout the synthesis except for the C-terminal histidine. Improved yields in the coupling steps in the N-terminal part of the molecule were obtained by coupling each amino acid both in dichloromethane and dimethylformamide. The use of acetamidomethyl as an S-protecting group for cysteine made it possible to isolate and purify the linear peptide. The deblocked and oxidized peptide was fractionated by ion-exchange chromatography (Bio-Rex 70) to obtain a highly purified apamin with full biological activity and with the same physical and chemical properties as the natural peptide. Circular dichroism (CD) spectra of the synthetic and natural apamin were identical.     

Liquid phase synthesis of arylamines and its application to the benzimidazolone via nucleophilic aryl substitution.

A method for soluble, inexpensive polymer-supported synthesis of aryl amines and benzimidazolone on the basis of nucleophilic aryl substitution (S(N)Ar) is described. This method involves a direct coupling reaction between resin bound aryl fluoride and amines at ambient temperature. The products are isolated in quantitative yields and excellent purity by simple precipitation and washing. This liquid phase method proves to be a useful tool for constructing combinatorial arylamine and benzimidazolone libraries.     

A suitable 1,2,4-oxadiazoles synthesis by microwave irradiation

One pot microwave-assisted synthesis of substituted 1,2,4-oxadiazoles in solvent and under solvent free condition was performed exploring the importance of some coupling reagents. Good yields and short reaction times were the main aspects of the methods.     

Kinetically controlled synthesis of dipeptides using ficin as biocatalyst.

The application of the sulfhydryl protease ficin as biocatalyst is proposed as a novel method for enzyme-catalyzed synthesis of dipeptides. The negligible peptidase but considerable esterase activity at alkaline pH facilitated the kinetically controlled formation of peptide bonds by coupling the ester substrates Z-Ala-OMe and Z-Gly-OMe with L-alanine, D-alanine, L-glutamine, D-glutamine and L-Cys(acetamidomethyl) respectively. The reaction is accomplished without the occurrence of secondary peptide hydrolysis. Under optimum reaction conditions (pH 9.2, high ratio nucleophile/carboxyl component, 4.8% ethanol, 40 degrees C), the peptide yields ranged from 5 to 91%, depending on the structure of the amino and/or carboxyl component. No racemization was observed in the enzymatic reaction. Application of short-chain peptides has been advocated recently in clinical nutrition. Ficin-catalyzed peptide synthesis might be an attractive biotechnological approach for the synthesis of suitable dipeptides in this respect.     

Solid-phase synthesis of endothelin receptor antagonists

A new solid-phase synthesis for ET receptor antagonists suitable for automation is presented. A support bound 2-hydroxybutyric acid derivative was converted to the corresponding ether derivatives using 4-halo-2-methylsulfonylpyrimidines. Subsequent Suzuki coupling with various aryl boronic acids gave the desired antagonists in good yields and purities. Highly potent antagonists with excellent selectivity for ET(A) were obtained.     

A new 2'-hydroxyl protecting group for the automated synthesis of oligoribonucleotides.

We have developed a new type of 2'-hydroxyl protecting group for the automated machine synthesis of RNA oligomers: a 2-hydroxyisophthalate formaldehyde acetal (HIFA). The unique feature of this protecting group is that, as the bis ester, it is relatively stable to the acidic conditions that are used for repeated removal of dimethoxytrityl groups during chain elongation, but the final deprotection step in alkali, which cleaves the chain from the support and removes the base and phosphate protecting groups, converts it to the bis carboxylate and this can be removed relatively rapidly by treatment with mild acid. Conversion of the bis ester to the bis carboxylic acid increases the rate of acid-catalyzed hydrolysis of the acetal by 42-fold at pH 1, and, possibly, by 1320-fold at pH 3. The bis ester is 112 times more stable than the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group (Fpmp) towards hydrolysis at pH 1, while the bis acid is only 2.35 times more stable than Fpmp at pH 3. In synthesis of the dimers UpU and UpG, with a coupling time of 5 min, the dimethoxytrityl cation assay indicated coupling yields of > 98%.     

Fmoc-amino acid chlorides in solid phase synthesis of opioid peptides.

Fmoc-amino acid chlorides were employed in the solid phase synthesis of the opioid peptides [Leu]enkephalin, [Leu]enkephalin amide, and dermorphin. The conventional polystyrene-based Merrifield resin or Wang's resin served as solid support. A binary salt of either triethylamine or diisopropylethylamine in the presence of 1-hydroxybenzotriazole or pivalic acid was used for acylation. The coupling rates were quite fast, being comparatively faster when 1-hydroxybenzotriazole was used along with triethylamine or diisopropylethylamine. The peptides obtained in good yields showed, after purification, biological and spectral properties identical with those of the natural peptides.     

The convenient synthesis and evaluation of the anticancer activities of new resveratrol derivatives

In the present study we report the simple synthesis and antitumour activity of novel stilbene derivatives 13-22. The key synthetic strategies involved Wadsworth-Horner-Emmons condensation and coupling reactions in high yields. All compounds showed significant growth inhibition on human tumour cell lines, with the most potent compound (19) exhibiting an IC(50) of 5.7 microM-11.4 microM in vitro.     

Recent advances in the high-speed solid phase synthesis of RNA

Development of rapid and reliable RNA synthesis strategies is fueled by the emergence of critical functional and regulatory roles for RNA, including RNA interference. Traditional methods are based on 5'-dimethoxytrityl-2'-silyl protection strategies which are derivatives of highly successful DNA synthesis methods. These approaches are limited in their ability to rapidly produce oligos of sufficient purity and length for genomic and pharmaceutical applications. Recently, new protection chemistries have been developed that circumvent the many limitations of 2'-silyl protection. The 5'-silyl-2'-bis(acetoxyethoxy)methyl strategy is the most notable, as it provides dramatic improvements--faster coupling rates, higher yields, greater product purity and superior post-synthetic ease-of-handling--affording a reliable high-speed chemical RNA synthesis technology.     

Solid phase synthesis of acylglycine human metabolites

Acylglycines represents a large and important class of human metabolites. They are often used in medicine to identify fatty acid oxidation disorders. A highly efficient solid phase synthesis approach to obtain these clinically important compounds is developed via coupling reaction between glycine-preloaded Wang resin and a set of carboxylic acids. The developed methodology facilitates the preparation of several structurally-diverse acylglycines with high yields and purity.     

Benzimidazolium triflate-activated synthesis of (6-4) photoproduct-containing oligonucleotides and its application.

In the solid-phase synthesis of oligonucleotides containing the pyrimidine(6-4)pyrimidone photoproduct using a dinucleotide building block, considerable amounts of by-products were found as the chain length increased. The by-products were the major product when a 49mer was synthesized on a 40 nmol scale. It was assumed that these by-products were formed by the coupling of phosphoramidites with the N3 imino function of the 5' component of the (6-4) photoproduct. We examined imidazolium triflate and benzimidazolium triflate to find an alternative activator for DNA synthesis. Imidazolium triflate prevented by-product formation to some extent, but the coupling yields were low. Benzimidazolium triflate was comparable to tetrazole in coupling efficiency and reduced by-product formation to a great extent, without modification of the synthesizer program. The obtained 49mer was used to detect proteins that recognize UV-damaged DNA in HeLa cell extracts. Two major protein-DNA complexes were found when a 49mer duplex was used as probe, while a 30mer duplex failed to detect one of them. This application showed the usefulness of long chain 'damaged' oligonucleotides in biochemical studies.     

(1H-benzotriazol-1-yloxy)-N,N-dimethylmethaniminium hexachloroantimonate (BOMI), a novel coupling reagent for solution and solid-phase peptide synthesis.

A HOBt-based immonium-type compound,(1H-benzotriazol-1-yloxy)-N,N-dimethyl methaniminium hexachloroantimonate (BOMI), was synthesized and successfully applied to the synthesis of various oligopeptides with good yields. The estimation of racemization and the influence of several reaction parameters such as solvents, bases and temperature were studied by HPLC using a model reaction. It was found that the reactivity of BOMI was much higher than that of HOBt-based phosphonium- and uronium-type coupling reagents. Moreover, its racemization was lower than that of other HOBt-derived coupling reagents. The effectiveness of BOMI was also demonstrated by the synthesis of Leu-enkephalin both in solution and in the solid-phase.     

Solvent effects on coupling yields during rapid solid-phase synthesis of CGRP(8-37) employing in situ neutralization.

The success of solid-phase peptide synthesis is often dependent upon solvation of the resin and the growing resin-bound peptide chain. We investigated the relationship between solvent properties and solvation of the resin and peptide-resin in order to obtain satisfactory coupling yields for the rapid solid-phase peptide synthesis, using butyloxycarbonyl-(Boc)-amino acid derivatives, of human-alpha-calcitonin gene-related peptide(8-37) (CGRP(8-37)). Solvation of (p-methylbenzhydrylamine)copoly(styrene-1% divinylbenzene (DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8-37) (peptide-resin) was correlated to solvent Hildebrand solubility (delta) and hydrogen-bonding (delta(h)) parameters. Contour solvation plots of delta(h) vs. delta revealed maximum solvation regions of resin and peptide-resin. Maximum resin solvation occurred with N-methylpyrrolidinone (NMP), NMP : dimethylsulfoxide (DMSO) (8 : 2) and DMSO. Inefficient solvation of the peptide-resin occurred with these solvents and resulted in poor syntheses with average coupling yields of 78.1, 88.9 and 91.8%, respectively. Superior peptide-resin solvation was obtained using dimethylacetamide (DMA) and dimethylformamide (DMF), resulting in significantly higher average coupling yields of 98.0 and 99.5%, respectively. Thus, the region of maximum peptide-resin solvation shifts to solvents with higher delta(h) values. DMF provided the most effective peptide-resin solvation and was the only solvent from which CGRP(8-37) was obtained as a single major product in the crude cleaved material.