Novel and Efficient Synthesis of Phenethyl Formate via Enzymatic Esterification of Formic Acid.

Current methods for the production of esters, including chemical synthesis and extraction from natural sources, are hindered by low yields and environmental pollution. The enzymatic synthesis of these compounds could help overcome these problems. In this study, phenethyl formate, a commercially valuable formate ester, was synthesized using commercial immobilized lipases. The effects of specific enzymes, enzyme concentration, formic acid:phenethyl alcohol molar ratio, temperature, and solvent were studied in order to optimize the synthesis conditions, which were identified as 15 g/L of Novozym 435 enzyme, a 1:5 formic acid:phenethyl alcohol molar ratio, a 40 °C reaction temperature, and 1,2-dichloroethane as the solvent. Under these conditions, phenethyl formate was obtained in a conversion yield of 95.92%. In addition, when 1,2-dichloroethane was replaced with toluene as the solvent, the enzyme could be recycled for at least 20 reactions with a steady conversion yield above 92%, testifying to the economic aspects of the process. The enzymatic synthesis of phenethyl formate using the proposed method is more environmentally friendly than methods currently employed in academic and laboratory settings. Moreover, the method has the potential to enhance the value-added properties of formic acid owing to its downstream use in the production of commercially essential esters.

HF-Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization.

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post-translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.

Low-energy electron-induced chemistry of condensed methanol: implications for the interstellar synthesis of prebiotic molecules.

In the interstellar medium, UV photolysis of condensed methanol (CH3OH), contained in ice mantles surrounding dust grains, is thought to be the mechanism that drives the formation of "complex" molecules, such as methyl formate (HCOOCH3), dimethyl ether (CH3OCH3), acetic acid (CH3COOH), and glycolaldehyde (HOCH2CHO). The source of this reaction-initiating UV light is assumed to be local because externally sourced UV radiation cannot penetrate the ice-containing dark, dense molecular clouds. Specifically, exceedingly penetrative high-energy cosmic rays generate secondary electrons within the clouds through molecular ionizations. Hydrogen molecules, present within these dense molecular clouds, are excited in collisions with these secondary electrons. It is the UV light, emitted by these electronically excited hydrogen molecules, that is generally thought to photoprocess interstellar icy grain mantles to generate "complex" molecules. In addition to producing UV light, the large numbers of low-energy (< 20 eV) secondary electrons, produced by cosmic rays, can also directly initiate radiolysis reactions in the condensed phase. The goal of our studies is to understand the low-energy, electron-induced processes that occur when high-energy cosmic rays interact with interstellar ices, in which methanol, a precursor of several prebiotic species, is the most abundant organic species. Using post-irradiation temperature-programmed desorption, we have investigated the radiolysis initiated by low-energy (7 eV and 20 eV) electrons in condensed methanol at - 85 K under ultrahigh vacuum (5 x 10(-10) Torr) conditions. We have identified eleven electron-induced methanol radiolysis products, which include many that have been previously identified as being formed by methanol UV photolysis in the interstellar medium. These experimental results suggest that low-energy, electron-induced condensed phase reactions may contribute to the interstellar synthesis of "complex" molecules previously thought to form exclusively via UV photons.

An improved reagent for determination of aliphatic amines with fluorescence and online atmospheric chemical ionization-mass spectrometry identification.

An improved reagent named 2-[2-(dibenzocarbazole)-ethoxy] ethyl chloroformate (DBCEC-Cl) for the determination of aliphatic amines by high-performance liquid chromatography (HPLC) with fluorescence detection and post-column online atmospheric chemical ionization-mass spectrometry (APCI-MS) identification has been developed. DBCEC-Cl could easily and quickly label aliphatic amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M+H](+) under APCI-MS in positive-ion mode. The ratios for fluorescence responses were I(DBCEC-amine)/I(BCEC-amine)=1.02-1.60; I(DBCEC-amine)/I(BCEOC-amine)=1.30-2.57; and I(DBCEC-amine)/I(FMOC-amine)=2.20-4.12 (here, I was relative fluorescence intensity). The ratios for MS responses were IC(DBCEC-amine)/IC(BCEC-amine)=4.16-29.31 and IC(DBCEC-amine)/IC(BCEOC-amine)=1.23-2.47 (Here, IC: APCI-MS ion current intensity). Detection limits calculated from 0.0244 pmol injection, at a signal-to-noise ratio of 3, were 0.3-3.0 fmol. The relative standard deviations for within-day determination (n=6) were 0.045-0.081% for retention time and 0.86-1.03% for peak area for the tested aliphatic amines. The mean intra- and inter-assay precision for all amine levels were <3.64% and 4.67%, respectively. The mean recoveries ranged from 96.9% to 104.7% with their standard deviations in the range of 1.80-2.70 (RSDs%). Excellent linear responses were observed with coefficients of >0.9991.

Fluorous-tagged carbamates for the pd-catalyzed amination of aryl halides.

A novel fluorous-tagged ammonia surrogate has been synthesized and its application to the synthesis of anilines by Buchwald-Hartwig palladium-catalyzed amidation-hydrolysis protocol is described. Primary anilines were obtained in moderate to good yields after a sequence of two reaction steps involving fluorous separation techniques for their purification. Preliminary results indicate that N-substituted anilines can also be obtained using just N-substituted-FBoc carbamates as the nitrogen source.

A simple, convenient and chemoselective formylation of sterols by Vilsmeier reagent.

Vilsmeier reagent (DMF-POCl(3)) was used as an efficient formylating agent. Several sterols having sec-hydroxyl group at 3/17-position have been modified into respective formate esters. The method is simple, mild, chemoselective and provides sec-alcoholic protection in good yields.

Toward the next-generation drug delivery vehicle: synthesis of a dendrimer with four orthogonally reactive groups.

The synthesis of a dendrimer based on melamine that displays multiple copies of four orthogonally reactive groups, three on the surface and one on the interior, is described. The three groups on the surface are nucleophilic and include four free hydroxyl groups, four hydroxyl groups masked as tert-butyldiphenylsilyl (TBDPS) ethers, and 16 amines masked as tert-butoxycarbonyl (BOC) groups. The core of the dendrimer displays two electrophilic monochlorotriazines. The dendrimer is available in seven linear steps (eight total steps) at 55% overall yield for the longest linear sequence.

Multiple isotope effect study of the acid-catalyzed hydrolysis of methyl formate.

Multiple isotope effects have been measured for the acid-catalyzed hydrolysis of methyl formate in 0.5 M HCl at 20 degrees C. The isotope effects in the present investigation include the carbonyl carbon (13k = 1.028 +/- 0.001), the carbonyl oxygen (18k = 0.9945 +/- 0.0009), the nucleophile oxygen (18k = 0.995 +/- 0.001), and the formyl hydrogen ((D)k = 0.81 +/- 0.02). Determination of the carbonyl carbon, carbonyl oxygen, and formyl hydrogen isotope effects was performed via isotopic analysis of residual substrate. However, determination of the oxygen nucleophile isotope effect required analysis of the oxygen atoms of the product (formic acid), which exchange with the solvent (water) under acid conditions. This necessitated measurement of the rate of exchange of these oxygen atoms under the conditions for hydrolysis (k(ex) = 0.0723 min(-1)) and correction of the raw isotope ratios measured during the nucleophile-O isotope effect experiment. These results, along with the previously reported isotope effect for the leaving oxygen (18k = 1.0009) and the ratio of the rate of hydrolysis to that of exchange of the carbonyl oxygen with water (k(h)/k(ex) = 11.3), give a detailed picture of the transition-state structure for the reaction.

Synthesis of labelled PNA oligomers by a post-synthetic modification approach.

The preparation of t-butoxycarbonyl (Boc)-protected O(4)-(o-nitrophenyl) thymine peptide nucleic acid (PNA) monomer is described. This PNA monomer was incorporated into PNA oligomer sequences. The post-synthetic modification of the oligomers to yield fluorescently-labelled PNA oligomers was studied before and after the removal of the protecting groups. In both cases, the desired fluorescently-labelled PNA oligomer was obtained in good yields.

Synthesis of of beta-amyloid precursor peptide and presenilin segments.

It seems likely that the beta-amyloid precursor protein (APP) and the presenilins (PS-1/2) play important roles in the development of Alzheimer's disease (AD). Attempts to mimic the biochemical actions of these proteins are often made by the application of fragments of these proteins. However, the synthesis of these segments by conventional methods of peptide synthesis is problematic. We have synthesized several C-terminal fragments of APP and PS-1/2 by solid-phase synthesis through combination of automatic and manual methods of synthesis. This permits solution of the 'difficult sequences' in the solid-phase synthesis of these peptides. Some details of the syntheses of nine segments are presented in this paper.

Synthesis of thyrotropin-releasing hormone-related peptides using N alpha-tert-butyloxycarbonyl-omega-(N-tert-butyloxycarbonylcarbamoyl)- alpha-amino acids.

Application of N alpha,Nca-di-tert-butyloxycarbonylhomoglutamine to synthesis of thyrotropin-releasing hormone (TRH) analogs was examined. The delta-lactam formation from homoglutaminylpeptides took place more easily than gamma-lactam formation from glutaminylpeptides in water or dioxane containing acetic acid. [pHgu1,Nva2]-TRH had dose-dependent antagonistic activity against pentobarbital anesthesia in mice, but almost no binding activity to TRH receptor in rat brain.

Formate ester formation in amide solutions.

Simple aliphatic alcohols, deoxynucleosides and nucleosides undergo reaction with formamide yielding formate esters. Formate ester formation was observed to occur slowly at 100 degrees C and more rapidly at 130 degrees C. As expected, formate esters were hydrolyzed to the alcohol and formic acid upon heating in aqueous solution. It was proposed to study the possibility that formate esters are formed initially in amide solvents, followed by displacement of formate by dihydrogen phosphate ion to form monophosphate esters. Experiments are described which demonstrate the formation and hydrolysis of formate esters, as well as their lack of reaction with hydrogen phosphate ion. Formate esters are not intermediates in the phosphorylation of nucleosides in formamide. Their formation has been observed and such an esterification is a side reaction during the phosphorylation of nucleosides in formamide.

Syntheses of 4-[N-(tert-butoxycarbonyl)aminoacyloxymethyl]-3-nitrobenzoic acids for use in the liquid-phase peptide synthesis.

The syntheses of 4-(Boc-aminoacyloxymethyl)-3-nitrobenzoic acids are described. These compounds are suitable reagents for coupling to polyethylene glycol or its derivatives by the dicyclohexylcarbodiimide method to obtain 4-(Boc-aminoacyloxymethyl)-3-nitrobenzoyl-polyethylene glycol support. This is demonstrated by the reaction of the 4-carboxy-2-nitrobenzyl ester of Boc-alanine with glycylpolyethylene glycol in excellent yield. This synthetic route produces a much better coupling yield and an easier purification procedure in comparison with the original method in which Boc-amino acid cesium salts are allowed to react with 4-(bromomethyl)-3-nitrobenzoyl-polyethylene glycol.

Synthesis of peptides by the solid-phase method. IV. Des-Arg(9)-bradykinin and analogs.

We have synthesized a series of 19 analogs of the octapeptide fragment of bradykinin (BK), des-Arg 9-bradykinin, in order to perform a structure-activity study of this peptide on the newly discovered B1 receptor of bradykinin. The first time, each residue of the octapeptide was replaced by L-alanine to pinpoint biologically important residues. Thereafter, both phenylalanine residues in positions 5 and 8 were substituted by L-tyrosine methyl ether, L-cyclohexylalanine, D-phenylalanine, and L-leucine. This paper describes the synthesis of the analogs by the solid phase method. A Beckman peptide synthesizer was used to assemble the peptides on the resin support. Couplings were performed by the symmetrical anhydribe procedure. After cleavage with liquid HF, the peptides were purified by ion-exchange chromatography on carboxymethyl-cellulose and by gel filtration on Bio-Gel P2 resin. The purity of the octapeptides was then checked by tic, paper electrophoresis, amino acid analysis, and elemental analysis. The new peptides were tested on the rabbit aorta in order to evaluate their kinin-like activities and to see if they act as antagonist. The results of the biological assays are discussed in terms of structure-activity relationships.