Micellar Mechanisms for Desymmetrization Reactions in Aqueous Media.

Water-mediated organic reactions significantly contribute to the protection of the environment. Desymmetrization reactions, which convert only one of the identical functional groups within one molecule, are cost-effective because of the low cost of the starting materials. In combination with these two merits, highly efficient and practical selective monohydrolysis reactions of symmetric diesters were previously reported. The products of these reactions are versatile building blocks. The mechanisms of this reaction are hypothesized to proceed through micellar aggregates in which the hydrophilic carboxylate anions formed by monohydrolysis are directed outward and the remaining hydrophobic groups are directed inward in governing the selectivities. Here, dynamic light scattering and electrophoretic light scattering experiments were performed for detection of the key intermediates in the reaction. These experiments revealed the existence of aggregates with negative charges on the surface in the mainly aqueous media, supporting the reaction mechanisms that control the high selectivities.

Synthesis of polynorbornadienes by ring-opening metathesis polymerization and their saturated derivatives bearing various ester groups and carboxyl groups.

Various symmetric and non-symmetric polynorbornadienes having a variety of ester groups and carboxyl groups were synthesized by ring-opening metathesis polymerization (ROMP) with Grubbs' third generation catalyst (G3 or [Ru]-III catalyst) in a controlled living manner from half-esters prepared by the selective monohydrolysis of symmetric diesters that we previously reported. The half-esters thus obtained can be directly submitted to ROMP with the G3 catalyst, leading to mostly the trans structure and narrow polydispersity indexes. The subsequent hydrogenation yielded saturated polymers, improving the thermostabilities according to the T 5 d results. Our selective monohydrolysis reactions combined with ROMP initiated by the G3 catalyst have proven to be an efficient tool for the production of a variety of homopolymers with well-controlled structures in a living manner.

Efficient and practical synthesis of monoalkyl oxalates under green conditions.

Monoalkyl oxalates are among the most important building blocks being applied to the synthesis of a variety of significant classes of compounds or applied to various cutting-edge reactions. However, their commercial availability is limited. Their synthetic methods are also limited because of the difficulty to synthesize them, and those hitherto reported are carried out in organic solvents often with the use of toxic reagents with mostly low to modest yields. Here we have developed practical synthesis of monoalkyl oxalates in aqueous media by applying the highly efficient selective monohydrolysis reactions of symmetric diesters which we reported previously. The best conditions apply an aqueous NaOH solution with relatively nontoxic THF or acetonitrile as a co-solvent at around 0-5 °C. The procedures are simple and environmentally friendly without requiring toxic or expensive reagents, yet yielding the corresponding half-esters in high yields with high purities. All the half-esters prepared here are stable over a long period of time. Therefore, our studies are expected to offer practical green methods for the synthesis of monoalkyl oxalates.

Kinetic Resolution of Nearly Symmetric 3-Cyclohexene-1-carboxylate Esters Using a Bacterial Carboxylesterase Identified by Genome Mining.

A new bacterial carboxylesterase (CarEst3) was identified by genome mining and found to efficiently hydrolyze racemic methyl 3-cyclohexene-1-carboxylate (rac-CHCM) with a nearly symmetric structure for the synthesis of (S)-CHCM. CarEst3 displayed a high substrate tolerance and a stable catalytic performance. The enantioselective hydrolysis of 4.0 M (560 g·L-1) rac-CHCM was accomplished, yielding (S)-CHCM with a >99% ee, a substrate to catalyst ratio of 1400 g·g-1, and a space-time yield of 538 g·L-1·d-1.

Syntheses of polynorbornadienes by ring-opening metathesis polymerizations of symmetric and non-symmetric 2,3-bis(alkoxycarbonyl)norbornadienes and their conversion to half-ester derivatives.

Libraries of polynorbornadienes were synthesized with good yields with a ruthenium-containing 2nd generation Grubbs catalyst by ring-opening metathesis polymerization (ROMP) of a variety of symmetric and non-symmetric 2,3-bis(alkoxycarbonyl)norbornadiene monomer units prepared from the half-esters obtained efficiently by the selective monohydrolysis reactions of symmetric diesters we reported earlier. Among these polymers, the polynorbornadienes with t-butoxycarbonyl groups derived from non-symmetric monomer units were converted to the half-ester derivatives by deprotection with trifluoroacetic acid, yielding amphiphilic polymers. The hydrogenation reactions of the obtained polymers were carried out to yield polymers having saturated structures in the main chains for improvement of the thermal stabilities. All these polymers were characterized by their molecular weights and thermal properties along with the spectroscopic data. Our selective monohydrolysis reactions have been proven to be a versatile tool for production of relatively homogeneous polymer libraries.

Synthesis of d-labeled and unlabeled ethyl succinic anhydrides and application to quantitative analysis of peptides by isotope differential mass spectrometry.

Ethyl succinic anhydride and its d5-labeled version have been synthesized and applied to quantitative analysis of peptides in combination with MALDI or ESI mass spectrometry. These modifiers react with amino groups in the N-termini and lysine side chains in proteins, and therefore the combination of these modifiers was shown to be a useful tool for quantification of peptides and hence for proteomics research.

Quantification of proteins using (13)C7-labeled and unlabeled iodoacetanilide by nano liquid chromatography/nanoelectrospray ionization and by selected reaction monitoring mass spectrometry.

The combination of cysteine-specific modifiers, iodoacetanilide (IAA) and (13)C7-labeled iodoacetanilide ((13)C7-IAA), has been applied to absolute quantification of proteins. The selected reaction monitoring (SRM) with the use of nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT-MS) analysis was applied to precise quantification of three commercial proteins. Good correlation was observed between the theoretical ratios and observed ratios for all these proteins both in a simple buffer solution and in a complex protein environment. Due to efficient tagging, this method does not require separate synthesis of isotope-labeled peptides for the SRM studies. Therefore, this method is expected to be a useful tool for proteomics research.

Quantitative protein analysis using (13)C7-labeled iodoacetanilide and d5-labeled N-ethylmaleimide by nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry.

We have developed a methodology for quantitative analysis and concurrent identification of proteins by the modification of cysteine residues with a combination of iodoacetanilide (IAA, 1) and (13)C7-labeled iodoacetanilide ((13)C7-IAA, 2), or N-ethylmaleimide (NEM, 3) and d5-labeled N-ethylmaleimide (d5-NEM, 4), followed by mass spectrometric analysis using nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT-MS). The combinations of these stable isotope-labeled and unlabeled modifiers coupled with LC separation and ESI mass spectrometric analysis allow accurate quantitative analysis and identification of proteins, and therefore are expected to be a useful tool for proteomics research.

Peptide peak intensities enhanced by cysteine modifiers and MALDI TOF MS.

Two cysteine-specific modifiers we reported previously, N-ethyl maleimide (NEM) and iodoacetanilide (IAA), have been applied to the labeling of cysteine residues of peptides for the purpose of examining the enhancement of ionization efficiencies in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The peak intensities of the peptides as a result of modification with these modifiers were compared with the peak intensities of peptides modified with a commercially available cysteine-specific modifier, iodoacetamide (IA). Our experiments show significant enhancement in the peak intensities of three cysteine-containing synthetic peptides modified with IAA compared to those modified with IA. The results showed a 4.5-6-fold increase as a result of modification with IAA compared to modification with IA. Furthermore, it was found that IAA modification also significantly enhanced the peak intensities of many peptides of a commercially available proteins, bovine serum albumin (BSA), compared to those modified with IA. This significant enhancement helped identify a greater number of peptides of these proteins, leading to a higher sequence coverage with greater confidence scores in identification of proteins with the use of IAA.

A pyrene maleimide with a flexible linker for sampling of longer inter-thiol distances by excimer formation.

Pyrene-containing compounds are commonly used in a number of fluorescence-based applications because they can form excited-state dimers (excimers) by stacking interaction between excited-state and ground-state monomers. Their usefulness arises from the facts that excimer formation requires close proximity between the pyrenes and that the excimer emission spectrum is very different from that of the monomers. One of many applications is to assess proximity between specific sites of macromolecules labeled with pyrenes. This has been done using pyrene maleimide, a reagent that reacts with reduced thiols of cysteines, but its use for structural studies of proteins has been rather limited. This is because the introduction of two cysteines at sufficiently close distance from each other to obtain excimer fluorescence upon labeling with pyrene maleimide requires detailed knowledge of the protein structure or extensive site-directed mutagenesis trials. We synthesized and tested a new compound with a 4-carbon methylene linker placed between the maleimide and the pyrene (pyrene-4-maleimide), with the aim of increasing the sampling distance for excimer formation and making the use of excimer fluorescence simpler and more widespread. We tested the new compound on thiol-modified oligonucleotides and showed that it can detect proximity between thiols beyond the reach of pyrene maleimide. Based on its spectroscopic and chemical properties, we suggest that pyrene-4-maleimide is an excellent probe to assess proximities between cysteines in proteins and thiols in other macromolecules, as well as to follow conformational changes.

Synthesis of d-labeled and unlabeled benzoyloxysuccinimides and application to quantitative analysis of peptides and a protein by isotope differential mass spectrometry.

Benzoyloxysuccinimide and its d(5)-labeled version, which react with amino groups in the N-termini and lysine side chains in proteins, were synthesized and applied to quantitative analysis of peptides and a commercially available protein in combination with a MALDI mass spectrometer.

Remote exo/endo selectivity in selective monohydrolysis of dialkyl bicyclo[2.2.1]heptane-2,3-dicarboxylate derivatives.

High exo-facial selectivity was observed in the selective monohydrolysis of a series of near-symmetric diesters that possess an exo-ester group and an endo-ester group attached on a norbornane or norbornene skeleton. The selectivities were found to be clear-cut, although the reaction center in these reactions is one covalent bond distant from the norbornane or norbornene ring, where the difference of the environment between the exo face and endo face is therefore expected to be negligible. The effect of the co-solvent we studied earlier for the selective monohydrolysis reaction was also confirmed and contributed to improvement of the yields of the half-esters.

Synthesis of 13C7-labeled iodoacetanilide and application to quantitative analysis of peptides and a protein by isotope differential mass spectrometry.

(13)C(7)-Labeled iodoacetanilide has been synthesized for specific labeling of sulfhydryl groups of cysteine residues and has been successfully applied to quantitative analysis of peptides and a commercial protein in combination with (13)C-unlabeled iodoacetanilide and a MALDI mass spectrometer. Subsequent tandem mass spectrum analysis revealed that (13)C(7)-labeled iodoacetanilide remained intact during the collision-induced dissociation (CID) conditions.

Solvation of dimethyl succinate in a sodium hydroxide aqueous solution. A computational study.

Molecular dynamics simulations were carried out to study dimethyl succinate/water/NaOH solutions. The potential of mean force method was used to determine the transport mechanism of a dimethyl succinate (a diester) molecule across the aqueous/vapor interface. The computed number density profiles show a strong propensity for the diester molecules to congregate at the interface, with the solubility of the diester increasing with increasing NaOH concentration. It is observed that the major contribution to the interfacial solvation free-energy minimum is from electrostatic interactions. Even at higher NaOH concentrations, the increasing electrostatic interaction between the diester and ions is not large enough to favor the solvation of diester in bulk solutions. The calculated solvation free energies are found to be -2.6 to -3.5 kcal/mol in variant concentrations of NaOH aqueous solutions. These values are in qualitative agreement with the corresponding experimental measurements. The computed surface potential indicates that the contribution of diester molecules to the total surface potential is about 25%, with the major contribution from interfacial water molecules.

Practical large scale synthesis of half-esters of malonic acid.

A practical large-scale synthesis of monomethyl malonate and monoethyl malonate, which are among the most commonly applied half-esters in organic synthesis, is described, applying the highly efficient selective monohydrolysis of symmetric diesters we reported before. The optimal conditions with regard to the type of base, equivalent, co-solvents, and the reaction time have been examined for large-scale reactions. Monomethyl malonate and monoethyl malonate were obtained in high yields with near 100% purity within only half a day. The conditions of this selective monohydrolysis reaction are environmentally benign and straightforward, as it requires only water, a small proportion of a volatile co-solvent, and inexpensive reagents, and produces no hazardous by-products, and therefore the synthetic utility of this reaction in process chemistry is expected.

Electrospray ionization mass spectroscopic analysis of peptides modified with N-ethylmaleimide or iodoacetanilide.

The cysteine-specific modifiers we reported previously, N-ethylmaleimide (NEM) and iodoacetanilide (IAA), have been applied to label cysteine residues of peptides in combination with electrospray ionization mass spectrometry (ESI-MS/MS), and their scope in proteomic studies was examined. Peptides modified with N-ethylmaleimide (NEM) or iodoacetanilide (IAA) showed significant enhancement in ionization efficiencies. These modifiers were also found to remain intact in tandem mass spectrometry. Both combinations of N-ethylmaleimide (NEM) and d(5)-N-ethylmaleimide (d(5)-NEM), and iodoacetanilide (IAA) and (13)C(6)-iodoacetanilide ((13)C(6)-IAA) were also shown to be applicable to quantitative analysis of a peptide.

Quantitative proteome analysis using D-labeled N-ethylmaleimide and 13C-labeled iodoacetanilide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A new methodology for quantitative analysis of proteins is described, applying stable-isotope labeling by small organic molecules combined with one- or two-dimensional electrophoresis and MALDI-TOF-MS, also allowing concurrent protein identification by peptide mass fingerprinting. Our method eliminates fundamental problems in other existing isotope-tagging methods requiring liquid chromatography and MS/MS, such as isotope effects, fragmentation, and solubility. It is also anticipated to be more practical and accessible than those LC-dependent methods.

Synthesis of D-labeled naphthyliodoacetamide and application to quantitative peptide analysis by isotope differential mass spectrometry.

D-labeled and -unlabeled N-beta-naphthyliodoacetamides have been synthesized for specific modification of the sulfhydryl groups of cysteine residues in proteins or peptides, and have been applied to quantitative analysis of several peptides. A combination of these reagents, coupled with mass spectrometry, is anticipated to serve as a useful tool for quantitative analysis of peptides and hence proteins.

Proteomics in medicinal chemistry.

Proteomics is becoming an important research area for studying protein expression patterns induced by different external stimuli. An important aspect of proteomics is to identify and quantify proteins. Many new technologies and techniques have been developed in this field and have been applied to various aspects of drug discovery.

Synthesis of 13C-labeled iodoacetanilide and application to quantitative peptide analysis by isotope differential mass spectrometry.

13C-Labeled and unlabeled iodoacetanilides have been synthesized for covalent modification of the sulfhydryl groups of cysteine residues in proteins or peptides. A combination of these reagents, coupled with mass spectrometry, is a powerful tool for quantitative analysis of peptides and hence proteins.