Monitoring solution-phase combinatorial library synthesis by capillary electrophoresis.

Capillary electrophoresis has been applied to monitor model reactions in solution-phase combinatorial chemistry. In particular, the simultaneous alkylation reactions of secondary amines with a series of benzyl halides has been investigated. Reactant and product concentrations were monitored using capillary electrophoresis in a non-aqueous buffer system. The simplified sample preparation was a key feature making this an attractive method of analysis. The results demonstrate that capillary electrophoresis is a useful tool for monitoring reactions to determine initial rates, rate constants, and extinction correlation coefficients for quantitative analysis in combinatorial chemistry, and is a broadly applicable technique for the analysis of a variety of organic and bioorganic transformations.     

Quarterly U.S. patent review: First quarter, 1999

The following are among the U.S. patents, issued in the first quarter of 1999, directed to combinatorial chemistry and related technologies. Patent issuances in the field are growing in number. Additionally, patents claiming libraries themselves, as opposed to synthetic methodologies, are becoming more common. It is impossible to be comprehensive. The author would be pleased to recognize additional patents and invites the use of his e‐mail address for this purpose. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:189–190, 1998/1999.     

Vancomycin‐based chiral stationary phases for micro‐column liquid chromatography

Vancomycin selectively immobilized to silica via either one of its two amino groups has been investigated and compared with columns made from native vancomycin. The chemical modification of vancomycin prior to immobilization involved protection of one amino group as a 9‐fluorenylmethyl carbamate. The immobilization and the subsequent cleavage of the protecting group was performed on‐column. The types of compounds that can be separated with the vancomycin chiral stationary phases resemble those separated previously by capillary electrophoresis and thin‐layer chromatography. The protected chiral stationary phases were also investigated and in some cases very high enantioselectivity were obtained. One example of this is a separation of thalidomide with an α‐value as high as 5.4. The soft immobilization procedure preserves the structure of native vancomycin, in contrast to other approaches. Good repeatability and stability of the columns have also been obtained. Chirality 11:121–128, 1999. © 1999 Wiley‐Liss, Inc.     

Design,synthesis and characterisation of affinity ligands for glycoproteins

The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein–carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar‐binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine‐based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself. Copyright © 1999 John Wiley & Sons, Ltd.     

FTICR-mass spectrometry for high-resolution analysis in combinatorial chemistry

The diversity of compound collections required for finding lead structures in pharmaceutical research can be provided by means of combinatorial organic chemistry. The resultant enormous number of single compounds but also of compound mixtures represents a challenge for the analyst. With the introduction of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS or FT-MS), a new and, as yet, not widespread mass spectrometric technique (a means of analysis of such compound libraries with a very high mass resolution) high mass accuracy and high sensitivity has become available. Moreover, in combination with electrospray ionization (ESI), not only high-throughput measurements via flow-injection analysis (FIA) but also coupling with separation techniques such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) is possible. Structural verification by way of decomposing ions (MS(n); n > or = 2) using a variety of different dissociation techniques can be performed by FTICR-MS. This is the first review specifically covering applications of FTICR-MS in the field of combinatorial chemistry.     

Techniques for high-throughput characterization of peptides, oligonucleotides and catalysis efficiency

Combinatorial processes have been widely applied to many disciplines in chemistry and biology. The vast numbers of unique entities generated by combinatorial synthesis have led to the development of high-throughput methods for characterizing samples, to avoid bottlenecks created by the application of conventional, serial-based analytical techniques. In recent years, high-throughput and novel methods utilizing mass spectrometry, multiplexed capillary electrophoresis, various forms of optical detection, and even sound waves have been investigated for a variety of applications.     

Establishment and Evaluation of the Novel Tetramethylammonium‐L‐Hydroxyproline Chiral Ionic Liquid Synergistic System Based on Clindamycin Phosphate for Enantioseparation by Capillary Electrophoresis

Much attention has been paid to chiral ionic liquids (ILs) in analytical chemistry, especially its application in capillary electrophoresis (CE) enantioseparation. However, the investigation of chiral ionic liquids synergistic systems based on antibiotic chiral selectors has been reported in only one article. In this work, a novel chiral ionic liquid, tetramethylammonium‐L‐hydroxyproline (TMA‐L‐Hyp), was applied for the first time in CE chiral separation to evaluate its potential synergistic effect with clindamycin phosphate (CP) as the chiral selector. As observed, significantly improved separation was obtained in this TMA‐L‐Hyp/CP synergistic system compared to TMA‐L‐Hyp or a CP single system. Several primary factors that might influence the separation were investigated, including CP concentration, TMA‐L‐Hyp concentration, buffer pH, types and concentrations of organic modifier, applied voltage, and capillary temperature. The best results were obtained with a 40 mM borax buffer (pH 7.6) containing 30 mM TMA‐L‐Hyp, 80 mM CP, and 20% (v/v) methanol, while the applied voltage and temperature were set at 20 kV and 20°C, respectively. Chirality 27:598–604, 2015. © 2015 Wiley Periodicals, Inc.     

Enantiomeric resolution of some nonsteroidal antiinflammatory and anticoagulant drugs using β‐cyclodextrins by capillary electrophoresis

β‐cyclodextrin (CD) and its derivatives HP‐β‐CD, DM‐β‐CD, and TM‐β‐CD have been employed as chiral selectors for the separation of three nonsteroidal antiinflammatory drugs (NSAIDs) and anticoagulant at relatively low concentration (8–15 mM) by capillary zone electrophoresis (CZE). In this study, baseline separation was achieved for ibuprofen, ketoprofen, naproxen, and warfarin. It was found that the addition of 0.1% hydroxypropyl methyl cellulose (HPMC) was effective for separation. Under these conditions, the S‐(+) enantiomer eluted before R‐(−) in terms of ibuprofen; the calculated energy values obtained from the molecular modeling correlated well with the elution order. An equation for calculating the pK_a values by capillary electrophoresis was introduced, and the pK_a values of the four chiral drugs at 25°C were obtained based on the equation. The value pK_a + 0.5 is proposed to be the suitable pH of the background electrolyte for the separation of chiral compounds containing a carboxylic group. Chirality 11:56–62, 1999. © 1999 Wiley‐Liss, Inc.     

Free solution mobility of oligomeric DNA

The free solution electrophoretic mobility of a charged oligomer in an ionic solvent that approximately takes into account relaxation field effects, screening of the velocity field, and the hydrodynamic interactions resulting from motions of the charges due to an electric field is described. For double‐stranded DNA, the free solution electrophoretic mobility under ionic strengths determined by the buffer and pH conditions relevant to capillary electrophoresis increases with increasing molecular weight up to few hundred base pairs. © 1999 John Wiley & Sons, Inc. Biopoly 49: 209–214, 1999     

Individual molecules of thermostable alkaline phosphatase support different catalytic rates at room temperature

Thermus thermophilus cells were grown at 70°C, lysed and the lysate subjected to single molecule alkaline phosphatase assays, using a capillary electrophoresis laser‐induced fluorescence detection‐based method. The enzyme was found to be heterogeneous with respect to catalytic rate when assayed at room temperature. Turnover numbers ranged 12‐fold, with an average of 400 ± 200 reactions/min for the 80 molecules assayed. Copyright © 2002 John Wiley & Sons, Ltd.     

Optical resolution and enantiomeric purity determination of the analgesic cizolirtine

The optical resolution of (±)‐cizolirtine was accomplished with excellent results (>99% ee) by means of crystallization with (+)‐ or (−)‐di‐p‐toluoyltartaric acid. The optical purity of the samples was controlled by three independent methods: ¹H NMR, capillary electrophoresis (CE) (using β‐cyclodextrins as chiral resolving agents), and HPLC (using a glycoproteic column). The use of a rapid analytical technique like ¹H NMR for estimating the relative amounts of each enantiomer, together with the high sensitivity of CE, afforded a convenient strategy for monitoring the entire process leading to enantiopure compounds. Chirality 11:63–69, 1999. © 1999 Wiley‐Liss, Inc.     

Microfluidic combinatorial chemistry

Microreactors are finding increasing application in the field of combinatorial chemistry. In the past few years, microreactor chemistry has shown great promise as a novel method on which to build new chemical technology and processes. It has been conclusively demonstrated that reactions performed within microreactors invariably generate relatively pure products in high yield. One of the immediate and obvious applications is therefore in combinatorial chemistry and drug discovery.     

Rational optimization of a HIV‐1 Tat inhibitor: Rapid progress on combinatorial lead structures

Lead molecules identified by combinatorial chemistry approaches are preferred starting points for straightforward improvements of compound profiles. Structure‐guided rationales can be supported and complemented by systematic variations based on the modular nature of the molecules. A peptoidic compound (CGP 64222), previously identified from a sequential unrandomization process, was shown to specifically inhibit the interaction between the HIV‐1 trans‐activator Tat and its RNA response element TAR. To improve the compound's pharmaceutical attractiveness an approach to reduce both, size and number of charges was pursued. Because this resulted in activity decrease, parallel synthesis with variations on one rationally defined position aimed at the identification of structural determinants was undertaken to regain in vitro activity in biochemical and cellular Tat‐TAR interaction assays. As a result CGP74026 was identified, a drastically simplified but highly active Tat antagonist, which is able to block HIV‐1 replication even in primary human cells. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:155–168, 1998/1999.     

Nonaqueous Capillary Electrophoretic Enantioseparation of Water Insoluble Tröger's Base Derivatives Using β‐Cyclodextrin as Chiral Selector

Racemic mixtures of six Tröger's base derivatives were separated by chiral nonaqueous capillary electrophoresis. The separation protocol was optimized first for suitable solvents. Then the applicability of various salts dissolved in organic solvents and their mixtures was evaluated. As chiral selectors β‐cyclodextrin and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin at various concentrations were used. The best enantioselectivity for the studied analytes was obtained utilizing formamide as organic nonaqueous solvent containing a mixture of sodium citrate and tris(hydroxymethyl)aminomethane acetate as electrolytes, and β‐cyclodextrin as chiral additive. The experimental results demonstrated the feasibility of nonaqueous capillary electrophoresis for enantioseparation of Tröger's base derivatives. This technique represents a suitable alternative to more commonly used capillary electrophoresis in aqueous environment. Chirality 25:810–813, 2013. © 2013 Wiley Periodicals, Inc.     

Role of Fourier transform infrared spectroscopy in the rehearsal phase of combinatorial chemistry: a thin-layer chromatography equivalent for on-support monitoring of solid-phase organic synthesis

The adaptation of diverse organic reactions to solid supports requires significant reaction optimization efforts. A convenient on-support analytical method functionally similar to TLC in solution chemistry is very advantageous. As a TLC-equivalent method, the single bead FTIR is a simple, sensitive, fast, and convenient analytical method to monitor SPOS without stopping the reaction or cleaving the product. As with TLC, single bead FTIR provides a wide range of information such as qualitative assessment, quantitative determination, and reaction kinetics. Studies with the single bead FTIR have not only provided a tool for daily monitoring of the solid-phase reactions, but a way to understand the properties of polymer-bound substrate and the nature of polymer-supported organic reactions. It has assisted in the selection of a wide range of reaction conditions rapidly for SPOS in the rehearsal phase of combinatorial chemistry. Due to its convenience and efficiency, FTIR internal reflection spectroscopy has evolved as a useful analytical methodology for monitoring of combinatorial chemistry reactions directly on polymer surface.     

Solving chemical problems through the application of evolutionary principles

Molecular evolution has been widely applied in the laboratory to generate novel biological macromolecules. The principles underlying evolution have more recently been used to address problems in the chemical sciences, including the discovery of functional synthetic small molecules, catalysts, materials and new chemical reactions. The application of these principles in dynamic combinatorial chemistry and in efforts involving small molecule-nucleic acid conjugates has facilitated the evaluation of large numbers of candidate structures or reactions for desired characteristics. These early efforts suggest the promise of pairing evolutionary approaches with synthetic chemistry.     

Digital fragment analysis of short tandem repeats by high‐throughput amplicon sequencing

High‐throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high‐throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high‐throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost‐effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence‐aware microsatellite data.     

Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi‐Enzyme Cascade Reactions – Synthesis of Nucleotide Sugars

Nucleotide sugars are considered as bottleneck and expensive substrates for enzymatic glycan synthesis using Leloir‐glycosyltransferases. Synthesis from cheap substrates such as monosaccharides is accomplished by multi‐enzyme cascade reactions. Optimization of product yields in such enzyme modules is dependent on the interplay of multiple parameters of the individual enzymes and governed by a considerable time effort when convential analytic methods like capillary electrophoresis (CE) or HPLC are applied. We here demonstrate for the first time multiplexed CE (MP‐CE) as fast analytical tool for the optimization of nucleotide sugar synthesis with multi‐enzyme cascade reactions. We introduce a universal separation method for nucleotides and nucleotide sugars enabling us to analyze the composition of six different enzyme modules in a high‐throughput format. Optimization of parameters (T, pH, inhibitors, kinetics, cofactors and enzyme amount) employing MP‐CE analysis is demonstrated for enzyme modules for the synthesis of UDP‐α‐D‐glucuronic acid (UDP‐GlcA) and UDP‐α‐D‐galactose (UDP‐Gal). In this way we achieve high space‐time‐yields: 1.8 g/L?h for UDP‐GlcA and 17 g/L?h for UDP‐Gal. The presented MP‐CE methodology has the impact to be used as general analytical tool for fast optimization of multi‐enzyme cascade reactions.     

Suppressing the epimerization of endothioamide peptides during Fmoc/t‐Bu‐based solid phase peptide synthesis

Despite a number of intriguing utilities associated with thioamide‐containing peptides and proteins in the context of biophysics, pharmacology and chemical biology, it has hitherto remained as one of the underexplored territories of peptidomimetics. The synthesis of long mono to multiply substituted endothioamide peptides is invariably accompanied with severe epimerization, oxoamide formation and various other undesired side reactions, resulting in messy product profiles. This has completely restrained their use as novel chemical tools for biological studies. During the chain elongation of an N‐terminally located thioamide peptide using the Fmoc/t‐Bu chemistry, it becomes vulnerable to the repetitive basic treatments as required for such chemistry. The incompatibility of thioamide moiety with bases as well as strong coupling reagents leads to epimerization as well as other side reactions due to its nucleophilicity, resulting in the loss of the stereochemical identity of the thioamidated amino acid residue. An easy‐to‐implement and efficient protocol to synthesize long (>10‐mer) endothioamide peptides, significantly suppressing epimerization and other side reactions using 10% piperidine/dimethylformamide for 1 min, is reported herein. The novelty of the protocol is shown through the efficient synthesis of a number of 10–12‐mer mono to multiply thioamide‐substituted peptides with broad substrate scopes. The utility of the protocol in the context of protein engineering and chemical protein synthesis is also shown through the synthesis of a thioamide version of the 16‐mer peptide from the B1 domain of protein G. Such a protocol to synthesize long endothioamide peptides would open up avenues toward engineering and accessing novel thiopeptide and thioprotein‐based chemical tools, the synthesis of which had been a serious hurdle thus far. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Following the lipase catalyzed enantioselective hydrolysis of amino acid esters with capillary electrophoresis using contactless conductivity detection

The progress of the enzymatic hydrolysis of racemic mixtures of the enantiomers of the methyl esters of serine and threonine was monitored. This was possible in a reaction vessel of 1.5 mL by direct sampling of volumes in the nanoliter‐range directly into an electrophoresis capillary. Contactless conductivity detection was used for quantification as the analytes are not accessible by UV‐detection in capillary electrophoresis. Porcine pancreatic lipase and wheat germ lipase both showed a preference for the L‐enantiomers of both amino acid esters. The selectivity of the porcine lipase between the two L‐esters of the two amino acids was also studied and it was found that the production of L ‐threonine had priority over L ‐serine. Chirality, 2010. © 2009 Wiley‐Liss, Inc.