Recent developments in capillary and microchip electroseparations of peptides (2021-mid-2023).

This review article brings a comprehensive survey of developments and applications of high-performance capillary and microchip electromigration methods (zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, micropreparation, and physicochemical characterization of peptides in the period from 2021 up to ca. the middle of 2023. Progress in the study of electromigration properties of peptides and various aspects of their analysis, such as sample preparation, adsorption suppression, electroosmotic flow regulation, and detection, are presented. New developments in the particular capillary electromigration methods are demonstrated, and several types of their applications are reported. They cover qualitative and quantitative analysis of synthetic or isolated peptides and determination of peptides in complex biomatrices, peptide profiling of biofluids and tissues, and monitoring of chemical and enzymatic reactions and physicochemical changes of peptides. They include also amino acid and sequence analysis of peptides, peptide mapping of proteins, separation of stereoisomers of peptides, and their chiral analyses. In addition, micropreparative separations and physicochemical characterization of peptides and their interactions with other (bio)molecules by the above CE methods are described.

The separation of cyclic diadenosine diphosphorothioate and the diastereomers of its difluorinated derivative and the estimation of the binding constants and ionic mobilities of their complexes with 2-hydroxypropyl-ß-cyclodextrin by affinity capillary electrophoresis.

The incorporation of phosphorothioate linkages has recently been extensively employed in therapeutic oligonucleotides. For their separation and quality control, new high-efficient and high-sensitive analytical methods are needed. In this work, a new affinity capillary electrophoresis method has been developed and applied for the separation of a potential anticancer drug, 2',3'-cyclic diadenosine diphosphorothioate (Rp , Rp ) (ADU-S100), and three recently newly synthesized diastereomers of its difluorinated derivative, 3',3'-cyclic di(2'-fluoro, 2'-deoxyadenosine phosphorothioate). The separation was performed in the various background electrolytes (BGEs) within a pH range 5-9 using several native and derivatized cyclodextrins (CDs) as chiral additives of the BGE. Relatively good separations were obtained with ß-, γ-, and 2-hydroxypropyl-γ-CDs in some of the BGEs tested. However, the best separation was achieved using the 2-hydroxypropyl-ß-CD chiral selector at 43.5 mM average concentration in the BGE composed of 40 mM Tris, 40 mM tricine, pH 8.1. Under these conditions, all the previous four cyclic dinucleotides (CDNs) were baseline separated within 4 min. Additionally, the average apparent binding constants and the average actual ionic mobilities of the complexes of all four CDNs with 2-hydroxypropyl-ß-CD in the above BGE were determined. The formed complexes were found to be relatively weak, with the average apparent binding constants in the range of 12.2-94.1 L mol-1 and with the actual ionic mobilities spanning the interval (-7.8 to -12.7) × 10-9  m2  V-1  s-1 . The developed method can be applied for the separation, analysis, and characterization of the above and similar CDNs.

Determination of physicochemical parameters of (bio)molecules and (bio)particles by capillary electromigration methods.

The review provides an overview of recent developments and applications of capillary electromigration (CE) methods for the determination of important physicochemical parameters of various (bio)molecules and (bio)particles. These parameters include actual and limiting (absolute) ionic mobilities, effective electrophoretic mobilities, effective charges, isoelectric points, electrokinetic potentials, hydrodynamic radii, diffusion coefficients, relative molecular masses, acidity (ionization) constants, binding constants and stoichiometry of (bio)molecular complexes, changes of Gibbs free energy, enthalpy and entropy and rate constants of chemical reactions and interactions, retention factors and partition and distribution coefficients. For the determination of these parameters, the following CE methods are employed: zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography. In the individual sections, the procedures for the determination of the above parameters by the particular CE methods are described.

Determination of the binding constants and ionic mobilities of diquat complexes with randomly sulfated cyclodextrins by affinity capillary electrophoresis.

The enantiomers of diquats (DQs), a new class of functional organic molecules, were recently separated by capillary electrophoresis (CE) with high resolution up to 11.4 within 5-7 min using randomly sulfated α-, ß-, and γ-cyclodextrins (CDs) as chiral selectors. These results indicated strong interactions between dicationic diquats and multiply negatively charged sulfated CDs (S-CDs). However, the binding strength of these interactions was not quantified. For that reason, in this study, affinity CE was applied for the determination of the binding constants and ionic mobilities of the complexes of DQ P- and M-enantiomers with CD chiral selectors in an aqueous medium. The non-covalent interactions of 10 pairs of DQ enantiomers with the above CDs were investigated in a background electrolyte (BGE) composed of 22 mM NaOH, 35 mM H3PO4, pH 2.5, and 0.0-1.0 mM concentrations of CDs. The average apparent binding constant and the average actual ionic mobility of the DQ-CD complexes were determined by nonlinear regression analysis of the dependence of the effective mobility of DQ enantiomers on the concentration of CDs in the BGE. The complexes were found to be relatively strong with the averaged apparent binding constants in the range 13 600-547 400 L/mol.

Acidity constants and protonation sites of cyclic dinucleotides determined by capillary electrophoresis, quantum chemical calculations, and NMR spectroscopy.

Cyclic dinucleotides (CDNs) are important second messengers in bacteria and eukaryotes. Detailed characterization of their physicochemical properties is a prerequisite for understanding their biological functions. Herein, we examine acid-base and electromigration properties of selected CDNs employing capillary electrophoresis (CE), density functional theory (DFT), and nuclear magnetic resonance (NMR) spectroscopy to provide benchmark pKa values, as well as to unambiguously determine the protonation sites. Acidity constants (pKa ) of the NH+ moieties of adenine and guanine bases and actual and limiting ionic mobilities of CDNs were determined by nonlinear regression analysis of the pH dependence of their effective electrophoretic mobilities measured by CE in aqueous background electrolytes in a wide pH range (0.98-11.48), at constant temperature (25°C), and constant ionic strength (25 mM). The thermodynamic pKa values were found to be in the range 3.31-4.56 for adenine and 2.28-3.61 for guanine bases, whereas the pKa of enol group of guanine base was in the range 10.21-10.40. Except for systematic shifts of ∼2 pKa , the pKa values calculated by the DFT-D3//COSMO-RS composite protocol that included large-scale conformational sampling and "cross-morphing" were in a relatively good agreement with the pKa s determined by CE and predict N1 atom of adenine and N7 atom of guanine as the protonation sites. The protonation of the N1 atom of adenine and N7 atom of guanine in acidic background electrolytes (BGEs) and the dissociation of the enol group of guanine in alkaline BGEs was confirmed also by NMR spectroscopy.

Chlorinated Cubane-1,4-dicarboxylic Acids.

Herein, we report radical chlorination of cubane-1,4-dicarboxylic acid leading preferentially to one monochlorinated cubane dicarboxylate (ca. 70%) that is accompanied by four dichlorinated derivatives (ca. 20% in total). The exact positions of the chlorine atoms have been confirmed by X-ray diffraction of the corresponding single crystals. The acidity constants of all dicarboxylic acids in water were determined by capillary electrophoresis (3.17 ± 0.04 and 4.09 ± 0.05 for monochlorinated and ca. 2.71 ± 0.05 and 3.75 ± 0.05 for dichlorinated cubanes). All chlorinated derivatives as well as the parent diacid showed high thermal stability (decomposition above 250 °C) as documented by differential scanning calorimetry. The probable reaction pathways leading to individual isomers were proposed, and the energies of individual transition states and intermediates were obtained using density functional theory calculations (B3LYP-D3BJ/6-311+G(d,p)). The relative strain energies for all newly prepared derivatives as well as for hypothetical hexahalogenated (fluorinated, chlorinated, brominated, and iodinated) derivatives of cubane-1,4-dicarboxylic acids were predicted using wavefunction theory methods. The hexafluorinated derivative was identified as the most strained compound (57.5 kcal/mol), and the relative strain decreased as the size of halogen atoms increased (23.7 for hexachloro, 16.7 for hexabromo, and 4.0 kcal/mol for the hexaiodo derivative).

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (mid-2018-2022).

This review gives a wide overview of recent advances and applications of capillary electrophoresis and microchip capillary electrophoresis methods in the fields of proteomics and peptidomics in the period from mid-2018 up to the end of 2022. The methodological topics covering sample preparation and concentration techniques, hyphenation of capillary electrophoresis methods with mass spectrometry, and multidimensional separations by on-line or off-line coupled different capillary electrophoresis and liquid chromatography techniques are described and new developments in both bottom-up and top-down approaches in proteomics are presented. In addition, various applications of capillary electrophoresis methods in proteomic and peptidomic studies are demonstrated. They include monitoring of protein posttranslational modifications and applications in biological and biochemical research, clinical peptidomics and proteomics, and food analysis.

The separation of the enantiomers of diquats by capillary electrophoresis using randomly sulfated cyclodextrins as chiral selectors.

Diquats, derivatives of the widely used herbicide diquat, represent a new class of functional organic molecules. A combination of their special electrochemical properties and axial chirality could potentially result in their important applications in supramolecular chemistry, chiral catalysis, and chiral analysis. However, prior to their practical applications, the diquats have to be prepared in enantiomerically pure forms and the enantiomeric purity of their P- and M-isomers has to be checked. Hence, a chiral capillary electrophoresis (CE) method has been developed and applied for separation of P- and M-enantiomers of 11 new diquats. Fast and better than baseline CE separations of enantiomers of all 11 diquats within a short time 5-7 min were achieved using acidic buffer, 22 mM NaOH, 35 mM H3 PO4 , pH 2.5, as a background electrolyte, and 6 mM randomly sulfated α-, ß-, and γ-cyclodextrins as chiral selectors. The most successful selector was sulfated γ-cyclodextrin, which baseline separated the enantiomers of all 11 diquats, followed by sulfated ß-cyclodextrin and sulfated α-cyclodextrin, which baseline separated enantiomers of 10 and nine diquats, respectively. Using this method, a high enantiopurity degree of the isolated P- and M-enantiomers of three diquats with a defined absolute configuration was confirmed and their migration order was identified.

Characterization of vestibular schwannoma tissues using liquid chromatography-tandem mass spectrometry analysis of specific peptide fragments separated by in-sample tryptic protein digestion followed by mathematical analysis.

Vestibular schwannoma is the most common benign neoplasm of the cerebellopontine angle. Its first symptoms include hearing loss, tinnitus, and vestibular symptoms, followed by cerebellar and brainstem symptoms, along with palsy of the adjacent cranial nerves. However, the clinical picture has unpredictable dynamics and currently, there are no reliable predictors of tumor behavior. Hence, it is desirable to have a fast routine method for analysis of vestibular schwannoma tissues at the molecular level. The major objective of this study was to verify whether a technique using in-sample specific protein digestion with trypsin would have the potential to provide a proteomic characterization of these pathological tissues. The achieved results showed that the use of this approach with subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of released peptides allowed a fast identification of a considerable number of proteins in two differential parts of vestibular schwannoma tissue as well as in tissues of control healthy samples. Furthermore, mathematical analysis of MS data was able to discriminate between pathological vestibular schwannoma tissues and healthy tissues. Thus, in-sample protein digestion combined with LC-MS/MS separation and identification of released specific peptides followed by mathematical analysis appears to have the potential for routine characterization of vestibular schwannomas at the molecular level. Data are available via ProteomeXchange with identifier PXD045261.

Recent developments in capillary and microchip electroseparations of peptides (2019-mid 2021).

The review provides a comprehensive overview of developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, microscale isolation, and physicochemical characterization of peptides from 2019 up to approximately the middle of 2021. Advances in the investigation of electromigration properties of peptides and in the methodology of their analysis, such as sample preparation, sorption suppression, EOF control, and detection, are presented. New developments in the individual CE and CEC methods are demonstrated and several types of their applications are shown. They include qualitative and quantitative analysis, determination in complex biomatrices, monitoring of chemical and enzymatic reactions and physicochemical changes, amino acid, sequence, and chiral analyses, and peptide mapping of proteins. In addition, micropreparative separations and determination of significant physicochemical parameters of peptides by CE and CEC methods are described.

Covalent anionic copolymer coatings with tunable electroosmotic flow for optimization of capillary electrophoretic separations.

We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40 × 10-9  m2 V-1 s-1 interval for PAMAMPS coatings ranging from 0 to 60 mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2-10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.

Nonaqueous capillary electrophoresis and quantum chemical calculations applied to investigation of acid-base and electromigration properties of azahelicenes.

Nonaqueous capillary electrophoresis (NACE) using methanol (MeOH) as a solvent of the BGEs and quantum mechanical density functional theory (DFT) have been applied to determine the thermodynamic acidity (ionization) constants (pKa ) of mono- and diaza[5]helicenes, mono- and diaza[6]helicenes, and their dibenzo derivatives in MeOH and water. First, the mixed acidity constants, pKa,MeOHmix${\rm{p}}K_{{\rm{a,MeOH}}}^{{\rm{mix}}}$ , of ionogenic pyridinium groups of azahelicenes and their derivatives in MeOH were obtained by nonlinear regression analysis of pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by NACE in a large series of methanolic BGEs within a wide conventional pH range (pHMeOH 1.6-12.0) and at ambient temperature (21-26°C) in a home-made CE device. Prior to mixed acidity constant calculation, the effective mobilities were corrected to reference temperature (25°C) and constant ionic strength (25 mM). Then, the mixed acidity constants were recalculated to the thermodynamic acidity constants pKa,MeOH by the Debye-Hückel theory of nonideality of electrolyte solutions. Finally, from the methanolic thermodynamic pKa,MeOH values, the aqueous thermodynamic pKa,H2O${\rm{p}}{K_{{\rm{a,}}{{\rm{H}}_{\rm{2}}}{\rm{O}}}}$ constants were estimated using the empirical relations between methanolic and aqueous acidity constants derived for structurally related pyridine derivatives. Depending on the number and position of the nitrogen atoms in their molecules, the analyzed azahelicenes were found to be weak to moderate bases with methanolic pKa,MeOH in the range 2.01-8.75 and with aqueous pKa,H2O${\rm{p}}{K_{{\rm{a,}}{{\rm{H}}_{\rm{2}}}{\rm{O}}}}$ in the range 1.67-8.28. The thermodynamic pKa,MeOH obtained by the DFT calculations were in a good agreement with those determined experimentally by NACE.

Peptide mapping of proteins by capillary electromigration methods.

This review article provides a wide overview of important developments and applications of capillary electromigration methods in the area of peptide mapping of proteins in the period 1997-mid-2022, including review articles on this topic. It deals with all major aspects of peptide mapping by capillary electromigration methods: i) precleavage sample preparation involving purification, preconcentration, denaturation, reduction and alkylation of protein(s) to be analyzed, ii) generation of peptide fragments by off-line or on-line enzymatic and/or chemical cleavage of protein(s), iii) postcleavage preparation of the generated peptide mixture for capillary electromigration separation, iv) separation of the complex peptide mixtures by one-, two- and multidimensional capillary electromigration methods coupled with mass spectrometry detection, and v) a large application of peptide mapping for variable purposes, such as qualitative analysis of monoclonal antibodies and other protein biopharmaceuticals, monitoring of posttranslational modifications, determination of primary structure and investigation of function of proteins in biochemical and clinical research, characterization of proteins of variable origin as well as for protein and peptide identification in proteomic and peptidomic studies.

Chiral analysis of ß-alanyl-d,l-tyrosine and its derivatives and estimation of binding constants of their complexes with 2-hydroxypropyl-ß-cyclodextrin by capillary electrophoresis.

Chiral CE methods were developed for the elucidation of l- or d-configuration of tyrosine residue in antimicrobial dipeptide ß-alanyl-tyrosine (ß-Ala-Tyr) isolated from the hemolymph of larvae of fleshfly Neobellieria bullata and for the evaluation of enantiopurity of its synthetic isomers (ß-Ala-d-Tyr and ß-Ala-l-Tyr), and enantiomers of their amidated and acetylated derivatives, ß-Ala-d,l-Tyr-NH2 and N-Ac-ß-Ala-d,l-Tyr, respectively. Baseline separations were achieved for all three pairs of enantiomers: (i) for ß-Ala-d,l-Tyr in acidic background electrolyte composed of 32/50 mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 2.5, and 20 mg/mL 2-hydroxypropyl-ß-cyclodextrin as chiral selector; (ii) for ß-Ala-d,l-Tyr-NH2 enantiomers in acidic background electrolyte consisting of 48/50 mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 3.5, and 30 mg/mL 2-hydroxypropyl-ß-cyclodextrin; and (iii) for enantiomers of N-Ac-ß-Ala-d,l-Tyr in alkaline background electrolyte composed of 50/49 mM Na2 B4 O7 /NaOH, pH 10.5, and 60 mg/mL 2-hydroxypropyl-ß-cyclodextrin. From CE analyses of mixed samples of isolated ß-Ala-Tyr and synthetic standards ß-Ala-l-Tyr and ß-Ala-d-Tyr, it turned out that isolated ß-Ala-Tyr was pure l-enantiomer. In addition, the average apparent binding constants, Kb , and average actual ionic mobilities of the complexes of ß-Ala-d,l-Tyr and its above derivatives with 2-hydroxypropyl-ß-cyclodextrin were determined. These complexes were weak, with Kb values ranging from 11.2 to 79.1 L/mol. Their cationic mobilities were equal to (5.6-9.2) × 10-9 m2 /V/s, and anionic mobilities to (-1.3-1.6) × 10-9 m2 /V/s.

Liquid chromatography-tandem mass spectrometry analysis of peptides separated from the insoluble matrix by in-sample tryptic protein digestion for rapid discrimination of various induced pathological states of human bone models in oral surgery.

For the understanding of pathological states of bone tissues in oral surgery, it would be desirable to have the possibility to simulate these processes on bone cell models in vitro. These cultures, similarly to bone tissues, contain numerous proteins entrapped in the insoluble matrix. The major goal of this study was to verify whether a method based on direct in-matrix protein digestion could be suitable for the discrimination between different induced pathological states of bone cell models cultivated in vitro. Using in-sample specific protein digestion with trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released peptides, 446 proteins (in average per sample) were identified in a bone cell in vitro model with induced cancer, 440 proteins were found in a model with induced inflammation, 451 proteins were detected in control in vitro culture, and 491 proteins were distinguished in samples of vestibular laminas of maxillary bone tissues originating from six different patients. Subsequent partial least squares - discrimination analysis of obtained liquid chromatography-tandem mass spectrometry data was able to discriminate among in vitro cultures with induced cancer, with induced inflammation, and control cultivation. Thus, the direct in-sample protein digestion by trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released specific peptide fragments from the insoluble matrix and mathematical analysis of the mass spectrometry data seems to be a promising tool for the routine proteomic characterization of in vitro human bone models with induced different pathological states.

Inhibitors of CA IX Enzyme Based on Polyhedral Boron Compounds.

This review describes recent progress in the design and development of inhibitors of human carbonic anhydrase IX (CA IX) based on space-filling carborane and cobalt bis(dicarbollide) clusters. CA IX enzyme is known to play a crucial role in cancer cell proliferation and metastases. The new class of potent and selective CA IX inhibitors combines the structural motif of a bulky inorganic cluster with an alkylsulfamido or alkylsulfonamido anchor group for Zn2+ ion in the enzyme active site. Detailed structure-activity relationship (SAR) studies of a large series containing 50 compounds uncovered structural features of the cluster-containing inhibitors that are important for efficient and selective inhibition of CA IX activity. Preclinical evaluation of selected compounds revealed low toxicity, favorable pharmacokinetics and ability to reduce tumor growth. Cluster-containing inhibitors of CA IX can thus be considered as promising candidates for drug development and/or for combination therapy in boron neutron capture therapy (BNCT).

Determination of acidity constants, ionic mobilities, and hydrodynamic radii of carborane-based inhibitors of carbonic anhydrases by capillary electrophoresis.

Capillary electrophoresis (CE) has been applied for determination of the thermodynamic acidity constants (pKa ) of the sulfamidoalkyl and sulfonamidoalkyl groups, the actual and limiting ionic mobilities and hydrodynamic radii of important compounds, eight carborane-based inhibitors of carbonic anhydrases, which are potential new anticancer drugs. Two types of carboranes were investigated, (i) icosahedral cobalt bis(dicarbollide)(1-) ion with sulfamidoalkyl moieties, and (ii) 7,8-nido-dicarbaundecaborate with sulfonamidoalkyl side chains. First, the mixed acidity constants, pKamix , of the sulfamidoalkyl and sulfonamidoalkyl groups of the above carboranes and their actual ionic mobilities were determined by nonlinear regression analysis of the pH dependences of their effective electrophoretic mobility measured by capillary electrophoresis in the pH range 8.00-12.25, at constant ionic strength (25 mM), and constant temperature (25°C). Second, the pKamix were recalculated to the thermodynamic pKa s using the Debye-Hückel theory. The sulfamidoalkyl and sulfonamidoalkyl groups were found to be very weakly acidic with the pKa s in the range 10.78-11.45 depending on the type of carborane cluster and on the position and length of the alkyl chain on the carborane scaffold. These pKa s were in a good agreement with the pKa s (10.67-11.27) obtained by new program AnglerFish (freeware at https://echmet.natur.cuni.cz), which provides thermodynamic pKa s and limiting ionic mobilities directly from the raw CE data. The absolute values of the limiting ionic mobilities of univalent and divalent carborane anions were in the range 18.3-27.8 TU (Tiselius unit, 1 × 10-9 m2 /Vs), and 36.4-45.9 TU, respectively. The Stokes hydrodynamic radii of univalent and divalent carborane anions varied in the range 0.34-0.52 and 0.42-0.52 nm, respectively.

In-bone protein digestion followed by LC-MS/MS peptide analysis as a new way towards the routine proteomic characterization of human maxillary and mandibular bone tissue in oral surgery.

Proteomic characterization of alveolar bones in oral surgery represents an analytical challenge due to their insoluble character. The implementation of a straightforward technique could lead to the routine use of proteomics in this field. This work thus developed a simple technique for the characterization of bone tissue for human maxillary and mandibular bones. It is based on the direct in-bone tryptic digestion of proteins in both healthy and pathological human maxillary and mandibular bone samples. The released peptides were then identified by the LC-MS/MS. Using this approach, a total of 1120 proteins were identified in the maxillary bone and 1151 proteins in the mandibular bone. The subsequent partial least squares-discrimination analysis (PLS-DA) of protein data made it possible to reach 100% discrimination between the samples of healthy alveolar bones and those of the bone tissue surrounding the inflammatory focus. These results indicate that the in-bone protein digestion followed by the LC-MS/MS and subsequent statistical analysis can provide a deeper insight into the field of oral surgery at the molecular level. Furthermore, it could also have a diagnostic potential in the differentiation between the proteomic patterns of healthy and pathological alveolar bone tissue. Data are available via ProteomeXchange with the identifier PXD026775.

Polyhalogenated Bicyclo[1.1.1]pentane-1,3-dicarboxylic Acids.

Herein we report the highly selective radical chlorination of 2,2-difluorobicyclo[1.1.1]pentane-1,3-dicarboxylic acid. Together with radical hydrodechlorination by TMS3SiH, four new bicyclo[1.1.1]pentane cages carrying two fluorine and one to three chlorine atoms in bridge positions have been obtained. The exact positions of all halogen atoms have been confirmed by X-ray diffraction. The acidity constants (pKa) for all new derivatives have been determined by capillary electrophoresis, and these experimental values show excellent agreement with pKas predicted by DFT methods. Extensive DFT calculations have been used to rationalize the selective formation of four out of nine possible F2Cl1-4 isomers of bridge-halogenated bicyclo[1.1.1]pentanes and to obtain relative strain energies for all possible isomers.

Multipodal insulin mimetics built on adamantane or proline scaffolds.

Multi-orthogonal molecular scaffolds can be applied as core structures of bioactive compounds. Here, we prepared four tri-orthogonal scaffolds based on adamantane or proline skeletons. The scaffolds were used for the solid-phase synthesis of model insulin mimetics bearing two different peptides on the scaffolds. We found that adamantane-derived compounds bind to the insulin receptor more effectively (Kd value of 0.5 µM) than proline-derived compounds (Kd values of 15-38 µM) bearing the same peptides. Molecular dynamics simulations suggest that spacers between peptides and central scaffolds can provide greater flexibility that can contribute to increased binding affinity. Molecular modeling showed possible binding modes of mimetics to the insulin receptor. Our data show that the structure of the central scaffold and flexibility of attached peptides in this type of compound are important and that different scaffolds should be considered when designing peptide hormone mimetics.