Enantiomeric separation of N‐protected amino acids by non‐aqueous capillary electrophoresis using quinine or Tert‐butyl carbamoylated quinine as chiral additive

A capillary electrophoretic (CE) method for the enantioseparation of N‐protected chiral amino acids was developed using quinine and tert‐butyl carbamoylated quinine as chiral selectors added to nonaqueous electrolyte solutions (NACE). A series of various N‐derivatized amino acids were tested as chiral selectands, and in order to optimize the CE enantioseparation of these compounds, different parameters were investigated: the nature of the organic solvent, the combination of different solvents, the nature and the concentration of the background electrolyte, the selector concentration, the capillary temperature, and the applied voltage. The influence of these factors on the separation of the analyte enantiomers and the electroosmotic flow was studied. Generally, with tert‐butyl carbamoylated quinine as chiral selector, better enantioseparations were achieved than with unmodified quinine. Optimum experimental conditions were found with a buffer made of 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM tert‐butyl carbamoylated quinine in an ethanol–methanol mixture (60:40 v/v). Under these conditions, DNB‐Leu enantiomers could be separated with a selectivity factor (α) of 1.572 and a resolution (Rs) of 64.3; a plate number (N) of 127,000 and an asymmetry factor (As) of 0.93 were obtained for the first migrating enantiomer. Chirality 11:622–630, 1999. © 1999 Wiley‐Liss, Inc.     

Simple Resolution of Enantiomeric NMR Signals of α‐Amino Acids by Using Samarium(III) Nitrate With L‐Tartarate

Readily available L‐tartaric acid, which is a bidentate ligand with two chiral centers forming a seven‐membered chelate ring, was applied to the chiral ligand for the chiral nuclear magnetic resonance (NMR) shift reagent of samarium(III) formed in situ. This simple method does not cause serious signal broadening in the high magnetic field. Enantiomeric ¹³C and ¹H NMR signals and enantiotopic ¹H NMR signals of α‐amino acids were successfully resolved at pH 8.0 and the 1:3 molar ratio of Sm(NO₃)₃:L‐tartaric acid. It is elucidated that the enantiomeric signal resolution is attributed to the anisotropic magnetic environment for the enantiomers induced by the chiral L‐tartarato samarium(III) complex rather than differences in stability of the diastereomeric substrate adducts. The present ¹³C NMR signal resolution was also effective for the practical simultaneous analysis of plural kinds of DL‐amino acids. Chirality 27:353–357, 2015.© 2015 Wiley Periodicals, Inc.     

Investigation of the Enantioselectivity of Tetramethylammonium L‐hydroxyproline Ionic Liquid as a Novel Chiral Ligand in Ligand‐Exchange CE and Ligand‐Exchange MEKC

Chiral ionic liquids (ILs) have drawn more and more attention in separation science; however, only a few papers focused on the application of chiral ILs as chiral ligands in LE‐CE. In this article, a novel amino acid ionic liquid (AAIL), tetramethylammonium L‐hydroxyproline ([TMA][L‐OH‐Pro]), was first applied as a chiral ligand to evaluate its enantioselectivity towards several aromatic amino acids in ligand‐exchange capillary electrophoresis (LE‐CE) and ligand‐exchange micellar electrokinetic capillary chromatography (LE‐MEKC). In the LE‐CE system, excellent separations were achieved for tryptophan (Rs = 3.03) and 3, 4‐dihydroxyphenylalanine (DOPA) (Rs = 4.35). Several parameters affecting the enantioseparation were systematically investigated, including AAIL concentration, type and concentration of central metal ion, buffer pH, as well as applied voltage. The optimum separation was obtained with 60 mM AAIL containing 30 mM Cu (II) at pH 4.5. Additionally, an LE‐MEKC system was established to further study the enantioselectivity of [TMA][L‐OH‐Pro] towards selected analytes. As observed, the separations of the enantiomers of tryptophan, phenylalanine, and histidine were all improved compared to the LE‐CE system. The results indicated that the application of AAILs as chiral ligands is a promising method in chiral separation science. Chirality 27:58–63, 2015. © 2014 Wiley Periodicals, Inc.     

Chiral HPLC Separation and Modeling of Four Stereomers of DL‐Leucine‐DL‐Tryptophan Dipeptide on Amylose Chiral Column

Chiral high‐performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL‐leucine‐tryptophan DL‐dipeptide on AmyCoat‐RP column are described. The mobile phase applied was ammonium acetate (10 mM)‐methanol‐acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL‐, DD‐, DL‐, and LD‐ stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0–2.3 and 5.6–14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π–π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL‐leucine‐DL‐tryptophan in unknown matrices. Chirality 28:642–648, 2016. © 2016 Wiley Periodicals, Inc.     

Efficient racemization of N‐phenylacetyl‐D‐glufosinate for L‐glufosinate production

Most amino acids contain chiral centres and exist as both D‐enantiomer and L‐enantiomer. The optically pure enantiomer is often more valuable than the racemate. Enzymatic resolution provides an effective strategy to obtain optically pure amino acids but often results in large amounts of unwanted isomer. In this study, optically pure L‐glufosinate (L‐PPT) was obtained by coupling amidase‐mediated hydrolysis of N‐phenylacetyl‐D,L‐glufosinate with racemization of N‐phenylacetyl‐D‐glufosinate (NPDG), which exclusively exhibits effective herbicidal properties compared with its D‐enantiomer. To improve the yield of L‐PPT, the racemization reaction conditions were optimized, and through single‐factor experiments, the optimal reaction temperature, reaction time, and mole ratio of phenylacetic acid to NPDG were determined to be 150°C, 30 minutes, and 1.5, respectively. The response surface methodology was applied to further optimize the racemization conditions, and the final yield of L‐PPT reached 96.13% with optimum reaction temperature of 154°C, reaction time of 23 minutes, and phenylacetic acid/NPDG mole ratio of 1.7, respectively. Moreover, adding a small amount of acetic anhydride further raised the yield of L‐PPT to 97.02%.     

Enantiomeric resolution and modeling of DL‐alanine‐DL‐tryptophan dipeptide on amylose stationary phase

The enantiomeric resolution of DL‐alanine‐DL‐tryptophan dipeptide is described on amylose stationary phase. The eluent used was CH₃OH─CH₃COONH₄ (10mM)─CH₃CN (50: 40, 10) at 0.8‐mL/min flow, 230‐nm detection, 25‐minute run time, and 25°C ± 1°C temperature. The chiral phase was amylose [AmyCoat RP (15 cm × 0.46 cm × 5 micron)]. The magnitudes of the retention factors (k) were 2.71, 3.52, 5.11, and 7.75. The magnitudes of separation factor (α) were 1.19, 1.57, and 1.51 while the resolution factors (Rs) were 3.25, 14.84, and 15.76. The limits of detection and quantitation were of 2.5 to 5.4 and 12.8 to 27.5 μg/mL. The enantiomeric resolution is controlled by hydrogen, hydrophobic, π‐π, steric, etc interactions. The elution order of the enantiomer was supported by the modeling data. The described method is fast, reproducible, precise, and selective, which can be used successfully for evaluating the enantiomers of the reported dipeptide.     

High‐performance liquid chromatography evaluation of the enantiomeric purity of amino acids by means of automated precolumn derivatization with ortho‐phthalaldehyde and chiral thiols

The use of ortho‐phthalaldehyde (OPA) for the derivatization of amino acids (AA) is well known. It enables the separation of the derivatives on common reversed phase columns and improves the sensitivity with fluorescence detection. With the use of a chiral thiol an indirect enantioseparation of chiral amines and AAs is feasible. The major drawback of the OPA‐derivatization is the poor stability of the products. Here, a method with an in‐needle derivatization procedure is optimized to facilitate a quantitative conversion of the AA with OPA and the chiral thiols N‐acetyl‐L‐cysteine or N‐isobutyryl‐L‐cysteine, followed by a subsequent analysis, eluding the stability issue. Both enantiomers of a single AA were separated as OPA‐derivatives with a pentafluorophenyl column and a gradient program consisting of 50 mM sodium acetate buffer pH = 5.0 and acetonitrile. Fluorescence detection is commonly used to achieve sufficient sensitivity. In this study, the enantiomeric impurity of an AA can be detected indirectly with common UV spectrophotometric detection with a limit of quantitation of 0.04%. Seventeen different L‐AAs were tested and the amount of D‐AA for each individual AA was calculated by means of area normalization, which ranged from not detectable up to 4.29%. The recovery of the minor enantiomer of L‐ and D‐AA was demonstrated for three AAs at a 0.04% level and ranged between 92.3 and 113.3%, with the relative standard deviation between 1.7 and 8.2%.     

Molecular signatures of long‐lived proteins: autolytic cleavage adjacent to serine residues

The centre of the human lens, which is composed of proteins that were synthesized prior to birth, is an ideal model for the evaluation of long‐term protein stability and processes responsible for the degradation of macromolecules. By analysing the sequences of peptides present in human lens nuclei, characteristic features of intrinsic protein instability were determined. Prominent was the cleavage on the N‐terminal side of serine residues. Despite accounting for just 9% of the amino acid composition of crystallins, peptides with N‐terminal Ser represented one‐quarter of all peptides. Nonenzymatic cleavage at Ser could be reproduced by incubating peptides at elevated temperatures. Serine residues may thus represent susceptible sites for autolysis in polypeptides exposed to physiological conditions over a period of years. Once these sites are cleaved, other chemical processes result in progressive removal or ‘laddering’ of amino acid residues from newly exposed N‐ and C‐termini. As N‐terminal Ser peptides originated from several crystallins with unrelated sequences, this may represent a general feature of long‐lived proteins.     

Enantioselective resolution of 4‐chloromandelic acid by liquid–liquid extraction using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid

A liquid–liquid extraction resolution of 4‐chloro‐mandelic acid (4‐ClMA) was studied by using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid (2‐Cl‐Z‐AA) as a chiral extractant. Important factors affecting the extraction efficiency were investigated, including the type of chiral extractant, pH value of aqueous phase, initial concentration of chiral extractant in organic phase, initial concentration of 4‐ClMA in aqueous phase, and resolution temperature. It was observed that the concentration of (R)‐4‐ClMA was much higher than that of (S)‐4‐ClMA in organic phase due to a higher stability of the complex formed between (R)‐4‐ClMA and 2‐Cl‐Z‐AA. A separation factor (α) of 3.05 was obtained at 0.02 mol/L 2‐Cl‐Z‐Valine dissolved in dichloromethane, pH of 2.0, concentration of 4‐ClMA of 0.11 mmol/Land T of 296.7K.     

Utilization of (18‐Crown‐6)‐2,3,11,12‐tetracarboxylic Acid as a Chiral NMR Solvating Agent for Diamines and β‐Amino Acids

The compound (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid was evaluated as a chiral nuclear magnetic resonance (NMR) solvating agent for a series of diamines and bicyclic β‐amino acids. The amine must be protonated for strong association with the crown ether. An advantage of (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid over many other crown ethers is that it undergoes a neutralization reaction with neutral amines to form the protonated species needed for binding. Twelve primary diamines in neutral and protonated forms were evaluated. Diamines with aryl and aliphatic groups were examined. Some are atropisomers with equivalent amine groups. Others have two nonequivalent amine groups. Association equilibria for these systems are complex, given the potential formation of 2:1, 1:1, and 1:2 crown‐amine complexes and given the various charged species in solution for mixtures of the crown ether with the neutral amine. The crown ether produced enantiomeric differentiation in the ¹H NMR spectrum of one or more resonances for every diamine substrate. Also, a series of five bicyclic β‐amino acids were examined and (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid caused enantiomeric differentiation in the ¹H NMR spectrum of three or more resonances of each compound. Chirality 27:708–715, 2015. © 2015 Wiley Periodicals, Inc.     

Crystal structure of human cytosolic aspartyl‐tRNA synthetase,a component of multi‐tRNA synthetase complex

Human cytosolic aspartyl‐tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi‐tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Ų which comprises 16.6% of the monomeric surface area. Our structure reveals the C‐terminal end of the N‐helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N‐helix for the transfer of tRNA^Asp to elongation factor 1α. From our analyses of the crystal structure and post‐translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.Proteins 2013; 81:1840–1846. © 2013 Wiley Periodicals, Inc.     

Two‐dimensional chromatography method applied to the enantiomeric determination of lansoprazole in human plasma by direct sample injection

A two‐dimensional HPLC method based on the direct injection of biological samples has been developed and validated for the determination of lansoprazole enantiomers in human plasma. The lansoprazole enantiomers were extracted from the biological matrix using an octyl restricted access media bovine serum albumin column (C₈ RAM BSA) and the enantioseparation was performed on an amylose tris(3,5‐dimethoxyphenylcarbamate) chiral column using acetonitrile:water (35:65 v/v) and UV detection at 285 nm. Analysis time was 25 min with no time spent on sample preparation. The method was applied to the analysis of the plasma samples obtained from nine Brazilian volunteers who received a 30 mg oral dose of racemic lansoprazole and was able to quantify the enantiomers of lansoprazole in the clinical samples analyzed. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Novel renin inhibitors containing derivatives of N‐alkylleucyl‐β‐hydroxy‐γ‐amino acids

In search for new drugs lowering arterial blood pressure, which could be applied in anti‐hypertensive therapy, research concerning agents blocking of renin‐angiotensin‐aldosteron system has been conducted. Despite many years of research conducted at many research centers around the world, aliskiren is the only one renin inhibitor, which is used up to now. Four novel potential renin inhibitors, having structure based on the peptide fragment 8–13 of human angiotensinogen, a natural substrate for renin, were designed and synthesized. All these inhibitors contain unnatural moieties that are derivatives of N‐methylleucyl‐β‐hydroxy‐γ‐amino acids at the P₂‐P₁' position: 4‐[N‐(N‐methylleucyl)‐amino]‐3‐hydroxy‐7‐(3‐nitroguanidino)‐heptanoic acid (AHGHA), 4‐[N‐(N‐methylleucyl)‐amino]‐3‐hydroxy‐5‐phenyl‐pentanoic acid (AHPPA) or 4‐[N‐(N‐methylleucyl)‐amino]‐8‐benzyloxycarbonylamino‐3‐hydroxyoctanoic acid (AAHOA). The previously listed synthetic β‐hydroxy‐γ‐amino acids constitute pseudodipeptidic units that correspond to the P₁‐P₁' position of the inhibitor molecule. An unnatural amino acid, 4‐methoxyphenylalanin (Phe(4‐OMe)), was introduced at the P₃ position of the obtained compounds. Three of these compounds contain isoamylamide of 6‐aminohexanoic acid (ε‐Ahx‐Iaa) at the P₂'‐P₃' position. The proposed modifications of the selected human angiotensinogen fragment are intended to increase bioactivity, bioavailability, and stability of the inhibitor molecule in body fluids and tissues. The inhibitor Boc‐Phe(4‐OMe)‐MeLeu‐AHGHA‐OEt was obtained in the form of an ethyl ester. The hydrophobicity coefficient, expressed as log P varied between 3.95 and 8.17. In vitro renin inhibitory activity of all obtained compounds was contained within the range 10^?6‐10^?9 M. The compound Boc‐Phe(4‐OMe)‐MeLeu‐AHPPA‐Ahx‐Iaa proved to be the most active (IC₅₀ = 1.05 × 10^?9 M). The compounds Boc‐Phe(4‐OMe)‐MeLeu‐AHGHA‐Ahx‐Iaa and Boc‐Phe(4‐OMe)‐MeLeu‐AHPPA‐Ahx‐Iaa are resistant to chymotrypsin. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Preparative Enantioseparation of β‐Substituted‐2‐Phenylpropionic Acids by Countercurrent Chromatography With Substituted β‐Cyclodextrin as Chiral Selectors

Preparative enantioseparation of four β‐substituted‐2‐phenylpropionic acids was performed by countercurrent chromatography with substituted β‐cyclodextrin as chiral selectors. The two‐phase solvent system was composed of n‐hexane‐ethyl acetate‐0.10 mol L‐1 of phosphate buffer solution at pH 2.67 containing 0.10 mol L^‐1 of hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) or sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD). The influence factors, including the type of substituted β‐cyclodextrin, composition of organic phase, concentration of chiral selector, pH value of the aqueous phase, and equilibrium temperature were optimized by enantioselective liquid–liquid extraction. Under the optimum separation conditions, 100 mg of 2‐phenylbutyric acid, 100 mg of tropic acid, and 50 mg of 2,3‐diphenylpropionic acid were successfully enantioseparated by high‐speed countercurrent chromatography, and the recovery of the (±)‐enantiomers was in the range of 90–91% for (±)‐2‐phenylbutyric acid, 91–92% for (±)‐tropic acid, 85–87% for (±)‐2,3‐diphenylpropionic acid with purity of over 97%, 96%, and 98%, respectively. The formation of 1:1 stoichiometric inclusion complex of β‐substituted‐2‐phenylpropionic acids with HP‐β‐CD was determined by UV spectrophotometry and the inclusion constants were calculated by a modified Benesi‐Hildebrand equation. The results showed that different enantioselectivities among different racemates were mainly caused by different enantiorecognition between each enantiomer and HP‐β‐CD, while it might be partially caused by different inclusion capacity between racemic solutes and HP‐β‐CD. Chirality 27:795–801, 2015. © 2015 Wiley Periodicals, Inc.     

Enantioselective Photolysis and Quantitative Chiral Analysis of Tryptophan Complexed With Alkali‐Metalized L‐Serine in the Gas Phase

The enantioselective photolysis of a cold gas‐phase noncovalent complex of tryptophan with alkali‐metalized L‐serine, M⁺(L‐Ser)(Trp) (M = Na and Li), was examined using a tandem mass spectrometer containing a variable‐temperature ion trap. CO₂ loss from Trp in the clusters was enantiomerically selective in ultraviolet excitation with linearly polarized light. M⁺(L‐Ser) promoted the enantioselective photolysis of Trp as a chiral auxiliary. The enantioselective photolysis of the D‐enantiomer was applied to a quantitative chiral analysis, in which the optical purity of tryptophan could be determined by measuring the relative abundance ratio R of the enantioselective CO₂ loss to the chiral‐independent evaporation of L‐Ser in a single photodissociation mass spectrum of M⁺(L‐Ser)(Trp). Chirality 27:349–352, 2015. © 2015 Wiley Periodicals, Inc.     

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N‐terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP‐like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β‐ d ‐galactosidase‐labeled Gly‐γAbu‐SDKP as a marker antigen, an anti‐rabbit IgG‐coated immunoplate as a bound/free separator and 4‐methylumbelliferyl‐β‐ d ‐galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP‐IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter‐assay and 13.3 and 7.8% for intra‐assay comparisons. Plasma AcSDKP‐IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Enantioselective liquid‐liquid extraction of DL‐mandelic acid using chiral diphosphine ligands as extractants

In order to expand the application range of chiral diphosphine ligands, (S)‐BINAP, (S)‐SEGPHOS, and (S)‐MeO‐BIPHEP were employed as extractants to recognize DL‐mandelic acid. The results indicated that (S)‐SEGPHOS‐Cu exhibited considerable ability to recognize DL‐mandelic acid with operational enantioselectivity (α) of 2.677. The process of extraction of DL‐mandelic acid using (S)‐SEGPHOS‐Cu as extractant was systematically investigated. Performance factor (pf) was adopted to comprehensively evaluate the extraction. After optimization by response surface methodology (RSM), the optimal extraction condition is temperature of 5.5°C, (S)‐SEGPHOS‐Cu concentration of 3.0 mmol/L, and pH of 8.0. And the predicted and experimental maximum values of pf were 0.26374 and 0.26839, respectively.     

N‐glycosylation microheterogeneity and site occupancy of an Asn‐X‐Cys sequon in plasma‐derived and recombinant protein C

Human protein C (hPC) is glycosylated at three Asn‐X‐Ser/Thr and one atypical Asn‐X‐Cys sequons. We have characterized the micro‐ and macro‐heterogeneity of plasma‐derived hPC and compared the glycosylation features with recombinant protein C (tg‐PC) produced in a transgenic pig bioreactor from two animals having approximately tenfold different expression levels. The N‐glycans of hPC are complex di‐ and tri‐sialylated structures, and we measured 78% site occupancy at Asn‐329 (the Asn‐X‐Cys sequon). The N‐glycans of tg‐PC are complex sialylated structures, but less branched and partially sialylated. The porcine mammary epithelial cells glycosylate the Asn‐X‐Cys sequon with a similar efficiency as human hepatocytes even at these high expression levels, and site occupancy at this sequon was not affected by expression level. A distinct bias for particular structures was present at each of the four glycosylation sites for both hPC and tg‐PC. Interestingly, glycans with GalNAc in the antennae were predominant at the Asn‐329 site. The N‐glycan structures found for tg‐PC are very similar to those reported for a recombinant Factor IX produced in transgenic pig milk, and similar to the endogenous milk protein lactoferrin, which may indicate that N‐glycan processing in the porcine mammary epithelial cells is more uniform than in other tissues.     

Enantioselective recognition of carboxylic acids by novel fluorescent triazine‐based thiazoles

Hydrogen bonding and π‐π interactions take special part in the enantioselectivity task. In this regard, because of having both hydrogen acceptor and hydrogen donor groups, melamine derivatives become more of an issue for enantioselectivity. In the light of such information, triazine‐based chiral, fluorescence active novel thiazole derivatives L1 and L2 were designed and synthesized from (S)‐(?)‐2‐amino‐1‐butanol and (1S,2R)‐(+)‐2‐amino‐1,2‐diphenylethanol. The structural establishment of these compounds was made by spectroscopic methods such as FTIR, ¹H, and ¹³C NMR. While the solution of these compounds in DMSO did not show any fluorescence emission, it was observed that the emission increased 44‐fold for L1 and 55‐fold for L2 in 95% water, similar to the aggregation‐induced emission (AIE) characterized compounds. In this regard, enantioselective capabilities of these compounds against carboxylic acids were tested, and in experiments carried out at a ratio of 40/60 DMSO/H₂O, it was determined that R‐2ClMA increased the fluorescence emission of L1 chiral receptor by 2.59 times compared to S‐isomer.     

Combined use of irreversible binding and MRM technology for low‐ and ultralow copy‐number protein detection and quantitation

In this paper, we present a method for the determination of low‐ and ultralow copy‐number proteins in biomaterials based on a combination of concentrating the protein from the sample onto cyanogen bromide‐activated Sepharose 4B (via nonspecific binding of free amino groups) and MRM. The detection limit and the dependence of the MRM peak areas on the concentration of protein in the sample were determined using the proteins CYP102 and BSA, as a model system, both in solution and after their addition to human plasma. Nonspecific protein enrichment of proteins from diluted sample volumes of 10–50 mL was found to increase the range of linear dependence of the chromatographic peak area on concentration by more than three orders of magnitude, allowing a lower LOD limit (LLOD) of as low as 10^?18 M. At this LLOD, at least two tryptic peptides of CYP102 and BSA could be detected with S/N of ≥7.0. The results were equally good for samples containing pure protein mixtures and proteins spiked into diluted depleted human blood plasma.