Search Results (18)

Cuscohygrine (CUS) and hygrine (HYG) are pyrrolidine alkaloids proposed as biomarkers of coca leaf consumption, a culturally accepted practice in some Latin American countries. Differentiating legal coca use from illicit cocaine consumption holds forensic importance. While LC-MS/MS is preferred, GC-MS remains widely used in Latin American toxicology labs due to accessibility. This study critically evaluates the analytical limitations of GC-MS for detecting CUS and HYG in biological matrices. Key parameters—injector temperature (180–290 °C), injection mode (split/splitless), solvent, liner condition, and matrix—were systematically studied. GC-MS showed significant limitations: low-abundance, non-specific fragments (m/z 42, 84, 98, 140) failed to meet the identification criteria in SIM mode. Thermal degradation of CUS to HYG and CUS-d6 to HYG-d3 was observed, especially with splitless injection and aged liners. Matrix effects produced signal enhancement ranging from +29% to +316%, meaning that analyte responses in biological samples were significantly higher than in neat standards, likely due to reduced degradation or adsorption. Although deuterated internal standards (CUS-d6) partially corrected signal variability and matrix enhancement, these corrections were not sufficient to overcome the fundamental limitations of GC-MS, including poor ion specificity and compound instability. These findings support the need for LC-MS/MS-based approaches for reliable alkaloid detection and question the suitability of GC-MS for CUS analysis in forensic toxicology contexts. Full article

Fluorinated organic compounds have demonstrated remarkable utility in medicinal chemistry due to their enhanced metabolic stability and potent therapeutic efficacy. Several examples exist of fluorinated non-steroidal anti-inflammatory drugs (NSAIDs), including diflunisal, flurbiprofen, and trifluoromethylated pyrazoles celecoxib and mavacoxib. These trifluoromethylated pyrazoles, which are most commonly constructed through the cyclocondensation of a trifluorinated 1,3-dicarbonyl and an aryl hydrazine, are also found in numerous other drug candidates. Here, we interrogate the effects of solvents and the presence of Brønsted or Lewis acid catalysts on catalyzing this process. We highlight the utility of benchtop ¹⁹F NMR spectroscopy in enabling the real-time quantification of reaction progress and the identification of fluorinated species present in crude reaction mixtures without the need for cost-prohibitive deuterated solvents. Ultimately, we find that the reaction solvent has the greatest impact on the rate and product yield, and also found that the relationship between the keto-enol equilibrium of the dicarbonyl starting material pyrazole formation rate is highly solvent-dependent. More broadly, we describe the optimization of the yield and kinetics of trifluoromethylpyrazole formation in the synthesis of celecoxib and mavacoxib, which is made possible through high-throughput reaction screening on benchtop NMR. Full article

Background/Objectives: Metabolic profiling of tissue samples via liquid-state nuclear magnetic resonance (NMR) requires the extraction of polar metabolites in a suitable deuterated solvent. Such methods often prioritise metabolite recovery over protein removal due to the relatively low sensitivity of NMR metabolomics and the routine use of methods able to supress residual protein signals. However, residual protein may impact metabolite integrity and the metabolite stability after NMR sample preparation is often overlooked. This study aimed to investigate the effect of residual protein contamination in rodent brain extracts and identify a reproducible extraction method that optimises metabolite recovery while ensuring sample stability. Methods: The performance of acetonitrile/water (50–100% MeCN), methanol/water (50–100% MeOH), and methanol/water/chloroform (MeOH/H₂O/CHCl₃) were assessed for extraction efficiency, reproducibility, residual protein contamination, and metabolite stability up to eight hours post NMR sample preparation. Results: Aspartate and glutamate deuteration were observed in 50% MeCN, 50% MeOH, and 67% MeOH extractions along with the conversion of N-acetyl aspartate to aspartate and acetate in 50% MeCN and 50% MeOH extractions. Both observations correlated with residual protein contamination and, thus, are a result of inadequate protein precipitation, as confirmed by ultrafiltration. MeOH/H₂O/CHCl₃ extraction preserved the stability of these metabolites while maintaining good extraction efficiency and reproducibility. Conclusions: Thus, we recommend MeOH/H₂O/CHCl₃ extraction for untargeted brain NMR metabolic profiling due to its effective protein precipitation and reliable performance. Nonetheless, the performance of detecting metabolites prone to oxidation such as ascorbate and glutathione is not improved by this method. Full article

Copper-catalyzed azide–alkyne cycloaddition click (CuAAC) reaction is widely used to synthesize drug candidates and other biomolecule classes. Homogeneous catalysts, which consist of copper coordinated to a ligand framework, have been optimized for high yield and specificity of the CuAAC reaction, but CuAAC reaction with these catalysts requires the addition of a reducing agent and basic conditions, which can complicate some of the desired syntheses. Additionally, removing copper from the synthesized CuAAC-containing biomolecule is necessary for biological applications but inconvenient and requires additional purification steps. We describe here the design and synthesis of a PNN-type pincer ligand complex with copper (I) that stabilizes the copper (I) and, therefore, can act as a CuAAC catalyst without a reducing agent and base under physiologically relevant conditions. This complex was immobilized on two types of resin, and one of the immobilized catalyst forms worked well under aqueous physiological conditions. Minimal copper leaching was observed from the immobilized catalyst, which allowed its use in multiple reaction cycles without the addition of any reducing agent or base and without recharging with copper ion. The mechanism of the catalytic cycle was rationalized by density functional theory (DFT). This catalyst’s utility was demonstrated by synthesizing coumarin derivatives of small molecules such as ferrocene and sugar. Full article

Gas chromatography-mass spectrometry (GC-MS) is suitable for the analysis of non-polar analytes. Free amino acids (AA) are polar, zwitterionic, non-volatile and thermally labile analytes. Chemical derivatization of AA is indispensable for their measurement by GC-MS. Specific conversion of AA to their unlabeled methyl esters (d₀Me) using 2 M HCl in methanol (CH₃OH) is a suitable derivatization procedure (60 min, 80 °C). Performance of this reaction in 2 M HCl in tetradeutero-methanol (CD₃OD) generates deuterated methyl esters (d₃Me) of AA, which can be used as internal standards in GC-MS. d₀Me-AA and d₃Me-AA require subsequent conversion to their pentafluoropropionyl (PFP) derivatives for GC-MS analysis using pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C). d₀Me-AA-PFP and d₃Me-AA-PFP derivatives of AA are readily extractable into water-immiscible, GC-compatible organic solvents such as toluene. d₀Me-AA-PFP and d₃Me-AA-PFP derivatives are stable in toluene extracts for several weeks, thus enabling high throughput quantitative measurement of biological AA by GC-MS using in situ prepared d₃Me-AA as internal standards in OMICS format. Here, we describe the development of a novel OMICS-compatible QC system and demonstrate its utility for the quality control of quantitative analysis of 21 free AA and metabolites in human plasma samples by GC-MS as Me-PFP derivatives. The QC system involves cross-standardization of the concentrations of the AA in their aqueous solutions at four concentration levels and a quantitative control of AA at the same four concentration levels in pooled human plasma samples. The retention time (t_R)-based isotope effects (IE) and the difference (δ(H/D) of the retention times of the d₀Me-AA-PFP derivatives (t_R(H)) and the d₃Me-AA-PFP derivatives (t_R(D)) were determined in study human plasma samples of a nutritional study (n = 353) and in co-processed QC human plasma samples (n = 64). In total, more than 400 plasma samples were measured in eight runs in seven working days performed by a single person. The proposed QC system provides information about the quantitative performance of the GC-MS analysis of AA in human plasma. IE, δ(H/D) and a massive drop of the peak area values of the d₃Me-AA-PFP derivatives may be suitable as additional parameters of qualitative analysis in targeted GC-MS amino acid-OMICS. Full article

The mild and effective preparation of deuterated organic molecules is an active area of research due to their important applications. Herein, we report an air-stable and easy to access copper(I) complex as catalyst for the deuteration of mono-substituted alkynes. Reactions were carried out in technical solvents and in the presence of air, to obtain excellent deuterium incorporation in a range of functionalised alkynes. Full article

Steroid hormone molecules may exhibit very different functionalities based on the associated functional groups and their 3D arrangements in space, i.e., absolute configurations and conformations. Infrared (IR) and vibrational circular dichroism (VCD) spectra of four different steroid hormones, namely dehydroepiandrosterone (DHEA), 17α-methyltestosterone (MTTT), (16α,17)-epoxyprogesterone (Epoxy-P4), and dehydroepiandrosterone acetate (AcO-DHEA), were measured in deuterated dimethyl sulfoxide and some also in carbon tetrachloride. Extensive conformational searches were carried out using the recent developed conformer-rotamer ensemble sampling tool (CREST) which also accounts for solvent effects using an implicit solvation model. All the CREST conformational candidates were then reoptimized at the B3LYP-D3BJ/def2-TZVPD with the PCM of solvent. The good agreements between the experimental IR and VCD spectra and the theoretical simulations provide a conclusive information about their conformational distribution and absolute configurations. The experimental and theoretical IR and VCD spectra of AcO-DHEA in the carbonyl and alkene stretching region showed some discrepancies, and the possible causes related to solvent effects, large amplitude motions and levels of theory used in the modelling were explored in detail. As part of the investigation, additional calculations at the B3LYP-D3BJ/6-31++G (2d,p) and B3LYP-D3BJ/cc-pVTZ levels, as well as some ‘mixed’ calculations with the double-hybrid functional B2PLYP-D3 were also carried out. The results indicate that the double-hybrid functional is important for predicting the correct IR band pattern in the carbonyl and alkene stretching region. Full article

Understanding of the nucleation process’s fundamental principles in saturated solutions is an urgent task. To do this task, it is necessary to control the formation of polymorphic forms of biologically active compounds. In certain cases, a compound can exist in a single polymorphic form, but have several solvates which can appear in different crystal forms, depending on the medium and conditions of formation, and show different pharmaceutical activity. In the present paper, we report on the analysis of Arbidol conformational preferences in two solvents of different polarities—deuterated chloroform and dimethyl sulfoxide—at 25 °C, using the 2D NOESY method. The Arbidol molecule has various solvate forms depending on the molecular conformation. The method based on the nuclear Overhauser effect spectroscopy was shown to be efficient in the analysis of complex heterocyclic compounds possessing conformation-dependent pseudo-polymorphism. It is one of the types of polymorphism observed in compounds forming crystal solvates. Combined use of NMR methods and X-ray data allowed determining of conformer populations of Arbidol in CDCl₃ and DMSO-d₆ which were found to be 8/92% and 37/63%, respectively. The preferred conformation in solution is the same that appears in stable crystal solvates of Arbidol. Full article

Brain tumors are neoplasms with one of the highest mortality rates. Therefore, the availability of methods that allow for the quick and effective diagnosis of brain tumors and selection of appropriate treatments is of critical importance for patient outcomes. In this study, coated blade spray-mass spectrometry (CBS-MS), which combines the features of microextraction and fast ionization methods, was applied for the analysis of brain tumors. In this approach, a sword-shaped probe is coated with a sorptive material to enable the extraction of analytes from biological samples. The analytes are then desorbed using only a few microliters of solvent, followed by the insertion of the CBS device into the interface on the mass spectrometer source. The results of this proof-of-concept experiment confirmed that CBS coupled to high-resolution mass spectrometry (HRMS) enables the rapid differentiation of two histologically different lesions: meningiomas and gliomas. Moreover, quantitative CBS-HRMS/MS analysis of carnitine, the endogenous compound, previously identified as a discriminating metabolite, showed good reproducibility with the variation below 10% when using a standard addition calibration strategy and deuterated internal standards for correction. The resultant data show that the proposed CBS-MS technique can be useful for on-site qualitative and quantitative assessments of brain tumor metabolite profiles. Full article

n-Octanol is the object of experimental and theoretical study of spectroscopic signatures and intermolecular interactions. The FTIR measurements were carried out at 293 K for n-octanol and its deuterated form. Special attention was paid to the vibrational features associated with the O-H stretching and the isotope effect. Density Functional Theory (DFT) in its classical formulations was applied to develop static models describing intermolecular hydrogen bond (HB) and isotope effect in the gas phase and using solvent reaction field reproduced by Polarizable Continuum Model (PCM). The Atoms in Molecules (AIM) theory enabled electronic structure and molecular topology study. The Symmetry-Adapted Perturbation Theory (SAPT) was used for energy decomposition in the dimers of n-octanol. Finally, time-evolution methods, namely classical molecular dynamics (MD) and Car-Parrinello Molecular Dynamics (CPMD) were employed to shed light onto dynamical nature of liquid n-octanol with emphasis put on metric and vibrational features. As a reference, CPMD gas phase results were applied. Nuclear quantum effects were included using Path Integral Molecular Dynamics (PIMD) and a posteriori method by solving vibrational Schrödinger equation. The latter applied procedure allowed to study the deuterium isotope effect. Full article

Using dynamic liquid-state NMR spectroscopy a degenerate double proton tautomerism was detected in tetramethyl reductic acid (TMRA) dissolved in toluene-d₈ and in CD₂Cl₂. Similar to vitamin C, TMRA belongs to the class of reductones of biologically important compounds. The tautomerism involves an intramolecular HH transfer that interconverts the peripheric and the central positions of the two OH groups. It is slow in the NMR time scale around 200 K and fast at room temperature. Pseudo-first-order rate constants of the HH transfer and of the HD transfer after suitable deuteration were obtained by line shape analyses. Interestingly, the chemical shifts were found to be temperature dependent carrying information about an equilibrium between a hydrogen bonded dimer and a monomer forming two weak intramolecular hydrogen bonds. The structures of the monomer and the dimer are discussed. The latter may consist of several rapidly interconverting hydrogen-bonded associates. A way was found to obtain the enthalpies and entropies of dissociation, which allowed us to convert the pseudo-first-order rate constants of the reaction mixture into first-order rate constants of the tautomerization of the monomer. Surprisingly, these intrinsic rate constants were the same for toluene-d₈ and CD₂Cl₂, but in the latter solvent more monomer is formed. This finding is attributed to the dipole moment of the TMRA monomer, compensated in the dimer, and to the larger dielectric constant of CD₂Cl₂. Within the margin of error, the kinetic HH/HD isotope effects were found to be of the order of 3 but independent of temperature. That finding indicates a stepwise HH transfer involving a tunnel mechanism along a double barrier pathway. The Arrhenius curves were described in terms of the Bell–Limbach tunneling model. Full article

Several classes of flavonoids, such as anthocyanins, flavonols, flavanols, and flavones, undergo a slow H/D exchange on aromatic ring A, leading to full deuteration at positions C(6) and C(8). Within the flavanol class, H-C(6) and H-C(8) of catechin and epicatechin are slowly exchanged in D₂O to the corresponding deuterated analogues. Even quercetin, a relevant flavonol representative, shows the same behaviour in a D₂O/DMSOd₆ 1:1 solution. Detailed kinetic measurements of these H/D exchange processes are here reported by exploiting the time-dependent changes of their peak areas in the ¹H-NMR spectra taken at different temperatures. A unifying reaction mechanism is also proposed based on our detailed kinetic observations, even taking into account pH and solvent effects. Molecular modelling and QM calculations were also carried out to shed more light on several molecular details of the proposed mechanism. Full article

Recently, the photoexcited triplet state of porphyrin was proposed as a promising spin-label for pulsed dipolar electron paramagnetic resonance (EPR). Herein, we report the factors that determine the electron spin echo dephasing of the photoexcited porphyrin in a water–glycerol matrix. The electron spin relaxation of a water-soluble porphyrin was measured by Q-band EPR, and the temperature dependence and the effect of solvent deuteration on the relaxation times were studied. The phase memory relaxation rate (1/T_m) is noticeably affected by solvent nuclei and is substantially faster in protonated solvents than in deuterated solvents. The T_m is as large as 13–17 μs in deuterated solvent, potentially expanding the range of distances available for measurement by dipole spectroscopy with photoexcited porphyrin. The 1/T_m depends linearly on the degree of solvent deuteration and can be used to probe the environment of a porphyrin in or near a biopolymer, including the solvent accessibility of porphyrins used in photodynamic therapy. We characterized the noncovalent binding of porphyrin to human serum albumin (HSA) from 1/T_m and electron spin echo envelope modulation (ESEEM) and found that porphyrin is quite exposed to solvent on the surface of HSA. The 1/T_m and ESEEM are equally effective and provide complementary methods to determine the solvent accessibility of a porphyrin bound to protein or to determine the location of the porphyrin. Full article

Hydrogen-bonded heterocomplexes formed by POOH-containing acids (diphenylphosphoric 1, dimethylphosphoric 2, diphenylphosphinic 3, and dimethylphosphinic 4) are studied by the low-temperature (100 K) ¹H-NMR and ³¹P-NMR using liquefied gases CDF₃/CDF₂Cl as a solvent. Formation of cyclic dimers and cyclic trimers consisting of molecules of two different acids is confirmed by the analysis of vicinal H/D isotope effects (changes in the bridging proton chemical shift, δH, after the deuteration of a neighboring H-bond). Acids 1 and 4 (or 1 and 3) form heterotrimers with very strong (short) H-bonds (δH ca. 17 ppm). While in the case of all heterotrimers the H-bonds are cyclically arranged head-to-tail, ···O=P–O–H···O=P–O–H···, and thus their cooperative coupling is expected, the signs of vicinal H/D isotope effects indicate an effective anticooperativity, presumably due to steric factors: when one of the H-bonds is elongated upon deuteration, the structure of the heterotrimer adjusts by shortening the neighboring hydrogen bonds. We also demonstrate the formation of cyclic tetramers: in the case of acids 1 and 4 the structure has alternating molecules of 1 and 4 in the cycle, while in case of acids 1 and 3 the cycle has two molecules of 1 followed by two molecules of 3. Full article

We used a combination of density functional theory (DFT) calculations and the implicit quantization of the acidic N–H and O–H bonds to assess the effect of deuteration on the binding of agonists (2-methylhistamine and 4-methylhistamine) and antagonists (cimetidine and famotidine) to the histamine H2 receptor. The results show that deuteration significantly increases the affinity for 4-methylhistamine and reduces it for 2-methylhistamine, while leaving it unchanged for both antagonists, which is found in excellent agreement with experiments. The revealed trends are interpreted in the light of the altered strength of the hydrogen bonding upon deuteration, known as the Ubbelohde effect, which affects ligand interactions with both active sites residues and solvent molecules preceding the binding, thus providing strong evidence for the relevance of hydrogen bonding for this process. In addition, computations further underline an important role of the Tyr250 residue for the binding. The obtained insight is relevant for the therapy in the context of (per)deuterated drugs that are expected to enter therapeutic practice in the near future, while this approach may contribute towards understanding receptor activation and its discrimination between agonists and antagonists. Full article