A GFP Inspired 8-Methoxyquinoline-Derived Fluorescent Molecular Sensor for the Detection of Zn2+ by Two-Photon Microscopy.

An effective, GFP-inspired fluorescent Zn2+ sensor is developed for two-photon microscopy and related biological application that features an 8-methoxyquinoline moiety. Excellent photophysical characteristics including a 37-fold fluorescence enhancement with excitation and emission maxima at 440 nm and 505 nm, respectively, as well as a high two-photon cross-section of 73 GM at 880 nm are reported. Based on the experimental data, the relationship between the structure and properties was elucidated and explained backed up by DFT calculations, particularly the observed PeT phenomenon for the turn-on process. Biological validation and detailed experimental and theoretical characterization of the free and the zinc-bound compounds are presented.

A Molecular Hybrid of the GFP Chromophore and 2,2'-Bipyridine: An Accessible Sensor for Zn2+ Detection with Fluorescence Microscopy.

The few commercially available chemosensors and published probes for in vitro Zn2+ detection in two-photon microscopy are compromised by their flawed spectroscopic properties, causing issues in selectivity or challenging multistep syntheses. Herein, we present the development of an effective small molecular GFP chromophore-based fluorescent chemosensor with a 2,2'-bipyridine chelator moiety (GFZnP BIPY) for Zn2+ detection that has straightforward synthesis and uncompromised properties. Detailed experimental characterizations of the free and the zinc-bound compounds within the physiologically relevant pH range are presented. Excellent photophysical characteristics are reported, including a 53-fold fluorescence enhancement with excitation and emission maxima at 422 nm and 492 nm, respectively. A high two-photon cross section of 3.0 GM at 840 nm as well as excellent metal ion selectivity are reported. In vitro experiments on HEK 293 cell culture were carried out using two-photon microscopy to demonstrate the applicability of the novel sensor for zinc bioimaging.

Microwave-Assisted Palladium Acetate-Catalyzed C-P Cross-Coupling of Arylboronic Acids and >P(O)H Reagents in the Absence of the Usual Mono- and Bidentate P-Ligands: Mechanistic Insights.

A less-studied halogen-free variation of the Hirao reaction involving the coupling of arylboronic acids with >P(O)H reagents, such as diarylphosphine oxides, diethyl phosphite, and ethyl phenyl-H-phosphinate, was investigated in detail using Pd(OAc)2 as the catalyst precursor and applying some excess of the P-reagent to supply the ligand via its trivalent tautomeric (>P-OH) form. The optimum conditions (1.2 equiv of the P-reagent, 135-150 °C, and air) were explored for the synthesis of diaryl-phenylphosphine oxides, aryl-diphenylphosphine oxides, diethyl arylphosphonates, ethyl diphenylphosphinate, and two bisphosphinoyl derivatives. In the reaction of 4-chlorophenyl- or 3-chlorophenylboronic acid with Ph2P(O)H, triphenylphosphine oxide was also formed as a byproduct. Theoretical calculations suggested that the catalytic cycle of the P-C coupling of PhB(OH)2 with Ph2P(O)H is different from that of the usual cross-coupling reactions. It comprises the addition of a phenyl anion and then the tautomeric form >P-OH of the >P(O)H reagent to the Pd2+ catalyst complex. This is then followed by reductive elimination affording Ph3PO that is accompanied with the conversion of Pd2+ to Pd0. There is a need for a subsequent stoichiometric oxidation of Pd(0) by molecular oxygen. The spontaneous formation of the self-assembling ligands around the Pd2+ center from the >P(O)H reactant plays a crucial role in the mechanism and promotes the efficiency of the catalyst.

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Arylnaphthalene Lignans with Anti-SARS-CoV-2 and Antiproliferative Activities from the Underground Organs of Linum austriacum and Linum perenne.

Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 µM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.

Preparation and Optical Study of 1-Formamido-5-Isocyanonaphthalene, the Hydrolysis Product of the Potent Antifungal 1,5-Diisocyanonaphthalene.

Aromatic isocyanides have gained a lot of attention lately as promising antifungal and anticancer drugs, as well as high-performance fluorescent analytical probes for the detection of toxic metals, such as mercury, even in vivo. Since this topic is relatively new and aromatic isocyanides possess unique photophysical properties, the understanding of structure-behavior relationships and the preparation of novel potentially biologically active derivatives are of paramount importance. Here, we report the photophysical characterization of 1,5-diisocyanonaphthalene (DIN) backed by quantum chemical calculations. It was discovered that DIN undergoes hydrolysis in certain solvents in the presence of oxonium ions. By the careful control of the reaction conditions for the first time, the nonsymmetric product 1-formamido-5-isocyanonaphthalene (ICNF) could be prepared. Contrary to expectations, the monoformamido derivative showed a significant solvatochromic behavior with a ~50 nm range from hexane to water. This behavior was explained by the enhanced H-bond-forming ability of the formamide group. The significance of the hydrolysis reaction is that the isocyano group is converted to formamide in living organisms. Therefore, ICNF could be a potential drug (for example, antifungal) and the reaction can be used as a model for the preparation of other nonsymmetric formamido-isocyanoarenes. In contrast to its relative 1-amino-5-iscyanonaphthalene (ICAN), ICNF is highly fluorescent in water, enabling the development of a fluorescent turnoff probe.

Tissue-Specific Accumulation and Isomerization of Valuable Phenylethanoid Glycosides from Plantago and Forsythia Plants.

A comparative phytochemical study on the phenylethanoid glycoside (PhEG) composition of the underground organs of three Plantago species (P. lanceolata, P. major, and P. media) and that of the fruit wall and seed parts of Forsythia suspensa and F. europaea fruits was performed. The leaves of these Forsythia species and six cultivars of the hybrid F. × intermedia were also analyzed, demonstrating the tissue-specific accumulation and decomposition of PhEGs. Our analyses confirmed the significance of selected tissues as new and abundant sources of these valuable natural compounds. The optimized heat treatment of tissues containing high amounts of the PhEG plantamajoside (PM) or forsythoside A (FA), which was performed in distilled water, resulted in their characteristic isomerizations. In addition to PM and FA, high amounts of the isomerization products could also be isolated after heat treatment. The isomerization mechanisms were elucidated by molecular modeling, and the structures of PhEGs were identified by nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HR-MS) techniques, also confirming the possibility of discriminating regioisomeric PhEGs by tandem MS. The PhEGs showed no cytostatic activity in non-human primate Vero E6 cells, supporting their safe use as natural medicines and allowing their antiviral potency to be tested.

Regio- and Diastereoselective Synthesis of 2-Arylazetidines: Quantum Chemical Explanation of Baldwin's Rules for the Ring-Formation Reactions of Oxiranes†.

A general, scalable two-step regio- and diastereoselective method has been described for the synthesis of versatile alkaloid-type azetidines from simple building blocks with excellent overall yields. In the kinetically controlled reaction, only the formation of the strained four-membered ring can be achieved instead of the thermodynamically favorable five-membered rings under appropriate conditions. Remarkable functional group tolerance has also been demonstrated. In this paper, we give a new scope of Baldwin's rules by density functional theory (DFT) calculations with an explicit solvent model, confirming the proposed reaction mechanisms and the role of kinetic controls in the stereochemical outcome of the reported transition-metal-free carbon-carbon bond formation reactions.

Experimental and Theoretical Study on the "2,2'-Bipiridyl-Ni-Catalyzed" Hirao Reaction of >P(O)H Reagents and Halobenzenes: A Ni(0) → Ni(II) or a Ni(II) → Ni(IV) Mechanism?

It was found by us that the P-C coupling reaction of >P(O)H reagents with PhX (X = I and Br) in the presence of NiCl2/Zn as the precursors for the assumed Ni(0) complexant together with 2,2'-bipyridine as the ligand took place only with PhI at 50/70 °C. M06-2X/6-31G(d,p)//PCM(MeCN) calculations for the reaction of Ph2P(O)H and PhX revealed a favorable energetics only for the loss of iodide following the oxidative addition of PhI on the Ni(0) atom. However, the assumed transition states with Ni(II) formed after P-ligand uptake and deprotonation could not undergo reductive elimination meaning a "dead-end route". Hence, it was assumed that the initial complexation of the remaining Ni2+ ions with 2,2'-bipyridine may move the P-C coupling forward via a Ni(II) → Ni(IV) transition. This route was also confirmed by calculations, and this mechanism was justified by preparative experiments carried out using NiCl2/bipyridine in the absence of Zn. Hence, the generally accepted Ni(0) → Ni(II) route was refuted by us, confirming the generality of the Ni(II) → N(IV) protocol, either in the presence of bipyridine, or using the excess of the >P(O)H reagent as the P-ligand. The results of the calculations on the complex forming ability of Ni(0) and Ni(II) with 2,2'-bipyridine or the P-reagents were in accord with our mechanistic proposition.

Focusing on the Catal. of the Pd- and Ni-Catalyzed Hirao Reactions.

The Hirao reaction involving the phosphinoylation or phosphonation of aryl halides by >P(O)H reagents is a P-C bond forming transformation belonging to the recently very hot topic of cross-couplings. The Pd- or Ni-catalyzed variations take place via the usual cycle including oxidative addition, ligand exchange, and reductive elimination. However, according to the literature, the nature of the transition metal catalysts is not unambiguous. In this feature article, the catalysts described for the Pd(OAc)2-promoted cases are summarized, and it is concluded that the "(HOY2P)2Pd(0)" species (Y = aryl, alkoxy) is the real catalyst. In our model, the excess of the >P(O)H reagent served as the P-ligand. During the less studied Ni(II)-catalyzed instances the "(HOY2P)(-OY2P)Ni(II)Cl-" form was found to enter the catalytic cycle. The newest conclusions involving the exact structure of the catalysts, and the mechanism for their formation explored by us were supported by our earlier experimental data and theoretical calculations.

Preparation of 2-phospholene oxides by the isomerization of 3-phospholene oxides.

A series of 1-substituted-3-methyl-2-phospholene oxides was prepared from the corresponding 3-phospholene oxides by double bond rearrangement. The 2-phospholene oxides could be obtained by heating the 3-phospholene oxides in methanesulfonic acid, or via the formation of cyclic chlorophosphonium salts. Whereas mixtures of the 2- and 3-phospholene oxides formed, when the isomerization of 3-phospholene oxides was attempted under thermal conditions, or in the presence of a base. The mechanisms of the various double bond migration pathways were elucidated by quantum chemical calculations.

Chemoselective Strategy for the Direct Formation of Tetrahydro-2,5-methanobenzo[ c]azepines or Azetotetrahydroisoquinolines via Regio- and Stereoselective Reactions.

The present study reports regio- and highly diastereoselective preparative methods for the synthesis of versatile alkaloid-type compounds from oxiranylmethyl tetrahydroisoquinolines. 2,5-Methanobenzo[ c]azepines or azetidine-fused heterocycles were synthesized in tandem reactions depending on the absence or presence of a BF3 co-reagent. A high functional group tolerance has also been demonstrated. DFT calculations with an explicit solvent model confirmed the proposed reaction mechanisms and the role of kinetic controls on the stereochemical outcome of the reported new methods.

Reply to the 'Comment on "Penicillin's catalytic mechanism revealed by inelastic neutrons and quantum chemical theory"' by S. A. Glover, Phys. Chem. Chem. Phys., 2019, 21, 18012.

Herein we comparatively comment on the molecular metric 'amidicity', a descriptor of amide reactivity, and differing methods to determining it; with focus on lactam-rings. Specifically, our established amidicity percentage (AM%) approach is quantitatively contrasted with the transamidation (TA) method. This comment is organised into two sections, firstly addressing the differing methods in context of the computational bases of amidicity. This is followed by the quantitative demonstration that although both the AM% and HRS methods provide estimates of resonance enthalpy (ΔHRE), the former is more reliable across a wider set of systems. The robustness of the AM% approach is affirmed by quantitative matching of experimental NMR and kinetics measurements tracking changes in amide reactivities, including in Penicillin arising from modulation of its amide group and environmental effects.

Physicochemical Profiling of Baicalin Along with the Development and Characterization of Cyclodextrin Inclusion Complexes.

Baicalin is a flavone glycoside extracted from Scutellaria baicalensis, a traditional Chinese herbal medicine. Numerous pharmacological effects of baicalin were reported (e.g. antioxidant, anxiolytic); nevertheless, the most important physicochemical properties influencing the pharmacokinetic behaviour and the concomitant oral bioavailability have not yet been described in a comprehensive study. The aim of this project was to characterize the acid-base, lipophilicity, biorelevant solubility and permeability properties of the drug substance and providing scientific data to support the dosage form design. Another important objective was the comparative evaluation of six various baicalin-cyclodextrin (CD) inclusion complexes along with the creation of a suitable Drug Delivery System (DDS) for this BCS IV drug. Biorelevant profiling was carried out by NMR-pH titrations, saturation shake-flask and distribution coefficients (logP) measurements, while CD inclusion studies were fulfilled by experimental methods (phase solubility, 1H/13C NMR, 2D ROESY) and computational approaches. Due to low aqueous solubility (67.03 ± 1.60 µg/ml) and low permeability (Papp = 0.037 × 10-6 cm/s), baicalin is classified as BCS IV. The γ-CD complexation significantly increased the solubility of baicalin (~ 5 times). The most promoted chemical shift change occurred in baicalin-γ-CD complex. Computational studies showed disparate binding pattern for baicalin in case of ß- and γ-CD; furthermore, the calculated complexation energy was - 162.4 kJ mol-1 for ß-CD, while it was significantly stronger for γ-CD (- 181.5 kJ mol-1). The physicochemical and structural information of baicalin and its CD complexes introduced herein can create molecular basis for a promising DDS with enhanced bioavailability containing a bioactive phytopharmacon.

High efficiency two-photon uncaging coupled by the correction of spontaneous hydrolysis.

Two-photon (TP) uncaging of neurotransmitter molecules is the method of choice to mimic and study the subtleties of neuronal communication either in the intact brain or in slice preparations. However, the currently available caged materials are just at the limit of their usability and have several drawbacks. The local and focal nature of their use may for example be jeopardized by a high spontaneous hydrolysis rate of the commercially available compounds with increased photochemical release rate. Here, using quantum chemical modelling we show the mechanisms of hydrolysis and two-photon activation, and synthesized more effective caged compounds. Furthermore, we have developed a new enzymatic elimination method removing neurotransmitters inadvertently escaping from their compound during experiment. This method, usable both in one and two-photon experiments, allows for the use of materials with an increased rate of photochemical release. The efficiency of the new compound and the enzymatic method and of the new compound are demonstrated in neurophysiological experiments.

Amide Activation in Ground and Excited States.

Not all amide bonds are created equally. The purpose of the present paper is the reinterpretation of the amide group by means of two concepts: amidicity and carbonylicity. These concepts are meant to provide a new viewpoint in defining the stability and reactivity of amides. With the help of simple quantum-chemical calculations, practicing chemists can easily predict the outcome of a desired process. The main benefit of the concepts is their simplicity. They provide intuitive, but quasi-thermodynamic data, making them a practical rule of thumb for routine use. In the current paper we demonstrate the performance of our methods to describe the chemical character of an amide bond strength and the way of its activation methods. Examples include transamidation, acyl transfer and amide reductions. Also, the method is highly capable for simple interpretation of mechanisms for biological processes, such as protein splicing and drug mechanisms. Finally, we demonstrate how these methods can provide information about photo-activation of amides, through the examples of two caged neurotransmitter derivatives.

The Palladium Acetate-Catalyzed Microwave-Assisted Hirao Reaction without an Added Phosphorus Ligand as a "Green" Protocol: A Quantum Chemical Study on the Mechanism.

It was proved by our experiments that on microwave irradiation, the mono- or bidentate phosphorus ligands generally applied in the palladium(II)-catalyzed P-C coupling reaction of aryl bromides and dialkyl phosphites or secondary phosphine oxides may be substituted by the excess of the >P(O)H reagent that exists under a tautomeric equilibrium. Taking into account that the reduction of the palladium(II) salt and the ligation of the palladium(0) so formed requires 3 equivalents of the P-species for the catalyst applied in a quantity of 5-10%, all together, 15-30% of the P-reagent is necessary beyond its stoichiometric quantity. In the coupling reaction of diphenylphosphine oxide, it was possible to apply diethyl phosphite as the reducing agent and as the P-ligand. The reactivities of the diethyl phosphite and diphenylphosphine oxide reagents were compared in a competitive reaction. The mechanism and the energetics of this new variation of the Hirao reaction of bromobenzene with Y2P(O)H reagents (Y=EtO and Ph) was explored by quantum chemical calculations. The first detailed study on simple reaction models justified our assumption that, under the conditions of the reaction, the trivalent form of the >P(O)H reagent may serve as the P-ligand in the palladium(0) catalyst, and shed light on the fine mechanism of the reaction sequence. The existence of the earlier described bis(palladium complex) {[H(OPh2P)2PdOAc]2} was refuted by high level theoretical calculations. This kind of complex may be formed only with chloride anions instead of the acetate anion. The interaction of palladium acetate and Y2P(O)H may result in only the formation of the [(HO)Y2P]2Pd complex that is the active catalyst in the Hirao reaction. The new variation of the Hirao reaction is of a more general value, and represents the greenest protocol, as there is no need for the usual P-ligands. Instead, the >P(O)H reagent should be used in an excess of up to 30%. Hence, the costs and environmental burdens may be decreased.

An experimental and theoretical study of reaction mechanisms between nitriles and hydroxylamine.

The industrially relevant reaction between nitriles and hydroxylamine yielding amidoximes was studied in different molecular solvents and in ionic liquids. In industry, this procedure is carried out on the ton scale in alcohol solutions and the above transformation produces a significant amount of unexpected amide by-product, depending on the nature of the nitrile, which can cause further analytical and purification issues. Although there were earlier attempts to propose mechanisms for this transformation, the real reaction pathway is still under discussion. A new detailed reaction mechanistic explanation, based on theoretical and experimental proof, is given to augment the former mechanisms, which allowed us to find a more efficient, side-product free procedure. Interpreting the theoretical results obtained, it was shown that the application of specific imidazolium, phosphonium and quaternary ammonium based ionic liquids could decrease simultaneously the reaction time while eliminating the amide side-product, leading to the targeted product selectively. This robust and economic procedure now affords a fast, selective amide free synthesis of amidoximes.

Excited state iminium form can explain the unexpected solvatochromic behavior of symmetric 1,5- and 1,8-diaminonaphthalenes.

A new model, based on the presence of the excited state iminium ion (Ar = NH2+), is proposed to interpret the solvatochromic behavior of symmetric 1,5- and 1,8-diaminonaphtahelenes (DANs) in aprotic to protic media. The importance of using explicit solvent models during DFT calculations in protic solvents to find the proper excited structure for symmetric molecules is also highlighted.

C(sp3)-H cyclizations of 2-(2-vinyl)phenoxy-tert-anilines.

1,5-hydride transfer-triggered cyclization reactions offering a robust method for C(sp3)-C(sp3) coupling and the synthesis of e.g. tetrahydroquinolines have been thoroughly investigated in the literature. Catalysts allowing milder reaction conditions or the development of enantioselective processes were important recent contributions to the field, as well as the studies on subtrates with oxygen or sulfur heteroatoms (besides the originally described nitrogen heterocycles). In a series of studies, we focused on expanded, higher order H-transfers/cyclizations by positioning the interacting substituents on distanced rings. Cyclizations of appropriately functionalized biaryl and fused bicyclic systems led to 7-9 membered rings. In the frame of this research, we set out to study the feasibility of the cyclization and the factors affecting it by in silico methods. The conclusions drawn from computational studies were complemented by cyclization screens on 2-(2-vinyl)phenoxy-tert-anilines and their CH2-expanded analogues, the results of which are presented here. Besides isolating the expected oxazonine products in several cases, we also observed a unique dimer formation, leading to an interesting 5-6-5 ring system.