Spectral signatures and molecular origin of acid dissociation intermediates

The existence of a broad, mid-infrared absorption ranging from 1000 to 3000 cm(-1) is usually interpreted as a signature for the existence of protonated water networks. Herein, we use cryogenic mixtures of water and hydrogen fluoride (HF) and show experimental and computational evidence that similarly wide absorptions can be generated by a broad distribution of proton-shared and ion pair complexes. In the present case, we demonstrate that the broadening is mainly inhomogeneous, reflecting the fact that the topology of the first solvation shell determines the local degree of ionization and the shared-proton asymmetric stretching frequency within H2O x HF complexes. The extreme sensitivity of the proton transfer potential energy hypersurface to local hydrogen bonding topologies modulates its vibrational frequency from 2800 down to approximately 1300 cm(-1), the latter value being characteristic of solvation geometries that yield similar condensed-phase proton affinities for H2O and fluoride. By linking the local degree of ionization to the solvation pattern, we are able to propose a mechanism of ionization for HF in aqueous solutions and to explain some of their unusual properties at large concentrations. However, an important conclusion of broad scientific interest is our prediction that spectral signatures that are normally attributed to protonated water networks could also reveal the presence of strong hydrogen bonds between un-ionized acids and water molecules, with important consequences to spectroscopic investigations of biologically relevant proton channels and pumps.     

Synthesis of a truncated tetradenolide

The enantiopure synthesis of a truncated tetradenolide is presented. Starting from the versatile Chiron 7,3-lactone-xylofuranose derivative (7,3-LXF), the enantiomerically pure synthesis of the title compound is obtained in six steps with a 40% overall yield.     

Synthesis of new thienocyclopenta[3,2-d]-oxazole and thiazole derivatives

Synthesis of thieno[2′,3′:5,4]cyclopenta[3,2-d]oxazole and thiazole derivatives are achieved by insertion of carbon dioxide and disulfide into 4-amino-5-chloro-5,6-dihydro-4H-cyclopenta[b]thiophen-6-one.     

Absolute Rate Constants for the Addition of the 1‐(tert‐Butoxy)carbonylethyl Radical onto Cyclic Alkenes in Solution

Absolute rate constants are reported for the addition of the 1‐[(tert‐butoxy)carbonyl]ethyl (= 2‐(1,1‐dimethylethoxy)‐1‐methyl‐2‐oxoethyl) radical ^.CHMeCO₂(t‐Bu) to several cyclic and monosubstituted alkenes in MeCN as obtained by time‐resolved electron paramagnetic resonance (EPR). The activation energies for the addition of this alkyl radical are mainly governed by the addition enthalpy but are also substantially lowered by the ambiphilic effect and by relief of cyclic strain.     

Automated linkage of proteins and payloads producing monodisperse conjugates

Controlled protein functionalization holds great promise for a wide variety of applications. However, despite intensive research, the stoichiometry of the functionalization reaction remains difficult to control due to the inherent stochasticity of the conjugation process. Classical approaches that exploit peculiar structural features of specific protein substrates, or introduce reactive handles via mutagenesis, are by essence limited in scope or require substantial protein reengineering. We herein present equimolar native chemical tagging (ENACT), which precisely controls the stoichiometry of inherently random conjugation reactions by combining iterative low-conversion chemical modification, process automation, and bioorthogonal trans-tagging. We discuss the broad applicability of this conjugation process to a variety of protein substrates and payloads.

Controlled protein functionalization holds great promise for a wide variety of applications.

Applications of protein conjugates are limitless, including imaging, diagnostics, drug delivery, and sensing.^1–4 In many of these applications, it is crucial that the conjugates are homogeneous.⁵ The site-selectivity of the conjugation process and the number of functional labels per biomolecule, known as the degree of conjugation (DoC), are crucial parameters that define the composition of the obtained products and are often the limiting factors to achieving adequate performance of the conjugates. For instance, immuno-PCR, an extremely sensitive detection technique, requires rigorous control of the average number of oligonucleotide labels per biomolecule (its DoC) in order to achieve high sensitivity.⁶ In optical imaging, the performance of many super-resolution microscopy techniques is directly defined by the DoC of fluorescent tags.⁷ For therapeutics, an even more striking example is provided by antibody–drug conjugates, which are prescribed for the treatment of an increasing range of cancer indications.⁸ A growing body of evidence from clinical trials indicates that bioconjugation parameters, DoC and DoC distribution, directly influence the therapeutic index of these targeted agents and hence must be tightly controlled.⁹Standard bioconjugation techniques, which rely on nucleophile–electrophile reactions, result in a broad distribution of different DoC species (Fig. 1a), which have different biophysical parameters, and consequently different functional properties.¹⁰Open in a separate windowFig. 1Schematic representation of the types of protein conjugates.To address this key issue and achieve better DoC selectivity, a number of site-specific conjugation approaches have been developed (Fig. 1b). These techniques rely on protein engineering for the introduction of specific motifs (e.g., free cysteines,¹¹ selenocysteines,¹² non-natural amino acids,^13,14 peptide tags recognized by specific enzymes^15,16) with distinct reactivity compared to the reactivity of the amino acids present in the native protein. These motifs are used to simultaneously control the DoC (via chemo-selective reactions) and the site of payload attachment. Both parameters are known to influence the biological and biophysical parameters of the conjugates,¹¹ but so far there has been no way of evaluating their impact separately.The influence of DoC is more straightforward, with a lower DoC allowing the minimization of the influence of payload conjugation on the properties of the protein substrate. The lowest DoC that can be achieved for an individual conjugate is 1 (corresponding to one payload attached per biomolecule). It is noteworthy that DoC 1 is often difficult to achieve through site-specific conjugation techniques due to the symmetry of many protein substrates (e.g., antibodies). Site selection is a more intricate process, which usually relies on a systematic screening of conjugation sites for some specific criteria, such as stability or reactivity.¹⁷Herein, we introduce a method of accessing an entirely new class of protein conjugates with multiple conjugation sites but strictly homogenous DoCs (Fig. 1c). To achieve this, we combined (a) iterative low conversion chemical modification, (b) process automation, and (c) bioorthogonal trans-tagging in one workflow.The method has been exemplified for protein substrates, but it is applicable to virtually any native bio-macromolecule and payload. Importantly, this method allows for the first time the disentangling of the effects of homogeneous DoC and site-specificity on conjugate properties, which is especially intriguing in the light of recent publications revealing the complexity of the interplay between payload conjugation sites and DoC for in vivo efficacy of therapeutic bioconjugates.¹⁸ Finally, it is noteworthy that this method can be readily combined with an emerging class of site-selective bioconjugation reagents to produce site-specific DoC 1 conjugates, thus further expanding their potential for biotechnology applications.¹⁹     

Synthesis of fluorinated trithioether compounds: X-ray structures of 1,3,5-(CH2SRf)3-2,4,6-(CH3)3C6, Rf = C6F5, 4-HC6F4, or 2-FC6H4

A series of three new trithioether compounds containing fluorinated phenyl moieties, 1,3,5-(CH₂SR_f)₃-2,4,6-(CH₃)₃C₆, R_f = C₆F₅ (1), 4-HC₆F₄ (2), or 2-FC₆H₄ (3), were prepared by treatment of 1,3,5-(CH₂Br)₃-2,4,6-(CH₃)₃C₆ with the corresponding Pb(SC₆F₅)₂ or NaSR_f. The new structures were verified by elemental analyses, IR, ¹H NMR, ¹⁹F NMR spectroscopies, and mass spectra. The single crystal X-ray diffraction studies of 1-3 show a similar cis,trans,trans-conformation for the three fluorophenylthiomethyl groups attached to the central benzene ring with all dihedral angles between planes of central ring and external rings close to 0°, giving flat molecules. Comparing, 1-3 with closely related tripodal molecules built-up on 2,4,6-trimethylbenzene, arrangement of one SR group respect to others seems to be defined by the nature of the R substituent. Then in the case of 1-3, a parallel arrangement of rings is favored over an orthogonal one, which would bring the ortho-F atoms close to H atoms of the methylene groups.     

Efficient and simple synthesis of 3-aryl-1H-pyrazoles

Efficient preparation of 3-aryl-1H-pyrazoles by reaction of 1-protected-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazoles with (het)aryl halides is described. The choice of THP protecting group is discussed.     

Persilylated phosphoranyl radicals: the first persistent noncyclic phosphoranyl radicals

Persistent noncyclic phosphoranyl radicals have been prepared and observed by electron paramagnetic resonance (EPR) for the first time. They were obtained by UV-photolysis of a solution containing a bis(trialkylsilyl) peroxide (R = Me, Et) and a tris(trialkylsilyl) phosphite (R = Me, Et, iPr). EPR parameters (a(P) approximately 100 mT) are typical of phosphoranyl radicals exhibiting a trigonal-bipyramidal structure, with the odd electron in an equatorial site. Analysis of the pseudo-first-order decay shows that these phosphoranyl radicals decay by S(H)2 homolytic substitution on the bis(trialkylsilyl) peroxide and by loss of a trialkylsilyloxyl radical (alpha-scission reaction). Both the S(H)2 and alpha-scission reactions depend on the steric bulk of the alkyl groups, that is, the bulkier the alkyl group, the slower the S(H)2 and alpha-scission reactions.     

Evidence of different stoichiometries for the limiting carbonate complexes across the lanthanide(III) series: a capillary electrophoresis-mass spectrometry study

The electrophoretic mobilities (mu ep,Ln) of twelve lanthanides (not Ce, Pr and Yb) were measured by CE-ICP-MS in 0.15 and 0.5 mol L(-1) Alk2 CO3 aqueous solutions for Alk+ = Li+, Na+, K+ and Cs+. In 0.5 mol L(-1) solutions, two different mu ep,Ln values were found for the light (La to Nd) and the heavy (Dy to Tm) lanthanides, which suggests two different stoichiometries for the carbonate limiting complexes. These results are consistent with a solubility study that attests the Ln(CO3)3(3-) and Ln(CO3)4(5-) stoichiometries for the heavy (small) and the light (big) lanthanides, respectively. The Alk+ counterions influence the mu ep,Ln Alk2 CO3 values, but not the overall shape of the mu ep,Ln Alk2 CO3 plots as a function of the lanthanide atomic numbers: the counterions do not modify the stoichiometries of the inner sphere complexes. The influence of the Alk+ counterions decreases in the Li+ > Na+ > K+ > Cs+ series. The K3,Ln stepwise formation constants of the Ln(CO3)3(3-) complexes slightly increase with the atomic numbers of the lanthanides while K4,Ln, the stepwise formation constants of Ln(CO3)4(5-) complexes, slightly decrease from La to Tb, and is no longer measurable for heavier lanthanides.     

Screening of pesticides in blood with liquid chromatography–linear ion trap mass spectrometry

In clinical or forensic toxicology, general unknown screening procedures are used to identify as many xenobiotics as possible, belonging to numerous chemical classes. We present here a general unknown screening procedure based on liquid chromatography coupled with use of a single linear ion trap mass spectrometer, and focus on the identification of pesticides and/or metabolites in whole blood. After solid-phase extraction (SPE), the compounds of interest were separated using a reversed-phase column and identified by the mass spectrometer operated first in the full-scan mass spectrometry (MS) mode, in the positive and negative polarities, followed by MS² and MS³ scanning of ions selected in data-dependent acquisition. The total scan time was 2.45 s. Two mass spectral libraries (MS² and MS³), each of 450 spectra, were created for the 320 pesticides and metabolites detected after injection of pure solutions. Robustness of the spectra and matrix effects were studied and were satisfactory for the present application. Detection limits for the 320 compounds were studied by extracting 1 mL spiked blood at concentrations between 10 μg/L and 10 mg/L. If necessary, it was possible to decrease the detection limits of some compounds by 10–100-fold by scanning MS² in only one polarity, owing to a shorter total scan time. However, at the same time, the detection specificity decreased as no confirmation could be recorded in the following MS³ scan and no information could be registered in the other polarity. So, in these rare cases, confirmation by another method was required.