Difluorination of α-(bromomethyl)styrenes via I(I)/I(III) catalysis: facile access to electrophilic linchpins for drug discovery

Simple α-(bromomethyl)styrenes can be processed to a variety of 1,1-difluorinated electrophilic building blocks via I(I)/I(III) catalysis. This inexpensive main group catalysis strategy employs p-TolI as an effective organocatalyst when combined with Selectfluor® and simple amine·HF complexes. Modulating Brønsted acidity enables simultaneous geminal and vicinal difluorination to occur, thereby providing a platform to generate multiply fluorinated scaffolds for further downstream derivatization. The method facilitates access to a tetrafluorinated API candidate for the treatment of amyotrophic lateral sclerosis. Preliminary validation of an enantioselective process is disclosed to access α-phenyl-β-difluoro-γ-bromo/chloro esters.

Simple α-(bromomethyl)styrenes can be processed to a variety of 1,1-difluorinated electrophilic building blocks via I(I)/I(III) catalysis.

Structural editing with fluorine enables geometric and electronic variation to be explored in functional small molecules whilst mitigating steric drawbacks.¹ This expansive approach to manipulate structure–function interplay continues to manifest itself in bio-organic and medicinal chemistry.² Of the plenum of fluorinated motifs commonly employed, the geminal difluoromethylene group³ has a venerable history.⁴ This is grounded in the structural as well as electronic ramifications of CH₂ → CF₂ substitution, as is evident from a comparison of propane and 2,2-difluoropropane (Fig. 1, upper). Salient features include localized charge inversion (C–H^δ+ to C–F^δ−) and a widening of the internal angle from 112° to 115.4°.⁵ Consequently, geminal difluoromethylene groups feature prominently in the drug discovery repertoire⁶ to mitigate oxidation and modulate physicochemical parameters. Catalysis-based routes to generate electrophilic linchpins that contain the geminal difluoromethylene unit have thus been intensively pursued, particularly in the realm of main group catalysis.^7–9 Motivated by the potential of this motif in contemporary medicinal chemistry, it was envisaged that an I(I)/I(III) catalysis platform could be leveraged to convert simple α-(bromomethyl)styrenes to gem-difluorinated linchpins: the primary C(sp³)–Br motif would facilitate downstream synthetic manipulations (Fig. 1, lower). To that end, p-TolI would function as a catalyst to generate p-TolIF₂in situ in the presence of an external oxidant¹⁰ and an amine·HF complex. Alkene activation (I) with subsequent bromonium ion formation (II)¹¹ would provide a pre-text for the first C–F bond forming process (III) with regeneration of the catalyst. A subsequent phenonium ion rearrangement¹²/fluorination sequence (III and IV) would furnish the geminal difluoromethylene group and liberate the desired electrophilic building block.Open in a separate windowFig. 1The geminal difluoromethylene group: bioisosterism, and catalysis-based access from α-(bromomethyl)styrenes via I(I)/I(III) catalysis.To validate this conceptual framework, a short process of reaction optimization (1a → 2a) was conducted to assess the influence of solvent, amine·HF ratio (Brønsted acidity)¹³ and catalyst loading (Table 1). Initial reactions were performed with p-TolI (20 mol%), Selectfluor® (1.5 equiv.) as an oxidant, and CHCl₃ as the reaction medium. Variation of the amine : HF ratio was conducted to explore the influence of Brønsted acidity on catalysis efficiency (entries 1–4). An optimal ratio of 1 : 6 was observed enabling the product 2a to be generated in >95% NMR-yield. Although reducing the catalyst loading to 10 and 5 mol% (entries 5 and 6, respectively) led to high levels of efficiency (79% yield with 5 mol%), the remainder of the study was performed with 20 mol% p-TolI. Notably, catalytic vicinal difluorination was not observed at any point during this optimization, in contrast with previous studies from our laboratory.^9d,i A solvent screen revealed the importance of chlorinated solvents (entries 7 and 8): in contrast, performing the reaction in ethyl trifluoroacetate (ETFA) and acetonitrile resulted in a reduction in yield (9 and 10). Finally, a control reaction in the absence of p-TolI confirmed that an I(I)/I(III) manifold was operational (entry 11). An expanded optimization table is provided in the ESI.^†Reaction optimization^a

Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography-tandem mass spectrometry

An SPE-LC-MS/MS method was developed, validated and applied to the determination of nicotine and five major metabolites in human urine: cotinine, trans-3'-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide. A 500 microL urine sample was pH-adjusted with phosphate buffer (1.5 mL) containing nicotine-methyl-d3, cotinine-methyl-d3 and trans-3'-hydroxycotinine-methyl-d3 internal standards. For the unconjugated metabolites, an aliquot (800 microL) of the buffered solution was applied to a 30 mg Oasis HLB-SPE column, rinsed with 2% NH4OH/H2O (3.0 mL) and H2O (3.0 mL) and eluted with methanol (500 microL). The eluate was analyzed isocratically (100% methanol) by LC-MS/MS on a diol column (50 x 2.1 mm). For the total metabolites, a beta-glucuronidase/buffer preparation (100 microL) was added to the remaining buffered solution and incubated at 37 degrees C (20 h). An aliquot (800 microL) of the enzymatically treated buffered solution was extracted and analyzed in the same manner. The conjugated metabolites were determined indirectly by subtraction. The quantitation range of the method (ng/mL) was 14-10,320 for nicotine, 15-9800 for cotinine and 32-19,220 for trans-3'-hydroxycotinine. The validated method was used to observe diurnal variations from a smoker's spot urine samples, elimination half-lives from a smoker's 24 h urine samples and metabolite distribution profiles in the spot and 24 h urine samples.     

LASER CROSS-LINKING OF PROTEINS TO NUCLEIC ACIDS: PHOTODEGRADATION AND ALTERNATIVE PHOTOPRODUCTS OF THE BACTERIOPHAGE T4 GENE 32 PROTEIN

Pulsed laser cross-linking provides a means of introducing a covalent bond between proteins and the nucleic acids to which they are bound. This rapid cross-linking effectively traps the equilibrium that exists at the moment of irradiation and thus allows examination of the protein-nucleic acid interactions that existed. Laser irradiation may also induce photodestruction of protein and we have used the bacteriophage T4 gene 32 protein to investigate this phenomenon. Our results show that both nonspecific and specific photoproducts can occur, specifically at wavelengths where the peptide backbone of proteins is known to absorb. These results demonstrate that nonspecific photodegradation can be correlated with the formation of a specific photodegradation product. The formation of this product was monitored to show that product yield is nonlinearly dependent on laser power and wavelength. We have also investigated an unexpected photoproduct whose formation is dependent on the length of the polynucleotide to which the gene 32 protein binds and that further demonstrates the complexities of analyzing protein-nucleic acid interactions through the use of UV laser cross-linking. These data provide essential information for the establishment of appropriate conditions for future studies that use UV cross-linking of protein-nucleic acid complexes.     

Aromatic annelation. I. Synthesis of pyridines.

The preparation of substituted pyridines from cycloalkanones by elaboration of an aromatic ring is described.     

Regiocontrolled [3+2] quinone-nitrile oxide entry to type II polyketide building blocks

Bromine substituents on naphthoquinones effectively activate and orient the [3+2] dipolar cycloaddition reaction with nitrile oxides to generate regiodefined type II polyketide building blocks.     

The role of the active site tyrosine in the mechanism of lytic polysaccharide monooxygenase

Catalytic breakdown of polysaccharides can be achieved more efficiently by means of the enzymes lytic polysaccharide monooxygenases (LPMOs). However, the LPMO mechanism has remained controversial, preventing full exploitation of their potential. One of the controversies has centered around an active site tyrosine, present in most LPMO classes. Recent investigations have for the first time obtained direct (spectroscopic) evidence for the possibility of chemical modification of this tyrosine. However, the spectroscopic features obtained in the different investigations are remarkably different, with absorption maximum at 420 and 490 nm, respectively. In this paper we use density functional theory (DFT) in a QM/MM formulation to reconcile these (apparently) conflicting results. By modeling the spectroscopy as well as the underlying reaction mechanism we can show how formation of two isomers (both involving deprotonation of tyrosine) explains the difference in the observed spectroscopic features. Both isomers have a [TyrO–Cu–OH]⁺ moiety with the OH in either the cis- or trans-position to a deprotonated tyrosine. Although the cis-[TyrO–Cu–OH]⁺ moiety is well positioned for oxidation of the substrate, preliminary calculations with the substrate reveal that the reactivity is at best moderate, making a protective role of tyrosine more likely.

With QM/MM, we investigate the mechanism of tyrosine deprotonation in lytic polysaccharide monooxygenases. Our results support deprotonation and our calculated UV-vis spectra show that two isomers must be formed to match recent experiments.     

The sublimation enthalpy of dimethyl oxalate

The sublimation enthalpy of dimethyl oxalate has been measured by calorimetric and head space analysis. These results along with vaporization enthalpy measured by correlation gas chromatography and fusion enthalpy measurements are compared to results predicted by two estimation techniques. A previous experimental measurement was found to be in error. A mean value of (75.2±0.5) kJ/mol was obtained which results in a corrected molar value of (–681.5±0.8) kJ/mol for the enthalpy of formation of gaseous dimethyl oxalate, _f H _m ^o (g, 298.15 K). This new value of _f H _m ^o (g, 298.15 K) for dimethyl oxalate, in combination with other enthalpies of formation, suggests that the ground state of oxalates are destabilized relative to -diketones by approximately 25 kJ/mol.     

Protein fluorescence measurements within electrospray droplets

The conformation of cytochrome c molecules within electrospray droplets is investigated by monitoring the laser induced fluorescence of its single tryptophan residue (Trp-59). By increasing the alcohol concentration of the electrosprayed solutions, protein denaturation is induced, giving rise to significant changes in the intensity of the detected fluorescence. Comparison with analogous denaturation experiments in solution provides information about the relative protein conformations and differences between the bulk-solution and droplet environments. Both electrospray-plume and bulk-solution fluorescence measurements using low methanol concentration solutions indicate the presence of folded protein structures. At high methanol content, fluorescence measurements are consistent with the presence of partly denatured or unfolded conformations. At intermediate methanol content, differences are observed between the extent of denaturation in solution and that within the droplets, suggesting electrosprayed proteins have more compact structures than those detected in bulk measurements using solutions of similar composition. This infers that some fraction of the proteins within the droplets have refolded relative to their bulk-solution conformation. Protein denaturation experiments using the low vapor pressure solvent 1-propanol indicate that differences between the droplet and solution measurements are not due to solvent evaporation effects. It is suggested that different droplet conformations are more likely the result of protein diffusion to the droplet surface and effects of the droplet/air interface. To our knowledge, these are the first reported measurements of protein fluorescence within electrospray droplets.     

Analysis of the 4800-Å absorption band of Cs2 by the classical method

The broad absorption band in Cs₂ having peak intensity near 4800 Å is analyzed through computational simulation of the experimental spectrum using the classical method. The absorption, which terminates in a weak satellite at 5223 Å, can be interpreted in terms of a single transition from the ground state (R_e = 4.65 Å, ω_e = 42 cm⁻¹) to an upper state having T_e = 20 470 cm⁻¹, ω_e = 33 cm⁻¹ and R_e = 5.28 Å. The absolute absorption strength is roughly consistent with published lifetime data, but its reliability is limited by thermodynamic uncertainties stemming from the remaining uncertainty in the Cs₂ ground state dissociation enegy. The paper includes a summary of diatomic radiation relations pertinent to the analysis of low-resolution spectra, and a brief discussion of the reduced potential method applied to the alkali dimer ground states.