Unlocking the function promiscuity of old yellow enzyme to catalyze asymmetric Morita-Baylis-Hillman reaction.

Exploring the promiscuity of native enzymes presents a promising strategy for expanding their synthetic applications, particularly for catalyzing challenging reactions in non-native contexts. In this study, we explore the promiscuous potential of old yellow enzymes (OYEs) to facilitate the Morita-Baylis-Hillman reaction (MBH reaction), leveraging substrate similarities between MBH reaction and reduction reaction. Using mass spectrometry and spectroscopic techniques, we confirm promiscuity of GkOYE in both MBH and reduction reactions. By blocking H- and H+ transfer pathways, we engineer GkOYE.8, which loses its reduction ability but enhances its MBH activity. The structural basis of MBH reaction catalyzed by GkOYE.8 is obtained through mutation studies and kinetic simulations. Furthermore, enantiocomplementary mutants GkOYE.11 and GkOYE.13 are obtained by directed evolution, exhibiting the ability to accept various aromatic aldehydes and alkenes as substrates. This study demonstrates the potential of leveraging substrate similarities to unlock enzyme functionalities, enabling the catalysis of new-to-nature reactions.

Substitution Effects of Alkene Dipeptide Isosteres on Adjacent Peptide Bond Rotation.

Alkene dipeptide isosteres (ADIs) are promising surrogates of peptide bonds that enhance the bioactive peptide resistance to enzymatic hydrolysis in medicinal chemistry. In this study, we investigated the substitution effects of an ADI on the energy barrier of cis-trans isomerization in the acetyl proline methyl ester (Ac-Pro-OMe) model. The (E)-alkene-type proline analog, which favors a cis-amide conformation, exhibits a lower rotational barrier than native Ac-Pro-OMe. A van't Hoff analysis suggests that the energy barrier is primarily reduced by enthalpic repulsion. It was concluded that although carbon-carbon double bonds and pyrrolidine rings individually increase the rigidity of the incorporation site, their combination can provide structural flexibility and disrupt bioactive conformations. This work provides new insights into ADI-based drug design.

Grubbs-Hoveyda catalysts conjugated to a ß-barrel protein: Effect of halide substitution on aqueous olefin metathesis activity.

The effect of halide substitution in Grubbs-Hoveyda II catalysts (GHII catalysts) embedded in the engineered ß-barrel protein nitrobindin (NB4exp) on metathesis activity in aqueous media was studied. Maleimide tagged dibromido and diiodido derivates of the GHII catalyst were synthesized and covalently conjugated to NB4exp. The biohybrid catalysts were characterized spectroscopically confirming the structural integrity. When the two chloride substituents at ruthenium center were exchanged against bromide and iodide, the diiodo derivative was found to show significantly higher catalytic activity in ring-closing metathesis of α,ω-diolefins, whereas the dibromido derivative was less efficient when compared with the parent dichlorido catalyst. Using the diiodido catalyst, high turnover numbers of up to 75 were observed for ring-closing metathesis (RCM) yielding unsaturated six- and seven-membered N-heterocycles.

Development of nitroalkene-based inhibitors to target STING-dependent inflammation.

Stimulator of Interferon Genes (STING) is essential for the inflammatory response to cytosolic DNA. Despite that aberrant activation of STING is linked to an increasing number of inflammatory diseases, the development of inhibitors has been challenging, with no compounds in the pipeline beyond the preclinical stage. We previously identified endogenous nitrated fatty acids as novel reversible STING inhibitors. With the aim of improving the specificity and efficacy of these compounds, we developed and tested a library of nitroalkene-based compounds for in vitro and in vivo STING inhibition. The structure-activity relationship study revealed a robustly improved electrophilicity and reduced degrees of freedom of nitroalkenes by conjugation with an aromatic moiety. The lead compounds CP-36 and CP-45, featuring a ß-nitrostyrene moiety, potently inhibited STING activity in vitro and relieved STING-dependent inflammation in vivo. This validates the potential for nitroalkene compounds as drug candidates for STING modulation to treat STING-driven inflammatory diseases, providing new robust leads for preclinical development.

Light-induced ß-hydroxy sulfone synthesis in DNA-encoded libraries.

We here describe a visible-light photooxidation of sulfinate salts with common alkenes to yield ß-hydroxy sulfones on DNA. This process demonstrates a broad substrate compatibility and achieves conversion rates ranging from moderate to excellent. Most importantly, it presents a straightforward, efficient, and metal-free approach for synthesizing Csp3-rich DNA-encoded libraries.

Oxidative Coupling of Hydroquinone Derivatives with Olefins to Dihydrobenzofurans.

Dihydrobenzofuran is an important skeleton for bioactive compounds and natural products. Hydroquinones can be easily modified into substituted hydroquinones, which effectively undergo oxidation to produce the corresponding benzoquinone derivatives. Benzoquinones are reactive electrophiles that are frequently utilized in coupling with olefins to dihydrobenzofurans. Herein, we report the one-pot oxidative coupling of hydroquinones bearing an electron-withdrawing group at the C2 position with olefins to dihydrobenzofurans in the presence of the Lewis acidic FeCl3 and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) oxidant. Furthermore, this method was applied to the oxidative coupling of N-electron-withdrawing group-substituted 4-aminophenol.

Applications of biocatalytic CC bond reductions in the synthesis of flavours and fragrances.

Industrial biotechnology and biocatalysis can provide very effective synthetic tools to increase the sustainability of the production of fine chemicals, especially flavour and fragrance (F&F) ingredients, the market demand of which has been constantly increasing in the last years. One of the most important transformations in F&F chemistry is the reduction of CC bonds, typically carried out with metal-catalysed hydrogenations or hydride-based reagents. Its biocatalytic counterpart is a competitive alternative, showcasing a range of advantages such as excellent chemo-, regio- and stereoselectivity, ease of implementation, mild reaction conditions and modest environmental impact. In the present review, the application of biocatalysed alkene reductions (from microbial fermentations with wild-type strains to engineered isolated ene-reductase enzymes) to synthetic processes useful for the F&F industry will be described, highlighting not only the exquisite stereoselectivity achieved, but also the overall improvement when chirality is not involved. Multi-enzymatic cascades involving CC bioreductions are also examined, which allow much greater chemical complexity to be built in one-pot biocatalytic systems.

Ultraviolet spectrophotometry as method to determine the concentration of ß-myrcene released from chitosan in aqueous medium.

Myrcene (ß-myrcene), found in essential oils from plant species such as hops and cannabis, has many advantageous properties, but its use is limited due to volatility and low solubility in water. One way to circumvent these limitations is to encapsulate the essential oils in a polymer matrix. However, these hydrophobic molecules are difficult to quantify when dispersed in water. Seeking to study the release of this terpene in drug release tests from polymeric matrices, this work aimed to develop an easy and cheap UV spectrophotometric method for the quantification of ß-myrcene in aqueous medium. To achieves this goal, samples were prepared in 0.05% (w/v) polysorbate 80 solution, with concentrations of ß-myrcene ranging from 0.01% to 0.1% (v/v), and were analyzed at 226 nm. Each sample was analyzed in triplicate and repeated on three different days, to evaluate the repeatability of the results. The results were subjected to Q, F and Student's t-tests. The regression parameters obtained for ß-myrcene were above 0.99 and through statistical analysis, it was possible to confirm the repeatability for the results. The values of the limits of detection and quantification indicated that the method is not affected by intrinsic factors of the equipment. The results of accuracy, robustness and selectivity showed recovery rates within acceptable limits. This demonstrates that the quantification of ß-myrcene in aqueous medium by UV spectrophotometry is feasible.

Alkylide derivatives of diphyllin: synthesis and preliminary anticancer evaluation.

Fourteen diphyllin 4-C-substituted alkylide derivatives were designed and synthesized using a Heck coupling and subsequent hydrogenation reaction. Olefins 3g and 3i exhibited the highest cytotoxicity on breast cancer cell lines MCF-7 with IC50 values of 0.08 and 0.07 µM, and they showed weaker V-ATPase inhibitory potency compared to diphyllin.

A directed enolization strategy enables by-product-free construction of contiguous stereocentres en route to complex amino acids.

Homochiral α-amino acids are widely used in pharmaceutical design as key subunits in chiral catalyst synthesis or as building blocks in synthetic biology. Many synthetic methods have been developed to access rare or unnatural variants by controlling the installation of the α-stereocentre. By contrast, and despite their importance, α-amino acids possessing ß-stereocentres are much harder to synthesize. Here we demonstrate an iridium-catalysed protocol that allows the direct upconversion of simple alkenes and glycine derivatives to give ß-substituted α-amino acids with exceptional levels of regio- and stereocontrol. Our method exploits the native directing ability of a glycine-derived N-H unit to facilitate Ir-catalysed enolization of the adjacent carbonyl. The resulting stereodefined enolate cross-couples with a styrene or α-olefin to install two contiguous stereocentres. The process offers very high levels of regio- and stereocontrol and occurs with complete atom economy. In broader terms, our reaction design offers a unique directing-group-controlled strategy for the direct stereocontrolled α-alkylation of carbonyl compounds, and provides a powerful approach for the synthesis of challenging contiguous stereocentres.

Molecular Basis for Chemoselectivity Control in Oxidations of Internal Aryl-Alkenes Catalyzed by Laboratory Evolved P450s.

P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond epoxidations and allylic C-H hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, a P450 from Labrenzia aggregata (P450LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-ß-methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the formation of a highly reactive carbocation intermediate. Here, additional library screening along this evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity of 94 % towards allylic C-H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered.

Theoretical investigation of the carbonyl-ene reaction between encapsulated formaldehyde and propylene over M-Cu-BTC paddlewheels (M= Be, Mg, and Ca): A DFT study.

Formaldehyde is a VOC gas that plays a key role in air pollution. To limit emissions into the environment, the utilization of this waste as a raw material is a promising way. In this work, the M06-L functional calculation was used to investigate the structure, electronic properties, and catalytic activity of group IIA metals (Be, Mg, and Ca) partial substitution on Cu-BTC paddlewheels for formaldehyde encapsulation and carbonyl-ene reaction with propylene. Formaldehyde is absorbed by the metal center of the paddlewheel via its oxygen atom. The adsorption of formaldehyde on the substituted metal sites increased as compared to the parent Cu-BTC which can facilitate formaldehyde to react with propylene. The adsorption free energies are predicted to be -15.1 (Be-Cu-BTC), -14.7 (Mg-Cu-BTC), and -14.5 (Ca-Cu-BTC) kcal mol-1, respectively. The substituted metal has a slight effect on the Lewis acidity of the Cu ion in the paddlewheel. The adsorption free energy of formaldehyde, similar to that found in the pristine Cu-BTC, is observed. For the carbonyl-ene reaction, the reaction is proposed via a single step involving the C-C bond formation between two reactants and one hydrogen of propylene methyl group moves to formaldehyde oxygen, simultaneously. It was found that the substituted metals do not affect the catalytic performance of the Cu center for this reaction. The activation energies for the reaction at the Cu center are in the range of 22.0-23.4 kcal mol-1, which are slightly different from Cu-BTC (21.5 kcal mol-1). Interestingly, the catalytic activity of this reaction on the substituted metal is greater than that on the Cu center. The catalytic activities are in the order Be-Cu-BTC (13.3 kcal mol-1) > Mg-Cu-BTC (15.9 kcal mol-1) > Ca-Cu-BTC (17.8 kcal mol-1). Among them, the Be site of the bimetallic Be-Cu-BTC paddlewheel is predicted as a promising candidate catalyst.

Harnessing alcohols as sustainable reagents for late-stage functionalisation: synthesis of drugs and bio-inspired compounds.

Alcohol is ubiquitous with unparalleled structural diversity and thus has wide applications as a native functional group in organic synthesis. It is highly prevalent among biomolecules and offers promising opportunities for the development of chemical libraries. Over the last decade, alcohol has been extensively used as an environmentally friendly chemical for numerous organic transformations. In this review, we collectively discuss the utilisation of alcohol from 2015 to 2023 in various organic transformations and their application toward intermediates of drugs, drug derivatives and natural product-like molecules. Notable features discussed are as follows: (i) sustainable approaches for C-X alkylation (X = C, N, or O) including O-phosphorylation of alcohols, (ii) newer strategies using methanol as a methylating reagent, (iii) allylation of alkenes and alkynes including allylic trifluoromethylations, (iv) alkenylation of N-heterocycles, ketones, sulfones, and ylides towards the synthesis of drug-like molecules, (v) cyclisation and annulation to pharmaceutically active molecules, and (vi) coupling of alcohols with aryl halides or triflates, aryl cyanide and olefins to access drug-like molecules. We summarise the synthesis of over 100 drugs via several approaches, where alcohol was used as one of the potential coupling partners. Additionally, a library of molecules consisting over 60 fatty acids or steroid motifs is documented for late-stage functionalisation including the challenges and opportunities for harnessing alcohols as renewable resources.

The Inhibition Mechanisms of Three Structurally Different Salvianolic Acids on the Non-Enzymatic Glycation of Bovine Serum Albumin.

The antiglycation mechanisms of three structurally different salvianolic acids (Sals) including salvianolic acid A (Sal-A), salvianolic acid B (Sal-B) and salvianolic acid C (Sal-C) were investigated using the bovine serum albumin (BSA)-fructose model. The results showed that the three compounds could inhibit the formation of glycation products, maintain protein structural stability, mitigate the development of amyloid fibrils and scavenge radicals. Notably, Sal-A possessed the highest anti-glycated activity compared with Sal-B and Sal-C. This may be related to the fact that Sal-A contained the most molecules of caffeic acid (Sal-A, Sal-B, and Sal-C possessing two, one, and zero caffeic acid units, respectively), and caffeic acid played a leading role in the antiglycation properties relative to Danshensu. Moreover, these compounds quenched the intrinsic fluorescence intensity of BSA in a static mode, with the binding constants in the order of Sal-A > Sal-B > Sal-C. Obviously, Sal-A possessed the strongest binding affinity among these compounds, which may be one of the reasons why it exhibited the optimal antiglycation capability. Furthermore, molecular docking demonstrated that the three Sals exerted protective effects on BSA by preventing glycation modification of lysine and arginine residues. These findings would provide valuable insights into the potential application of Sals for alleviating non-enzymatic glycation of protein.

Alkene dialkylation by triple radical sorting.

The development of bimolecular homolytic substitution (SH2) catalysis has expanded cross-coupling chemistries by enabling the selective combination of any primary radical with any secondary or tertiary radical through a radical sorting mechanism1-8. Biomimetic9,10 SH2 catalysis can be used to merge common feedstock chemicals-such as alcohols, acids and halides-in various permutations for the construction of a single C(sp3)-C(sp3) bond. The ability to sort these two distinct radicals across commercially available alkenes in a three-component manner would enable the simultaneous construction of two C(sp3)-C(sp3) bonds, greatly accelerating access to complex molecules and drug-like chemical space11. However, the simultaneous in situ formation of electrophilic and primary nucleophilic radicals in the presence of unactivated alkenes is problematic, typically leading to statistical radical recombination, hydrogen atom transfer, disproportionation and other deleterious pathways12,13. Here we report the use of bimolecular homolytic substitution catalysis to sort an electrophilic radical and a nucleophilic radical across an unactivated alkene. This reaction involves the in situ formation of three distinct radical species, which are then differentiated by size and electronics, allowing for regioselective formation of the desired dialkylated products. This work accelerates access to pharmaceutically relevant C(sp3)-rich molecules and defines a distinct mechanistic approach for alkene dialkylation.

An oxidative photocyclization approach to the synthesis of Securiflustra securifrons alkaloids.

Securines and securamines are cytotoxic alkaloids that contain reactive alkene and heterocyclic residues embedded in skeletons comprising four to six oxidized rings. This structural complexity imparts a rich chemistry to the isolates but has impeded synthetic access to the structures in the nearly three decades since their isolation. We present a flexible route to eight isolates that exemplify the three skeletal classes of metabolites. The route proceeds by the modular assembly of the advanced azides 38 and 49 (13 steps, 6 to 10% yield), sequential oxidative photocyclizations, and late-stage functional group manipulations. With this approach, the targets were obtained in 17 to 19 steps, 12 to 13 purifications, and 0.5 to 3.5% overall yield. The structure of an advanced intermediate was elucidated by microcrystal electron diffraction (MicroED) analysis. The route will support structure-function and target identification studies of the securamines.

Analytical Method for Quantifying Monoterpenoids (p-Cymene and Myrcene) in Nanoemulsions Using High-Performance Liquid Chromatography.

BACKGROUND: Myrcene and cymene, aromatic monoterpenes found in plants and essential oils, possess distinctive aromatic qualities. However, their volatility and limited solubility pose challenges in precise handling and formulation. Meanwhile, nanoemulsions emerge as promising drug delivery systems, improving the bioavailability and stability of these active ingredients. OBJECTIVE: This article aimed to develop an HPLC method for the quantification of two monoterpenoids, p-cymene and myrcene, in nanoemulsions. METHOD: The method used a Phenomenex® Synergi™ Fusion-RP column (150 mm × 4.6 mm id, 4 µm particle size) on an HPLC system with isocratic elution. The mobile phase was composed of acetonitrile and water (60:40, v/v) and was validated in terms of specificity, linearity, accuracy, precision, robustness, and selectivity. RESULTS: The method provided accurate and precise results with a correlation coefficient of 0.999 and RSD values of less than 2%. The method can be used for quality control of nanoemulsions containing these monoterpenoids and as a reference for future studies on their efficacy and stability. CONCLUSIONS: The study demonstrates the feasibility of using HPLC for the quantification of monoterpenoids in nanoemulsions and its potential as a quality control tool for nanoemulsion-based drug delivery systems. HIGHLIGHTS: The method's accuracy, precision, and reliability, as evidenced by high correlation coefficients and low RSD values, underscore its suitability for ensuring the consistent formulation of these monoterpenoid-containing nanoemulsions, while also serving as a reference point for future research endeavors in this field.

Atmospheric Degradation of Ecologically Important Biogenic Volatiles: Investigating the Ozonolysis of (E)-ß-Ocimene, Isomers of α and ß-Farnesene, α-Terpinene and 6-Methyl-5-Hepten-2-One, and Their Gas-Phase Products.

Biogenic volatile organic compounds (bVOCs), synthesised by plants, are important mediators of ecological interactions that can also undergo a series of reactions in the atmosphere. Ground-level ozone is a secondary pollutant generated through sunlight-driven reactions between nitrogen oxides (NOx) and VOCs. Its levels have increased since the industrial revolution and reactions involving ozone drive many chemical processes in the troposphere. While ozone precursors often originate in urban areas, winds may carry these hundreds of kilometres, causing ozone formation to also occur in less populated rural regions. Under elevated ozone conditions, ozonolysis of bVOCs can result in quantitative and qualitative changes in the gas phase, reducing the concentrations of certain bVOCs and resulting in the formation of other compounds. Such changes can result in disruption of bVOC-mediated behavioural or ecological interactions. Through a series of gas-phase experiments using Gas Chromatography Mass Spectrometry (GC-MS) and Proton Transfer Reaction Mass Spectrometry (PTR-MS), we investigated the products and their yields from the ozonolysis of a range of ubiquitous bVOCs, which were selected because of their importance in mediating ecological interactions such as pollinator and natural enemy attraction and plant-to-plant communication, namely: (E)-ß-ocimene, isomers of α and ß-farnesene, α-terpinene and 6-methyl-5-hepten-2-one. New products from the ozonolysis of these compounds were identified, and the formation of these compounds is consistent with terpene-ozone oxidation mechanisms. We present the degradation mechanism of our model bVOCs and identify their reaction products. We discuss the potential ecological implications of the degradation of each bVOC and of the formation of reaction products.

Enantio- and Diastereodivergent Cyclopropanation of Allenes by Directed Evolution of an Iridium-Containing Cytochrome.

Alkylidene cyclopropanes (ACPs) are valuable synthetic intermediates because of their constrained structure and opportunities for further diversification. Although routes to ACPs are known, preparations of ACPs with control of both the configuration of the cyclopropyl (R vs S) group and the geometry of the alkene (E vs Z) are unknown. We describe enzymatic cyclopropanation of allenes with ethyl diazoacetate (EDA) catalyzed by an iridium-containing cytochrome (Ir(Me)-CYP119) that controls both stereochemical elements. Two mutants of Ir(Me)-CYP119 identified by 6-codon (6c, VILAFG) saturation mutagenesis catalyze the formation of (E)-ACPs with -93% to >99% ee and >99:1 E/Z ratio with just three rounds of 96 mutants. By four additional rounds of mutagenesis, an enzyme variant was identified that forms (Z)-ACPs with up to 94% ee and a 28:72 E/Z ratio. Computational studies show that the orientation of the carbene unit dictated by the mutated positions accounts for the stereoselectivity.

H2 O ⋠B(C6 F5 )3 -Catalyzed para-Alkylation of Anilines with Alkenes Applied to Late-Stage Functionalization of Non-Steroidal Anti-Inflammatory Drugs.

Anilines are core motifs in a variety of important molecules including medicines, materials and agrochemicals. We report a straightforward procedure that allows access to new chemical space of anilines via their para-C-H alkylation. The method utilizes commercially available catalytic H2 O ⋅ B(C6 F5 )3 and is highly selective for para-C-alkylation (over N-alkylation and ortho-C-alkylation) of anilines, with a wide scope in both the aniline substrates and alkene coupling partners. Readily available alkenes are used, and include new classes of alkene for the first time. The mild reaction conditions have allowed the procedure to be applied to the late-stage-functionalization of non-steroidal anti-inflammatory drugs (NSAIDs), including fenamic acids and diclofenac. The formed novel NSAID derivatives display improved anti-inflammatory properties over the parent NSAID structure.