Narrow Substrate Scope Research Articles

New Strategies for Activation of Phosphonates/Phosphates to Forge Functional Phosphorus Compounds

Organophosphonate analogues are important structural motifs that are present in bioactive natural products, pharmaceuticals, and agrochemicals. Because they are useful in a broad range of applications, much effort has focused on developing efficient synthetic methods that enable access to organophosphonates and their derivatives. However, currently available synthetic procedures rely on harsh reaction conditions and are limited to a narrow substrate scope. Our lab has recently made important advances in leveraging inert phosphonates/phosphates into functional phosphorus compounds such as mixed phosphonates, phosphates, and mixed aryl phosphate derivatives. Presented herein is an overview of recent achievements in the synthesis of phosphonate/phosphate derivatives and a summary of our recent accomplishments in Tf2O-promoted activating strategy of phosphonate analogues.1 Introduction2 Direct Activating Strategy of Phosphonate Analogues3 Late-stage Phosphonylation of Natural Compounds4 Potential Applications5 Summary

Transition-Metal (Cu, Pd, Ni)-Catalyzed Difluoroalkylation via Cross-Coupling with Difluoroalkyl Halides.

Difluoroalkylated compounds play a remarkably important role in life and materials sciences because of the unique characteristics of the difluoromethylene (CF2) group. In particular, precise introduction of a CF2 group at the benzylic position can dramatically improve the biological properties of the corresponding molecules. As a consequence, difluoroalkylation of aromatic compounds has become a powerful strategy in modulating the bioactivities of organic molecules. However, efficient strategies to selectively synthesize difluoroalkylated arenes had been very limited before 2012. Traditional synthetic methods in this regard suffer from either harsh reaction conditions or narrow substrate scope, significantly restricting their widespread applications, particularly for late-stage difluoroalkylation of bioactive molecules. To overcome these limitations, a straightforward route to access these valuable difluoroalkylated skeletons is the direct introduction of the difluoroalkylated group (CF2R) onto aromatic rings through transition-metal-catalyzed cross-coupling. However, because of the instability of some difluoroalkylated metal species, which are prone to protonation, dimerization, and/or generation of other unknown byproducts, it is difficult to selectively control the catalytic cycle to suppress these side reactions. In this context, we proposed the use of low-cost and widely available difluoroalkyl halides as fluoroalkyl sources for transition-metal-catalyzed difluoroalkylation reactions via cross-coupling. In this Account, we summarize our major efforts on copper-, palladium-, and nickel-catalyzed difluoroalkylations of aromatics with low-cost and widely available difluoroalkyl halides as fluoroalkyl sources. Four modes of catalytic difluoroalkylation reactions, including nucleophilic difluoroalkylation, electrophilic difluoroalkylation, radical difluoroalkylation, and metal-difluorocarbene coupling (MeDiC), have been demonstrated through careful modulation of the catalytic systems. Among these reactions, the MeDiC reaction represents a new mode of fluoroalkylation. These processes enable difluoroalkylation of a variety of aryl halides and arylboron reagents under mild reaction conditions. A wide range of difluoroalkyl halides, including activated difluoroalkyl halides (Cl/BrCF2R, R = π system), unactivated difluoroalkyl halides (BrCF2R, R = alkyl, H), and especially the inert and inexpensive industrial chemical chlorodifluoromethane (ClCF2H), are applicable to these reactions, providing straightforward and facile routes to a diverse range of difluoroalkylated (hetero)arenes. These difluoroalkyl halide-based strategies can also be applied to prepare difluoroalkylated alkenes, alkynes, and alkanes and feature impressive advantages over conventional methods for the synthesis of difluoroalkylated compounds in terms of synthetic efficiency, functional group tolerance, and structural diversity. In particular, the late-stage difluoroalkylation of bioactive molecules through these processes offers good opportunities for the synthesis and development of new medicinal agents without the need for multistep de novo syntheses.

Computational redesign of enzymes for regio- and enantioselective hydroamination.

Introduction of innovative biocatalytic processes offers great promise for applications in green chemistry. However, owing to limited catalytic performance, the enzymes harvested from nature's biodiversity often need to be improved for their desired functions by time-consuming iterative rounds of laboratory evolution. Here we describe the use of structure-based computational enzyme design to convert Bacillus sp. YM55-1 aspartase, an enzyme with a very narrow substrate scope, to a set of complementary hydroamination biocatalysts. The redesigned enzymes catalyze asymmetric addition of ammonia to substituted acrylates, affording enantiopure aliphatic, polar and aromatic β-amino acids that are valuable building blocks for the synthesis of pharmaceuticals and bioactive compounds. Without a requirement for further optimization by laboratory evolution, the redesigned enzymes exhibit substrate tolerance up to a concentration of 300 g/L, conversion up to 99%, β-regioselectivity >99% and product enantiomeric excess >99%. The results highlight the use of computational design to rapidly adapt an enzyme to industrially viable reactions.

Highly Stereoselective Electrocatalytic Semihydrogenation of Alkynes to Z-Alkenes Using a Proton Exchange Membrane Reactor

Z-alkenes are versatile building blocks in organic synthesis and often found in pharmaceuticals, perfumes, and natural products. Although a number of methods for stereoselective synthesis of Z-alkene have been developed so far, the Lindlar semihydrogeneation of alkynes is a long-standing synthetic transformation and still remains a privileged system. However, it used toxic Pb additive and a large amount of quinolone, and has other limitations such as narrow substrate scope. Therefore, development of a powerful and reliable system for stereoselective semihydrogenation of alkyne to Z-alkene as alternative to the Lindlar catalytic system is still challenging target. Under these backgrounds, in this work, we have demonstrated electrocatalytic hydrogenation in a proton exchange membrane reactor so called PEM reactor to develop a powerful and reliable stereoselective semihydrogenation system (Figure 1). This system has several merits such as easy separation and recycle use of catalysts, operation under mild and benign conditions, and easy reaction control by applied voltage. Indeed, the use of PEM reactor with Pd catalysts gave Z-alkenes with excellent current efficiencies in our demonstrations. Figure 1

Enzyme Engineering Based on X-ray Structures and Kinetic Profiling of Substrate Libraries: Alcohol Dehydrogenases for Stereospecific Synthesis of a Broad Range of Chiral Alcohols

The narrow substrate scope of naturally occurring alcohol dehydrogenases (ADHs) greatly limits the enzymatic synthesis of important chiral alcohols. On the basis of X-ray crystal structures and kinetic profiling of a substrate library, we engineered variants of the stereospecific alcohol dehydrogenase from Candida parapsilopsis. This resulted in a set of four mutant enzymes which enable the asymmetric reduction of a broad range of prochiral ketones, including valuable pharmaceuticals and fine chemicals. The engineering strategy of this study paves the way for creating additional ADHs tailored for production of complex chiral alcohols.

Comparative Analysis of Bacterial Cytochromes P450 Involved in the Biosynthesis of 16‐ Membered Ring Macrolide Antibiotics

Members of the cytochrome P450 superfamily of monooxygenases (P450s) are some of nature's most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize C–H bonds has led to their growing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present work, we biochemically and structurally characterized diverse bacterial P450s involved in the biosynthesis of 16-membered ring macrolide antibiotics with significant potential for development into robust biocatalysts for the late-stage functionalization of complex molecules. Following exploratory efforts to probe the reactivity properties of the P450 MycCI from the mycinamicin biosynthetic pathway, we discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding and kinetics experiments along with X-ray crystallographic analysis of MycCI bound to its native substrate. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared with that of MycCI. Turnover and equilibrium binding experiments with substrate analogs revealed that TylHI exhibits a strict preference for 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. These results were partially explained through analysis of the X-ray crystal structure of TylHI in complex with its native substrate together with biochemical characterization of several sitedirected mutants. Comparative analysis of the MycCI/TylHI homolog ChmHI from the chalcomycin biosynthetic pathway provided a basis for constructing MycCI/TylHI chimeras in order to gain further insight into the features dictating the differences in the reactivity profiles of these two related P450s. These experiments unveiled the central role of the BC loop region in influencing the binding of 16-membered ring substrates to MycCI and TylHI. Overall, our studies have shed light on the molecular-level details underpinning the unique catalytic divergence of two homologous biosynthetic P450s and point toward specific regions in each protein that may be targeted in future efforts to rationally engineer novel P450-based biocatalysts. Support or Funding Information NSF under the CCI Center for Selective C–H Functionalization (CHE-1205646 and CHE-1700982), NIH (R01-GM078553, R35-GM118101, and T32-GM008353), University of Michigan Rackham Predoctoral Fellowship Physiological reactions catalyzed by P450s MycCI and TylHI in their respective biosynthetic pathways. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expanding the Scope of Sortase-Mediated Ligations by Using Sortase Homologues.

Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site-specific modification of the N-terminal serine residue in the naturally occurring antimicrobial peptide DCD-1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.

Synthesis of Sebacic Acid Using a De Novo Designed Retro‐Aldolase as a Key Catalyst

AbstractThe production of new organic compounds from natural feedstocks drives the construction of new biosynthetic cascades. To develop and generate valuable complex chemicals, it is necessary to go beyond natural enzymes towards de novo designed and engineered variants for both natural and non‐natural compounds. Herein, we exploit the applicability of a de novo retro‐aldolase for the synthesis of sebacic acid, a valuable precursor for bio‐based polymers. From ricinoleic acid, a route composed of four individual enzyme‐catalyzed steps was developed. As a result of the narrow substrate scope of natural aldolases, this enzymatic route could only be realized by the design and engineering of a de novo retro‐aldolase. We further highlight the outstanding potential of this technology for the realization of non‐natural reactions and pathways.

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway.

Cytochrome P450 monooxygenases (P450s) are some of nature's most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize inert C-H bonds has led to their increasing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present study, we investigated the reactivity of the P450 MycCI from the mycinamicin biosynthetic pathway toward a variety of macrocyclic compounds and discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding experiments, steady-state kinetics studies, and X-ray crystallographic analysis of MycCI bound to its native substrate mycinamicin VIII. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared to MycCI. Thus, the ability of the latter to hydroxylate both macrocyclic aglycones and macrolides sets it apart from related biosynthetic P450s and highlights its potential for developing novel P450 biocatalysts with broad substrate scope and high regioselectivity.

Evolution of the Aza-Diels–Alder Reaction of 2H-Azirines

The aza-Diels–Alder reaction of 2<i>H</i>-azirines with 1,3-dienes provides facile access to the structurally intriguing and difficult to access 1-azabicyclo[4.1.0]hept-3-ene scaffold. Its application in organic synthesis and medicinal chemistry is heavily hampered by the narrow substrate scope for both reacting partners. Herein, the discovery and exploration of this reaction are summarized and discussed. A recent breakthrough is an intramolecular version that overcomes the limitations of conventional methods.

What are the Limitations of Enzymes in Synthetic Organic Chemistry?

Enzymes have been used in organic chemistry and biotechnology for 100 years, but their widespread application has been prevented by a number of limitations, including the often-observed limited thermostability, narrow substrate scope, and low or wrong stereo- and/or regioselectivity. Directed evolution provides a means to address and generally solve these problems, especially since recent methodology development has made this protein engineering method faster, more efficient, and more reliable than in the past. This Darwinian approach to asymmetric catalysis has led to a number of industrial applications. Metabolic-pathway engineering, mutasynthesis, and fermentation are likewise enzyme-based techniques that enrich chemistry. This account outlines the scope, and particularly, the limitations, of biocatalysis. The complementary nature of enzymes and man-made catalysts is emphasized.

Biochemical properties of a new nitrile reductase cloned from Pectobacterium carotovorum

Nitrile reductase is a newly discovered enzyme class that can catalyze the reduction of nitriles directly to amines, a very important reaction in synthetic organic chemistry. However, little progress has been made towards the application of this biocatalysis reaction for organic synthesis since the initial discovery of nitrile reductase for over ten years. One of the potential reasons may be due to the lack of structural information and biochemical properties, in addition to its inherent narrow substrate scope. Meanwhile, the highly conserved active site residues make it a great challenge to widen the substrate spectrum of nitrile reductase by means of protein engineering approaches. To explore more nitrile reductases with different characteristics (e.g. specific activity, thermostability, etc) for potential synthetic applications, a new nitrile reductase, designated as PcNRed, was discovered from the genome of Pectobacterium carotovorum, with a moderate specific activity of 366U/g protein towards its natural substrate, preQ0. The PcNRed was subsequently overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity and then its biochemical properties were examined. The optimal reaction temperature and pH were determined to be 40°C and 7.4, respectively. The thermal deactivation process of PcNRed obeys first-order kinetics, with half-lives of 47.2, 13.6 and 3.9h measured at 30, 40, and 50°C, respectively. The kinetic constants were also determined with respect to both of the substrates NADPH and preQ0, giving kcat of 0.025s−1 and 0.024s−1, and KM of 0.229mM and <0.25μM. To shed some light on the structural information of PcNRed, a homology model was built and compared with its counterparts, which is helpful for further engineering of this novel but less-elucidated enzymes.

Lipase-initiated one-pot synthesis of spirooxazino derivatives: redesign of multicomponent reactions to expand substrates scope and application potential

Enzymatic multicomponent reactions (MCR) are very powerful for complex organic synthesis with environmentally friendly, highly efficient and selective characters, while sometimes having some unavoidable shortages such as narrow substrate scope and so on. Herein, this work demonstrated how to redesign one previously reported lipase-catalyzed MCR to achieve more broad substrates and more efficient synthesis. Twelve new spirooxazino derivatives with different substitutions were obtained in moderate to good yield, while all of them could not be synthesized using the previous route. Reaction conditions such as enzymes, enzyme concentration, amides and ratio of substrates were screened. Furthermore, particularly interesting is that chiral spirooxazino could also be obtained through a further developed two-enzymatic MCR process in one pot. As a domino process simultaneously constructing six new C-C/N bonds and two rings in only one step, the work will remarkably expand the application scope of enzymatic MCR for simple and green synthesis of complex compounds.

Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases.

Understanding the metabolic potential of organisms or a bacterial community based on their (meta) genome requires the reliable prediction of an enzyme's function from its amino acid sequence. Besides a remarkable development in prediction algorithms, the substrate scope of sequences with low identity to well-characterized enzymes remains often very elusive. From a recently conducted structure function analysis study of PLP-dependent enzymes, we identified a putative transaminase from Bacillus anthracis (Ban-TA) with the crystal structure 3N5M (deposited in the protein data bank in 2011, but not yet published). The active site residues of Ban-TA differ from those in related (class III) transaminases, which thereby have prevented function predictions. By investigating 50 substrate combinations its amine and ω-amino acid:pyruvate transaminase activity was revealed. Even though Ban-TA showed a relatively narrow amine substrate scope within the tested substrates, it accepts 2-propylamine, which is a prerequisite for industrial asymmetric amine synthesis. Structural information implied that the so-called dual substrate recognition of chemically different substrates (i.e. amines and amino acids) differs from that in formerly known enzymes. It lacks the normally conserved 'flipping' arginine, which enables dual substrate recognition by its side chain flexibility in other ω-amino acid:pyruvate transaminases. Molecular dynamics studies suggested that another arginine (R162) binds ω-amino acids in Ban-TA, but no side chain movements are required for amine and amino acid binding. These results, supported by mutagenesis studies, provide functional insights for the B. anthracis enzyme, enable function predictions of related proteins, and broadened the knowledge regarding ω-amino acid and amine converting transaminases.

Gem-Difluoroolefination of Diazo Compounds with TMSCF3 or TMSCF2Br: Transition-Metal-Free Cross-Coupling of Two Carbene Precursors.

A new olefination protocol for transition-metal-free cross-coupling of two carbene fragments arising from two different sources, namely, a nonfluorinated carbene fragment resulting from a diazo compound and a difluorocarbene fragment derived from Ruppert-Prakash reagent (TMSCF3) or TMSCF2Br, has been developed. This gem-difluoroolefination proceeds through the direct nucleophilic addition of diazo compounds to difluorocarbene followed by elimination of N2. Compared to previously reported Cu-catalyzed gem-difluoroolefination of diazo compounds with TMSCF3, which possesses a narrow substrate scope due to a demanding requirement on the reactivity of diazo compounds and in-situ-generated CuCF3, this transition-metal-free protocol affords a general and efficient approach to various disubstituted 1,1-difluoroalkenes, including difluoroacrylates, diaryldifluoroolefins, as well as arylalkyldifluoroolefins. In view of the ready availability of diazo compounds and difluorocarbene reagents and versatile transformations of 1,1-difluoroalkenes, this new gem-difluoroolefination method is expected to find wide applications in organic synthesis.

ChemInform Abstract: Asymmetric Synthesis of Axially Chiral Anilides by Pd‐Catalyzed Allylic Substitutions with P/Olefin Ligands.

As an attractive class of non-biaryl atropisomeric compounds, C–N axially chiral anilides have received considerable attention, and several methods have been successfully developed for their synthesis. Pd-catalyzed asymmetric allylic amination was proved to be an effective approach for the chiral anilide synthesis, although only moderate enantioselectivity and relatively narrow substrate scope have been achieved in the previous work. Searching for highly efficient methods for the synthesis of axially chiral anilides is therefore of great interest in synthetic and pharmaceutical chemistry. In this paper, a palladium-catalyzed asymmetric allylic substitution of ortho-substituted anilides using phosphorus amidite–olefin ligands was successfully achieved to afford a variety of axially chiral anilides in high yields with up to 84% ee. The absolute configurations of chiral anilides were also determined from X-ray and CD spectra.

ChemInform Abstract: Biocatalysis in Organic Chemistry and Biotechnology: Past, Present, and Future

AbstractReview: &lt; 100 refs.

Co‐immobilization of an Enzyme and a Metal into the Compartments of Mesoporous Silica for Cooperative Tandem Catalysis: An Artificial Metalloenzyme

Co‐immobilization of an Enzyme and a Metal into the Compartments of Mesoporous Silica for Cooperative Tandem Catalysis: An Artificial Metalloenzyme

Biocatalysis in Organic Chemistry and Biotechnology: Past, Present, and Future

Enzymes as catalysts in synthetic organic chemistry gained importance in the latter half of the 20th century, but nevertheless suffered from two major limitations. First, many enzymes were not accessible in large enough quantities for practical applications. The advent of recombinant DNA technology changed this dramatically in the late 1970s. Second, many enzymes showed a narrow substrate scope, often poor stereo- and/or regioselectivity and/or insufficient stability under operating conditions. With the development of directed evolution beginning in the 1990s and continuing to the present day, all of these problems can be addressed and generally solved. The present Perspective focuses on these and other developments which have popularized enzymes as part of the toolkit of synthetic organic chemists and biotechnologists. Included is a discussion of the scope and limitation of cascade reactions using enzyme mixtures in vitro and of metabolic engineering of pathways in cells as factories for the production of simple compounds such as biofuels and complex natural products. Future trends and problems are also highlighted, as is the discussion concerning biocatalysis versus nonbiological catalysis in synthetic organic chemistry. This Perspective does not constitute a comprehensive review, and therefore the author apologizes to those researchers whose work is not specifically treated here.

Progress on the Catalytic Asymmetric Hydrogenation of Imines

Chiral amines are important synthetic intermediates in the preparation of many physiologically active compounds. Catalytic asymmetric hydrogenation of prochiral imines represents one of the most direct and convenient route to chiral amines and their derivatives. During the past two decades, significant progress has been made in the asymmetric hydrogenation of imines. Many highly enantioselective chiral catalysts have been disclosed, some new methods and strategies have been developed, and some challenging imines substrates have been efficiently hydrogenated in high enantioselectivities. Despite these achievements, catalytic asymmetric hydrogenation of imines still have challenges of low reactivity, narrow substrate scope, harsh hydrogenation conditions and difficulty in catalyst reuse. Therefore, development of new catalyst system with high reactivity and enantioselectivity as well as environmental friendship in the asymmetric hydrogenation of imines is of great interest. This review presents a brief overview on the progress in catalytic asymmetric hydrogenation of cyclic and acyclic imines.