Deciphering complexity in Pd-catalyzed cross-couplings.

Understanding complex reaction systems is critical in chemistry. While synthetic methods for selective formation of products are sought after, oftentimes it is the full reaction signature, i.e., complete profile of products/side-products, that informs mechanistic rationale and accelerates discovery chemistry. Here, we report a methodology using high-throughput experimentation and multivariate data analysis to examine the full signature of one of the most complicated chemical reactions catalyzed by palladium known in the chemical literature. A model Pd-catalyzed reaction was selected involving functionalization of 2-bromo-N-phenylbenzamide and multiple bond activation pathways. Principal component analysis, correspondence analysis and heatmaps with hierarchical clustering reveal the factors contributing to the variance in product distributions and show associations between solvents and reaction products. Using robust data from experiments performed with eight solvents, for four different reaction times at five different temperatures, we correlate side-products to a major dominant N-phenyl phenanthridinone product, and many other side products.

Stereoselective synthesis of an advanced trans-decalin intermediate towards the total synthesis of anthracimycin.

Progress towards the total synthesis of the macrolide natural product anthracimycin is described. This new approach utilises an intermolecular Diels-Alder strategy followed by epimeirsation to form the key trans-decalin framework. The route culminates in the stereoselective synthesis of an advanced tricyclic lactone intermediate, containing five contiguous sterogenic centres with the correct relative and absolute stereochemistry required for the anthracimycin core motif.

Evidence for Suzuki-Miyaura cross-couplings catalyzed by ligated Pd3-clusters: from cradle to grave.

Pdn clusters offer unique selectivity and exploitable reactivity in catalysis. Understanding the behavior of Pdn clusters is thus critical for catalysis, applied synthetic organic chemistry and greener outcomes for precious Pd. The Pd3 cluster, [Pd3(µ-Cl)(µ-PPh2)2(PPh3)3][Cl] (denoted as Pd3Cl2), which exhibits distinctive reactivity, was synthesized and immobilized on a phosphine-functionalized polystyrene resin (denoted as immob-Pd3Cl2). The resultant material served as a tool to study closely the role of Pd3 clusters in a prototypical Suzuki-Miyaura cross-coupling of 4-fluoro-1-bromobenzene and 4-methoxyphenyl boronic acid at varying low Pd ppm concentrations (24, 45, and 68 ppm). Advanced heterogeneity tests such as Hg poisoning and the three-phase test showed that leached mononuclear or nanoparticulate Pd are unlikely to be the major active catalyst species under the reaction conditions tested. EXAFS/XANES analysis from (pre)catalyst and filtered catalysts during and after catalysis has shown the intactness of the triangular structure of the Pd3X2 cluster, with exchange of chloride (X) by bromide during catalytic turnover of bromoarene substrate. This finding is further corroborated by treatment of immob-Pd3Cl2 after catalyzing the Suzuki-Miyaura reaction with excess PPh3, which releases the cluster from the polymer support and so permits direct observation of [Pd3(µ-Br)(µ-PPh2)2(PPh3)3]+ ions by ESI-MS. No evidence is seen for a proposed intermediate in which the bridging halogen on the Pd3 motif is replaced by an aryl group from the organoboronic acid, i.e. formed by a transmetallation-first process. Our findings taken together indicate that the 'Pd3X2' motif is an active catalyst species, which is stabilized by being immobilized, providing a more robust Pd3 cluster catalyst system. Non-immobilized Pd3Cl2 is less stable, as is followed by stepwise XAFS of the non-immobilized Pd3Cl2, which gradually changes to a species consistent with 'Pdx(PPh3)y' type material. Our findings have far-reaching future implications for Pd3 cluster involvement in catalysis, showing that immobilization of Pd3 cluster species offers advantages for rigorous mechanistic examination and applied chemistries.

Unveiling Mechanistic Complexity in Manganese-Catalyzed C-H Bond Functionalization Using IR Spectroscopy Over 16 Orders of Magnitude in Time.

ConspectusAn understanding of the mechanistic processes that underpin reactions catalyzed by 3d transition metals is vital for their development as potential replacements for scarce platinum group metals. However, this is a significant challenge because of the tendency of 3d metals to undergo mechanistically diverse pathways when compared with their heavier congeners, often as a consequence of one-electron transfer reactions and/or intrinsically weaker metal-ligand bonds. We have developed and implemented a new methodology to illuminate the pathways that underpin C-H bond functionalization pathways in reactions catalyzed by Mn-carbonyl compounds. By integrating measurements performed on catalytic reactions with in situ reaction monitoring and state-of-the-art ultrafast spectroscopic methods, unique insight into the mode of action and fate of the catalyst have been obtained.Using a combination of time-resolved spectroscopy and in situ low-temperature NMR studies, we have shown that photolysis of manganese-carbonyl precatalysts results in rapid (<5 ps) CO dissociation─the same process that occurs under thermal catalytic conditions. This enabled the detection of the key states relevant to catalysis, including solvent and alkyne complexes and their resulting transformation into manganacycles, which results from a migratory insertion reaction into the Mn-C bond. By systematic variation of the substrates (many of which are real-world structurally diverse substrates and not simple benchmark systems) and quantification of the resulting rate constants for the insertion step, a universal model for this migratory insertion process has been developed. The time-resolved spectroscopic method gave insight into fundamental mechanistic pathways underpinning other aspects of modern synthetic chemistry. The most notable was the first direct experimental observation of the concerted metalation deprotonation (CMD) mechanism through which carboxylate groups are able to mediate C-H bond activation at a metal center. This step underpins a host of important synthetic applications. This study demonstrated how the time-resolved multiple probe spectroscopy (TRMPS) method enables the observation of mechanistic process occurring on time scales from several picoseconds through to µs in a single experiment, thereby allowing the sequential observation of solvation, ligand substitution, migratory insertion, and ultimate protonation of a Mn-C bond.These studies have been complemented by an investigation of the "in reaction flask" catalyst behavior, which has provided additional insight into new pathways for precatalyst activation, including evidence that alkyne C-H bond activation may occur before heterocycle activation. Crucial insight into the fate of the catalyst species showed that excess water played a key role in deactivation to give higher-order hydroxyl-bridged manganese carbonyl clusters, which were independently found to be inactive. Traditional in situ IR and NMR spectroscopic analysis on the second time scale bridges the gap to the analysis of real catalytic reaction systems. As a whole, this work has provided unprecedented insight into the processes underpinning manganese-catalyzed reactions spanning 16 orders of magnitude in time.

Broad Spectrum Enantioselective Amide Bond Synthetase from Streptoalloteichus hindustanus.

The synthesis of amide bonds is one of the most frequently performed reactions in pharmaceutical synthesis, but the requirement for stoichiometric quantities of coupling agents and activated substrates in established methods has prompted interest in biocatalytic alternatives. Amide Bond Synthetases (ABSs) actively catalyze both the ATP-dependent adenylation of carboxylic acid substrates and their subsequent amidation using an amine nucleophile, both within the active site of the enzyme, enabling the use of only a small excess of the amine partner. We have assessed the ability of an ABS from Streptoalloteichus hindustanus (ShABS) to couple a range of carboxylic acid substrates and amines to form amine products. ShABS displayed superior activity to a previously studied ABS, McbA, and a remarkable complementary substrate specificity that included the enantioselective formation of a library of amides from racemic acid and amine coupling partners. The X-ray crystallographic structure of ShABS has permitted mutational mapping of the carboxylic acid and amine binding sites, revealing key roles for L207 and F246 in determining the enantioselectivity of the enzyme with respect to chiral acid and amine substrates. ShABS was applied to the synthesis of pharmaceutical amides, including ilepcimide, lazabemide, trimethobenzamide, and cinepazide, the last with 99% conversion and 95% isolated yield. These findings provide a blueprint for enabling a contemporary pharmaceutical synthesis of one of the most significant classes of small molecule drugs using biocatalysis.

Opening a Pandora's Flask on a Prototype Catalytic Direct Arylation Reaction of Pentafluorobenzene: The Ag2CO3/Pd(OAc)2/PPh3 System.

Direct C-H functionalization reactions have opened new avenues in catalysis, removing the need for prefunctionalization of at least one of the substrates. Although C-H functionalization catalyzed by palladium complexes in the presence of a base is generally considered to proceed by the CMD/AMLA-6 mechanism, recent research has shown that silver(I) salts, frequently used as bases, can function as C-H bond activators instead of (or in addition to) palladium(II). In this study, we examine the coupling of pentafluorobenzene 1 to 4-iodotoluene 2a (and its analogues) to form 4-(pentafluorophenyl)toluene 3a catalyzed by palladium(II) acetate with the commonplace PPh3 ligand, silver carbonate as base, and DMF as solvent. By studying the reaction of 1 with Ag2CO3/PPh3 and with isolated silver (triphenylphosphine) carbonate complexes, we show the formation of C-H activation products containing the Ag(C6F5)(PPh3)n unit. However, analysis is complicated by the lability of the Ag-PPh3 bond and the presence of multiple species in the solution. The speciation of palladium(II) is investigated by high-resolution-MAS NMR (chosen for its suitability for suspensions) with a substoichiometric catalyst, demonstrating the formation of an equilibrium mixture of Pd(Ar)(κ1-OAc)(PPh3)2 and [Pd(Ar)(µ-OAc)(PPh3)]2 as resting states (Ar = Ph, 4-tolyl). These two complexes react stoichiometrically with 1 to form coupling products. The catalytic reaction kinetics is investigated by in situ IR spectroscopy revealing a two-term rate law and dependence on [Pdtot/nPPh3]0.5 consistent with the dissociation of an off-cycle palladium dimer. The first term is independent of [1], whereas the second term is first order in [1]. The observed rates are very similar with Pd(PPh3)4, Pd(Ph)(κ1-OAc)(PPh3)2, and [Pd(Ph)(µ-OAc)(PPh3)]2 catalysts. The kinetic isotope effect varied significantly according to conditions. The multiple speciation of both AgI and PdII acts as a warning against specifying the catalytic cycles in detail. Moreover, the rapid dynamic interconversion of AgI species creates a level of complexity that has not been appreciated previously.

Direct Cyclopalladation of Fluorinated Benzyl Amines by Pd3(OAc)6: The Coexistence of Multinuclear Pdn Reaction Pathways Highlights the Importance of Pd Speciation in C-H Bond Activation.

Palladacycles are key intermediates in catalytic C-H bond functionalization reactions and important precatalysts for cross-couplings. It is commonly believed that palladacycle formation occurs through the reaction of a substrate bearing a C-H bond ortho to a suitable metal-directing group for interaction with, typically, mononuclear "Pd(OAc)2" species, with cyclopalladation liberating acetic acid as the side product. In this study, we show that N,N-dimethyl-fluoro-benzyl amines, which can be cyclopalladated either ortho or para to fluorine affording two regioisomeric products, can occur by a direct reaction of Pd3(OAc)6, proceeding via higher-order cyclopalladated intermediates. Regioselectivity is altered subtly depending on the ratio of substrate:Pd3(OAc)6 and the solvent used. Our findings are important when considering mechanisms of Pd-mediated reactions involving the intermediacy of palladacycles, of particular relevance in catalytic C-H bond functionalization chemistry.

Understanding Precatalyst Activation and Speciation in Manganese-Catalyzed C-H Bond Functionalization Reactions.

An investigation into species formed following precatalyst activation in Mn-catalyzed C-H bond functionalization reactions is reported. Time-resolved infrared spectroscopy demonstrates that light-induced CO dissociation from precatalysts [Mn(C^N)(CO)4] (C^N = cyclometalated 2-phenylpyridine (1a), cyclometalated 1,1-bis(4-methoxyphenyl)methanimine (1b)) in a toluene solution of 2-phenylpyridine (2a) or 1,1-bis(4-methoxyphenyl)methanimine (2b) results in the initial formation of solvent complexes fac-[Mn(C^N)(CO)3(toluene)]. Subsequent solvent substitution on a nanosecond time scale then yields fac-[Mn(C^N)(CO)3(κ1-(N)-2a)] and fac-[Mn(C^N)(CO)3(κ1-(N)-2b)], respectively. When the experiments are performed in the presence of phenylacetylene, the initial formation of fac-[Mn(C^N)(CO)3(toluene)] is followed by a competitive substitution reaction to give fac-[Mn(C^N)(CO)3(2)] and fac-[Mn(C^N)(CO)3(η2-PhC2H)]. The fate of the reaction mixture depends on the nature of the nitrogen-containing substrate used. In the case of 2-phenylpyridine, migratory insertion of the alkyne into the Mn-C bond occurs, and fac-[Mn(C^N)(CO)3(κ1-(N)-2a)] remains unchanged. In contrast, when 2b is used, substitution of the η2-bound phenylacetylene by 2b occurs on a microsecond time scale, and fac-[Mn(C^N)(CO)3(κ1-(N)-2b)] is the sole product from the reaction. Calculations with density functional theory indicate that this difference in behavior may be correlated with the different affinities of 2a and 2b for the manganese. This study therefore demonstrates that speciation immediately following precatalyst activation is a kinetically controlled event. The most dominant species in the reaction mixture (the solvent) initially binds to the metal. The subsequent substitution of the metal-bound solvent is also kinetically controlled (on a ns time scale) prior to the thermodynamic distribution of products being obtained.

Mechanically Robust Hybrid Gel Beads Loaded with "Naked" Palladium Nanoparticles as Efficient, Reusable, and Sustainable Catalysts for the Suzuki-Miyaura Reaction.

The increase in demand for Pd and its low abundance pose a significant threat to its future availability, rendering research into more sustainable Pd-based technologies essential. Herein, we report Pd scavenging mechanically robust hybrid gel beads composed of agarose, a polymer gelator (PG), and an active low-molecular-weight gelator (LMWG) based on 1,3:2,4-dibenzylidenesorbitol (DBS), DBS-CONHNH 2 . The robustness of the PG and the ability of the LMWG to reduce Pd(II) in situ to generate naked Pd(0) nanoparticles (PdNPs) combine within these gel beads to give them potential as practical catalysts for Suzuki-Miyaura cross-coupling reactions. The optimized gel beads demonstrate good reusability, green metrics, and most importantly the ability to sustain stirring, improving reaction times and energy consumption compared to previous examples. In contrast to previous reports, the leaching of palladium from these next-generation beads is almost completely eliminated. Additionally, for the first time, a detailed investigation of these Pd-loaded gel beads explains precisely how the nanoparticles are formed in situ without a stabilizing ligand. Further, detailed catalytic investigations demonstrate that catalysis occurs within the gel beads. Hence, these beads can essentially be considered as robust "nonligated" heterogeneous PdNP catalysts. Given the challenges in developing ligand-free, naked Pd nanoparticles as stable catalysts, these gel beads may have future potential for the development of easily used systems to perform chemical reactions in "kit" form.

Coumarin C-H Functionalization by Mn(I) Carbonyls: Mechanistic Insight by Ultra-Fast IR Spectroscopic Analysis.

Mn(I) C-H functionalization of coumarins provides a versatile and practical method for the rapid assembly of fused polycyclic pyridinium-containing coumarins in a regioselective manner. The synthetic strategy enables application of bench-stable organomanganese reagents in both photochemical- and thermal-promoted reactions. The cyclomanganated intermediates, and global reaction system, provide an ideal testing ground for structural characterization of the active Mn(I) carbonyl-containing species, including transient species observable by ultra-fast time-resolved spectroscopic methods. The thermodynamic reductive elimination product, solely encountered from reaction between alkynes and air-stable organometallic cyclomanganated coumarins, has enabled characterization of a critical seven-membered Mn(I) intermediate, detected by time-resolved infrared spectroscopy, enabling the elucidation of the temporal profile of key steps in the reductive elimination pathway. Quantitative data are provided. Manganated polycyclic products are readily decomplexed by AgBF4 , opening-up an efficient route to the formation of π-extended hybrid coumarin-pyridinium compounds.

A comprehensive understanding of carbon-carbon bond formation by alkyne migratory insertion into manganacycles.

Migratory insertion (MI) is one of the most important processes underpinning the transition metal-catalysed formation of C-C and C-X bonds. In this work, a comprehensive model of MI is presented, based on the direct observation of the states involved in the coupling of alkynes with cyclometallated ligands, augmented with insight from computational chemistry. Time-resolved spectroscopy demonstrates that photolysis of complexes [Mn(C^N)(CO)4] (C^N = cyclometalated ligand) results in ultra-fast dissociation of a CO ligand. Performing the experiment in a toluene solution of an alkyne results in the initial formation of a solvent complex fac-[Mn(C^N)(toluene)(CO)3]. Solvent substitution gives an η2-alkyne complex fac-[Mn(C^N)(η2-R1C2R2)(CO)3] which undergoes MI of the unsaturated ligand into the Mn-C bond. These data allowed for the dependence of second order rate constants for solvent substitution and first order rate constants for C-C bond formation to be determined. A systematic investigation into the influence of the alkyne and C^N ligand on this process is reported. The experimental data enabled the development of a computational model for the MI reaction which demonstrated that a synergic interaction between the metal and the nascent C-C bond controls both the rate and regiochemical outcome of the reaction. The time-resolved spectroscopic method enabled the observation of a multi-step reaction occurring over 8 orders of magnitude in time, including the formation of solvent complexes, ligand substitution and two sequential C-C bond formation steps.

Pd-Catalyzed Cross-Couplings: On the Importance of the Catalyst Quantity Descriptors, mol % and ppm.

This Review examines parts per million (ppm) palladium concentrations in catalytic cross-coupling reactions and their relationship with mole percentage (mol %). Most studies in catalytic cross-coupling chemistry have historically focused on the concentration ratio between (pre)catalyst and the limiting reagent (substrate), expressed as mol %. Several recent papers have outlined the use of "ppm level" palladium as an alternative means of describing catalytic cross-coupling reaction systems. This led us to delve deeper into the literature to assess whether "ppm level" palladium is a practically useful descriptor of catalyst quantities in palladium-catalyzed cross-coupling reactions. Indeed, we conjectured that many reactions could, unknowingly, have employed low "ppm levels" of palladium (pre)catalyst, and generally, what would the spread of ppm palladium look like across a selection of studies reported across the vast array of the cross-coupling chemistry literature. In a few selected examples, we have examined other metal catalyst systems for comparison with palladium.

Reactivity of a Dinuclear PdI Complex [Pd2(µ-PPh2)(µ2-OAc)(PPh3)2] with PPh3: Implications for Cross-Coupling Catalysis Using the Ubiquitous Pd(OAc)2/nPPh3 Catalyst System.

[PdI 2(µ-PPh2)(µ2-OAc)(PPh3)2] is the reduction product of PdII(OAc)2(PPh3)2, generated by reaction of 'Pd(OAc)2' with two equivalents of PPh3. Here, we report that the reaction of [PdI 2(µ-PPh2)(µ2-OAc)(PPh3)2] with PPh3 results in a nuanced disproportionation reaction, forming [Pd0(PPh3)3] and a phosphinito-bridged PdI-dinuclear complex, namely [PdI 2(µ-PPh2){κ2-P,O-µ-P(O)Ph2}(κ-PPh3)2]. The latter complex is proposed to form by abstraction of an oxygen atom from an acetate ligand at Pd. A mechanism for the formal reduction of a putative PdII disproportionation species to the observed PdI complex is postulated. Upon reaction of the mixture of [Pd0(PPh)3] and [PdI 2(µ-PPh2){κ2-P,O-µ-P(O)Ph2}(κ-PPh3)2] with 2-bromopyridine, the former Pd0 complex undergoes a fast oxidative addition reaction, while the latter dinuclear PdI complex converts slowly to a tripalladium cluster, of the type [Pd3(µ-X)(µ-PPh2)2(PPh3)3]X, with an overall 4/3 oxidation state per Pd. Our findings reveal complexity associated with the precatalyst activation step for the ubiquitous 'Pd(OAc)2'/nPPh3 catalyst system, with implications for cross-coupling catalysis.

A Dichotomy in Cross-Coupling Site Selectivity in a Dihalogenated Heteroarene: Influence of Mononuclear Pd, Pd Clusters, and Pd Nanoparticles-the Case for Exploiting Pd Catalyst Speciation.

Site-selective dihalogenated heteroarene cross-coupling with organometallic reagents usually occurs at the halogen proximal to the heteroatom, enabled by intrinsic relative electrophilicity, particularly in strongly polarized systems. An archetypical example is the Suzuki-Miyaura cross-coupling (SMCC) of 2,4-dibromopyridine with organoboron species, which typically exhibit C2-arylation site-selectivity using mononuclear Pd (pre)catalysts. Given that Pd speciation, particularly aggregation, is known to lead to the formation of catalytically competent multinuclear Pdn species, the influence of these species on cross-coupling site-selectivity remains largely unknown. Herein, we disclose that multinuclear Pd species, in the form of Pd3-type clusters and nanoparticles, switch arylation site-selectivity from C2 to C4, in 2,4-dibromopyridine cross-couplings with both organoboronic acids (SMCC reactions) and Grignard reagents (Kumada-type reactions). The Pd/ligand ratio and the presence of suitable stabilizing salts were found to be critically important in switching the site-selectivity. More generally, this study provides experimental evidence that aggregated Pd catalyst species not only are catalytically competent but also alter reaction outcomes through changes in product selectivity.

Direct Observation of the Microscopic Reverse of the Ubiquitous Concerted Metalation Deprotonation Step in C-H Bond Activation Catalysis.

The ability of carboxylate groups to promote the direct functionalization of C-H bonds in organic compounds is unquestionably one of the most important discoveries in modern chemical synthesis. Extensive computational studies have indicated that this process proceeds through the deprotonation of a metal-coordinated C-H bond by the basic carboxylate, yet experimental validation of these predicted mechanistic pathways is limited and fraught with difficulty, mainly as rapid proton transfer is frequently obscured in ensemble measures in multistep reactions (i.e., a catalytic cycle consisting of several steps). In this paper, we describe a strategy to experimentally observe the microscopic reverse of the key C-H bond activation step underpinning functionalization processes (viz. M-C bond protonation). This has been achieved by utilizing photochemical activation of the thermally robust precursor [Mn(ppy)(CO)4] (ppy = metalated 2-phenylpyridine) in neat acetic acid. Time-resolved infrared spectroscopy on the picosecond-millisecond time scale allows direct observation of the states involved in the proton transfer from the acetic acid to the cyclometalated ligand, providing direct experimental evidence for the computationally predicted reaction pathways. The power of this approach to probe the mechanistic pathways in transition-metal-catalyzed reactions is demonstrated through experiments performed in toluene solution in the presence of PhC2H and HOAc. These allowed for the observation of sequential displacement of the metal-bound solvent by the alkyne, C-C bond formation though insertion in the Mn-C bond, and a slower protonation step by HOAc to generate the product of a Mn(I)-catalyzed C-H bond functionalization reaction.

Light- and Manganese-Initiated Borylation of Aryl Diazonium Salts: Mechanistic Insight on the Ultrafast Time-Scale Revealed by Time-Resolved Spectroscopic Analysis.

Manganese-mediated borylation of aryl/heteroaryl diazonium salts emerges as a general and versatile synthetic methodology for the synthesis of the corresponding boronate esters. The reaction proved an ideal testing ground for delineating the Mn species responsible for the photochemical reaction processes, that is, involving either Mn radical or Mn cationic species, which is dependent on the presence of a suitably strong oxidant. Our findings are important for a plethora of processes employing Mn-containing carbonyl species as initiators and/or catalysts, which have considerable potential in synthetic applications.

Correction: Time-resolved infra-red spectroscopy reveals competitive water and dinitrogen coordination to a manganese(i) carbonyl complex.

Correction for 'Time-resolved infra-red spectroscopy reveals competitive water and dinitrogen coordination to a manganese(i) carbonyl complex' by Jonathan B. Eastwood et al., Dalton Trans., 2020, DOI: .

Direct Measurement of the Visible to UV Photodissociation Processes for the PhotoCORM TryptoCORM.

PhotoCORMs are light-triggered compounds that release CO for medical applications. Here, we apply laser spectroscopy in the gas phase to TryptoCORM, a known photoCORM that has been shown to destroy Escherichia coli upon visible-light activation. Our experiments allow us to map TryptoCORM's photochemistry across a wide wavelength range by using novel laser-interfaced mass spectrometry (LIMS). LIMS provides the intrinsic absorption spectrum of the photoCORM along with the production spectra of all of its ionic photoproducts for the first time. Importantly, the photoproduct spectra directly reveal the optimum wavelengths for maximizing CO ejection, and the extent to which CO ejection is compromised at redder wavelengths. A series of comparative studies were performed on TryptoCORM-CH3 CN which exists in dynamic equilibrium with TryptoCORM in solution. Our measurements allow us to conclude that the presence of the labile CH3 CN facilitates CO release over a wider wavelength range. This work demonstrates the potential of LIMS as a new methodology for assessing active agent release (e.g. CO, NO, H2 S) from light-activated prodrugs.

Biocatalytic Synthesis of Moclobemide Using the Amide Bond Synthetase McbA Coupled with an ATP Recycling System.

The biocatalytic synthesis of amides from carboxylic acids and primary amines in aqueous media can be achieved using the ATP-dependent amide bond synthetase McbA, via an adenylate intermediate, using only 1.5 equiv of the amine nucleophile. Following earlier studies that characterized the broad carboxylic acid specificity of McbA, we now show that, in addition to the natural amine substrate 2-phenylethylamine, a range of simple aliphatic amines, including methylamine, butylamine, and hexylamine, and propargylamine are coupled efficiently to the native carboxylic acid substrate 1-acetyl-9H-ß-carboline-3-carboxylic acid by the enzyme, to give amide products with up to >99% conversion. The structure of wild-type McbA in its amidation conformation, coupled with modeling and mutational studies, reveal an amine access tunnel and a possible role for residue D201 in amine activation. Amide couplings were slower with anilines and alicyclic secondary amines such as pyrrolidine and piperidine. The broader substrate specificity of McbA was exploited in the synthesis of the monoamine oxidase A inhibitor moclobemide, through the reaction of 4-chlorobenzoic acid with 1.5 equiv of 4-(2-aminoethyl)morpholine, and utilizing polyphosphate kinases SmPPK and AjPPK in the presence of polyphosphoric acid and 0.1 equiv of ATP, required for recycling of the cofactor.