FIELD OF THE INVENTION

The present disclosure relates to a method of coupling a first carbon atom with a second carbon atom to form a C-C bond and a kit suitable for said method. The method comprises contacting carbon atoms with microbially supported metal nanoparticles. The microbially supported metal nanoparticles are prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions and the carbon atoms and microbially supported metal nanoparticles are contacted in a liquid in the presence of a surfactant. The kit comprises microbially supported metal nanoparticles prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions; and a surfactant.

BACKGROUND OF THE INVENTION

Palladium is an important metal with unique chemical reactivity and a growing annual demand (The European Commission, Study on the review of the list of Critical Raw Materials, 2017, DOI: 10.2873/876644). However, the increasing scarcity and unsustainability of current recycling technologies (e.g. pyro/hydrometallurgy) has brought the remediation of Pd to the forefront of chemical research in recent years (M. Bihani et al., Current Opinion in Green and Sustainable Chemistry, 2018, 11 , 45). Microorganisms offer an intriguing solution to this challenge, where native metal reduction pathways in bacteria can be used to convert soluble Pd waste into high-value metal nanoparticles (MNPs) (A. J. Murray, et al., Miner. Eng., 2017, 113, 102). In particular, the sulfate-reducing bacterium Desulfovibrio spp. has emerged as an attractive choice owing to its remarkable tolerance to various metals (see M. J. Capeness, M. C. Edmundson and L. E. Horsfall, New Biotechnol., 2015, 32, 727) and increasing genetic tractability (see M. N. Price et al., Front. Microbiol., 2014, 5, 577). Reduction of soluble metals by Desulfovibrio spp. occurs in the periplasm and generates biogenic nanoparticles that are transported to the cell surface and tightly associated to the outer membrane (M. J. Capeness, V. Echavarri- Bravo and L. E. Horsfall, Front. Microbiol., 2019, 10, 997). However, despite research into the mechanism and scalability of this process for remediation purposes, the catalytic chemistry of metal nanoparticles generated by this microorganism is only now beginning to be explored (see, for example, WO2011086343). Desulfovibrio spp. has the unique capability to generate small nanoparticles in high yield (>95%) from a variety of feedstocks in the absence of external reductants. The biosynthesis of MNPs also offers distinct advantages over chemically synthesized equivalents from a product uniformity and sustainability viewpoint. The chemical production of Pd nanoparticles on the surface of E. coli has been reported by Deplanche et al. (see K. Deplanche et al., Appl. Catal. B, 2014, 147, 651-665). The Pd nanoparticles were used in Suzuki coupling reactions and, in the presence of the John-Phos ligand afforded 4-methoxybiphenyl (62% yield) after 18h incubation in 67% ethanol at 80°C. A ligand-free reaction was attempted however resulted in no conversion.

The bio-production of palladium nanoparticles on cells of Gram-negative proteobacteria is reported by L. S. Sbjerg et al. in Green Chemistry, 2009, 11 , 2041-2043. The bacterially supported nanoparticles are used to catalyse Suzuki-Miyaura or Mizoroki-Heck reactions. Surfactants are not used in these reactions.

The synthesis of bacterially-supported palladium nanoparticles is also reported by J. A. Bennett et al. in Applied Catalysis B: Environmental, 2013, 140-141 , 700-707. The palladium nanoparticles are synthesised by biosorption of palladium^ I) complexes onto the bacterial cells, followed by reduction of the palladium^ I) complexes to palladium(O). The bacterially- supported nanoparticles are used to catalyse Heck reactions. Surfactants are not used in these reactions.

Assisted by a TPGS-750-M surfactant, Handa et al., in Science, 2015, 349, 1087-1091 , describe the chemical synthesis of Fe-ppm Pd nanoparticles. These were identified to promote a range of Suzuki coupling reactions under mild condition (in water at 45°C for 24 h). All successful reactions were carried out in the presence of metal binding ligands.

The production of Pd/Ag and Pd/Au nanoparticles using Shewanella oneidensis is described in by R. Kimber et al. in Microb. BiotechnoL, 2021 , 1751-7915. The nanoparticles were subsequently used in a Suzuki-Miyaura cross-coupling reaction in a mixed solvent (iPrOH/HaO) at 75°C.

The use of micelles to deliver reactants to palladium-containing nanoparticles for Suzuki- Miyaura cross-coupling is described in B. H. Lipshutz et ai, Chemistry - A European Journal, 2018, 1-25. Microbially supported metal nanoparticles are not described.

SUMMARY OF THE INVENTION

The present disclosure is based on studies by the inventors showing that biogenic Pd nanoparticles generated by Desulfovibrio alaskensis G20 are highly active catalysts for the ligand-free Suzuki Miyaura cross-coupling reaction under ambient conditions and for copper- free Sonogashira cross-coupling reactions also under ambient conditions. The reaction has broad scope, can be dramatically accelerated within membrane associated surfactant micelles and can outperform other heterogeneous NP catalysts generated microbially or via chemical synthesis. Overall, this suggests a unique feature of the metal reduction pathway in bacteria, such as Desulfovibrio spp. that is especially suited to the generation of highly active MNP catalysts for use in organic synthesis.

Therefore, viewed from a first aspect, there is provided a method of coupling a first carbon atom with a second carbon atom to form a C-C bond, the method comprising contacting the carbon atoms with microbially supported metal nanoparticles, wherein the microbially supported metal nanoparticles are prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions and wherein the carbon atoms and microbially supported metal nanoparticles are contacted in a liquid in the presence of a surfactant.

Viewed from a second aspect, there is provided a kit for use in coupling a first carbon atom with a second carbon atom to form a C-C bond, said kit comprising:

(i) microbially supported metal nanoparticles prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions; and

(ii) a surfactant.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Approaches to the Suzuki Miyaura cross-coupling reactions in TPGS micelles using chemically synthesised or biogenic palladium nanoparticles.

Figure 2. Structures of P-/N- ligands and surfactants used in screening studies. JohnPhos 4, SPhos 5, XPhos 6, sSPhos 7, AmPyol 8, TMG 9.

Figure 3. Examining the effect of TPGS micelles on the reaction. A) TEM images of DaPdNPs in the presence and absence of TPGS-1000. B) Time-course analysis showing the production of 3 and the effect of micelle addition. C) Concentration of 3 in reactions after 4 h. The % conversion to 3 is shown above each dataset. D) The effect of micelles on Pd activity at low catalyst loading. 2% w/v TPGS was added in all cases. Pd concentrations were determined by ICP-OES. Error bars represent the standard deviation of values from three independent experiments. Figure 4. Substrate scope. Yields in parenthesis are from reactions containing 2% w/v TPGS- 1000. Product concentrations determined by ¹ H NMR spectroscopy relative to an internal standard of TMB (10 mM).

Figure 5. Catalyst, ligand and base screen for the DaPdNP catalyzed Sonogashira reaction.

[a] Reactions were performed using 1 or 2 (30 mM), 3 (60 mM), base, Pd catalyst (0.3 mM, 1 mol%), ligand (3 mM) and TPGS-1000 (2% w/vol) in sealed tubes under an atmosphere of air.

[b] aryl bromide 1 was used as the substrate [c] aryl iodide 2 was used as the substrate [d] 60 mM. [e] 90 mM. [f] 30 mM. [g] 45 mM. cPdNPs refers to nanoparticulate Pd black (<25 nm particle size). Product concentrations were determined by ¹ H NMR spectroscopy relative to an internal standard of TMB. All data is shown as an average of replicate experiments to one standard deviation.

Figure 6: Initial screening data. A) Structures of phosphine ligands and surfactants. XPhos 7, RuPhos 8, tBuXPhos 9, SPhos 10, JohnPhos 11 , Cy- JohnPhos 12, dtbpf 13, PPh3 14, P(tBu)3 15, Cy-cBRIDP 16. B) Base-free reactivity in M9 growth media. C) Control reactions examining the components of the reaction. Reactions were set up and analyzed as outlined in Figure 6. [a] 5 mM substrate concentration. All data is shown as an average of replicate experiments to one standard deviation.

Figure 7: Substrate scope of the DaPdNP catalyzed Sonogashira reaction in the presence and absence of TPGS-1000 micelles. Reactions were performed using aryl iodides (25 mM), alkynes (30 mM), K2CO3 (30 mM), DaPdNPs (0.25 mM, 1 mol%), JohnPhos ligand (2.5 mM) with/without TPGS-1000 (2% w/vol). Data are reported as percentage yields and values in parenthesis are from reactions containing 2% w/vol TPGS-1000. Product conversions were determined by quantitative ¹ H NMR analysis relative to an internal standard of TMB (10 mM). [a] isolated yield from 0.6 g scale reaction.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that microbially supported metal nanoparticles prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions are highly active cross-coupling catalysts, particularly when used in the presence of a surfactant. It was thought that surfactants would be incompatible with biogenic nanoparticles. Accordingly, the ability to use surfactants with microbially-supported nanoparticles in cross- coupling reactions and the enhanced yields that resulted from this were unexpected. The carbon cross-coupling method is disclosed in detail below.

In the discussion that follows, reference is made to a number of terms, which are to be understood to have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds described herein, is intended to be in accordance with the rules of the International Union of Pure and Applied Chemistry (IUPAC) for chemical compounds, specifically the “IUPAC Compendium of Chemical Terminology (Gold Book)” (see A. D. Jenkins et aL, Pure & AppL Chem., 68, 2287- 2311 (1996)). For the avoidance of doubt, if an IUPAC rule is contrary to a definition provided herein, the definition herein is to prevail.

The term “comprising” or variants thereof will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term “consisting” or variants thereof will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

The term “about” herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified. For example, if the average (mean) number of repeat units referred to within square parentheses of formulae (IV) or (V) (n) is specified to be about 10 to about 25, n values of 9.5 to 26.25 are included.

The term “coupling”, when used to refer to the coupling of two carbon atoms, is well known in the field and means the binding of one carbon atom to another carbon atom to form a covalent bond between the two.

Where one or more elements are “contacted”, said elements are positioned so that they interact with one another, for example through the formation of bonds. Accordingly, where a first and second carbon atom are contacted with microbially supported metal nanoparticles, the first and second carbon atoms interact with the microbially supported metal nanoparticles, for example via the formation of bonds to the metal nanoparticles.

The term “microbially supported metal nanoparticles”, is used herein to refer to metal nanoparticles that are associated with a microbe, such as a bacterium. The metal nanoparticles may be associated with the surface of the microbe. Where the microbe is a bacterium, the metal nanoparticles may be associated with the surface of the bacterium, may lie within the cell wall or the cell membrane of the bacterium, or may lie within the plasma or organelles within the plasma of the bacterium.

Conditions described to be “anaerobic conditions” are conditions lacking in oxygen, i.e. where the amount of oxygen is less than usually found in the atmosphere (about 21 %). Typically, anaerobic conditions refer to an atmosphere comprising less than 10% oxygen, such as less than 5% or less than 1 % oxygen.

The term “hydrophilic” refers to an affinity for water, e.g. functional groups that have a high solubility in water and/or functional groups that have a negative logP value. By a negative logP value is meant that when the compound is contacted with a mixture of an aqueous solvent and a lipid solvent, the compound has a higher affinity for the aqueous phase than the lipid phase. Typically, hydrophilic groups have a solubility in water of greater than 300 mg/mL and/or a logP value in the range of about -8 to about -2 (such as about -7 to about -3). For the avoidance of doubt, solubility values are at 20 °C and at atmospheric pressure. LogP values are at 25 °C in octanol/water mixtures and may be calculated using, for example, the software freely available at (method for logP prediction developed at Molinspiration (miLogP2.2 - November 2005) based on group contributions, which have been obtained by fitting calculated logP with experimental logP for a training set more than twelve thousand, mostly drug-like molecules) or using Advanced Chemistry Development (ACD/Labs) Software V11.02 (© 1994-2021 ACD/Labs) (see htps://w .acdlabs.com products/percepta/predictors/loap/index.pho for further information).

The term “hydrophobic” refers to a lack of affinity for water, e.g. functional groups that have a low solubility in water and/or functional groups that have a positive logP value (a higher affinity for the lipid phase than the aqueous phase). Typically, hydrophobic groups have a solubility in water of less than 150 mg/mL and/or logP values of about 8 to about 13 (such as about 10 to about 12).

The term “alkoxy” defines univalent groups derived from alcohols by removal of the hydrogen atom of the hydroxy group. The term “alcohols” refers to alkanes wherein one hydrogen atom has been replaced with a hydroxy group. Methoxy is an example of an Cialkoxy group. The term “alkane” is intended to define acyclic branched or unbranched hydrocarbons having the general formula C _n H2n+2, wherein n is an integer >1. Examples of alkanes include methane, ethane, n-propane, iso-propane, n-butane, sec-butane, iso-butane and tert-butane.

The term “arene” refers to monocyclic and polycyclic aromatic hydrocarbons, such as benzene or naphthalene.

The term “heteroarene” refers to heterocyclic compounds derived from arenes by replacement of one or more methine (-C=) and/or vinylene (-CH=CH-) groups by trivalent or divalent heteroatoms, respectively, in such a way as to retain aromaticity. Examples of heteroarenes include pyridine, thiophene and quinoline.

The term “electron withdrawing” refers to groups that are able to pull or draw electron density away from the species to which they are bound, e.g. more effectively than a proton bound to the same position of the same species. Examples of electron withdrawing groups include halo groups, nitriles, carbonyls and nitro groups.

The term “electron donating” refers to groups that are able to release or donate electron density into the species to which they are bound, e.g. more effectively than a proton bound to the same position of the same species. Examples of electron donating groups include alkyl groups, hydroxyl and alkoxy groups, and amino groups.

The term “stereoisomer” is used herein to refer to isomers that possess identical molecular formulae and sequence of bonded atoms, but which differ in the arrangement of their atoms in space.

The term “enantiomer” defines one of a pair of molecular entities that are mirror images of each other and non-superimposable, i.e. cannot be brought into coincidence by translation and rigid rotation transformations. Enantiomers are chiral molecules, i.e. are distinguishable from their mirror image.

The term “racemic” is used herein to pertain to a racemate. A racemate defines a substantially equimolar mixture of a pair of enantiomers.

The term “diastereoisomers” (also known as diastereomers) defines stereoisomers that are not related as mirror images. The term “solvate” is used herein to refer to a complex comprising a solute, such as a compound or salt of the compound, and a solvent. If the solvent is water, the solvate may be termed a hydrate, for example a mono-hydrate, di-hydrate, tri-hydrate etc, depending on the number of water molecules present per molecule of substrate.

The term “isotope” is used herein to define a variant of a particular chemical element, in which the nucleus necessarily has the same atomic number but has a different mass number owing to it possessing a different number of neutrons.

As described above, the first aspect provides a method of coupling a first carbon atom with a second carbon atom to form a C-C bond, the method comprising contacting the carbon atoms with microbially supported metal nanoparticles, wherein the microbially supported metal nanoparticles are prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions and wherein the carbon atoms and microbially supported metal nanoparticles are contacted in a liquid in the presence of a surfactant.

In some embodiments, the metal reducing microbes and the source of metal are contacted in the absence of an external reducing agent, i.e. an external reducing agent (such as H2) is not added to the reaction. Sometimes, the metal reducing microbes and the source of metal are contacted in the presence of one or more external reducing agents. Examples of external reducing agents include hydrogen gas (H2), formic acid, nicotinamide adenine dinucleotide (NADH), NaBH4, N2H4, aldehydes such as formaldehyde, CO, ascorbic acid, hydroquinone, citrate and polyols. Often, the one or more external reducing agents are hydrogen gas and/or formic acid.

In some embodiments, the surfactant is capable of forming micelles in the liquid in which the method is conducted. Typically, the surfactant self-assembles in the liquid to form micelles. Micelles are known in the art to consist of an aggregate of surfactant molecules, thereby forming a colloidal suspension of the micelle within the liquid.

The liquid in which the carbon atoms and microbially supported metal nanoparticles are contacted is typically polar. By “polar” is meant that the liquid has a dielectric constant at 25 °C of 17 or more, such as 25 or more or 50 or more. Examples of polar liquids include water, ethanol, methanol, n-butyl alcohol, /so-propyl alcohol and n-propyl alcohol. The liquid in which the carbon atoms and microbially supported metal nanoparticles are contacted usually comprises one or more of the liquids listed above. Conveniently, the liquid is an aqueous based liquid and in some embodiments, the method is conducted in an aqueous solution, which is substantially water. By substantially is understood to mean that water is present in an amount of at least 90%, 95%, 97%, 98%, 99% or 100%. In some embodiments, the method is conducted in water.

Without being bound by theory, surfactants that self-assemble to form micelles in polar liquids are able to co-localise reactants (such as those comprising the first and second carbon atoms) in the hydrophobic interior (typically favoured by organic reactants). Thus, in some embodiments, the micelles are capable of emulsifying the first and second carbon atoms. For example, where the first and second carbon atoms are not fully soluble in the liquid used in the method, a suspension may result and the contacting of the first and second carbon atoms with microbially supported metal nanoparticles may be inefficient. Where the micelles are able to co-localise the first and second carbon atoms in the micelle interior, the first and second carbon atoms may be emulsified and may dissolve within the micelle interior. Surfactants that selfassemble to form micelles in polar liquids are also able to associate with cell membranes of bacteria and accelerate flux through engineered metabolic pathways by sequestering hydrophobic metabolites. When the metal nanoparticles are associated with the surface of bacteria, co-localization of the first and second carbon atoms to the surface of the bacteria results in more efficient contacting with the metal nanoparticles and accelerated product formation.

In some embodiments, the surfactant is a cyclodextrin (such as an a-, p- or y- cyclodextrin, e.g. Me- -cyclodextrin) or a compound of formula (I)

A-B(l) wherein A is a hydrophilic moiety and B is a hydrophobic moiety. Often, the surfactant is of formula (I). Without being bound by theory, where the method is conducted in a polar liquid such as water, the surfactant may form micelles in which the hydrophobic moieties (B) point inwards and form a hydrophobic micelle interior (in which the first and second carbon atoms may be co-localised), and the hydrophilic moieties (A) point outwards towards the surrounding polar liquid, thereby stabilising the micelle within the polar liquid.

In some embodiments, the hydrophilic moiety comprises any one selected from the group consisting of a polyether (such as PEG), quaternary ammonium and sulfate. Examples of surfactants comprising polyether hydrophilic moieties include TPGS-1000, TPGS-750-M, Triton X-100, polyoxyethylene (20) sorbitan monooleate (Tween-80), polyoxyethanyl-a- tocopheryl sebacate (PTS) and SPGS-550-M. Examples of surfactants comprising quaternary ammonium hydrophilic moieties include (C8-i8)alkyldimethylbenzylammonium salts such as benzyldimethylhexadecylammonium salts, e.g. benzyldimethylhexadecylammonium chloride. Examples of surfactants comprising sulfate hydrophilic moieties include (Cs-is)alkyl sulfate salts such as dodecyl sulfate salts, e.g. sodium dodecyl sulfate. Often, the hydrophilic moiety comprises a polyether (such as PEG).

As described above, the hydrophilic moieties typically have a solubility in water of greater than 300 mg/mL. Sometimes, the solubility in water of the hydrophilic moieties is greater than 350 mg/mL, such as greater than 400 mg/mL or greater than 500 mg/mL.

As described above, the hydrophilic moieties typically have a logP value in the range of about -8 to about -2 (such as about -7 to about -3). For the avoidance of doubt, this range includes PEG and methyl-PEG polymers with average (mean) numbers of repeat units ranging from about 14 to about 25. LogP values calculated for such polymers using Advanced Chemistry Development (ACD/Labs) Software V11.02 (© 1994-2021 ACD/Labs) are as follows: PEG (n=21) is -5.877 ± 0.943, PEG (n=24) is -6.582 ± 0.970, methyl-PEG (n=15) is -3.958 ± 0.887, and methyl-PEG (n=22) is -5.630 ± 0.958. The logP value calculated for PEG (n=22) using molinspiration.com is -4.8.

The hydrophobic moieties typically have a solubility in water of less than 150 mg/mL, such as less than 125 mg/mL or less than 110 mg/mL.

As described above, the hydrophobic moieties typically have logP values of about 8 to about 13 (such as about 10 to about 12). For the avoidance of doubt, this range includes a- Tocopherol (the logP value calculated for a-Tocopherol using Advanced Chemistry Development (ACD/Labs) Software V11.02 (© 1994-2021 ACD/Labs) is 10.962 ± 0.355).

Typically, the surfactant comprises a vitamin E derivative. In some embodiments, the surfactant is according to formula (II): wherein A is a hydrophilic moiety and L is a hydrophobic linker capable of bonding A to O.

L typically has a solubility in water of less than 150 mg/mL, such as less than 125 mg/mL or less than 110 mg/mL. L is typically according to formula (Ila): wherein the wavy lines intersect the bonds to A and O, and X is 1 to 10. X is often 1 to 5, such as 2 or 3. X is typically 2.

In some embodiments, the surfactant is according to formula (III):

In some embodiments, the surfactant is according to formula (IV): wherein R is H or Ci-ealkyloxy, n is 5 to 30 and B is a hydrophobic moiety. Typically, B comprises a vitamin E derivative.

In some embodiments, the surfactant is the structure represented by formula (V): wherein R is H or Ci-ealkyloxy and n is 5 to 30. n of formulae (IV) or (V), may be about 10 to about 25, such as about 15 to about 23. Typically, n is about 15 to about 17 or about 22 to about 23. For the avoidance of doubt, “n” refers to the average (mean) number of the repeat units referred to within square parentheses.

R of formulae (IV) or (V) may be H or methoxy. When R is methoxy, n is typically about 15.

In some embodiments, R of formulae (IV) or (V) is H and n of formulae (IV) or (V) is about 22.

In some embodiments, the surfactant is according to formulae (VI) or (VII):

where 22 and 15 are the average number of repeat units.

The surfactants of formulae (VI) and (VII) are known in the field as TPGS-1000 and TPGS- 750-M, respectively. Typically, the surfactant is according to formula (VI) (known in the field as TPGS-1000).

The method comprises contacting the carbon atoms with microbially supported metal nanoparticles. The metal nanoparticles may, for example, be found on, or attached to the surface of the microbe, such as bacterium. Various metal reducing microbes are described by Pantidos N. and Horsfall L.E. in J. Nanomed. Nanotechnol., 2014, 5(5):2339. The metal reducing microbe may be any one of the microbes described in this paper. The microbe may be a sulphate-reducing bacterium, such as from the genus Desulfovibrio, such as Desulfovibrio alaskensis, Desulfovibrio desulfuricans, or Shewanella, such as Shewanella oneidensis, or Shewanella algae. Alternatively, the microbe may be a bacterium from the Pseudomonas genus, such as Pseudomonas aeruginosa’, from the Escherichia genus, such as Escherichia coir, or from the Morganella genus. Such suitable bacteria are capable of reducing soluble palladium (II) into insoluble palladium (0), or platinum (IV) ions, into insoluble platinum (0), generating nanoparticles. Without being bound by theory, nanoparticles produced by bacteria are typically formed at the inner periplasmic membrane via reduction of metal ions by respiratory cytochromes and are then exported to the surface of the bacterial cell.

The metal nanoparticles may be formed from a single or a combination of metal(s). That is, the metal nanoparticles, may be nanoparticles formed of palladium alone, for example, or palladium in combination with any other metal, such as silver or gold. Often the metal (of the source of metal and the metal nanoparticles) is one or more d-block metal(s), i.e. the metal is often one or more transition metal(s). Typically, the metal is any one or a combination selected from the group consisting of palladium, platinum and ruthenium, such as palladium and platinum. In some embodiments, the metal is palladium. Cross coupling reactions are typically catalysed by palladium species, but catalysts comprising other metals, such as ruthenium, are also common (see, for example, T. Akayama at al., Green Chem., 2017,19, 3357-3369; and Y. Na et a!., J. Am. Chem. Soc. 2004, 126, 1 , 250-258).

In particular embodiments, the metal reducing microbe is Desulfovibrio alaskensis and the metal nanoparticles are palladium nanoparticles. The inventors have found that Desulfovibrio alaskensis supported palladium nanoparticles outperform other chemically- and biologically- synthesized Pd nanoparticles from plants and bacteria. See, for example, entry 6 of Table 5 of K. Deplanche et al., Applied Catalysis B: Environmental, 2014, 147, 651-665, in which palladium bio-supported nanoparticles on E. coli were used in Suzuki Miyaura reactions. Coupling of 4-bromoanisole and phenylboronic acid in the absence of ligand provided 0% yield, despite the use of relatively harsh reaction conditions (in 75% EtOH at 80 °C). As described above, the microbially supported metal nanoparticles are prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions. Typically, the microbe is capable of producing metal nanoparticles from a metal salt. The metal salt may comprise one or more counterions selected from the group consisting of chloride, bromide, cyanide, iodide, acetate, nitrate and sulfate, such as chloride, acetate, nitrate or sulfate. In addition to the metal cations, the metal salt may comprise further cations, such as one or more cations selected from the group consisting of sodium, potassium, calcium and magnesium, such as sodium. Often, the metal salt comprises sodium and chloride; or acetate, nitrate or sulfate counterions. For example, when the metal is palladium, the metal source may be a palladium salt such as Na2PdCl4, Pd(OAc)2, where Ac is acetate (-OC(O)CHs), Pd(NOs)2 or PdSO ₄ .

In some embodiments, the source of metal may be obtained from a waste stream, i.e. the source of metal may be recycled.

The method couples a first carbon atom with a second carbon atom to form a C-C bond. In some embodiments, the first carbon atom is bonded to a leaving group, and the C-C bond is formed by substituting the leaving group for the second carbon atom. The leaving group may be any one of the leaving groups typically used in carbon coupling reactions, such as halo, triflate or tosylate. In some embodiments, the leaving group is halo (such as bromo, iodo or chloro). The leaving group is often iodo or bromo, such as bromo.

In some embodiments, the method is a method of Suzuki-Miyaura, Sonogashira, Heck, Stille or Negishi coupling. Comprehensive reviews of such reactions are given by A. Suzuki in J. Organomet. Chem., 1999, 579, 147; and Shi et al. in Chem. Soc. Rev., 2011 , 40, 2761. Further examples of Stille and Negishi cross-coupling reactions are given in B. S. Takali et al., Angew.Chem. I nt. Ed. 2021 , 60, 4158-4163 and A. Krasovskiy, C. Duplais and B. H. Lipschutz, J. Am. Chem. Soc. 2009, 131 , 43, 15592-15593. In some embodiments, the method is a method of Suzuki-Miyaura or Sonogashira coupling, such as Suzuki-Miyaura coupling.

Where the method is a method of Suzuki-Miyaura coupling, the second carbon atom may be bonded to BR ¹ 2 (where R ¹ is OH or Ci-ealkyloxy or R ¹ 2 is pinacolato, i.e. the second carbon atom may be part of a boronic acid), the first carbon atom may be sp ² or sp ³ hybridised, the C-C bond may be formed by substituting the B(R ¹ )2 for the first carbon atom, and the coupling may be carried out in the presence of a base.

By “sp ² ” is meant that one s-orbital and two p-orbitals of the carbon atom combine to produce three identical sp ² hybrid orbitals, which lie in the same plane at an angle of about 120° (trigonal). Carbon atoms that are sp ² hybridised form a double bond to another atom, such as another carbon atom, and two single bonds to two other atoms. In some embodiments, sp ² hybridised carbon atoms are vinylic or form part of an arene or heteroarene.

By “sp ³ ” is meant that one s-orbital and three p-orbitals of the carbon atom combine to produce four identical sp ³ hybrid orbitals, with a tetrahedral geometry at an angle of about 109.5°. Carbon atoms that are sp ³ hybridised form a single bond to four other atoms.

By “base” is meant a Lewis base, i.e. a compound capable of donating a pair of electrons to an electron pair acceptor. The conjugate acids of suitable bases typically have pKa values of about 4 to about 20. Often, the conjugate acids of suitable bases have pKa values of about 5 to about 18, e.g. about 9 to about 17. Suitable bases include carbonates (such as potassium, sodium or calcium carbonate), amines (such as trimethylamine), tert-butoxides (such as potassium tert-butoxide), methoxides (such as sodium methoxide), hydroxides (such as potassium or sodium hydroxide) and potassium phosphates (such as potassium phosphate tribasic).

As described above, where the method is a method of Suzuki-Miyaura coupling, the C-C bond may be formed by substituting the B(R ¹ )2, bound to the second carbon atom, for the first carbon atom. Typically, a leaving group on the first carbon atom is substituted for the second carbon atom. Typically, R ¹ of BR ¹ 2 is OH or R ¹ 2 is pinacolato. In some embodiments, R ¹ is OH.

In some embodiments, the second carbon atom is sp ² hybridised. Often, the second carbon atom is vinylic or forms part of an optionally substituted arene or heteroarene.

Often, cross-coupling reactions are carried out in the presence of external metal-binding ligands, which are typically used to tune the reactivity or increase the stability of the metal catalyst. In some embodiments, the coupling is carried out in the presence of an external metal-binding ligand. Examples of external metal-binding ligands include phosphines such as triphenylphosphine, tri-terf-butylphosphine, 2-biphenylyl)di-terf-butylphosphine, 2-(di-terf- butylphosphino)biphenyl, (2-biphenyl)di-terf-butylphosphine (JohnPhos), (2-biphenyl)di- cyclohexylphosphine (Cy-JohnPhos), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos), 2-di-terf- butylphosphino-2',4',6'-triisopropylbiphenyl (‘BuXPhos), 2-dicyclohexylphosphino-2',6'- diisopropylbiphenyl (RuPhos), 1 ,1 '-bis(di-terf-butylphosphino)ferrocene (dtbpf), 1- (dicyclohexylphosphino)-2,2-diphenyl-1-methylcyclopropane (Cy-cBRIDP), and sodium 2'- dicyclohexylphosphino-2,6-dimethoxy-1 ,1 '-biphenyl-3-sulfonate hydrate (sSPhos); 2-amino- 4,6-pyrimidinediol (AmPyol); and 1 ,1 ,3,3-tetramethylguanidine (TMG). Particular examples of phosphines include triphenylphosphine, tri-terf-butylphosphine, 2-biphenylyl)di-terf- butylphosphine, 2-(di-terf-butylphosphino)biphenyl, (2-biphenyl)di-terf-butylphosphine (JohnPhos), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos), 2- dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos), 2-di-terf-butylphosphino-2',4',6'- triisopropylbiphenyl (‘BuXPhos), 2-dicyclohexylphosphino-2',6'-diisopropylbiphenyl (RuPhos), 1-(dicyclohexylphosphino)-2,2-diphenyl-1-methylcyclopropane (Cy-cBRIDP), and sodium 2'- dicyclohexylphosphino-2,6-dimethoxy-1 ,1 '-biphenyl-3-sulfonate hydrate (sSPhos). Even more particular examples of phosphine include triphenylphosphine, 2-biphenylyl)di-terf- butylphosphine, 2-(di-terf-butylphosphino)biphenyl, (2-biphenyl)di-terf-butylphosphine (JohnPhos), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos), 2- dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos), and sodium 2'- dicyclohexylphosphino-2,6-dimethoxy-1 ,1 '-biphenyl-3-sulfonate hydrate (sSPhos).

The inventors have found that the yield of cross-coupled product from Suzuki-Miyaura crosscoupling reactions catalysed by D. alaskensis supported metal nanoparticles increases when the reaction is conducted in the presence of TMG. Without being bound by theory, it is thought that, since the cell membrane of D. alaskensis is negatively charged and tightly associates to Pd-NPs, the co-localisation of cationic TMG to the cell surface under the reaction conditions might account for this observation. Therefore, in some embodiments, the external metalbinding ligand is TMG.

The inventors have found that where the method of coupling is a method of Suzuki-Miyaura coupling, wherein the carbon atoms and microbially supported metal nanoparticles are contacted in a liquid in the presence of a surfactant, the method proceeds effectively in the absence of an external metal-binding ligand, making the process cheaper, simpler and less wasteful. Thus, in some embodiments, the coupling is carried out in the absence of an external metal-binding ligand.

Where the method is a method of Sonogashira coupling, the second carbon atom may be sp hybridised and may be bonded to a hydrogen atom, the first carbon atom may be sp ² or sp ³ hybridised, the C-C bond may be formed by substituting the hydrogen atom bonded to the second carbon atom for the first carbon atom, and the coupling may be carried out in the presence of a base.

Sonogashira reactions are often carried out in the presence of copper co-catalysts (typically copper salts such as copper halide, e.g. copper iodide). In the case of the method described herein, a copper co-catalyst may be used to trans-metallate the sp hybridised second carbon atom to the metal nanoparticles. However, the inventors have found that copper co-catalysts are not necessary in order to carry out the Sonogashira coupling of the invention, making the process cheaper, simpler and less wasteful. Therefore, in some embodiments, where the method is a method of Sonogashira coupling, it is carried out in the absence of a co-catalyst (such as a copper co-catalyst).

By “sp” is meant that one s-orbital and one p-orbital of the carbon atom combine to produce two identical sp hybrid orbitals, which lie in the same plane at an angle of about 180° (digonal). Carbon atoms that are sp hybridised form a triple bond to another atom, such as another carbon atom, and one single bond to one other atom. In some embodiments, sp hybridised carbon atoms form a triple bond to another carbon atom and a single bond to hydrogen.

In some embodiments, where the method of coupling is a method of Sonogashira coupling, it is carried out in the presence of a base, such as a carbonate (e.g. potassium, sodium or calcium carbonate), an amine (e.g. trimethylamine) or a potassium phosphate (e.g. tri-basic potassium phosphate).

As described above, where the method is a method of Sonogashira coupling, the C-C bond may be formed by substituting the hydrogen atom bonded to the second carbon atom for the first carbon atom. Typically, a leaving group, such as iodo, on the first carbon atom is substituted for the second carbon atom.

In some embodiments, where the method is a method of Sonogashira coupling, the second carbon atom is sp hybridised and is bonded to a hydrogen atom, and the first carbon atom is sp ² hybridised. Often, the first carbon atom is vinylic or forms part of an optionally substituted arene or heteroarene. Often, the second carbon atom is the CH of a terminal alkyne.

As described previously, cross-coupling reactions are often carried out in the presence of external metal-binding ligands, which are typically used to tune the reactivity or increase the stability of the metal catalyst. In some embodiments, where the method is a method of Sonogashira coupling, it is carried out in the presence of an external metal-binding ligand, such as a phosphine. In some examples, the phosphine is any one selected from the group consisting of (2-biphenyl)di-terf-butylphosphine (JohnPhos), tri-terf-butylphosphine, 2- dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos), 2-dicyclohexylphosphino-2',4',6'- triisopropylbiphenyl (XPhos), 2-di-terf-butylphosphino-2',4',6'-triisopropylbiphenyl (‘BuXPhos), 2-dicyclohexylphosphino-2',6'-diisopropylbiphenyl (RuPhos), and l-(dicyclohexylphosphino)- 2,2-diphenyl-1-methylcyclopropane (Cy-cBRIDP). For example, the phosphine may be (2- biphenyl)di-terf-butylphosphine (JohnPhos). Where the method is a method of Heck coupling, the second carbon atom may be sp ² hybridised and may be bonded to two hydrogen atoms, the first carbon atom may be sp or sp ² hybridised, the C-C bond may be formed by substituting the hydrogen atom bonded to the second carbon atom for the first carbon atom, and the coupling may be carried out in the presence of a base.

Where the method is a method of Stille coupling, the second carbon atom may be bonded to Sn(Ci-6alkyl)3, the first carbon atom may be sp ² or sp ³ hybridised, and the C-C bond may be formed by substituting the Sn(Ci.ealkyl)3 for the first carbon atom. Often, the Ci-ealkyl is butyl.

Where the method is a method of Negishi coupling, the second carbon atom may be bonded to ZnX ¹ , the first carbon atom may be sp ² or sp ³ hybridised, and the C-C bond may be formed by substituting the ZnX ¹ for the first carbon atom, wherein X ¹ is halo. Typically, X ¹ is chloro or bromo.

In some embodiments, the first carbon atom is sp ² hybridised. Often, the first carbon atom forms a double bond to another carbon atom, and two single bonds to two other atoms. Typically, the first carbon atom forms a double bond to another carbon atom, a single bond to a further carbon atom and a single bond to a leaving group. The leaving group may be as described above (e.g. a halo). In some embodiments, the first carbon atom forms part of a substituted arene or heteroarene.

The inventors have shown that the method disclosed herein couples a range of different first and second carbon atoms including first and second carbon atoms that are part of optionally substituted arenes or heteroarenes comprising a range of electron withdrawing or electron donating functional groups. For the avoidance of doubt, the first and second carbon atoms need not be within the ring of the optionally substituted arene or heteroarene, for example the second carbon atom may be the CH of the alkynyl group of an optionally substituted arylalkyne or heteroarylalkyne). Accordingly, the first and second carbon atoms need not be limited to carbon atoms comprised within specific compounds. Nevertheless, the first carbon atom may be part of a substituted benzene. The substituted benzene may be substituted with a leaving group (wherein the leaving group may be as described above, e.g. halo) and a second substituent. The second substituent may be an electron withdrawing or an electron donating group. In some embodiments, the second substituent is any one of the group consisting of CO2(Ci-6alkyl), C(O)(Ci-6alkyl), NO2, hydroxy and Ci-ealkoxy. In some cases, the second substituent is any one of the group consisting of CC>2(Ci.6alkyl), NO2, hydroxy and Ci-ealkoxy. In other cases, the second substituent is any one of the group consisting of C(O)(Ci-6alkyl), NO2, hydroxy and Ci-ealkoxy. Oxidative addition of the first carbon atom to the catalyst (the first step in the carbon coupling reaction) is promoted when the first carbon atom is more acidic (more electropositive). Advantageously, therefore, the second substituent is an electron withdrawing group, such as any one of the group consisting of CC>2(Ci-6alkyl), C(O)(Ci-ealkyl) and NO2 (such as CC>2(Ci-6alkyl) and NO2 or C(O)(Ci-ealkyl) and NO2). In some embodiments, the second substituent is CO2Me, COMe or NO2 (such as CO2Me or NO2, or COMe or NO2). Oxidative addition of the first carbon atom to the catalyst may be hindered when the first carbon atom is blocked by bulky groups positioned adjacent to the first carbon atom. Advantageously, therefore, the second substituent is positioned meta or para to the first carbon atom.

The second carbon atom may be part of a substituted benzene or pyridine, for example a substituted phenylalkyne or a substituted pyridylalkyne. The substituted benzene or pyridine may be substituted with a first and a second substituent. Where the method is a method of Suzuki-Miyaura, Stille or Negishi coupling, the first substituent may be BR ¹ 2, Sn(Ci-ealkyl)3 or ZnX ¹ , respectively. Where the method is a method of Sonogashira coupling, there may be no first substituent, i.e. the corresponding carbon atom of the substituted benzene or pyridine may be bonded to hydrogen. The second substituent may be an electron withdrawing or an electron donating group. Typically, the second substituent is an electron donating group, such as Ci-ealkyl (e.g. methyl or ethyl) or Ci-ealkoxy (e.g. methoxy or ethoxy). Transmetallation of the second carbon atom to the catalyst may be hindered when the second carbon atom is blocked by bulky groups positioned adjacent to the second carbon atom. Advantageously, therefore, where the second carbon atom is part of a substituted benzene, the second substituent is positioned meta or para to the second carbon atom. Where the method is a method of Sonogashira coupling, the second carbon is typically the CH of a substituted terminal alkyne. The terminal alkyne need not be part of an optionally substituted arene or heteroarene. In some cases, the terminal alkyne is itself bonded to a saturated moiety such as an aliphatic moiety (e.g. a Ci -ealkyl) or an alkyl silyl (e.g. a trialkylsilyl such as trimethylsilyl). The inventors have demonstrated that the method is highly efficient under ambient conditions, thus the high temperatures (50 to 100 °C) often employed in crosscoupling reactions need not be applied here. Often, the coupling disclosed herein is carried out at temperatures of about 20 to about 40 °C, such as about 25 to about 40 °C. Without being bound by theory, the inventors have found that the reaction can occur in vivo. Surfactants that self-assemble to form micelles in polar liquids are able to associate with the cell membranes of bacteria. When the metal nanoparticles are associated with the surface of bacteria, co-localization of the first and second carbon atoms to the surface of the bacteria results in more efficient contacting with the metal nanoparticles and accelerated product formation. Advantageously, the coupling disclosed herein is carried out at the optimum temperature range of the microbe employed, e.g. when the microbe is Desulfovibrio alaskensis, the optimum temperature range is about 25 to about 40 °C, such as about 37 °C.

The inventors have shown that the method disclosed herein is highly efficient, and that relatively low loadings of the microbially supported metal nanoparticles may be used. Loadings of about 0.01 to about 2 mol% Pd relative to the amount (in moles) of the first carbon atom may be employed, such as loadings of about 0.1 to about 1 mol% (e.g. loadings of about 0.5 mol% Pd).

The inventors have found that, whilst the reaction procedure works unexpectedly effectively in the presence of microbially supported metal nanoparticles, the reaction does not proceed effectively if the microbially supported metal nanoparticles are replaced with chemically synthesised nanoparticles. The inventors have observed (by transmission electron microscopy) distinct interactions between the metal reducing microbes in the presence of micelles. Without being bound by theory, the inventors hypothesise that C-C bond formation occurs in membrane-associated micelles and that association between the metal reducing microbes and micelles allows the reactants and reagents to more easily combine. Provided in the second aspect is a kit for use in coupling a first carbon atom with a second carbon atom to form a C-C bond, said kit comprising:

(i) microbially supported metal nanoparticles prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions; and

(ii) a surfactant.

For the avoidance of doubt, the embodiments described above in relation to the first aspect of the invention apply mutatis mutandis to the second aspect. For example, the microbially supported metal nanoparticles may be found on, or attached to the surface of a bacterium; the bacterium may be Desulfovibrio alaskensis’, the metal nanoparticles may be formed from a single or a combination of metal(s) selected from the group consisting of palladium, platinum, ruthenium and neodymium; and/or the surfactant may be capable of forming micelles in the liquid and/or be of formula (V).

Any discussion herein of documents, acts, materials, devices, articles or the like is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application. It will be appreciated by those skilled in the art that numerous variations and/or modifications may be made to the invention as described herein without departing from the scope of the invention as described. The present embodiments are therefore to be considered for descriptive purposes and are not restrictive, and are not limited to the extent of that described in the embodiment. The person skilled in the art is to understand that the present embodiments may be read alone, or in combination, and may be combined with any one or a combination of the features described herein.

The subject-matter of each patent and non-patent literature reference cited herein is hereby incorporated by reference in its entirety.

The invention may be further understood with reference to the following non-limiting clauses:

1 . A method of coupling a first carbon atom with a second carbon atom to form a C-C bond, the method comprising contacting the carbon atoms with microbially supported metal nanoparticles, wherein the microbially supported metal nanoparticles are prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions and wherein the carbon atoms and microbially supported metal nanoparticles are contacted in a liquid in the presence of a surfactant.

2. The method of clause 1 , wherein the microbially supported metal nanoparticles are associated with the surface of the microbe.

3. The method of clause 1 or clause 2, wherein the metal reducing microbes and the source of metal are contacted in the absence of an external reducing agent.

4. The method of any one of clauses 1 to 3, wherein the surfactant is capable of forming micelles in the liquid.

5. The method of clause 4, wherein the micelles are capable of emulsifying the first and second carbon atoms.

6. The method of any one of clauses 1 to 5, wherein the surfactant is of formula (I) A-B(l) wherein A is a hydrophilic moiety and B is a hydrophobic moiety.

The method of clause 6, wherein A-B is of formula (II) wherein A is a hydrophilic moiety and L is a hydrophobic linker capable of bonding A to O. The method of clause 6 or clause 7, wherein A-B is of formula (III) wherein A is a hydrophilic moiety. The method of any one of clauses 6 to 8, wherein A-B is of formula (IV) wherein R is H or Ci-ealkyloxy, n is about 5 to about 30 and B is a hydrophobic moiety. The method of any one of clauses 1 to 5, wherein the surfactant is the structure represented by formula (V) wherein R is H or Ci-ealkyloxy and n is about 5 to about 30. The method of clause 9 or clause 10, wherein n is about 10 to about 25. The method of clause 9 or clause 10, wherein n is about 15 to about 23. The method of any one of clauses 9 to 12, wherein Ci-ealkyloxy is methoxy. The method of any one of clauses 9 to 12, wherein R is H and n is about 22 to about 23. The method of any one of clauses 1 to 14, wherein the microbially supported metal nanoparticles are bacterially supported metal nanoparticles and the metal reducing microbes are metal reducing bacteria. The method of clause 15, wherein the metal reducing bacteria are of the Desulfovibrionaceae Family. The method of clause 15, wherein the metal reducing bacteria are of the Desulfovibrio Genus. The method of clause 15, wherein the metal reducing bacteria are Desulfovibrio alaskensis. The method of any one of clauses 1 to 18, wherein the metal is any one or a combination selected from the group consisting of palladium, platinum and ruthenium. The method of any one of clauses 1 to 18, wherein the metal is palladium. The method of clause 20, wherein the source of palladium is Na2PdCl4, Pd(OAc)2, Pd(NO ₃ ) ₂ or PdSO ₄ . 22. The method of any one of clauses 1 to 21, wherein the first carbon atom is bonded to a leaving group, and the C-C bond is formed by substituting the leaving group for the second carbon atom.

23. The method of clause 22, wherein the leaving group is any one selected from halo, triflate and tosylate.

24. The method of clause 22, wherein the leaving group is halo.

25. The method of any one of clauses 1 to 24, wherein the second carbon atom is bonded to BR ¹ 2, the first carbon atom is sp ² or sp ³ hybridised, the C-C bond is formed by substituting the B(OR ¹ )2 for the first carbon atom, and the coupling is carried out in the presence of a base; wherein

R ¹ is OH or Ci-ealkyloxy or R ¹ 2 is pinacolato.

26. The method of clause 25, wherein the coupling is carried out in the absence of an external metal-binding ligand.

27. The method of any one of clauses 1 to 26, wherein the second carbon atom is sp ² hybridised

28. The method of any one of clauses 1 to 24, wherein the second carbon atom is sp hybridised and is bonded to a hydrogen atom, the first carbon atom is sp ² or sp ³ hybridised, the C-C bond is formed by substituting the hydrogen atom bonded to the second carbon atom for the first carbon atom, and the coupling is carried out in the presence of a base. The method of clause 28, wherein the first carbon atom is bonded to a leaving group, which is iodo. The method of clause 28 or clause 29, wherein the method is carried out in the presence of an external metal-binding ligand. The method of clause 30, wherein the external metal-binding ligand is any one selected from the group consisting of (2-biphenyl)di-terf-butylphosphine (JohnPhos), tri-terf- butylphosphine, 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos), 2- dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos), 2-di-terf-butylphosphino- 2',4',6'-triisopropylbiphenyl (‘BuXPhos), 2-dicyclohexylphosphino-2',6'- diisopropylbiphenyl (RuPhos), and 1-(dicyclohexylphosphino)-2,2-diphenyl-1- methylcyclopropane (Cy-cBRIDP). The method of clause 30, wherein the external metal-binding ligand is (2-biphenyl)di- terf-butylphosphine (JohnPhos). The method of any one of clauses 1 to 24, wherein the second carbon atom is sp ² hybridised and is bonded to two hydrogen atoms, the first carbon atom is sp or sp ² hybridised, the C-C bond is formed by substituting the hydrogen atom bonded to the second carbon atom for the first carbon atom, and the coupling is carried out in the presence of a base. The method of any one of clauses 1 to 24, wherein the second carbon atom is bonded to Sn(Ci.ealkyl)3, the first carbon atom is sp ² or sp ³ hybridised, and the C-C bond is formed by substituting the Sn(Ci.ealkyl)3 for the first carbon atom. The method of any one of clauses 1 to 24, wherein the second carbon atom is bonded to ZnX ¹ , the first carbon atom is sp ² or sp ³ hybridised, and the C-C bond is formed by substituting the ZnX ¹ for the first carbon atom; wherein X ¹ is halo. The method of any one of clauses 1 to 35, wherein the first carbon atom is sp ² hybridised. The method of clause 36, wherein the first carbon atom is part of a substituted arene or heteroarene. The method of any one of clauses 1 to 37, wherein the liquid comprises water. The method of any one of clauses 1 to 38, wherein the coupling is carried out at temperatures of about 20 to about 40 °C. The method of any one of clauses 1 to 38, wherein the coupling is carried out at temperatures of about 37 °C. The method of any one of clauses 1 to 40, wherein the microbially supported metal nanoparticles are present at loadings of about 0.1 to about 1 mol%. A kit for use in coupling a first carbon atom with a second carbon atom to form a C-C bond, said kit comprising:

(i) microbially supported metal nanoparticles prepared by contacting metal reducing microbes with a source of metal under anaerobic conditions; and

(ii) a surfactant. The kit of clause 42, wherein the microbially supported metal nanoparticles are as defined in any one of clauses 15 to 21. 44. The kit of clause 42 or clause 43, wherein the surfactant is as defined in any one of clauses 4 to 14.

The present disclosure will now be further described by way of example and with reference to the attached figures. The following non-limiting examples below serve to illustrate the invention further.

EXAMPLES

General materials and methods

Unless otherwise noted, starting materials and reagents were obtained from commercial suppliers and were used without further purification. All water used experimentally was purified with a Suez Select purification system (18 mQ/cm, 0.2 pM filter).

NMR and IR: Proton nuclear magnetic resonance spectra ( ¹ H NMR) were recorded using an AVA 500 NMR spectrometer (Bruker) at the specified frequency at 298K. NMR solvents were used as purchased from commercial suppliers. For all quantitative NMR measurements, 1 ,3,5- trimethoxybenzene (TMB) was used as an internal standard. Infrared (IR) spectroscopy was performed using a Spectrum Two IR spectrometer (Perkin Elmer).

XRD: X-ray diffraction experiments were recorded on a MiniFlex 600 benchtop X-ray diffractometer (Rigaku) with D/teX Ultra 1 D silicon strip detector (Rigaku) using CuKa radiation. Scanning was run at a 28 angle from 20 to 100° with a 2° step size (40 kV, 15 mA).

ICP-OES: Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) was performed using an Optima 8300 instrument (Perkin Elmer). Experiments were performed using the following conditions: samples were sonicated for 30 min at 21 °C in a water bath and then centrifuged (20,000 x g, 2 h). The supernatant (the ionic fraction) was added to a solution of aqua regia (HCkHNOs = 14:10%) and heated to 80°C for 8 h. The resulting sample was diluted in 2% nitric acid to a final volume of 3 mL and used immediately. Unspun samples (whole fraction) were prepared using an analogous method. When quantifying by ICP a standard curve was constructed over a range of analyte concentrations (0.04-10 ppm) providing linear relationships of Intensity = 41356cpd + 1573.4.

TEM: Transmission electron microscopy (TEM) was performed using a JEM-1400 Plus (JEOL) with an accelerating voltage of 80 kV. TEM images were captured using a GATAN OneView camera. Image processing was carried out using Imaged software. Experiments were performed using the following conditions: suspensions of DaPdNPs were drop cast on to a 200 mesh carbon-coated copper grid and dried for 5 min under air. The excess liquid was then removed and the samples visualised immediately, using a TEM (JEM-1400 Plus, JEOL) with an accelerating voltage of 80 kV.

Strain, media, buffer and culture condition

Desulfovibrio alaskensis G20 (DSM 17464) was obtained from DSMZ. Desulfovibrio alaskensis G20 (DSM 17464) was grown statically on de-gassed Postgate Medium C at 30 °C in an anaerobic chamber fed with 10% H2 and 10% CO2 in nitrogen. Optical densities of D. alaskensis cultures were determined using a WPA C08000 spectrophotometer (Biochorm) by measuring absorbance at 600 nm.

Postgate Medium C was prepared according to the following procedure: KH2PO4 (0.5 g), NH4CI (1.0 g), Na ₂ SO ₄ (4.5 g), CaCI ₂ *6H ₂ O (0.06 g), MgSO ₄ *7H ₂ O (0.06 g), 60% sodium lactate (10 mL) yeast extract (1.0 g), FeSO4*7H2O (0.004 g) and sodium citrate- hW (0.3 g) were dissolved in 990 mL ultrapure water and its pH was adjusted to 7.5 using 2 M NaOH. The medium was autoclaved at 121 °C for 20 min and cooled to room temperature. Autoclaved Postgate Medium C was stored at room temperature and degassed in the anaerobic chamber overnight before the use.

MOPs buffer was prepared according to the following procedure: MOPs free acid (41.86 g) was dissolved in 1 L ultrapure water and its pH was adjusted to 7.0 using 1 M H2SO4. The buffer was autoclaved at 121 °C for 20 min and cooled to room temperature. Autoclaved MOPs buffer was stored at room temperature and degassed in the anaerobic chamber overnight before the use.

DaPdNPs production by Desulfovibrio alaskensis

D. alaskensis cells were pre-grown following previously reported methods (M. J. Capeness, V. Echavarri- Bravo, L. E. Horsfall, Front. Microbiol. 2019, 10, 997), recovered and washed with MOPs buffer three times prior to inoculation (to a final ODeoo of 1.0) in 50 mL centrifuge tubes containing 40 mL MOPs buffer. Freshly made Na2PdCl4 stock solution (40 mM in H2O) was added to the cell suspension to a final concentration of 2 mM. The centrifuge tubes were incubated at 30 °C for 20 h in an anaerobic chamber. The biogenic nanoparticles (DaPdNPs) were harvested by centrifugation at 15 min, 4,500 x g) and washed with 50% acetone in H2O (1 x vol equiv.). Subsequently, the DaPdNPs were freeze-dried overnight, resuspended in ultrapure water and sonicated in a water bath for 30 min. The resulting nanoparticles were analysed by XRD and TEM. The recovery of Pd as DaPdNPs was determined by ICP-OES. Suzuki Coupling

General Suzuki coupling reaction

Cross coupling reactions were carried out using the following procedure: DaPdNPs were added to a 15 mL Hungate tube containing aryl halide (25 mM), phenyl boronic acid (30 mM), K2CO3 (30 mM) and TPGS-1000/TPGS-750-M (2% w/v) in H2O (5 mL). The tubes were sealed with butyl rubber septa and screw-caps and incubated at 37 °C (200 rpm) for 20 h in a New Brunswick Innova 44 incubator shaker (Eppendorf). After this time, the reactions were cooled to room temperature, extracted with methyl terf-butyl ether (MTBE, 3 x 1.7 mL) and concentrated under reduced pressure. The crude residue was dissolved in 1 mL CDCI3 containing 10 mM TMB and analysed by ¹ H NMR spectroscopy.

Coupling reaction optimisation in agueous solution

Unless otherwise noted, model reaction (DaPdNPs (0.25 mM), 4-bromanisole (25 mM), phenylboronic acid (30 mM), K2CO3 (30 mM) in H2O (5 mL)) was used by following the general Suzuki-Miyaura coupling reaction protocol, described above. For ligand/additive screening, triphenylphosphine (PPhs), JohnPhos, SPhos, XPhos, sSPhos, 4-aminopyridine-2,6-diol (AmPyol), 1 ,1 ,3,3-tetramethylguanidine (TMG) or green tea polyphenols was added to the model reaction (2.5 mM for ligands and 0.1/ or 0.01 % w/v for green tea polyphenols). For halide screening, 4-chloroanisole/4-iodoanisole (25 mM) was used as the coupling partner instead of 4-bromoanisole.

Coupling reaction with chemically produced Pd catalysts

Chemically produced Pd nanoparticle (cPdNP, 0.25 mM) or Pd on activated carbon (Pd/C, 0.25 mM) was added to the model reaction with/without ligands (XPhos or TMG, 2.5 mM) in place of DaPdNPs. The reactions were conducted and analysed by following the general Suzuki- Miyaura coupling reaction protocol, described above.

Micellar coupling reaction in the presence of various ligands

The model reaction containing no ligand, JohnPhos, SPhos, XPhos or TMG (2.5 mM) was conducted in the presence of TPGS-750-M or TPGS-1000 (2% w/v). The reactions were conducted and analysed by following the general Suzuki-Miyaura coupling reaction protocol, described above.

Effect of TPGS-1000 on the reaction speed and the catalyst loading minimisation

The reactions were conducted and analysed by following the general Suzuki-Miyaura coupling reaction protocol, described above. Reactions containing TPGS-1000 (2% w/v) or no surfactant and either no ligand, XPhos or TMG (2.5 mM) was sealed and incubated at 37 °C (200 rpm) in a New Brunswick Innova 44 incubator shaker (Eppendorf). Reactions were collected at 1 , 2, 4, 20 and 40 h, and extracted with MTBE (3 x 1.7 mL) immediately. For the catalyst loading minimisation experiment, the concentration of DaPdNPs was adjusted to 0.01 , 0.05, 0.1 , 0.5 or 1.0 in the ligand-free model reaction with/without TPGS-1000 (2% w/v). The reactions and catalyst-free control reaction were sealed and incubated at 37 °C (200 rpm) for 20 h.

Investigation of the reaction scope

Aryl bromide (4-bromoacetophenone, 3-bromoacetophenone, 2-bromoacetophenone, 1- bromo-4-nitrobenzene or 4-bromophenol: 25 mM) and boronic acid (p-tolylboronic acid, 2- methoxy-3-pyridinylboronic acid, 4-ethoxyphenylboronic acid, 3-ethoxyphenylboronic acid, 3- ethoxyphenylboronic acid :30 mM) were added to Hungate tube containing DaPdNPs (0.25 mM) and K2CO3 (30 mM) in H2O (5 mL). Reactions of 25 different cross-coupling combination were carried out and analysed by following the general Suzuki-Miyaura coupling reaction protocol, described above. Subsequently, for the reactions resulting in less than 80% yield, the identical coupling reactions were conducted in the presence of TPGS-1000 (2% w/v).

Three-phase test

For the solid support, the Wang resin, a polystyrene based resin with a p-benzyloxybenzyl alcohol linker often used in solid phase peptide synthesis was chosen (S. S. Wang, J. Am. Chem. Soc. 1973, 95,1328-1333). lodobenzoic acid bound to a Wang resin was synthesised according to the following procedure: Wang resin (2.0 g, 1.0 mmol/g loading) was added to a solution of dichloromethane:/V,/V-dimethylformamide (9:1 , 20 mL) and stirred for 15 min. N, N'- diisopropylcarbodiimide (930 pL, 300 mM), 4-iodobenzoic acid (2.5 g, 500 mM) and 4- (dimethylamino)pyridine (1.0 mg, 0.4 mM) were added to the mixture and the reaction was stirred for 3 days at room temperature (21 °C). The resin was recovered by filtration, washed with dichloromethane (3 x 20 mL) and dried under vacuum. Formation of the target compound was confirmed by the appearance of a characteristic ester stretch in the IR spectrum of the product at 1615 cm ^-1 . For the three-phase test, polymer-supported iodobenzoic acid was added in place of haloaryls following standard cross-coupling reaction conditions. After incubation, the solid was collected by vacuum filtration, washed with dichloromethane (4 x 5.0 mL), dried under vacuum, dissolved in TFA:dichloromethane (9:1 , 2.0 mL) and stirred at room temperature for 2 h. The resin was collected by vacuum filtration and washed with dichloromethane (4 x 5.0 mL). The combined organic layers were concentrated under reduced pressure. Control reactions were conducted using methyl 4-iodobenzoate (33 mg, 25 mM, 1.0 equiv) instead of the polymer-supported iodobenzoic acid by following the general Suzuki-Miyaura coupling reaction protocol, described above.

TEM analysis of DaPdNPs and TPGS micelles

Samples were prepared for TEM analysis by incubating DaPdNPs (0.25 mM) and TPGS-1000 or TPGS-750-M (0.4% w/v) in H2O at 37 °C (220 rpm) for 12 h. One drop was then transferred on to a Formvar/Carbon 200 mesh Copper grid and left to dry in air for 10 min. Excess solution was removed by touching the grid edge with filter paper. A drop of 1% aqueous uranyl acetate was applied for 1 minute and then removed by touching the grid edge with filter paper. TEM observation was carried out as described above.

Product and Pd separation

The model reaction containing TPGS-1000 was conducted, extracted and concentrated as described above. The crude residue was dissolved in 10% ethanol (5 mL). An aliquot of the filtered solution (1 mL) was diluted in 1-methoxy-2-propanol to a final volume of 5 mL and analysed by ICP-OES immediately. As a result, no detectable amount of Pd was quantified in the sample (the calculated LoD ^[3] : 0.02 pM). This demonstrated the cross-coupling product can be easily separated from DaPdNPs catalyst in the extraction process, meaning no extra separation steps are required.

Results and Discussion

The present studies began by investigating whether bacteriogenic Pd-NPs from Desulfovibrio alaskensis G20 (DaPdNPs) could catalyse C-C bond formation. We chose the Suzuki Miyaura reaction, inspired by previous studies by Deplanche et al. who demonstrated the use of E. coli- supported Pd-NPs to catalyse the cross-coupling of aryl halides and phenylboronic acid (62% conversion, EtOH( _aq ), 80 °C) (K. Deplanche, et al. Appl. Catal., B, 2014, 147, 651). To this end, DaPdNPs were prepared from anaerobic cultures of D. alaskensis G20 grown in the presence of Na2PdCL (30 °C, 20 h) and isolated via centrifugation in 97% yield, as determined by ICP- OES. Analysis of the nanoparticles by X-ray diffraction (XRD) confirmed the presence of zero- valent Pd in a standard cubic arrangement. To test the reactivity of DaPdNPs we used the substrates 4-bromoanisole 1 and phenylboronic acid 2. Reactions were carried out in deionised water at 37 °C for 20 h in the presence of ligands that are known to increase the reactivity of Pd in aqueous media. Encouragingly, 4-methoxybiphenyl 3 was detected in all reactions and also in the absence of any additional ligand (see Table 1 , entry 1). The ligand-free activity of biogenic MNPs at ambient temperature is rare and suggested to us that DaPdNPs possess unique catalytic properties. No product was observed using 4-chloroanisole, and 4-iodoanisole showed no increase in reactivity when compared to 1. Interestingly, the addition of triphenylphosphine decreased the cross coupling of 1 and 2 to 24%, whereas the bis- phenylphosphine ligands JohnPhos 4 (see Fig. 2) and XPhos 6 (see Fig. 2) typically used in Buchwald-Hartwig amination reactions increased the yield to 38% and 40%, respectively. The water-soluble ligands sSPhos 7 (see Fig. 2) and AmPyol 8 (see Fig. 2) decreased the yield 2- 3-fold, despite being widely used to enhance the reactivity of Pd catalysed reactions in aqueous and/or biological conditions. However, the addition of tetramethylguanidine (TMG, 9) (see Fig. 2) increased the yield of 3 to 62%. As the cell membrane of D. alaskensis is negatively charged and tightly associates to Pd-NPs the co-localisation of cationic TMG to the cell surface under the reaction conditions could account for this observation. No further improvement in yield was observed using green tea polyphenols, despite the use of these plant-derived compounds being reported to enable ligand-free cross coupling reactions under aqueous conditions (H. Veisi, A. Rostami and M. Shirinbayan, Appl. Organomet. Chern., 2017, 31 , e3609). Finally, we examined the mechanism of the reaction and compared the activity of biogenic nanoparticles to chemically synthesised equivalents. We confirmed the reaction is heterogeneous using a three-phase test by observing no product formation when using polymer-supported iodobenzoic acid (Table 1 , entry 10), eliminating the possibility that catalysis occurs via the leaching of soluble Pd from DaPdNPs into solution. Interestingly, 3 was formed in <1 % yield when using chemically synthesised heterogeneous Pd catalysts under the same reaction conditions. For example, Pd nanoparticles generated by chemical vapor deposition (cPdNP) and Pd on activated carbon (Pd/C) both afforded 3 in <1% yield, increasing to 5% for Pd/C in the presence of 9 (Table 1 , entries 11-16). This intriguing result suggests a unique feature of nanoparticle biosynthesis in D. alaskensis that is especially suited to the generation of highly active heterogeneous Pd catalysts.

Table 1 Catalyst, ligand and additive screen for the DaPdNP catalysed Suzuki Miyaura reaction. ^[al

[a] Reactions were performed using 1 (25 mM), 2 (30 mM), K2CO3 (30 mM), Rd catalyst (0.25 mM) and ligand (2.5 mM) in sealed tubes under an atmosphere of air. Product concentrations were determined by ¹ H NMR spectroscopy relative to an internal standard of TMB. All data shown is an average of three experiments to one standard deviation, [b] polymer-supported iodobenzoic acid was used, [c] 2% w/vol.

Having confirmed the activity of DaPdNPs under aqueous conditions we moved on to examine methods to increase their activity in vivo. As Pd nanoparticles associate to the outer membrane of D. alaskensis, we hypothesised that co-localisation of 1 and 2 to this region might also accelerate product formation. To begin, we examined the effect of co-localising the reaction within TPGS micelles (see Fig. 2). These vitamin E-derived surfactants are known to selfassemble in aqueous solution and promote organic reactions by co-localising reactants in the hydrophobic micelle interior (M. Cortes-Clerget et al., Nat. Commun., 2019, 10, 2169; and S. C. Cosgrove et al., Angew. Chem. Int. Ed. Engl., 2020, 59, 18156). They also associate with cell membranes and accelerate flux through engineered metabolic pathways by sequestering hydrophobic metabolites (S. Wallace and E. P. Balskus, Angew. Chem. Int. Ed. Engl., 2016, 55, 6023). To our surprise, the addition of 2% w/v TPGS-750-M and TPGS-1000 significantly increased the reactivity of DaPdNPs in the absence of ligand, affording 3 in 75% and 99% yield, respectively.

Table 2 Micellar coupling reaction in the presence of various ligands

The yield shown above is the average of triplicate experiments ([a]: duplicate).

In all cases, the hydroxylated surfactant TPGS-1000 outperformed the O-methylated congener TPGS-750-M. To explore the reasons for this, we examined cells by transmission electron microscopy (TEM). Interestingly, the addition of TPGS-1000 produced highly-ordered micelles at the cell surface (Fig. 3A), whereas TPGS-750-M produced disordered agglomerates that appeared to disrupt cell morphology. Although the increased membrane penetration of TPGS- 750-M has been observed in E. collar^ attributed to the increased hydrophobicity of the micelle surface (Fig. 2, R=CHs, n=17), this was shown to have no effect on the rate of reactions in the micelle interior (S. Wallace, 2016, supra). For D. alaskensis, however, TPGS-750-M micelles embedded in the cell membrane are distanced from MNPs bound at the cell surface and could therefore explain the reduced yield of 3. Extracellular presentation of DaPdNPs could also combine with favourable hydrogen bonding interactions between TPGS-1000 (Fig. 2, R=H, n=23) and outer-cell matrix polysaccharides surrounding the Pd nanoparticles to co-localise the substrates and catalyst. Altogether, this provides evidence to support the hypothesis that TPGS-1000 and TPGS-750-M enhance the reactivity of biogenic Pd nanoparticles from D. alaskensis by localising the reaction components at the outer cell membrane. To further assess the extent to which TPGS micelles accelerate the reaction we moved on to measure product formation over time (Fig. 3B). In the absence of TPGS-750-M or TPGS-1000, the reaction reached 40 and 60% conversion after 20 h in the presence of ligand 6 or 9, respectively. In contrast, TPGS-1000 accelerated the formation of 3 to 90% after 4 h in the absence of ligand and to >99% and 93% yield using 6 and 9, respectively (Fig. 3C). High reactivity was also maintained at low catalyst loading. For example, reducing DaPdNPs to 1 mol% reduced product conversion to 25% in the absence of micelles, whereas quantitative conversion was observed in the presence of TPGS-1000 at catalyst loadings of >0.5 mol% (Fig. 3D). This reduced to 31% and 4% yield at 0.1 mol% Pd in the presence and absence of TPGS-1000, respectively. Overall, this dramatic effect (>5-fold) highlights the unique benefits of micellar catalysis in D. alaskensis cells and how this can be combined with organic ligands designed for use in synthetic chemistry to enhance the reactivity of biological metals in vivo.

Using these optimised conditions, we next investigated the reaction scope using a range of aryl bromide and aryl boronic acid substrates (Fig. 4). This included arenes containing a range of electron withdrawing and donating function groups at ortho-, meta- and para-positions and included the heterocyclic (methoxy)pyridyl boronic acid (MeOPyB, 16). To our delight, DaPdNPs were effective catalysts for most substrates tested and when combined with TPGS- 1000 enabled the formation of challenging sp ² C-C bonds. Electron withdrawing acyl groups were tolerated at all positions in the aryl bromide substrate. Reactivity was reduced for orthoacyl and ortho-ethoxy substrates 12 and 19 due to steric hindrance, but could be increased through the addition of TPGS-1000. For the cross coupling of 11 and 16, 12 and 17, 12 and 19, 13 and 16 and 14 and 16, the use of TPGS-1000 increased product yield >2-fold. This is particularly impressive for heteroaromatic substrates and the cross-coupling of two orthosubstituted aromatics. Notably, 14 and 19 were unreactive in the presence of DaPdNPs but product formation was increased to 42% in the presence of TPGS-1000 micelles. Overall, this shows that TPGS micelles not only accelerate product formation in the presence of cells but also increase the overall reaction scope of biogenic PdNPs

Conclusions

In summary, we have demonstrated that Pd° nanoparticles synthesised by Desulfovibrio alaskensis are highly active heterogeneous catalysts for the Suzuki Miyaura reaction of aryl bromides and phenylboronic acids. These biological catalysts can be readily prepared from bacterial cell culture using common Pd salts in high yield (>97%) and outperform other available heterogenous Pd catalysts generated via chemical or biological methods. We show that reactions catalysed by these nanoparticles can be enhanced using organic ligands and/or designer micelles to co-localise substrates at the cell membrane. The combination of organic chemistry and microbiology tools results in an overall reaction that is highly efficient (>99% yield, 0.5 mol% Pd), occurs under ambient conditions (aqueous media, 37 °C) and can be applied to a wide range of substrates to generate products containing challenging sp ² C-C bonds. To the best of our knowledge, this is the first report of a ligand-free Suzuki-Miyaura reaction catalysed by a biological Pd catalyst in micellar nanoreactors.

Sonogashira Coupling

General Sonogashira coupling reaction

Cross coupling reactions were carried out using the following procedure: DaPdNPs were added to a 15 mL Hungate tube containing aryl halide (25/30 mM), phenylacetylene (30/60 mM), base and surfactant (2% w/vol) in 5 mL H2O. The tubes were sealed with butyl rubber septa and screw-caps and incubated at 37 °C (200 rpm) for 20 h in a New Brunswick Innova 44 incubator shaker (Eppendorf). After this time, the reactions were cooled to room temperature, extracted with dichloromethane (DCM, 3 x 1.7 mL) and concentrated under reduced pressure. The crude residue was dissolved in 1 mL CDCI3 containing 10 mM TMB and analyzed by ¹ H NMR spectroscopy.

Coupling reaction optimisation

Unless otherwise noted, model reaction (DaPdNPs (0.3 mM), halide (30 mM), phenylacetylene (60 mM), base and surfactant (2% w/vol) in H2O (5 mL)) was used by following the above general reaction protocol for reaction optimization and screening experiments.

For halide screening, 4-bromoanisole or 4-iodoanisole were used as the aryl halide. For base screening, KsPO^S W, CS2CO3, EtsN, /P^NEt or K2CO3 was added to the model reaction at various concentration (1.0, 1.5, 2.0 or 3.0 eq to halide concentration). For ligand screening, the model reaction was performed in the presence of XPhos, RuPhos, tBuXPhos, SPhos, JohnPhos, Cy-JohnPhos, 1 ,1 '-Bis(di-tert-butylphosphino)ferrocene (dtbpf), triphenylphosphine (PPhs), tri-terf-butylphosphine (P(tBu)3) or Cy-cBRIDP or in the absence of ligands. For surfactant screening, TPGS-1000, TPGS-750-M, PS-750-M or PTS was added to the model reaction at 2% w/vol. The reactions were conducted and analyzed by following the general Sonogashira coupling reaction protocol.

Coupling reaction with other Pd catalysts Chemically produced Pd nanoparticle (<25 nm, >99.5%, Sigma-Aldrich product no. 686468) (cPdNP), or Pd on activated carbon (Pd/C) was added to the optimized model reaction at 0.3 mM in place of DaPdNPs. The reactions were conducted and analyzed by following the general Sonogashira coupling reaction protocol.

Investigation of the reaction scope

Optionally substituted aryl iodide (4-iodoacetophenone, 3-iodoacetophenone, 1-iodo-4- nitrobenzene or 4-iodophenol: 25 mM) and optionally substituted phenylacetylene (p- tolylacetylene, 3-pyridinylphenylacetylene, 4-methoxyphenylacetylene, 3- methoxyphenylacetylene, 2-methoxyphenylacetylene, trimethylsilylphenylacetylene :30 mM) were added to Hungate tube containing DaPdNPs (0.25 mM, 1 mol%), K2CO3 (30 mM), JohnPhos ligand (2.5 mM) and TPGS-1000 (2% w/v) in H2O (5 mL). Reactions of 22 different cross-coupling combinations were carried out and analysed by following the general Sonogashira coupling reaction protocol, described above. Product conversions were determined by quantitative ¹ H NMR analysis relative to an internal standard of TMB (10 mM).

Control Experiments

Negative control experiments were performed in the optimized model reaction (DaPdNPs (0.25 mM), 4- bromoanisole/4-iodoanisole (25 mM), phenylacetylene (30 mM) and K2COs (30 mM)) in the presence/absence of JohnPhos (2.5 mM) or TPGS-1000 (2% w/vol). The reactions were conducted and analyzed by following the general Sonogashira coupling reaction protocol.

Base-free reactions

The optimized model reaction was performed in M9-glucose in the absence of base. The reaction was conducted and analyzed by following the general Sonogashira coupling reaction protocol.

Results and Discussion

Our studies began by investigating whether Pd-NPs from Desulfovibrio alaskensis G20 could catalyze a Sonogashira cross coupling reaction under biorelevant conditions.

Palladium nanoparticles were prepared, as reported previously, by anaerobic culturing of D. alaskensis G20 in the presence of Na2PdCk followed by centrifugation and Pd quantification by ICP-OES. Following reports by Lipshutz et al., in Org. Lett., 2008, 10, 3793-3796, and ourselves, we chose 4-haloanisoles and phenylacetylene as substrates, tribasic potassium phosphate as the base and XPhos as the ligand for Pd. Reactions were conducted in aqueous media in the presence of the Vitamin E-derived surfactant TPGS-1000, which is known to form micelles that co-localize DaPdNPs and reactants, and associate with the cell membrane. Initial reactions using bromoanisole 1 afforded trace amounts of 1-methoxy-4- (phenylethynyl)benzene 4 by ¹ H NMR. Pleasingly, the use of iodoanisole 2 increased product conversion to 20%. Altering the base to cesium carbonate reduced the yield 2-fold to 9%, however the use of triethylamine increased the yield of 4 to 37%. This is in line with previous reports that triethylamine increases the yields of other C-C cross-coupling reactions catalyzed by Pd(PfBu) ₃ ) ₂ , PdCI ₂ (dtbpf) and PdCI ₂ (CH ₃ CN) ₂ in TPGS-750-M and PTS micelles (Lipshutz et al., 2008, supra). Further increasing the concentration of Et ₃ N to 90 mM resulted in a moderate increase in 4 to 51% yield. Under these conditions, eliminating the ligand decreased the yield to 27% so we next screened various phosphine ligands with the aim of further increasing product conversion (Figure 5, entries 7-15 and Figure 6A).

The use of the less-substituted RuPhos ligand 8 decreased the yield to 35%, whereas modification of the phosphine substituent on the XPhos ligand from Cy ₂ to ^Bu) ₂ in 'BuXPhos 9 had no effect. Significantly altering the electronics of the biphenyl ring through use of 2,6- dimethoxy groups in SPhos 10 increased the yield to 60%. However, use of the unsubstituted biphenylphosphine ligand JohnPhos 11 increased the yield of 4 to >99%, indicating that electronics in addition to ligand planarity was key to increasing the reactivity of DaPdNPs in TPGS-1000 micelles. Interestingly, use of the Cy-JohnPhos ligand 12 reduced reactivity, as did the use of the ferrocene-based ligand dtbpf 13. The simple phosphine ligands PPh ₃ 14 decreased the yield to 12%, whereas P('Bu) ₃ 15 and the Takasago Cy-cBRIDP ligand 16 only moderately decreased the yield of 4 to 77% and 74%, respectively.

Under these optimized conditions we found that the concentration of Et ₃ N could be reduced to 30-45 mM whilst retaining >90% conversion and could also be replaced entirely with K ₂ CO ₃ or M9 growth media (Figure 5, entries 16-18 and Figure 6B). This latter result was especially promising as it suggests that the DaPdNP catalyzed Sonogashira reaction could be interfaced with microbial metabolism and developed as a new biocompatible reaction. Together, the combined use of JohnPhos, base and TPGS-1000 increased product formation 12-fold using microbial Pd nanoparticle catalysts. Intriguingly, the use of JohnPhos alone was sufficient to increase the yield of 4 from 27% to 48% yield, whereas the use of TPGS-1000 in the absence of JohnPhos had no effect (9% yield of 4; Figure 7C). Without being bound by theory, this suggests that JohnPhos directly binds to DaPdNPs to form an active Pd complex that is sequestered into the micelle interior. Hydrogen bonding interactions between the terminal hydroxyl group of TPGS-1000 and cell surface glycans have been hypothesized to facilitate the Suzuki-Miyaura reactivity of DaPdNPs (see Y. Era et a/., Green Chem., 2021 , 23, 8886-8890). This new observation indicates that this interaction can be combined with direct activation of Pd at the cell membrane to tune and/or activate the chemistry of biogenic Pd towards new modes of reactivity. Finally, we assessed the scope of the DaPdNP catalyzed Sonogashira reaction under our optimized conditions using a range of alkyne and aryl iodide substrates containing heteroatoms, electron-withdrawing and electron-donating functional groups (Figure 7). Product formation was observed for all substrates in up to 99% yield and improved up to 6- fold by the presence of TPGS-1000. This was particularly effective for the coupling of poorly reactive electron-deficient aryl iodides containing para-NC>2 substituents (3- to 6-fold increase) and their coupling to heterocyclic 3-ethynylpyridine (Figure 7).

Conclusions

In summary, we have reported that biogenic Pd nanoparticles generated by the anaerobic bacterium Desulfovibrio alaskensis G20 catalyze the Sonogashira cross coupling of phenylacetylenes and aryl iodides in membrane associated TPGS micelles. The reaction occurs under mild conditions (aqueous media, 37 °C, pH 7.4) using a range of substrates, and outperforms other heterogenous Pd catalysts generated via chemical methods. The combined use of transition metal catalysts from bacteria with new ligands and surfactants from the field of organic chemistry promises to enable the sustainable synthesis of novel compounds that are currently inaccessible to engineered biological systems.