De Novo Resistance to Arg10-Teixobactin Occurs Slowly and Is Costly.

Bacterial pathogens are rapidly evolving resistance to all clinically available antibiotics. One part of the solution to this complex issue is to better understand the resistance mechanisms to new and existing antibiotics. Here, we focus on two antibiotics. Teixobactin is a recently discovered promising antibiotic that is claimed to "kill pathogens without detectable resistance" (L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, et al., Nature 517:455-459, 2015, https://doi.org/10.1038/nature14098). Moenomycin A has been extensively used in animal husbandry for over 50 years with no meaningful antibiotic resistance arising. However, the nature, mechanisms, and consequences of the evolution of resistance to these "resistance-proof" compounds have not been investigated. Through a fusion of experimental evolution, whole-genome sequencing, and structural biology, we show that Staphylococcus aureus can develop significant resistance to both antibiotics in clinically meaningful timescales. The magnitude of evolved resistance to Arg10-teixobactin is 300-fold less than to moenomycin A over 45 days, and these are 2,500-fold and 8-fold less than evolved resistance to rifampicin (control), respectively. We have identified a core suite of key mutations, which correlate with the evolution of resistance, that are in genes involved in cell wall modulation, lipid synthesis, and energy metabolism. We show the evolution of resistance to these antimicrobials translates into significant cross-resistance against other clinically relevant antibiotics for moenomycin A but not Arg10-teixobactin. Lastly, we show that resistance is rapidly lost in the absence of antibiotic selection, especially for Arg10-teixobactin. These findings indicate that teixobactin is worth pursuing for clinical applications and provide evidence to inform strategies for future compound development and clinical management.

The C-Type Lysozyme from the upper Gastrointestinal Tract of Opisthocomus hoatzin, the Stinkbird.

Muramidases/lysozymes are important bio-molecules, which cleave the glycan backbone in the peptidoglycan polymer found in bacterial cell walls. The glycoside hydrolase (GH) family 22 C-type lysozyme, from the folivorous bird Opisthocomus hoazin (stinkbird), was expressed in Aspergillus oryzae, and a set of variants was produced. All variants were enzymatically active, including those designed to probe key differences between the Hoatzin enzyme and Hen Egg White lysozyme. Four variants showed improved thermostability at pH 4.7, compared to the wild type. The X-ray structure of the enzyme was determined in the apo form and in complex with chitin oligomers. Bioinformatic analysis of avian GH22 amino acid sequences showed that they separate out into three distinct subgroups (chicken-like birds, sea birds and other birds). The Hoatzin is found in the "other birds" group and we propose that this represents a new cluster of avian upper-gut enzymes.

The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes.

The pathogenic bacterium Group A Streptococcus pyogenes produces several extracellular DNases that have been shown to facilitate invasive infection by evading the human host immune system. DNases degrade the chromatin in neutrophil extracellular traps, enabling the bacterium to evade neutrophil capture. Spd1 is a type I, nonspecific ßßα/metal-dependent nuclease from Streptococcus pyogenes, which is encoded by the SF370.1 prophage and is likely to be expressed as a result of prophage induction. We present here the X-ray structure of this DNase in the wild-type and Asn145Ala mutant form. Through structural and sequence alignments as well as mutagenesis studies, we have identified the key residues His121, Asn145 and Glu164, which are crucial for Spd1 nucleolytic activity and shown the active site constellation. Our wild-type structure alludes to the possibility of a catalytically blocked dimeric form of the protein. We have investigated the multimeric nature of Spd1 using size-exclusion chromatography with multi-angle light scattering (SEC-MALLS) in the presence and absence of the divalent metal ion Mg(2+), which suggests that Spd1 exists in a monomeric form in solution.

Rational design of a cyclohexanone dehydrogenase for enhanced α,ß-desaturation and substrate specificity.

The selective α,ß-desaturation of cyclic carbonyl compounds, which are found in the core of many steroid and bioactive molecules, using green chemistry is highly desirable. To achieve this task, we have for the first time described and solved the de novo structure of a member of the cyclohexanone dehydrogenase class of enzymes. The breadth of substrate specificity was investigated by assaying the cyclohexanone dehydrogenase, from Alicycliphilus denitrificans, against several cyclic ketones, lactones and lactams. To investigate substrate binding, a catalytic variant, Y195F, was generated and used to obtain a crystallographic complex with the natural substrate, cyclohexanone. This revealed substrate-active site interactions, as well as the proximity of the cofactor, flavin adenine dinucleotide, and enabled us to propose a mechanistic function to key amino acids. We then used molecular dynamic simulations to guide design to add functionality to the cyclohexanone dehydrogenase enzyme. The resulting W113A variant had overall improved enzyme activity and substrate scope, i.e., accepting the bulkier carbonyl compound, dihydrocoumarin. Structural analysis of the W113A variant revealed a broader, more open active site, which helped explain the modified substrate specificity. This work paves the way for future bespoke regioselective α,ß-desaturation in the synthesis of important bioactive molecules via rational enzyme engineering.

Structure and activity of the Streptococcus pyogenes family GH1 6-phospho-ß-glucosidase SPy1599.

The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide-processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 α-1,6- and GH38 α-1,3-mannosidases (SPy1603 and SPy1604), a GH84 ß-hexosaminidase (SPy1600) and a putative GH2 ß-galactosidase (SPy1586), as well as SPy1599, a family GH1 `putative ß-glucosidase'. Here, the solution of the three-dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (ß/α)(8)-barrel, consistent with CAZy family GH1 and other members of the GH-A clan. SPy1599 has been annotated in sequence depositions as a ß-glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three-dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate-binding pocket in the active site and has possible 6-phospho-ß-glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6-phospho-ß-glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6-phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP-PTSs).

The copper active site of CBM33 polysaccharide oxygenases.

The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61.

Edible flowers of Helichrysum italicum: Composition, nutritive value, and bioactivities.

Helichrysum italicum (H. italicum) is a halophyte shrub with bright yellow flowers with a strong curry-like aroma. The essential oils of H. italicum have been used in the production of cosmetics and pharmaceuticals, due to their antiallergic and anti-inflammatory properties. In the agri-food sector, H. italicum flowers can be used for seasoning and flavoring food, and as natural food preservatives. Here, we report on the composition, bioactive compounds, and nutritive value of H. italicum flowers. Flowers were mainly composed of carbohydrates (>80 % dry weight), followed by minerals (6.31 ± 0.95 % dw), protein (5.44 ± 0.35 % dw), and lipids (3.59 % ± 0.53 % dw). High percentages of Fe, Zn, Ca, and K were found in the flower material, along with a high content in antioxidants, polyphenols, and carotenoids, as corroborated by the nuclear magnetic resonance (NMR) data. Flowers were mainly composed of saturated fatty acids (SFAs) (54.50 ± 0.95 % of total FA), followed by polyunsaturated fatty acids (PUFAs) (37.73 ± 1.25 % of total FA) and monounsaturated fatty acids (MUFAs) (7.77 ± 0.34 %), as detected by gas chromatography mass spectrometry (GC-MS). The omega-6 PUFA linoleic acid (22.55 ± 0.76 % of total FA) was the most abundant fatty acid found. Flower extracts showed antimicrobial activity against Saccharomyces cerevisiae and Komagataella phaffii, as well as against Gram-negative (Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus) bacteria. H. italicum flower material was nontoxic to human intestinal Caco-2 model cells at concentrations up to 1.0 % w/v.

Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations.

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α-helices, the α-domain, and another predominantly composed of ß-sheets, the ß-domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate-binding cleft between the α- and ß-domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X-ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate-binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site-directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate-binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases.

Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity.

O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.

The structure of a family GH25 lysozyme from Aspergillus fumigatus.

Lysins are important biomolecules which cleave the bacterial cell-wall polymer peptidoglycan. They are finding increasing commercial and medical application. In order to gain an insight into the mechanism by which these enzymes operate, the X-ray structure of a CAZy family GH25 ;lysozyme' from Aspergillus fumigatus was determined. This is the first fungal structure from the family and reveals a modified alpha/beta-barrel-like fold in which an eight-stranded beta-barrel is flanked by three alpha-helices. The active site lies toward the bottom of a negatively charged pocket and its layout has much in common with other solved members of the GH25 and related GH families. A conserved active-site DXE motif may be implicated in catalysis, lending further weight to the argument that this glycoside hydrolase family operates via a ;substrate-assisted' catalytic mechanism.

Structures of two truncated phage-tail hyaluronate lyases from Streptococcus pyogenes serotype M1.

The crystal structures of truncated forms of the Streptococcus pyogenes phage-encoded hyaluronate lyases HylP2 and HylP3 were determined by molecular replacement to 1.6 and 1.9 A resolution, respectively. The truncated forms crystallized in a hexagonal space group, forming a trimer around the threefold crystallographic axis. The arrangement of the fold is very similar to that observed in the structure of the related hyaluronate lyase HylP1. The structural elements putatively involved in substrate recognition are found to be conserved in both the HylP2 and HylP3 fragments.

Cysteines and Disulfide-Bridged Macrocyclic Mimics of Teixobactin Analogues and Their Antibacterial Activity Evaluation against Methicillin-Resistant Staphylococcus Aureus (MRSA).

Teixobactin is a highly potent cyclic depsipeptide which kills a broad range of multi-drug resistant, Gram-positive bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA) without detectable resistance. In this work, we describe the design and rapid synthesis of novel teixobactin analogues containing two cysteine moieties, and the corresponding disulfide-bridged cyclic analogues. These analogues differ from previously reported analogues, such as an Arg10-teixobactin, in terms of their macrocyclic ring size, and feature a disulfide bridge instead of an ester linkage. The new teixobactin analogues were screened against Methicillin-resistant Staphylococcus aureus and Methicillin-sensitive Staphylococcus aureus. Interestingly, one teixobactin analogue containing all l-amino acid building blocks showed antibacterial activity against MRSA for the first time. Our data indicates that macrocyclisation of teixobactin analogues with disulfide bridging is important for improved antibacterial activity. In our work, we have demonstrated the unprecedented use of a disulfide bridge in constructing the macrocyclic ring of teixobactin analogues.

Design and Syntheses of Highly Potent Teixobactin Analogues against Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), and Vancomycin-Resistant Enterococci (VRE) in Vitro and in Vivo.

The cyclic depsipeptide, teixobactin, kills a number of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium tuberculosis without detectable resistance. To date, teixobactin is the only molecule in its class that has shown in vivo antibacterial efficacy. In this work, we designed and synthesized 10 new in vivo ready teixobactin analogues. These analogues showed highly potent antibacterial activities against Staphylococcus aureus, MRSA, and vancomycin-resistant enterococci (VRE) in vitro. One analogue, d-Arg4-Leu10-teixobactin, 2, was found to be noncytotoxic in vitro and in vivo. Moreover, topical instillation of peptide 2 in a mouse model of S. aureus keratitis decreased the bacterial bioburden (>99.0% reduction) and corneal edema significantly as compared to untreated mouse corneas. Collectively, our results have established the high therapeutic potential of a teixobactin analogue in attenuating bacterial infections and associated severities in vivo.

FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells.

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.

Structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum.

The digestion of the plant cell wall requires the concerted action of a diverse repertoire of enzyme activities. An important component of these hydrolase consortia are arabinofuranosidases, which release L-arabinofuranose moieties from a range of plant structural polysaccharides. The anaerobic bacterium Clostridium thermocellum, a highly efficient plant cell wall degrader, possesses a single alpha-L-arabinofuranosidase (EC 3.2.1.55), CtAraf51A, located in GH51 (glycoside hydrolase family 51). The crystal structure of the enzyme has been solved in native form and in 'Michaelis' complexes with both alpha-1,5-linked arabinotriose and alpha-1,3 arabinoxylobiose, both forming a hexamer in the asymmetric unit. Kinetic studies reveal that CtAraf51A, in contrast with well-characterized GH51 enzymes including the Cellvibrio japonicus enzyme [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607-614], catalyses the hydrolysis of alpha-1,5-linked arabino-oligosaccharides and the alpha-1,3 arabinosyl side chain decorations of xylan with equal efficiency. The paucity of direct hydrogen bonds with the aglycone moiety and the flexible conformation adopted by Trp(178), which stacks against the sugar at the +1 subsite, provide a structural explanation for the plasticity in substrate specificity displayed by the clostridial arabinofuranosidase.

Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a beta-N-acetylglucosaminidase and not a hyaluronidase.

Group A streptococcus (Streptococcus pyogenes) is the causative agent of severe invasive infections such as necrotizing fasciitis (the so-called 'flesh eating disease') and toxic-shock syndrome. Spy1600, a glycoside hydrolase from family 84 of the large superfamily of glycoside hydrolases, has been proposed to be a virulence factor. In the present study we show that Spy1600 has no activity toward galactosaminides or hyaluronan, but does remove beta-O-linked N-acetylglucosamine from mammalian glycoproteins--an observation consistent with the inclusion of eukaryotic O-glycoprotein 2-acetamido-2-deoxy-beta-D-glucopyranosidases within glycoside hydrolase family 84. Proton NMR studies, structure-reactivity studies for a series of fluorinated analogues and analysis of 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline as a competitive inhibitor reveals that Spy1600 uses a double-displacement mechanism involving substrate-assisted catalysis. Family 84 glycoside hydrolases are therefore comprised of both prokaryotic and eukaryotic beta-N-acetylglucosaminidases using a conserved catalytic mechanism involving substrate-assisted catalysis. Since these enzymes do not have detectable hyaluronidase activity, many family 84 glycoside hydrolases are most likely incorrectly annotated as hyaluronidases.

Syntheses of potent teixobactin analogues against methicillin-resistant Staphylococcus aureus (MRSA) through the replacement of l-allo-enduracididine with its isosteres.

The recently discovered cyclic depsipeptide, teixobactin, is a highly potent antibiotic against multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Mycobaterium tuberculosis. It comprises of 4 D amino acids and a rare l-allo-enduracididine amino acid. The synthesis of a properly protected l-allo-enduracididine amino acid and its incorporation into teixobactin is time consuming, synthetically challenging and low yielding and is therefore a major bottleneck in the development of potent analogues of teixobactin. In this article, we have synthesised 8 analogues of teixobactin using commercially available building blocks by replacing the l-allo-enduracididine amino acid with its isosteres. Furthermore, we have tested all the compounds against a panel of Gram positive bacteria including MRSA and explained the observed trend in biological activity. Although all the analogues were active, three analogues from this work, showed very promising activity against MRSA (MIC 1 µg mL-1). We can conclude that amino acids which are the closest isosteres of l-allo-enduracididine are the key to synthesising simplified potent analogues of teixobactin using rapid syntheses and improved yields.

Defining the molecular structure of teixobactin analogues and understanding their role in antibacterial activities.

The discovery of the highly potent antibiotic teixobactin, which kills the bacteria without any detectable resistance, has stimulated interest in its structure-activity relationship. However, a molecular structure-activity relationship has not been established so far for teixobactin. Moreover, the importance of the individual amino acids in terms of their l/d configuration and their contribution to the molecular structure and biological activity are still unknown. For the first time, we have defined the molecular structure of seven teixobactin analogues through the variation of the d/l configuration of its key residues, namely N-Me-d-Phe, d-Gln, d-allo-Ile and d-Thr. Furthermore, we have established the role of the individual d amino acids and correlated this with the molecular structure and biological activity. Through extensive NMR and structural calculations, including molecular dynamics simulations, we have revealed the residues for maintaining a reasonably unstructured teixobactin which is imperative for biological activity.

Teixobactin analogues reveal enduracididine to be non-essential for highly potent antibacterial activity and lipid II binding.

Teixobactin is a highly promising antibacterial depsipeptide consisting of four d-amino acids and a rare l-allo-enduracididine amino acid. l-allo-Enduracididine is reported to be important for the highly potent antibacterial activity of teixobactin. However, it is also a key limiting factor in the development of potent teixobactin analogues due to several synthetic challenges such as it is not commercially available, requires a multistep synthesis, long and repetitive couplings (16-30 hours). Due to all these challenges, the total synthesis of teixobactin is laborious and low yielding (3.3%). In this work, we have identified a unique design and developed a rapid synthesis (10 min µwave assisted coupling per amino acid, 30 min cyclisation) of several highly potent analogues of teixobactin with yields of 10-24% by replacing the l-allo-enduracididine with commercially available non-polar residues such as leucine and isoleucine. Most importantly, the Leu10-teixobactin and Ile10-teixobactin analogues have shown highly potent antibacterial activity against a broader panel of MRSA and Enterococcus faecalis (VRE). Furthermore, these synthetic analogues displayed identical antibacterial activity to natural teixobactin (MIC 0.25 µg mL-1) against MRSA ATCC 33591 despite their simpler design and ease of synthesis. We have confirmed lipid II binding and measured the binding affinities of individual amino acid residues of Ala10-teixobactin towards geranyl pyrophosphate by NMR to understand the nature and strength of binding interactions. Contrary to current understanding, we have shown that a cationic amino acid at position 10 is not essential for target (lipid II) binding and potent antibacterial activity of teixobactin. We thus provide strong evidence contrary to the many assumptions made about the mechanism of action of this exciting new antibiotic. Introduction of a non-cationic residue at position 10 allows for tremendous diversification in the design and synthesis of highly potent teixobactin analogues and lays the foundations for the development of teixobactin analogues as new drug-like molecules to target MRSA and Mycobacterium tuberculosis.

Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus.

The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 A (1 A=0.1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code 1OYG) as a search model. LsdA displays a five-bladed beta-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys339-Cys395 disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.