Crystal structure of a phenoxyl radical complex relevant to the metal site of the galactose oxidase enzyme: A facile one-pot synthesis, evidence for hydrogen atom transfer and DNA cleavage via self-activation.

Copper complexes [Cu(L1H)ClO4] (1) and [Cu(L2)NO3] (2), which are relevant to the metal site of the galactose oxidase enzyme, were synthesized and characterized by different spectroscopic methods. L1H2 and L2H2 [where L1H2 stands for 2,2'-((1E,1'E)(2,2'-(pyridine-2,6-diyl)bis(2-phenylhydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol and L2H2 stands for 6,6'-((1E,1'E)-(2,2'-(pyridine-2,6-diyl)bis(2-phenylhydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(2,4-di-tert-butylphenol), H stands for dissociable proton] are pentadentate ligands. These ligands provide pyridyl N, two imine N, and two non-innocent phenoxyl and phenolato O donors, forming complex 1 as a non-radical complex, while complex 2 is a phenoxyl radical complex. The molecular structures of complexes 1 and 2 were authenticated by X-ray crystallography. Benzyl alcohol oxidation was investigated, and the conversion of 9,10-dihydroanthracene to anthracene was examined to scrutinize the H-atom abstraction reaction. Nuclease activity with complexes 1 and 2 was investigated by self-activated plasmid DNA (pBR322) cleavage. Non-innocent properties of the ligand-containing phenolato function were investigated by DFT calculations.

Tailoring Galactose Oxidase for Self-Powered Benzyl Alcohol Sensing.

Benzyl alcohol (BnOH) is a widely-used preservative in a variety of cosmetics, but the excess addition (≥1.0 %) may cause strong symptoms such as nausea, gastrointestinal irritation, convulsion, even death, making it crucial to monitor and control the addition quantity. Herein, we have developed a test-strip-like BnOH detection method via tailoring a galactose oxidase (GOase) towards BnOH oxidation and preparing a self-powered electrochromic strip for BnOH concentration visualization. A double-substituted GOase variant (Y329S/R330F), on the basis of the reported GOase M1 , has been obtained by semi-rational design with a 24.6-fold improved activity towards BnOH compared to GOase M1 . The GOase Y329S/R330F electrode has a response to BnOH with a linear range of 0.04 to 3.25 mM (R2 =0.9985), a sensitivity of 122.78 µA mM-1 cm-2 , and a detection limit of 0.03 mM (S/N=3). Coupling an electrochromic Prussian blue (PB) cathode helps the successful sensing visualization without any further power supply. The present sensing is more convenient and user-friendly than the generally used gas chromatography (GC) and high performance liquid chromatography (HPLC), and brings a more accessible solution to the field of quality controlling.

Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity.

Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5_2 enzymes towards galactose-containing saccharides.

Copper radical oxidases: galactose oxidase, glyoxal oxidase, and beyond!

The copper radical oxidases (CROs) are an evolutionary and functionally diverse group of enzymes established by the historically significant galactose 6-oxidase and glyoxal oxidase from fungi. Inducted in 2013, CROs now constitute Auxiliary Activity Family 5 (AA5) in the Carbohydrate-Active Enzymes (CAZy) classification. CROs catalyse the two-electron oxidation of their substrates using oxygen as the final electron acceptor and are particularly distinguished by a cross-linked tyrosine-cysteine co-factor that is integral to radical stabilization. Recently, there has been a significant increase in the biochemically and structurally characterized CROs, which has revealed an expanded natural diversity of catalytic activities in the family. This review provides a brief historical introduction to CRO biochemistry and structural biology as a foundation for an update on current advances in CRO enzymology, biotechnology, and biology across kingdoms of life.

One step cascade detection of galactose based on a galactose oxidase-composited peroxidase nanozyme.

Abnormal galactose metabolism is the main cause of galactosemia, which makes the accurate and rapid analysis of galactose levels in food and organism the key issue at present. In this study, a novel strategy for one-step galactose determination was proposed based on galactose oxidase and copper-based metal-organic framework complexes (GAOx@MOF) with dual catalytic activities at neutral pH. Typically, GAOx catalyzes the oxidation of the C6 hydroxyl group of D-galactose to generate an aldehyde (D-galactose-hexanedial), and coupled with the reduction of dioxygen to H2O2, which was immediately transformed to ˙OH by mimicking peroxidase activity and at the same time oxidized ABTS to a green product with a clear colorimetric signal. The whole process was completed using one buffer, which simplified the procedure and increased the sensitivity. Moreover, the proposed method can also be used for the quantitative analysis of galactose. It showed a good linear relationship at 20-1000 µM, while the LOD was 6.67 µM. Furthermore, the strategy has been successfully utilized for galactose determination in milk samples, which proved its promising applications in clinical analysis and the food industry.

Harnessing galactose oxidase in the development of a chemoenzymatic platform for glycoconjugate vaccine design.

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.

Toward scalable biocatalytic conversion of 5-hydroxymethylfurfural by galactose oxidase using coordinated reaction and enzyme engineering.

5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. Toward addressing the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, here we report coordinated reaction and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The biocatalyst and reaction conditions presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive blueprint for further development, giving hope for the creation of a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.

Kß X-ray Emission Spectroscopy as a Probe of Cu(I) Sites: Application to the Cu(I) Site in Preprocessed Galactose Oxidase.

Cu(I) active sites in metalloproteins are involved in O2 activation, but their O2 reactivity is difficult to study due to the Cu(I) d10 closed shell which precludes the use of conventional spectroscopic methods. Kß X-ray emission spectroscopy (XES) is a promising technique for investigating Cu(I) sites as it detects photons emitted by electronic transitions from occupied orbitals. Here, we demonstrate the utility of Kß XES in probing Cu(I) sites in model complexes and a metalloprotein. Using Cu(I)Cl, emission features from double-ionization (DI) states are identified using varying incident X-ray photon energies, and a reasonable method to correct the data to remove DI contributions is presented. Kß XES spectra of Cu(I) model complexes, having biologically relevant N/S ligands and different coordination numbers, are compared and analyzed, with the aid of density functional theory (DFT) calculations, to evaluate the sensitivity of the spectral features to the ligand environment. While the low-energy Kß2,5 emission feature reflects the ionization energy of ligand np valence orbitals, the high-energy Kß2,5 emission feature corresponds to transitions from molecular orbitals (MOs) having mainly Cu 3d character with the intensities determined by ligand-mediated d-p mixing. A Kß XES spectrum of the Cu(I) site in preprocessed galactose oxidase (GOpre) supports the 1Tyr/2His structural model that was determined by our previous X-ray absorption spectroscopy and DFT study. The high-energy Kß2,5 emission feature in the Cu(I)-GOpre data has information about the MO containing mostly Cu 3dx2-y2 character that is the frontier molecular orbital (FMO) for O2 activation, which shows the potential of Kß XES in probing the Cu(I) FMO associated with small-molecule activation in metalloproteins.

Formation of Monofluorinated Radical Cofactor in Galactose Oxidase through Copper-Mediated C-F Bond Scission.

Galactose oxidase (GAO) contains a Cu(II)-ligand radical cofactor. The cofactor, which is autocatalytically generated through the oxidation of the copper, consists of a cysteine-tyrosine radical (Cys-Tyr•) as a copper ligand. The formation of the cross-linked thioether bond is accompanied by a C-H bond scission on Tyr272 with few details known thus far. Here, we report the genetic incorporation of 3,5-dichlorotyrosine (Cl2-Tyr) and 3,5-difluorotyrosine (F2-Tyr) to replace Tyr272 in the GAOV previously optimized for expression through directed evolution. The proteins with an unnatural tyrosine residue are catalytically competent. We determined the high-resolution crystal structures of the GAOV, Cl2-Tyr272, and F2-Tyr272 incorporated variants at 1.48, 1.23, and 1.80 Šresolution, respectively. The structural data showed only one halogen remained in the cofactor, indicating that an oxidative carbon-chlorine/fluorine bond scission has occurred during the autocatalytic process of cofactor biogenesis. Using hydroxyurea as a radical scavenger, the spin-coupled hidden Cu(II) was observed by EPR spectroscopy. Thus, the structurally defined catalytic center with genetic unnatural tyrosine substitution is in the radical containing form as in the wild-type, i.e., Cu(II)-(Cl-Tyr•-Cys) or Cu(II)-(F-Tyr•-Cys). These findings illustrate a previously unobserved C-F/C-Cl bond cleavage in biology mediated by a mononuclear copper center.

Proximity Enzymatic Glyco-Remodeling Enables Direct and Highly Efficient Lipid Raft Imaging on Live Cells.

Lipid rafts, highly ordered cell membrane domains mainly composed of cholesterol, sphingolipids, and protein receptors, serve as important functional platforms for regulation of lipid/protein interactions. The major predicament in lipid raft study is the lack of direct and robust visualization tools for in situ tracking raft components. To solve this issue, we herein report a proximity enzymatic glyco-remodeling strategy for direct and highly efficient lipid raft labeling and imaging on live cells. Through cofunctionalization of raft-specific recognition motif and glycan-remodeling enzyme on gold nanoparticles, the fabricated nanoprobe can be specifically guided to the raft domains to perform catalytic remodeling on neighboring glycans. Taking advantage of the abundant glycoconjugates enriched in lipid rafts, this elaborate design achieves the translation of one raft-recognition event to multiple raft-confined labeling operations, thus, significantly increasing the labeling efficiency and imaging sensitivity. The direct covalent labeling also enables in situ and long-term tracking of raft components in live cells. The method possesses broad applicability and potential expansibility, thus, will greatly facilitate the investigations on the complex composition, organization, and dynamics of lipid rafts.

Harnessing the active site triad: merging hemilability, proton responsivity, and ligand-based redox-activity.

Metalloenzymes catalyze many important reactions by managing the proton and electron flux at the enzyme active site. The motifs utilized to facilitate these transformations include hemilabile, redox-active, and so called proton responsive sites. Given the importance of incorporating and understanding these motifs in the area of coordination chemistry and catalysis, we highlight recent milestones in the field. Work incorporating the triad of hemilability, redox-activity, and proton responsivity into single ligand scaffolds will be described.

Novel electrochemical sensing of galactose using GalOxNPs/CHIT modified pencil graphite electrode.

For the construction of galactose biosensor, chitosan was electropolymerised onto the pencil graphite electrode. This chitosan modified pencil graphite electrode acts as good matrix for immobilization of enzyme nanoparticles of galactose oxidase. Development of this nanocomposite was further confirmed by Fourier transform infrared spectroscopy and scanning electron microscopy. The presence of chitosan makes the present galactose biosensor more efficient, reproducible and stable. The sensitivity was reported 7 × 10-3 mA/mM/cm2 with linear range from 0.05 to 25 mM and better detection limit of 0.05 mM. When the solution of galactose was spiked with 0.5 mM and 1 mM, the analytical recoveries were found 98.6% and 97.6%. A better storage stability was achieved (90days) when compared to earlier reported biosensors.

Oxygen-Depleted Calixarenes as Ligands for Molecular Models of Galactose Oxidase.

A calix[4]arene ligand, in which two of the phenol functions are replaced by pyrazole units has been employed to mimic the His2 -Tyr2 (His: histidine, Tyr: tyrosine) ligand sphere within the active site of the galactose oxidase (GO). The calixarene backbone forces the corresponding copper(II) complex into a see-saw-type structure, which is hitherto unprecedented in GO modelling chemistry. It undergoes a one-electron oxidation that is centered at the phenolate donor leading to a copper-coordinated phenoxyl radical like in the GO. Accordingly, the complex was tested as a functional model and indeed proved capable of oxidizing benzyl alcohol to the respective aldehyde using two phenoxyl-radical equivalents as oxidants. Finally, the results show that the calixarene platform can be utilized to arrange donor functions to biomimetic binding pockets that allow for the creation of novel types of model compounds.

Production of Galactose Oxidase Inside the Fusarium fujikuroi Species Complex and Recombinant Expression and Characterization of the Galactose Oxidase GaoA Protein from Fusarium subglutinans.

Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of D-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s-1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M-1 s-1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides D-(+)-galactose, the purified enzyme also acted against D-(+)-raffinose, α-D-(+)-melibiose, and methyl-α-D-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.

The crystal and molecular structures of three copper-containing complexes and their activities in mimicking galactose oxidase.

The structures of three copper-containing complexes, namely (benzoato-κ2O,O')[(E)-2-({[2-(diethylamino)ethyl]imino}methyl)phenolato-κ3N,N',O]copper(II) dihydrate, [Cu(C7H5O2)(C13H19N2O)]·2H2O, 1, [(E)-2-({[2-(diethylamino)ethyl]imino}methyl)phenolato-κ3N,N',O](2-phenylacetato-κ2O,O')copper(II), [Cu(C8H7O2)(C13H19N2O)], 2, and bis[µ-(E)-2-({[3-(diethylamino)propyl]imino}methyl)phenolato]-κ4N,N',O:O;κ4O:N,N',O-(µ-2-methylbenzoato-κ2O:O')copper(II) perchlorate, [Cu2(C8H7O2)(C12H17N2O)2]ClO4, 3, have been reported and all have been tested for their activity in the oxidation of D-galactose. The results suggest that, unlike the enzyme galactose oxidase, due to the precipitation of Cu2O, this reaction is not catalytic as would have been expected. The structures of 1 and 2 are monomeric, while 3 consists of a dimeric cation and a perchlorate anion [which is disordered over two orientations, with occupancies of 0.64 (4) and 0.36 (4)]. In all three structures, the central Cu atom is five-coordinated in a distorted square-pyramidal arrangment (τ parameter of 0.0932 for 1, 0.0888 for 2, and 0.142 and 0.248 for the two Cu centers in 3). In each species, the environment about the Cu atom is such that the vacant sixth position is open, with very little steric crowding.

Mannich base Cu(II) complexes as biomimetic oxidative catalyst.

Galactose Oxidase (GOase) and catechol oxidase (COase) are the metalloenzymes of copper having monomeric and dimeric sites of coordination, respectively. This paper summarizes the results of our studies on the structural, spectral and catalytic properties of new mononuclear copper (II) complexes [CuL(OAc)] (1), and [CuL2] (2), (HL = 2,4­dichloro­6­{[(2'­dimethyl­aminoethyl)methylamino]methyl}­phenol) which can mimic the functionalities of the metalloenzymes GOase and COase. The structure of the compounds has been elucidated by X-ray crystallography and the mimicked Cu(II) catalysts were further characterized by EPR. These mimicked models were used for GOase and COase catalysis. The GOase catalytic results were identified by GC-MS and, analyzed by HPLC at room temperature. The conversion of benzyl alcohol to benzaldehyde were significant in presence of a strong base, Bu4NOMe in comparison to the neutral medium. Apart from that, despite of being monomeric in nature, both the homogeneous catalysts are very prone to participate in COase mimicking oxidation reaction. Nevertheless, during COase catalysis, complex 1 was found to convert 3,5­ditertarybutyl catechol (3,5-DTBC) to 3,5­ditertarybutyl quinone (3,5-DTBQ) having greater rate constant, kcat or turn over number (TON) value over complex 2. The generation of reactive intermediates during COase catalysis were accounted by electrospray ionization mass spectrometry (ESI-MS). Through mechanistic approach, we found that H2O2 is the byproduct for both the GOase and COase catalysis, thus, confirming the generation of reactive oxygen species during catalysis. Notably, complex 1 having mono-ligand coordinating atmosphere has superior catalytic activity for both cases in comparison to complex 2, that is having di-ligand environment.

π-π Stacking Interaction in an Oxidized CuII -Salen Complex with a Side-Chain Indole Ring: An Approach to the Function of the Tryptophan in the Active Site of Galactose Oxidase.

In order to gain new insights into the effect of the π-π stacking interaction of the indole ring with the CuII -phenoxyl radical as seen in the active form of galactose oxidase, we have prepared a CuII complex of a methoxy-substituted salen-type ligand, containing a pendent indole ring on the dinitrogen chelate backbone, and characterized its one-electron-oxidized forms. The X-ray crystal structures of the oxidized CuII complex exhibited the π-π stacking interaction of the indole ring mainly with one of the two phenolate moieties. The phenolate moiety in close contact with the indole moiety showed the characteristic phenoxyl radical structural features, indicating that the indole ring favors the π-π stacking interaction with the phenoxyl radical. The UV/Vis/NIR spectra of the oxidized CuII complex with the pendent indole ring was significantly different from those of the complex without the side-chain indole ring, and the absorption and CD spectra exhibited a solvent dependence, which is in line with the phenoxyl radical-indole stacking interaction in solution. The other physicochemical results and theoretical calculations strongly support that the indole ring, as an electron donor, stabilizes the phenoxyl radical by the π-π stacking interaction.

Functionalized carbon nanotube adsorption interfaces for electron transfer studies of galactose oxidase.

Modified electrodes featuring specific adsorption platforms able to access the electrochemistry of the copper containing enzyme galactose oxidase (GaOx) were explored, including interfaces featuring nanomaterials such as nanoparticles and carbon nanotubes (CNTs). Electrodes modified with various self-assembled monolayers (SAMs) including those with attached nanoparticles or amide-coupled functionalized CNTs were examined for their ability to effectively immobilize GaOx and study the redox activity related to its copper core. While stable GaOx electrochemistry has been notoriously difficult to achieve at modified electrodes, strategically designed functionalized CNT-based interfaces, cysteamine SAM-modified electrode subsequently amide-coupled to carboxylic acid functionalized single wall CNTs, were significantly more effective with high GaOx surface adsorption along with well-defined, more reversible, stable (≥ 8 days) voltammetry and an average ET rate constant of 0.74 s-1 in spite of increased ET distance - a result attributed to effective electronic coupling at the GaOx active site. Both amperometric and fluorescence assay results suggest embedded GaOx remains active. Fundamental ET properties of GaOx may be relevant to biosensor development targeting galactosemia while the use functionalized CNT platforms for adsorption/electrochemistry of electroactive enzymes/proteins may present an approach for fundamental protein electrochemistry and their future use in both direct and indirect biosensor schemes.

Chemoenzymatic Approach for the Proteomics Analysis of Mucin-Type Core-1 O-Glycosylation in Human Serum.

Human serum is a complex body fluid that contains various N-linked and O-linked glycoproteins. Compared with N-linked glycoproteins, the serum O-linked glycoproteins are not well-studied due to their high heterogeneity and their low abundance. Herein, we presented a novel chemoenzymatic method to analyze core-1 type of O-GalNAcylation in human serum. In this approach, the tryptic digest of serum was first subjected to PNGase F treatment to release the N-glycan and was then treated with strong acid to release sialic acid residues from mucin-type O-glycans. In this way, the internal Gal/GalNAc residues were exposed and were oxidized by the galactose oxidase to carry the aldehyde groups. The oxidized O-GalNAcylated peptides were then captured by hydrazide beads and eluted with methoxylamine for LC-MS/MS analysis. The de-N-deglycosylation decreased the abundance of N-glycopeptides, the desialylation simplified the O-glycans and the enzymatic oxidization conferred the enrichment specificity. We have demonstrated that this method was fitted to analyze O-GalNAcylated peptides with high confidence. This method was applied to analyze human serum, which resulted in the identification of 59 O-GalNAc modified peptide sequences corresponding to 38 glycoproteins from 50 µL of serum. This method is expected to have broad applications in the analysis of O-glycoproteome.

Catalytic Promiscuity of Galactose Oxidase: A Mild Synthesis of Nitriles from Alcohols, Air, and Ammonia.

We report an unprecedented catalytically promiscuous activity of the copper-dependent enzyme galactose oxidase. The enzyme catalyses the one-pot conversion of alcohols into the related nitriles under mild reaction conditions in ammonium buffer, consuming ammonia as the source of nitrogen and dioxygen (from air at atmospheric pressure) as the only oxidant. Thus, this green method does not require either cyanide salts, toxic metals, or undesired oxidants in stoichiometric amounts. The substrate scope of the reaction includes benzyl and cinnamyl alcohols as well as 4- and 3-pyridylmethanol, giving access to valuable chemical compounds. The oxidation proceeds through oxidation from alcohol to aldehyde, in situ imine formation, and final direct oxidation to nitrile.