Vinyl-pyrazole as a biomimetic acetaldehyde surrogate

Inspired by the enzyme acetylene hydratase, we investigated the reactivity of acetylene with tungsten(ii) pyrazole complexes. Our research revealed that the complex [WBr2(pz-NHCCH3)(CO)3] (pz = 3,5-dimethyl-pyrazolate) facilitates the stochiometric reaction between pzH and acetylene to give N-vinyl-pz. This vinyl compound readily hydrolyzes to acetaldehyde, mirroring the product of acetylene hydration in the enzymatic process. The formation of the vinyl compound likely involves a reactive intermediate complex where acetylene acts as a two-electron donor, in contrast to isolable acetylene complexes that are inert to nucleophilic attack by water. Results suggest an alternative mechanism for the enzyme, including vinylation of a neighboring amino acid by acetylene in the active site prior to hydration.


General considerations
All experiments were carried out under inert atmosphere employing standard Schlenk and glovebox techniques unless otherwise stated.Commercially available chemicals were used as received.Air and moisture-sensitive chemicals were stored in Schlenk flasks or under N2 atmosphere in a glovebox; liquids were additionally stored over molecular sieves.The tungsten complexes [WBr2(MeCN)2(CO)3] 1 and [WBr2(C2H2)(MeCN)2(CO)]/[WBr2(C2H2)2(MeCN)(CO)] 1,2 were prepared by published procedures using Schlenk and glovebox techniques.C2D2 was generated in situ by adding D2O to CaC2 and was passed through CaCl2 for drying.Oxygen-free solutions of NaOH were obtained by flushing the solution with N2 for 1 h.All solvents were purified by a Pure Solv Solvent Purification System and stored over activated molecular sieves (3 or 4 Å).NMR spectra were recorded using Bruker Avance III and Bruker Avance NEO 500 MHz spectrometers. 1 H NMR spectra were recorded at 300 MHz for room temperature or at 500 MHz for low-temperature measurements and referenced to residual protons of the NMR solvents. 13C NMR spectra were obtained at 75 MHz and spectra were referenced to the deuterated solvent peak.The chemical shifts δ are given in ppm.The multiplicity of peaks is denoted as broad singlet (bs), singlet (s), doublet (d), triplet (t), quadruplet (q), and multiplet (m).Coupling constants J are given in Hertz.IR spectra were recorded in the solid state at a resolution of 2 cm -1 on a Bruker ALPHA-P Diamant ATR-FTIR.HPLC-MS was carried out on an Agilent Technologies 1260 Infinity II machine on a reverse phase C18 column.Gradient elution was carried out with acetonitrile and water with 0.1 % formic acid added.A linear gradient from 30 % MeCN to 100 % MeCN over 20 min was maintained with a flow rate of 0.8 mL/min.
Samples were ionized by electrospray ionization and detected by mass spectrometer.
Elemental analyses (C, H, N) were carried out by the Department of Inorganic Chemistry at the Graz University of Technology (Heraeus Vario Elementar automatic analyzer).

Formation of aldehyde over time
A stock solution of 1 (105 mg, 169 µmol) and mesitylene (9.47 µmol, 56 µmol) as an internal standard in CD3CN (5950 µL; 7 x 850 µL) was prepared and left standing at room temperature for 12 h.Aliquots of the stock solution (850 µL each) were then placed in 25 mL Schlenk flasks, and at -10 °C (ice/NaCl) under acetylene counterflow, H2O (2.18 µL, 121 µmol, 3 equiv.)was added quickly before closing the flask and allowing for saturation of the solution with acetylene under vigours stirring for 5 min.The sealed flasks were then placed in an oil bath preheated to 60 °C and heated for the specific time stated below in Table S1.Flasks that were removed from the oil bath were quickly cooled to 0 °C and under N2 counterflow samples for 1 H NMR spectroscopy were taken directly.The 1 H NMR samples were stored at -30 °C prior to measurement.The amount of formed aldehyde is evaluated by comparing the relative integral of the internal mesitylene standard at 6.80 ppm with the integral of the aldehyde signals at 9.69 ppm for acetaldehyde, and at 9.46 ppm for crotonaldehyde, respectively, in the 1 H NMR spectrum (Fig. S17).
a The signal-to-noise ratio did not allow for quantification.
Control experiment with acetaldehyde.A stock solution of 1 (15 mg/850 µL, 24 µmol/850 µL) and mesitylene (1.12 µL/850 µL, 8 µmol/850 µL, 1/3 equiv) was prepared in CD3CN and left standing at room temperature for 12 h.A Schlenk flask was then charged with 850 µL of this stock and under N2-counterflow at 0 °C, acetaldehyde (2.71 µL, 48 µmol) followed by water (2.18 µmol, 121 µmol) was added.The flask was closed and placed in an oil bath preheated to 60 °C for 4 h.The sample was subsequently cooled to 0 °C and a sample for 1 H NMR spectroscopy was taken directly.No crotonaldehyde could be detected in the 1 H NMR spectrum (Fig. S21).
Formation of vinyl-pyrazole and subsequent hydrolysis.A J. Young NMR tube containing a solution of 1 (15 mg, 24 µmol), mesitylene (1.12 µL, 8 µmol), and C2H2 in CD3CN (600 µL) was left standing at room temperature for 24 h before recording a 1 H NMR spectrum.The presence of N-vinyl-3,5-dimethyl pyrazole and the absence of aldehyde were detected.Subsequently, 1 drop of degassed H2O was added to the mixture under inert conditions and the NMR tube was placed into an oil bath and heated to 60 °C for 24 h before recording another 1 H NMR spectrum.Acetaldehyde was detected while signals corresponding to N-vinyl-3,5-dimethyl pyrazole were absent from the spectrum.The stacked spectra are shown in Fig. S18.
Preparation of 1,2-dideutero acetaldehyde.C2D2 was prepared from calcium carbide and D2O at 0 °C and was passed through a CaCl2 column to remove residual water.

Crystal structure determination
A single orange plate-shaped crystal of 1 and a single blue block-shaped crystal of 2i were selected and mounted on a glass fiber on a Bruker APEX-II CCD diffractometer.A single orange plate-shaped crystal of 1i and a dark violet block-shaped crystal of 3 were selected and mounted on a glass fiber on an XtaLAB Synergy, Dualflex, HyPix-Arc 100 diffractometer.All the measurements were performed using monochromatized Mo K radiation (0.71073 Å) at 100 K. Data reduction, scaling, and absorption corrections were performed for the four structures using CrysAlisPro (Rigaku, V1.171.43,2023). 4For structures 1 and 2i, empirical absorption correction was performed using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm.For 1i and 3, a numerical absorption correction based on gaussian integration over a multifaceted crystal model and an empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm were performed.
The structures of 1, 1i, and 2i were solved with the ShelXT 2018/2 structure solution program 5 using the intrinsic phasing solution method and by using Olex2 6 as the graphical interface.The models were refined with version 2019/3 of ShelXL 7 using full-matrix least-squares techniques against F 2 .All non-hydrogen atoms were refined anisotropically.Hydrogen atom positions were calculated geometrically and refined using a riding model, except for the positions of the H atoms of the NH groups which were taken from a difference Fourier map, the N-H distances were fixed to 0.88 Å, and the H atoms were refined with individual isotropic displacement parameters without any constraints to the bond angles.The structure of 3 was solved with the ShelXT 2018/2 structure solution program 5 using the intrinsic phasing solution method and by using Olex2 6 as the graphical interface.The model was refined with version 2019/3 of ShelXL 8 using least squares minimization as a 2-component inversion twin with a BASF factor
Scheme S1.Derivatization of aldehydes with DNPH under acidic conditions gives AA-DNPH and CA-DNPH, respectively, which were analyzed by HPLC-MS.

Table S2 .
Data collection and structure refinement details for compounds 1 and 1i.

Table S3 .
Data collection and structure refinement details for compounds 2i and 3.