Nmr tube

NMR spectroscopy was used to observe the presence of small molecules in crude SvV and ShV. Crude venoms were pooled from multiple spines of each individual fish for analysis. In addition, seven individual spines from SvV were also analyzed to investigate any differences in venom composition between spines.
NMR data were obtained as previously reported following standard procedures [27 ]. Spectra were recorded at 290 and 298 K on a Bruker Avance III 600 MHz spectrometer (Bruker, Billerica, MA, USA) equipped with a cryoprobe. Crude venom samples were prepared in 90% H₂O/10% D₂O (v/v) (99.9%, Cambridge Isotope Labs, Tewksbury, Massachusetts, United States of America), vortexed, and centrifuged prior to transfer to an NMR tube (Wilmad, 5 mm). One‐dimensional (1D) and two‐dimensional (2D) spectra were collected using standard Bruker pulse programs, and referenced to external 4,4‐dimethyl‐4‐silapentane‐1‐sulfonic acid (DSS; Cambridge Isotope Laboratories). The 1D data included a cpmgpr1d experiment to suppress the larger peptide and protein signals. The 2D spectra included TOCSY, NOESY, COSY, HMBC, ¹³C‐HSQC, and ¹⁵N‐HSQC experiments with TOCSY and NOESY mixing times of 80 and 500 ms, respectively. Spectra of crude SvV were also collected in 100% MeOD (Cambridge Isotope Labs, methanol‐d₄) to observe peaks otherwise obscured by the water suppression. Spectra were analyzed with topspin v3.6.3 (Bruker).

Commercially available unsaturated substrates (propargyl alcohol (Sigma-Aldrich, 99%), 3-butyn-2-ol (Alfa Aesar, 98%), 2-methyl-3-butyn-2-ol (Sigma-Aldrich, 98%)), bis(norbornadiene)rhodium(I) tetrafluoroborate ([Rh(nbd)₂]BF₄, nbd = norbornadiene, Umicore, 99.9%), 1,4-bis(diphenylphosphino)butane (dppb, Sigma-Aldrich, 98%), methanol-d₄ (Zeotope, 99.8% D) and ultrapure hydrogen ( > 99.999%) were used as received. Propargyl pyruvate was synthesized according to previously published procedure^{46 (link)}. At first, the mixture of [Rh(nbd)₂]BF₄ and dppb was dissolved in methanol-d₄. The amounts of reactants were calculated to get the 10 mM concentration of Rh complex; dppb was taken in ~2% molar excess with respect to [Rh(nbd)₂]BF₄. The solution was left for ~30 min with periodic mixing to ensure formation of [Rh(nbd)(dppb)]BF₄ complex. The resultant solution (0.5 mL aliquots) was placed in standard 5 mm Wilmad NMR tubes tightly connected with ¼ in. outer diameter PTFE tubes. Before conducting the experiment, a portion of one of the unsaturated precursors 1'–4' was added to an NMR tube; the amount of an unsaturated substrate was calculated so that its concentration in the resultant solution was 0.8 M (neglecting the slight dilution due to own volume of the added liquid). The solution was thoroughly mixed afterwards to ensure homogeneous distribution of the added substrate.

Each serum sample (100 μL) was mixed with 500 μL of deuterium oxide (²H₂O) (ISOTEC, Sigma-Aldrich, St. Louis, MO, USA) and pipetted into a 5-mm (outer diameter) NMR tube (Wilmad-LabGlass, Vineland, NJ, USA) for NMR analysis [23 (link)]. Solution-state NMR analyses were performed at a proton resonance frequency of 400 MHz (9.4 Tesla) using an ECZ^™ NMR spectrometer (JEOL Ltd., Tokyo, Japan) interfaced with a probe (digital auto-tunable type [NM-03812RO5S]) and equipped with Delta^™ NMR processing and control software, version 5.3.2 (JEOL Ltd.). The field was locked to the ²H resonance of the ²H₂O solvent. One-dimensional proton NMR signals were automatically acquired at a probe temperature of 30°C using the program supplied by JEOL that supported the macro function in Delta^™. Free induction decay (FID) data were acquired using a single pulse with a 2.0-s relaxation delay between repeated pulse sequences. The strong signal arising from free water was suppressed using DANTE presaturation. Other conditions were as follows: radio-frequency pulse width, 2.93 μs; acquisition time, 1.636 s; repetition time, 3.636 s; spectral width, 10,016 Hz; number of data points, 16,384; and number of steady-state transients, 400. The FID data were saved in JEOL Delta format (JDF).