Ascend 400 mhz

Isoquinolone derivatives were synthesized as described previously [11 (link),12 (link),13 (link),14 (link),15 (link),16 (link),17 (link),18 (link),19 (link)]. The structure of newly synthesized compounds 7–10 and 21 was confirmed by 300 MHz (Varian Unity Plus 300 MHz; Varian Inc., Palo Alto, CA, USA) and 400 MHz (Bruker Ascend 400 MHz; Bruker Daltonik, Bremen, Germany) nuclear magnetic resonance (NMR) spectrometry. Purity was estimated to be ≥99%.

Solution state NMR experiments were performed using a Bruker Avance III HD and Ascend^TM 400 MHz spectrometer with a z-gradient system (Bruker Biospin, Billerica, MA, USA). Samples (0.5 mM) of the three final purified tIK series peptides labeled with ¹⁵N were dissolved in 400 μL of D₂O/H₂O 1:9 solvent, and a solution state 2D ¹H-¹⁵N HSQC experiment with uniformly ¹⁵N-labeled and selectively ¹⁵N-labeled tIK series was performed at pH 2 considering the isoelectric point. The time domain (TD) of F2 was set to 2048, F1 was set to 320, and the number of scans (NS) was set to 72.

All solution-state NMR experiments were carried out using Bruker Avance III HD and Ascend^TM 400 MHz spectrometer (Bruker Biospin, Billerica, MA, USA) with z-gradient system. Micelle samples for solution-state experiments were prepared by dissolving 1 mg uniformly ¹⁵N-labeled hAPP-TM with 0.1 M DPC-d₃₈ (Cambridge Isotope Laboratories, Andover, MA, USA) micelles in 400 μL H₂O/D₂O (90%/10%) at pH 4.0. The hAPP-TM powder samples were prepared at different concentrations (1.0 mM, 2.0 mM and 5.0 mM) to demonstrate multimer formation. Additionally, peptide samples for identification of zinc ion blockade effect were mixed with ZnCl₂ (Junsei Chemical Co., Tokyo, Japan) at concentrations of 0 mM, 20.0 mM, 70.0 mM, 100.0 mM, respectively. The 2D ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) data were recorded at 313 K with 256 increments in F1 and 128 increments in F2 with 2048 complex points. Results were processed by TOPSPIN 4.0.6 (Bruker Biospin, Rheinstetten, Germany).

All manipulations were conducted under an N₂ atmosphere using Schlenk techniques. The compounds (η⁵-C₅H₄CHO)Re(CO)₃²¹ , (η⁵-C₅H₄COCH₃)Re(CO)₃²² , (η⁵-C₅H₄CHO)Mn(CO)₃²³ and 4-hydrazinyl-benzenesulphonamide²⁴ (link) were prepared according to published procedures. Ferrocene carboxaldehyde (98%), acetyl ferrocene (95%), acetylcymantrene (98%) and sulphanilamide (99%) were obtained from Sigma-Aldrich (Chicago, IL) and used without additional purification. Solvents such as CH₂Cl₂, hexane, acetone, EtOH, DMSO and THF were obtained commercially and purified using standard methods. Infra-red spectra were recorded in solid state (KBr pellet) on a Jasco FT-IR 4600 spectrophotometer. ¹H NMR spectra were measured on a Bruker spectrometer model ASCEND TM 400 MHz. All NMR spectra are reported in parts per million (ppm, δ) relative to tetramethylsilane (Me₄Si), with the residual solvent proton resonances used as internal standards. Coupling constants (J) are reported in Hertz (Hz), and integrations are reported as number of protons. The following abbreviations were used to describe the peak patterns: s = singlet, d = doublet, t = triplet and m = multiplet. Mass spectra were obtained on a Shimadzu model QP5050A GC-MS at the Laboratorio de Servicios Analíticos, Pontificia Universidad Católica de Valparaíso. Elemental analyses were measured on a Perkin Elmer CHN Analyser 2400.

A solution of AX or AX-B with butylamine in deuterated solvent was added to appropriate NMR tubes, as described the following: each amoxicillin (15·10⁻⁶ mol) was dissolved in 0.5 ml of deuterated solvent (D₂O or deuterated PBS), and an excess of butylamine (5·10⁻⁶ l, 50·10⁻⁶ mols) was added. The reactions were monitored by ¹H NMR on a Bruker ASCENDTM 400 MHz.

The ¹H and ¹³C NMR spectra of all compounds in this paper were obtained by Bruker Ascend^TM-400 MHz Fourier transform NMR tester. The solvents used were CDCl₃-d, DMSO-d₆, Methanol-d₄ and Acetic Acid-d₄, the chemical shifts (δ) are in ppm and the coupling constants (J) are in Hz. The ionization source for low-resolution mass spectrometry was electrospray ionization (ESI), measured by a Waters ACQUITY UPLC SQ MS. The ionization source for high-resolution mass spectrometry was electrospray ionization (ESI), measured by an IonSpec 4.7 Tesla FTMA mass spectrometer. The HPLC (High Performance Liquid Chromatography) detection instruments involved in this paper include Agilent 1200 and DGU-20A5R High Performance Liquid Chromatography. Melting points were determined using the SGW X-4 micro melting point instrument.

The propolis extract at a concentration of 20 mg was solubilized in 600 µL of deuterated dimethyl sulfoxide (DMSO-D6). The ¹H and ¹³C spectra were obtained on a Bruker spectrometer (model Ascend™ 400MHz, Boston, MA, USA). The TopSpin 3.6.2 software was used for control and data processing, with manual calibration by suppressing the residual H₂O signal, and adjusting the ¹H and ¹³C spectra by the residual solvent signal, DMSO-H6, respectively, at 2.49 ppm and 39.5 ppm [84 (link)].