Sciex tripletof 4600

Optical rotations were
acquired using a Jasco P-2000 polarimeter. UV spectra were measured
using a Beckman Coulter DU-800 spectrophotometer. NMR spectra were
recorded using a Varian 500 MHz instrument. The chemical shifts reported
for 1, 2, and micropeptin 996 were referenced
to the residual solvent peaks of DMSO-d₆ (δ_H 2.50 and δ_C 39.5). HRESIMS
analysis was performed using an AB SCIEX TripleTOF 4600 mass spectrometer
with Analyst software. MS/MS data were recorded on this same instrument
using the product ion function. LC-MS/MS was performed using a ThermoFisher
LTQ XL mass spectrometer with an electrospray ionization (ESI) source.
Semi-preparative and analytical HPLC was carried out using a Dionex
UltiMate 3000 HPLC system equipped with a micro vacuum degasser, an
autosampler, and a diode-array detector.

The in vitro stability of lixisenatide and 1a–1i were determined using a previously described method with small modification.^{34 (link)} In brief, plasma were obtained from SD rats (male, 200–250 g) and stored at −20 °C until use. Lixisenatide and 1a–1i were dissolved in PBS (pH 7.4) and 0.5 mL of the compound were added to the 1 mL plasma to achieve concentration of 1000 ng mL⁻¹. The mixture was vortex mixed for 60 s and then incubated at 37 °C for 6, 12, 24, and 48 h. At each time point, 100 μL of sample was removed and mixed with acetonitrile (200 μL) containing 2% formic acid for the precipitation of plasma protein. Then the mixture was centrifuged at 14 000 rpm for 15 min, and the supernatant (50 μL) was analyzed via liquid chromatography-mass spectrometry (LC-MS/MS) system (AB SCIEX TripleTOF 4600, USA). The degradation curves of each tested compounds were determined in triplicate.