Micromass zq instrument

Unless otherwise noted, reagents and solvents were obtained from commercial suppliers and were used without further purification. ¹H NMR spectra were recorded on a 500 MHz Bruker A500, and chemical shifts are reported in parts per million (ppm, δ) downfield from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin multiplicities are described as s (singlet), br (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Mass spectra were obtained on a Waters Micromass ZQ instrument. Preparative HPLC was performed on a Waters Sunfire C18 column (19 × 50 mm, 5 μM) using a gradient of 15–95% methanol in water or acetonitrile in water containing 0.05% trifluoroacetic acid (TFA) over 22 min (28 min run time) or 35 min (45 min run time) at a flow rate of 20 mL/min. The purity of all the final compounds is ≥95%, and it is measured by analytical reverse-HPLC using a gradient of 15–85% acetonitrile in water containing TFA over 2.5 min at a flow rate of 2 mL/min.

We describe the interaction of three small molecules, referred to as compounds A, B, and C, with ZAP-70 or Syk. These compounds were identified in a high throughput screen for ZAP-70 inhibitors; the detail of which will be published separately (Visperas et al. in preparation). The chemical structures of these compounds are shown in Figure 2. The compound suppliers are as follows: Compound A was obtained from InterBioScreen Ltd. (Supplier ID: STOCK5S-74623). Compound B was obtained from Pharmeks Ltd. (Supplier ID: PHAR025826). Compound C was obtained from ChemBridge Corp. (Supplier ID 7997218). The compounds were obtained in powdered form and dissolved at 50 mM in DMSO. The purity of all compounds was assessed and their correct masses verified by LC/MS. All compounds submitted for testing were judged to be of 95% or higher purity based on analytical LC-MS analysis on a Waters Micromass ZQ instrument, through UV absorbance at 254 nm and light scattering. Separations were carried out on an XTerra® MS C18 5μm 4.6×50mm column at ambient temperature using a mobile phase of water-methanol containing 0.1% formic acid. Chemical identity was confirmed in positive ion mode by the presence of the anticipated molecular weight M+1 ion. No purification was performed on the compound solutions.

Solvents and reagents were obtained from commercial sources and were used without further purification. NMR spectra were recorded on a Varian Oxford AS600 600 MHz instrument. NMR chemical shifts are expressed in ppm relative to internal solvent peaks, and coupling constants are measured in Hz (br = broad). Mass spectra were recorded on a Waters Micromass ZQ instrument using an ESI source coupled to a Waters 2525 HPLC system operating in reverse mode with Waters SunfireTM C185 uM 4.6×50 mm column. Flash chromatography was performed using a Biotage Isolera One flash purification system.