Deuterium oxide

Thymotrinan (Arg-Lys-Asp, TP3), thymocartin (Arg-Lys-Asp-Val, TP4), thymopentin (Arg-Lys-Asp-Val-Tyr, TP5) and their metabolites were provided by GL Biochem Ltd. (Shanghai, China). All peptide purities were higher than 98% as evidenced by RP-HPLC assay (Agilent Technologies, Santa Clara, CA, USA). Calcein, cobalt hydrodichloride, N-hydroxysuccinimide (NHS), leucine aminopeptidase (EC 3.4.11.2, microsomal from porcine kidney), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC), ethylene diamine tetraacetic acid (EDTA), and glycol chitosan (GCS, degree of deacetylation ca. 75.0%, 250 KDa) were purchased from Sigma (St. Louis, MO, USA). Deuterium oxide was provided by Cambridge Isotope Laboratories, Inc. (Cambridge Isotope Laboratories, Tewksbury, MA, USA). These peptides and compounds were used as received without further purification.

SCID mice bearing HCC70 tumors were treated with indicated drugs for 2 days and then injected with 99% deuterium oxide (Cambridge Isotope Laboratories, 23.3 mg/g; i.p.). After 2 h, the mice were terminated under anesthesia; the liver and tumor were excised. Samples (10 mg) were extracted with 1 mL of extraction solvent, 3:3:2 acetonitrile, isopropanol, and water. The extraction solvent was pre-cooled at −20 °C. Samples were homogenized by using Genogrinder at 500 × g for 30 s, shaken for 5 min at 4 °C, and centrifuged for 2 min at 20,000 × g. Five-hundred microliters of supernatant was dried overnight. Ten microliters of methoxyamine hydrochloride was added to each dried sample followed by shaking at 30 °C for 1.5 h. Ninety-one microliters of N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA, Sigma-Aldrich, St. Louis, MO) was added to each sample followed by shaking at 70 °C for 60 min for tert butyldimethylsilylation. Agilent 7200 GC-accurate-mass QTOF (Agilent, Santa Clara, CA) was used for data acquisition. Agilent J&W DB-5ms Ultra Inert column was used (Agilent, Santa Clara, CA). The column was held at 60 °C for 0.5 min, ramped to 325 °C at 10 °C/min, and held at 325 °C for 10 min. Raw data were processed by using Agilent Mass Hunter Quantitative Analysis software for QTOF (B. 07.00).

The NMR heteronuclear single quantum coherence spectra were acquired using a Bruker 600 MHz magnet at the Central Alabama High Field NMR Facility at the University of Alabama at Birmingham. Samples were freshly prepared on the day of collection in 1× TBS with 2 mM BME, pH 6.8, supplemented with sodium azide (Fisher BioReagents) and deuterium oxide (99%; Cambridge Isotope Laboratories, Inc). The collected spectra were analyzed using computer-aided resonance assignment, and the peak lists were exported into Microsoft Excel for subsequent analysis. Briefly, CSPs identified using a combination of nearest neighbor and comparison to previous analysis of rBH3 binding and were quantified by calculating the ΔΔppm of each amino acid residue using the formula: √Δδ_H² + (Δδ_N/5)². Mean and standard deviation of the CSP for each residue was calculated in Microsoft Excel, and any residues demonstrating CSP >1 SD were considered significant. All spectra were collected in biological triplicate, and residues that demonstrated significant CSP in all three spectra were mapped to the ribbon model of RRM1 on PyMOL (pymol.org), using Protein Data Bank file 1SJQ: NMR structure of RRM1 from human polypyrimidine tract binding protein isoform 1 (PTB1). In Figure 4, A–C, CSPs are plotted by residue, and examples of overlaid raw data, respectively, are shown from a representative spectra.

TCEP (tris-(2-carboxyethyl) phosphine), formic acid (MS grade ~98%), sodium phosphate monobasic, sodium phosphate di-basic and sodium chloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deuterium oxide (99.9%) was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Guanidine hydrochloride (8.0 M) was purchased from Thermo Scientific (Waltham, MA, USA). Acetonitrile and water were UPLC/MS grade and purchased from BioSolve B.V. (Valkenswaard, The Netherlands). All chemicals were used without further purification unless otherwise specified.

The 3D-enriched fragment library was purchased from Life Chemicals and divided into mixtures of 4–5 fragments as previously described [11 (link)]. Amino acids, uracil, thiamine-HCl, LB broth, biotin, and nicotinic acid were purchased from RPI Corp. The 5-fluoroindole, magnesium sulfate, succinic acid, calcium chloride, Iron(III) chloride, ¹⁵N ammonium chloride, L-Moses, and imidazole were purchased from Millipore Sigma. Potassium diphosphate, potassium monophosphate, sodium phosphate, manganese (II) chloride, zinc(I) chloride, and sodium chloride were purchased from Fischer Scientific. Cobalt (II) chloride, Ethylenediaminetetraacetic acid, and copper (II) chloride was purchased from Acros Organics. Boric acid was purchased from Mallinckrodt. Deuterium oxide and dimethyl sulfoxide-d6 were purchased from Cambridge Isotope Laboratories. GSK4027 was purchased from Cayman Chemicals and TP238 was synthesized previously [55 (link)].

l-Alanine ethyl ester hydrochloride (l-Ala-OEt) and d-alanine ethyl ester hydrochloride (d-Ala-OEt) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). l-Proline benzyl ester (Pro-OBzl) was purchased from Tokyo Chemical Industry (Japan). Deuterium oxide was purchased from Cambridge Isotope Laboratories, Inc. Standard chemicals were purchased from Wako Chemical Co. (Kanagawa, Japan).