Natural Products

LC-MS/MS acquisition for all in house generated libraries was performed using a Bruker Daltonics Maxis qTOF mass spectrometer equipped with a standard electrospray ionization source (ESI). The mass spectrometer was tuned by infusion of Tuning Mix ES-TOF (Agilent Technologies) at a 3 μL/min flow rate. For accurate mass measurements, lock mass internal calibration used a wick saturated with hexakis (1H,1H,3H-tetrafluoropropoxy) phosphazene ions (Synquest Laboratories, m/z 922.0098) located within the source. Samples were introduced by a Thermo Scientific UltraMate 3000 Dionex UPLC using a 20 μL injection volume. A Phenomenex Kinetex 2.6 μm C18 column (2.1 mm × 50 mm) was used. Compounds from NIH Prestwick Phytochemical Library, NIH Natural Product Library, and NIH Small Molecule Pharmacologically Active Library were separated using a seven minute linear water-acetonitrile gradient (from 98:2 to 2:98 water:acetonitrile) containing 0.1% formic acid. Compounds from NIH Clinical Collections and FDA Library part 2 Library employed a step gradient for chromatographic separation [5% solvent B (2:98 water:acetonitrile) containing 0.1% formic acid for 1.5 min, a step gradient of 5% B-50% B in 0.5 min, held at 50% B for 2 min, a second step of 50% B-100% B in 6 min, held at 100% B for 0.5 min, 100%-5 % B in 0.5 min and kept at 5% B for 0.5 min]. The flow rate was 0.5 mL/min. The mass spectrometer was operated in data dependent positive ion mode; automatically switching between full scan MS and MS/MS acquisitions. Full scan MS spectra (m/z 50 – 1500) were acquired in the TOF and the top ten most intense ions in a particular scan were fragmented using collision induced dissociation (CID) utilizing stepping.

We loaded catalogs from over 320 commercial vendors and 130 annotated catalogs. Some sources such as HMDB and DrugBank were loaded as several distinct catalogs in ZINC allowing us to leverage the curation of metabolite origin such as plant metabolites in HMDB or drug status such as investigational drugs in DrugBank. All catalogs in ZINC are categorized by their biogenic and bioactivity status, if any.⁹⁵ Only descriptions that characterized the entire catalog contents were applied. For instance, the “Approved” subset of DrugBank was categorized as “World Drugs” since it contains over 100 drugs approved in other countries but not by FDA, and the “Endogenous” subset of HMDB was categorized as having a biogenic type of “endogenous human metabolite”. Molecules inherit biogenic and bioactive properties from the catalogs they are found in. These values are computed and stored, and are accessible in the interface as molecular features. There are four biogenic catalog levels: 1) Endogenous human metabolites, i.e. compounds that are synthesized in man. Interestingly, this may include compounds produced by our bacterial flora; 2) Metabolites of any species, i.e. small molecules that are involved in metabolism, development and reproduction, but not metabolites of xenobiotics; 3) Biogenic compounds, often called natural products; 4) Unknown biogenic status. Likewise, ZINC supports seven levels of bioactivity annotation as follows. 1) FDA approved; 2) World drugs; 3) Investigational, compounds reported to be used in clinical trials; 4) In Man, which including nutraceuticals, for instance; 5) In vivo, which includes DrugBank experimental compounds that have been in animals; 6) In cells, which includes compounds reported active in cell based assays; 7) In vitro, compounds active or assumed active at 10 μM or better in a direct binding assay. All other compounds are marked as having unknown biological activity. The categories are ordered to be progressively inclusive within each series, thus all FDA approved drugs are also world drugs and all compounds active in cells are also active in vitro. We annotate as building blocks those catalogs of compounds available in preparative quantities, typically 250 mg or more. Commercial vendors are categorized by the speed and cost of compound acquisition, allowing the best purchasability of every compound to be computed based on its current catalog membership. Catalog categorizations are refined continually by purchasing experience in our lab and reports from colleagues, as follows:⁹⁵ 1) In stock, delivery in under two weeks, 95% typical acquisition success rate; 2) Procurement agent, in stock, delivery in 2 weeks, 95% typical acquisition success rate; 3) Make-on-demand, delivery typically within 8 to 10 weeks, 70% typical acquisition success rate; 4) Boutique, where the cost may be high, but still likely cheaper than making it yourself, 70% typical acquisition success rate.