Robert Brkljača

RECENT ADVANCEMENTS IN HPLC-NMR AND APPLICATIONS FOR NATURAL PRODUCT PROFILING AND IDENTIFICATION

This review provides a summary of the recent advances in high pressure liquid chromatography (HPLC) coupled to nuclear magnetic resonance (NMR) spectroscopy. Significant improvements have included the miniaturization of NMR components as well as the use of cryogenically cooled probes. Further hyphenated applications such as HPLC-SPE-NMR, column trapping and HPLC-NMR-MS are also discussed. Finally, an overview of the application of these methodologies to the profiling and dereplication of natural products is provided.

Surface Water Dependent Properties of Sulfur-Rich Molybdenum Sulfides: Electrolyteless Gas Phase Water Splitting

Sulfur-rich molybdenum sulfides are an emerging class of inorganic coordination polymers that are predominantly utilized for their superior catalytic properties. Here we investigate surface water dependent properties of sulfur-rich MoSx (x = 32/3) and its interaction with water vapor. We report that MoSx is a highly hygroscopic semiconductor, which can reversibly bind up to 0.9 H2O molecule per Mo. The presence of surface water is found to have a profound influence on the semiconductors properties, modulating the materials photoluminescence by over 1 order of magnitude, in transition from dry to moist ambient. Furthermore, the conductivity of a MoSx-based moisture sensor is modulated in excess of 2 orders of magnitude for 30% increase in humidity. As the core application, we utilize the discovered properties of MoSx to develop an electrolyteless water splitting photocatalyst that relies entirely on the hygroscopic nature of MoSx as the water source. The catalyst is formulated as an ink that can be coated onto insulating substrates, such as glass, leading to efficient hydrogen and oxygen evolution from water vapor. The concept has the potential to be widely adopted for future solar fuel production.

Phytochemical investigation of the constituents derived from the Australian plant macropidia fuliginosa

A phytochemical study of the flowers and bulbs derived from the Australian plant Macropidia fuliginosa, involving hyphenated spectroscopic methodologies (HPLC-NMR and HPLC-MS), together with conventional isolation strategies, resulted in the identification of 16 constituents (1, 2, 4-17) representative of six different structural classes. Six new compounds (12-17) were identified from the bulbs of the plant. The isolated compounds were assessed for antimicrobial activity, and compound 8 was found to be more potent against P. aeruginosa than ampicillin.

Chemical Profiling (HPLC-NMR & HPLC-MS), Isolation, and Identification of Bioactive Meroditerpenoids from the Southern Australian Marine Brown Alga Sargassum paradoxum

A phytochemical investigation of a southern Australian marine brown alga, Sargassum paradoxum, resulted in the isolation and identification of four new (5, 9, 10, and 15) and nine previously reported (1, 2, 6–8, and 11–14) bioactive meroditerpenoids. HPLC-NMR and HPLC-MS were central to the identification of a new unstable compound, sargahydroquinal (9), and pivotal in the deconvolution of eight (1, 2, 5–7, and 10–12) other meroditerpenoids. In particular, the complete characterization and identification of the two main constituents (1 and 2) in the crude dichloromethane extract was achieved using stop-flow HPLC-NMR and HPLC-MS. This study resulted in the first acquisition of gHMBCAD NMR spectra in the stop-flow HPLC-NMR mode for a system solely equipped with a 60 μL HPLC-NMR flow cell without the use of a cold probe, microcoil, or any pre-concentration.

Dereplication and chemotaxonomical studies of marine algae of the Ochrophyta and Rhodophyta phyla.

Dereplication and chemotaxonomic studies of six marine algae of the Ochrophyta and one of the Rhodophyta phyla resulted in the detection of 22 separate compounds. All 16 secondary metabolites, including four new compounds (16–19), could be rapidly dereplicated using HPLC-NMR and HPLC-MS methodologies in conjunction with the MarinLit database. This study highlights the advantages of using NMR data (acquired via HPLC-NMR) for database searching and for the overall dereplication of natural products.

HPLC-NMR and HPLC-MS Profiling and Bioassay-Guided Identification of Secondary Metabolites from the Australian Plant Haemodorum spicatum

Phytochemical dereplication was undertaken on the bioactive crude CH2Cl2 extract of the bulbs of the Australian plant Haemodorum spicatum employing HPLC-NMR and HPLC-MS methodologies. Subsequent bioassay-guided isolation resulted in the identification of two new phenylphenalenones [haemoxiphidone (8) and haemodoronol (17)] and two new chromenes [haemodordione (13) and haemodordiol (16)], together with seven previously described compounds. Antimicrobial testing showed that the compounds displayed selective antibacterial activity. Most noteworthy were the activities displayed by several of the compounds against multi-drug-resistant Pseudomonas aeruginosa.

Phenylphenalenones and oxabenzochrysenones from the Australian plant Haemodorum simulans.

Chemical investigation of the Australian plant Haemodorum simulans (Haemodoraceae) resulted in the isolation of two new phenylphenalenones, haemoxiphidone and haemodordioxolane from the bulbs together with the first report of an oxabenzochrysenone glycoside, haemodoroxychrysenose from the aerial parts of the plant. Also isolated were two previously described phenylphenalenones 5,6-dimethoxy-7-phenyl-1H,3H-naphtho[1,8-cd]pyran-1,3-dione and haemodorone and two oxabenzochrysenones 5-hydroxyl-2-methoxy-1H-naphtho[2,1,8-mna]xanthen-1-one and 5-methoxy-1H-naphtho[2,1,8-mna]xanthen-1-one. The X-ray structure of the phenylphenalenone 5,6-dimethoxy-7-phenyl-1H,3H-naphtho[1,8-cd]pyran-1,3-dione was secured for the first time. All compounds were deduced by detailed spectroscopic analyses. HPLC-NMR chemical profiling of an enriched fraction containing a mixture of haemodordioxolane and 5,6-dimethoxy-7-phenyl-1H,3H-naphtho[1,8-cd]pyran-1,3-dione facilitated the partial identification of these secondary metabolites. The structure previously assigned as xiphidone in our initial studies of this plant was re-assigned as the new isomer haemoxiphidone.

Application of the Crystalline Sponge Method to Revise the Structure of the Phenalenone Fuliginone

The structure of fuliginone was revised from a phenyl substituted phenalenone to a hydroxyl substituted phenalenone as a result of its re-purification via HPLC with subsequent NMR analysis together with an independent synthesis and analysis of the crystal structure, which was secured via the crystalline sponge method. On-flow High Performance Liquid Chromatography coupled to Nuclear Magnetic Resonance spectroscopy (HPLC-NMR) was employed to confirm the presence of the natural product in the plant extract and to monitor for any possible degradation or conversion of the compound.

Chemical Constituents of Hoya buotii Kloppenb.

Chemical investigation of the dichloromethane extracts of Hoya buotii Kloppenb. afforded taraxerone (1), taraxerol (2), a mixture of β-sitosterol (3a) and stigmasterol (3b) in about 2:1 ratio, and a mixture of α-amyrin cinnamate (4a) and β-amyrin cinnamate (4b) in about 1:2 ratio from the stems; 1, 2, and 3a from the roots; a mixture of 4a and 4b in about 3:2 ratio from the flowers; and 3a, squalene (5) and saturated hydrocarbons from the leaves. The structures of 1-5 were identified by comparison of their NMR data with those reported in the literature.

Limit of detection studies for application to natural product identification using high performance liquid chromatography coupled to nuclear magnetic resonance spectroscopy

In the pursuit of new natural products, the demand to rapidly identify compounds present, in ever decreasing amounts, in complex crude extracts has become a limiting factor. Despite improvements in HPLC-NMR hardware and pulse sequences, no extensive limit of detection (LOD) investigations have been reported for the acquisition of 2D NMR spectroscopic experiments acquired through HPLC-NMR. In this study the LOD for five key 1D and 2D NMR spectroscopic experiments have been established, using two reference compounds, including the on-flow (WET 1D proton), stop-flow (WET1D proton), gCOSY, HSQCAD and gHMBCAD NMR experiments. The LOD for all of the NMR experiments were within the range of 700ng to 1mg for the set of fixed experimental parameters implemented. For principle components in a complex multi-component mixture, this would allow for in situ compound identification. HPLC-NMR analysis was employed to investigate the principle components present in a marine brown alga crude extract, Cystophora subfarcinata.