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. Author manuscript; available in PMC: 2018 Jan 27.
Published in final edited form as: J Nat Prod. 2016 Dec 28;80(1):12–18. doi: 10.1021/acs.jnatprod.6b00949

Identification of Neuroprotective Spoxazomicin and Oxachelin Glycosides via Chemoenzymatic Glycosyl-Scanning

Jianjun Zhang †,‡, Ryan R Hughes †,‡, Meredith A Saunders §, Sherif I Elshahawi †,‡, Larissa V Ponomareva †,‡, Yinan Zhang †,‡, Sydney R Winchester §, Samantha A Scott §, Manjula Sunkara ||, Andrew J Morris ||, Mark A Prendergast §, Khaled A Shaaban †,‡,*, Jon S Thorson †,‡,*
PMCID: PMC5337260  NIHMSID: NIHMS849646  PMID: 28029796

Abstract

The assessment of glycosyl-scanning to expand the molecular and functional diversity of metabolites from the underground coal mine fire-associated Streptomyces sp. RM-14-6 is reported. Using the engineered glycosyltransferase OleD Loki and a 2-chloro-4-nitrophenylglycoside-based screen, six metabolites were identified as substrates of OleD Loki, from which 12 corresponding metabolite glycosides were produced and characterized. This study highlights the first application of the 2-chloro-4-nitrophenylglycoside-based screen toward an unbiased set of unique microbial natural products and the first reported application of the 2-chloro-4-nitrophenylglycoside-based transglycosylation reaction for the corresponding preparative synthesis of target glycosides. Bioactivity analysis (including antibacterial, antifungal, anticancer, and EtOH damage neuroprotection assays) revealed glycosylation to attenuate the neuroprotective potency of 4, while glycosylation of the structurally related inactive spoxazomicin C (3) remarkably invoked neuroprotective activity.

Graphical Abstract

graphic file with name nihms849646u1.jpg

Natural products (NPs) discovery efforts continue to expose pharmacophores of notable scientific, clinical, and commercial value1–5 that often inspire synthetic organic chemists6–12 and reveal unique metabolic pathways that offer new opportunities in mechanistic enzymology, synthetic biology, and biocatalysis.13–22 For the latter, permissive catalysts involved in late-stage NP tailoring modifications (acylation, alkylation, glycosylation, oxidation) have been particularly enabling in NP core scaffold diversification.5,15,23–30 The demonstrated impact of glycosylation within this context is diverse and includes influencing NP or small-molecule drug potency, mechanism, pharmacodynamics, pharmacokinetics, and ADME properties29–39 where the advent of permissive chemoenzymatic strategies complement and/or circumvent key limitations of conventional chemical glycosylation as a medicinal chemistry tool.29,40–51 Such chemoenzymatic strategies have benefited from the development of donor/acceptor permissive glycosyltransferases via directed evolution and a corresponding screening platform enabled by the use of simple activated glycoside donors that drive the equilibrium of glycosyltransferase-catalyzed reactions toward product formation and provide real-time indicators of sugar-nucleotide utilization in parallel (Figure 1).52–60 In the current study, we assess the potential of this permissive chemoenzymatic platform for glycosylation of oxazole carboxamide and oxachelin analogues isolated from the Ruth Mullins underground coal mine isolate Streptomyces sp. RM-14-6.61 This analysis revealed the permissive engineered OleD Loki to catalyze the glycosylation of spoxazomicin C [an antitrypanosomal NP first reported from Streptosporangium oxazolinicum K07-0460(T)]62 and oxachelin (a metal chelator first reported from Streptomyces sp. GW9/1258),63 providing the first reported corresponding glycosides of naturally occurring phenol/oxazolines. Subsequent evaluation of the glycosides produced revealed 2-O-β-D-glucosylspoxazomicin C (3a) as neuroprotective to EtOH-induced damage in rat hippocampal-derived primary cell cultures, while the parent NP (3) and glycosides 2-O-(2′-amino-2′-deoxy-β-D-glucosyl)spoxazomicin C (3c) and 12-O-β-D-glucosylspoxazomicin C (3b) lacked activity. In addition, glycosylation of spoxazomicin D led to marked decrease in potency over the parent NP comparator in the same assay, while oxachelin (5) glycosylation had no apparent effect on bioactivity. Cumulatively, this study further extends the demonstrated substrate scope of OleD Loki and, given the impact of glucose conjugation on improving blood–brain barrier transport,64–68 may also offer a convenient strategy to modulate the properties of novel NPs to treat neurological deficits resulting from alcohol abuse.61,69–71

Figure 1.

Figure 1

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General glycosyl-scanning scheme. In this strategy, activated 2-chloro-4-nitrophenylglycoside donors help drive the OleD Loki-catalyzed transglycosylation reaction toward desired product formation where the resulting production of colorimetric 2-chloro-4-nitrophenol affords a real-time indicator of sugar-nucleotide production and utilization as an indirect measure of target glycoside production.

RESULTS AND DISCUSSION

Using a 2-chloro-4-nitrophenylglycoside-driven colorimetric screen for the identification of putative OleD Loki substrates (Figure 1),56,57,60 preliminary assays were accomplished with 2 mM (2-chloro-4-nitrophenyl)-β-D-glucoside (1) or (2-chloro-4-nitrophenyl)-2-amino-2-deoxy-β-D-glucoside (2) as donor, 1 mM acceptor, 0.1 mM UDP, and enzyme (0.26 μM Loki for donor 1; 2.6 μM Loki for donor 2) in a 384-well plate format. Putative substrates evaluated included 17 recently characterized metabolites from Streptomyces sp. RM-14-6 (3–7 and 9; 10–20; Figures 2 and S1),61 five of which [spoxazomicin C (3), spoxazomicin D (4), N-salicyloyl-2-aminopropane-1,3-diol (6) (2R)-N-salicyloyl-2-aminopropan-1-ol (7), and o-hydroxybenzamide (9); Figure 2] were identified as putative substrates (Figure 3A and B). Subsequent hit validation confirmed the results from the colorimetric screen with turnovers ranging from 6% to 68% based on HPLC integration, where, in some cases (e.g., 3), the multiple products observed were attributed to putative variant glycoside regioisomers as previously reported for OleD-catalyzed reactions.72,73 Interestingly, this secondary analysis also revealed oxachelin (5) as a false negative with 45% turnover indicated by HPLC peak integration, the product of which was also confirmed by HRMS. Importantly, this analysis highlights the utility of the 2-chloro-4-nitrophenylglycoside-based screen as a rapid first-pass assay to identify putative substrates for engineered OleDs and highlights the first application of donor 2 (2-chloro-4-nitrophenyl-GlcNH2) within this context.

Figure 2.

Figure 2

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Structures of validated OleD Loki substrates identified and corresponding glycosides generated via preparative OleD Loki-catalyzed chemoenzymatic synthesis.

Figure 3.

Figure 3

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OleD Loki-catalyzed colorimetric glycosyl-scanning results using 2-chloro-4-nitrophenyl-Glc (1) (A) or 2-chloro-4-nitrophenyl-GlcNH2 (2) (B) as donor for all Streptomyces sp. RM-14-6 metabolites confirmed to turnover based on LC-MS [positive control, 4-methylumbelliferone (4-MeU); negative control, no acceptor (DMSO)]. No turnover was observed with compounds 10–20 (Supporting Information, Figure S1). The turnover of compound 8 (the synthetic enantiomer of metabolite 7) was identical to 7.

For full product characterization and biological evaluation, chemoenzymatic scale-up reactions were conducted with spoxazomicin C (3), oxachelin (5), N-salicyloyl-2-aminopropane-1,3-diol (6), (2R)-N-salicyloyl-2-aminopropan-1-ol (7), and (2S)-N-salicyloyl-2-aminopropan-1-ol (8) to give 2-O-β-D-glucosylspoxazomicin C (3a, 1.2 mg), 12-O-β-D-glucosylspoxazomicin C (3b, 0.5 mg), 2-O-(2′-amino-2′-deoxy-β-D-glucosyl)spoxazomicin C (3c, 3.9 mg), 23-O-β-D-glucosyloxachelin (5a, 1.5 mg), 23-O-(2′-amino-2′-deoxy-β-D-glucosyl)-oxachelin (5b, 2.0 mg), N-(6-O-β-D-glucosyl)salicyloyl-2-aminopropane-1,3-diol (6a, 2.0 mg), N-[6-O-(2′-amino-2′-deoxy-β-D-glucosyl)]salicyloyl-2-aminopropane-1,3-diol (6b, 0.8 mg), (2R)-N-(6-O-β-D-glucosyl)salicyloyl-2-aminopropan-1-ol (7a, 1.6 mg), (2R)-N-[6-O-(2′-amino-2′-deoxy-β-D-glucosyl)]salicyloyl-2-aminopropan-1-ol (7b, 4.6 mg), (2S)-N-(6-O-β-D-glucosyl)salicyloyl-2-aminopropan-1-ol (8a, 1.8 mg), and (2S)-N-[6-O-(2′-amino-2′-deoxy-β-D-glucosyl)]salicyloyl-2-aminopropan-1-ol (8b; 4.8 mg), respectively. This study is among the first to highlight the utility of the 2-chloro-4-nitrophenyl-glycoside-based platform as a means for preparative synthesis of novel glycosides.

Structure Elucidation

The physicochemical properties of the new glycosylated compounds 3a–c, 4a, 5a/b, 6a/b, 7a/b, and 8a/b are summarized in the Experimental Section. Compounds 3a–c were obtained as white solids. HRMS of 3a–c revealed a common molecular formula for 3a and 3b (C16H21NO8; consistent with 1 as donor) and a distinct molecular formula for 3c (C16H22N2O7; consistent with 2 as donor). Comparison of 1H and 13C NMR spectral features of 3a–c to those of the parent aglycone [spoxazomicin C (3)] and, in particular, observed 3J HMBC sugar H-1′ to aglycon C-2/C-12 correlations (for 3a and 3c) and large sugar anomeric proton coupling constants (δH 5.02–5.30 ppm, d; J = 7.8–9.0 Hz) confirmed the corresponding 3a C2-O-β-D-glucosyl (β-D-Glc; Figure 2, GI), 3b C12-O-β-D-glucosyl, and 3c C2-O-2′-amino-2′-deoxy-β-D-glucosyl (β-D-GlcNH2; Figure 2, GII) moieties. Similarly, compounds 5a/b were obtained as white solids with molecular formulas consistent with monoglycosides (C33H47N7O16, C33H48N8O15, respectively), where NMR confirmed the C23 phenolic oxygen of 5 as the point of attachment for β-D-Glc (Figure 2; GI, 5a) and β-D-GlcNH2 (Figure 2; GII, 5b). Likewise, compounds 6a/b, 7a/b, and 8a/b were confirmed by HRMS and 1D and 2D NMR to be β-D-Glc- and β-D-GlcNH2-derived C6-O-monoglycosides of aglycones 6, 7, and 8, respectively (Figure 2). Given the lack of observed OleD Loki turnover with spoxazomicin D (4), the corresponding C2-O-β-D-Glc comparator for biological evaluation (Figure 2; 4a) was generated via Koenigs–Knorr glycosylation following conventional protocols.74,75

Biological Activity

All glycosides were evaluated in standard antibacterial, antifungal, and anticancer activity assays, and, based on the reported EtOH damage neuroprotective activity of spoxazomicin D (4),61 a select set of glycosides (3a–c and 4a, Figure 2) were also evaluated in an EtOH damage neuroprotection assay.71,76,77 For the latter, compounds (0.01–1.0 μM) were assessed for their ability to protect against EtOH cytotoxicity of rat hippocampal-derived primary cell cultures exposed to 100 mM EtOH as determined by propidium iodide uptake, a highly polar nucleic acid intercalating agent that labels compromised cells (Figure 4A–D). Mean increases of approximately 70% of ethanol-naïve control levels were observed with each replication. Compound 3a was found to reduce EtOH-induced increases in propidium iodide uptake. Post hoc analyses revealed that this reversal of propidium iodide uptake by compound 3a was observed at 0.01 and 0.10 μM (Figure 4E). In EtOH-naïve medium, coexposure to compound 3a was also found to induce significant propidium iodude uptake at 1 μM. Notably, coexposure of compounds 3b and 3c did not significantly attentuate EtOH-induced cytotoxicity. Compound 4a was also found to decrease EtOH-induced increases in propidium iodide uptake. Post hoc analyses demonstrated this reduction in propidium iodide by 4a was observed at 0.10 and 1.0 μM (Figure 4F; ~103 less potent than the parent 461). In EtOH-naïve medium, coexposure to compound 4a did reveal significant increases in prodidium iodide uptake at 0.01 and 0.10 μM. In contrast, glycosylation had no apparent effect on antibacterial, antifungal, or anticancer activity, as both the parental NPs and all corresponding glycosides were inactive at the concentrations tested (≤60–120 μM antibacterial/fungal; ≤20 μM anticancer). This study is the first to demonstrate glycosylation as a means to modulate the neuroprotective properties of small molecules in the context of EtOH-induced cytotoxicity.

Figure 4.

Figure 4

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EtOH damage neuroprotection assay (propidium iodide uptake in rat-derived organotypic hippocampal slice primary cell cultures). (A) DMSO control; (B) 48 h exposure to 100 mM EtOH; (C) 48 h exposure to 100 mM EtOH and 10 nM 3a; (D) 48 h exposure to 100 mM EtOH and 1 μM 4a; (E) dose–response with 48 h exposure to EtOH (100 mM) in the absence or presence of 3a on propidium iodide uptake; (F) Ddose–response with 48 h exposure to EtOH (100 mM) in the absence or presence of 4a on propidium iodide uptake. *p < 0.001 vs control; **p < 0.001 vs EtOH.

Discussion

Permissive engineered OleDs in conjunction with UDP-Glc as the donor have been successfully used in the preparative synthesis of select pharmacophore glucosides where product distribution highlighted the catalyst’s ability to afford distinct glycosidic regioisomers and, in some cases, disaccharide-substituted analogues via iterative glycosylation.72,78,79 The advent of a 2-chloro-4-nitrophenylglycoside-based platform within this context extended the utility of engineered OleDs for sugar nucleotide synthesis and rapid identification of new substrates (referred to as “glycosyl-scanning”).55–57,59,60 The current study notably builds on the prior precedent in two primary ways. First, this study is the first application of the 2-chloro-4-nitrophenylglycoside-based screen toward an unbiased set of unique microbial natural products as well as the first application of 2-chloro-4-nitrophenyl-GlcNH2 as a donor in “glycosyl-scanning”. The corresponding 35% hit rate further highlights the unique permissivity of the OleD Loki GT and its potential in natural product diversification. Second, this study is the first application of a 2-chloro-4-nitrophenylglycoside-based transglycosylation reaction for the preparative synthesis of target glycosides where the recycling of the nucleotide diphosphate (UDP) byproduct (a known glycosyltransferase inhibitor) to drive reaction equilibrium and the use of a simple inexpensive synthetic donor (i.e., 2) are advantageous. Consistent with prior studies of engineered OleDs,53,54,72,73,78–80 analysis of the glycosides formed in this study reveals a propensity toward phenolic glycosylation, where the observed aliphatic β-D-O-Glc 3b stands as an exception. Lack of OleD turnover with 4 (and the corresponding poor reactivity of the 4 C2-OH based on synthetic yields in chemical glycosylation) may implicate prohibitive acceptor intramolecular interactions not present in extended structures such as 5. Finally, this study also further highlights the potential of glycosylation to modulate the bioactivity of a given pharmacophore, where glycosylation invokes (in the case of 3) or slightly reduces (in the case of 4) the desired neuroprotective effects of the parent natural products.29–39 While the fundamental mechanism(s) of neuroprotection remain to be determined, this study presents important structure–activity relationships that may support future target identification (e.g., via incorporation of sugars bearing reactive handles for affinity pull-down studies) and/or, given the impact of glucose conjugation on improving blood–brain barrier activity transport,64–68 may also enhance biodistribution in the context of advancing leads to treat neurological deficits resulting from alcohol abuse.61,69–71

EXPERIMENTAL SECTION

General Experimental Procedures

All reagents were reagent grade and purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used without purification unless otherwise noted. All solvents used were of ACS grade and purchased from Pharmco-AAPER (Brookfield, CT, USA). Donors (2-chloro-4-nitrophenyl)-β-D-glucoside (1) and (2-chloro-4-nitrophenyl)-2-amino-2-deoxy-β-D-glucoside (2) were synthesized as previously described.55,57 All studies used pure OleD Loki (≥95%; an engineered P67T/I112P/T113M/S132F/A242I OleD variant), the overproduction and affinity purification for which have been previously described.56 The isolation, purification, and characterization of Streptomyces sp. RM-14-6 metabolites (3–7 and 9–20; Figures 2 and S1) used for this study are reported elsewhere.61 Analytical TLC was performed using Sorbent Technologies silica gel glass TLC plates (EMD Chemical Inc., PA, USA). Visualization was accomplished with UV light (254 nm) followed by staining with a diluted sulfuric acid solution (5% in EtOH) and heating. NMR spectra were measured using Varian (Palo Alto, CA, USA) Vnmr 500 (1H, 500 MHz; 13C, 125.7 MHz) and Vnmr 400 (1H, 399.8 MHz; 13C, 100.5 MHz) spectrometers using 99.8% D2O with 0.05% v/v tetramethylsilane (TMS) or 99.8% CD3OD from Cambridge Isotopes (Cambridge Isotope Laboratories, MA, USA), where δ-values were referenced to TMS. Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), and m (multiplet). Chemical shifts are reported in parts per million (ppm), and coupling constants J are given in Hz. High-resolution electrospray ionization (HRESI) mass spectra were obtained on a Waters (Milford, MA, USA) LCT time-of-flight (TOF) spectrometer or an AB SCIEX Triple TOF 5600 system (AB Sciex, Framingham, MA, USA). Staphylococcus aureus, Salmonella enterica, and Saccharomyces cerevisiae strains and A549 cells were obtained from ATCC (Manassas, VA, USA); Micrococcus luteus and Escherichia coli were obtained from NRRL (Peoria, IL, USA).

Chromatography Methods

Analytical reversed-phase HPLC was accomplished using an Agilent 1260 system (Santa Clara, CA, USA) equipped with a photodiode diode array detector [method A: Phenomenex Luna C18 (Torrance, CA, USA), 4.6 × 250 mm, 5 μm; solvent A: H2O/0.1% TFA, solvent B: CH3CN; flow rate: 1 mL min−1; 0–20 min, 1–75% B; 20–23 min, 75–100% B; 23–31 min, 100% B; 31–32 min, 100–5% B; 32–34 min, 5% B; A215/254]. Semipreparative reversed-phase HPLC was accomplished using a Varian ProStar model 210 equipped with a photodiode diode array detector [method B: Phenomenex Gemini C18, 10 × 250 mm, 5 μm; solvent A: H2O/0.1% TFA, solvent B: CH3CN; flow rate: 4.5 mL min−1; 0–25 min, 5–55% B; 25–27 min, 55–100% B; 27–33 min, 100% B; 33–34 min, 100–5% B; 34–38 min, 5% B; A254].

Colorimetric Glycosyl-Scanning Assays

Reactions were conducted in triplicate in a 384-well plate format containing 2 mM donor [(2-chloro-4-nitrophenyl)-β-D-glucoside (1) or (2-chloro-4-nitrophenyl)-2-amino-2-deoxy-β-D-glucoside (2)], 1 mM acceptor, 0.1 mM UDP, and enzyme (0.26 μM Loki for donor 1; 2.6 μM Loki for donor 2) in a final total volume of 20 μL of Tris-HCl buffer (25 mM, 5 mM MgCl2, pH 8.0). Reactions were initiated with the addition of enzyme and incubated at 30 °C for 8 h with continuous monitoring (ΔA410) using a Fluostar Omega microplate reader (BMG LABTECH GmbH, Ortenberg, Germany). Reactions that displayed a ΔA410 of three standard deviations above that of controls lacking aglycone (DMSO vehicle) were identified as preliminary substrates and subsequently quenched via the addition of 100 μL of 50% MeOH and filtered (MultiScreen filter plate; Millipore, Billerica, MA, USA) according to manufacturer’s instructions. Then the filtrate was analyzed by HPLC (method A) and LC-MS.

General Procedure for Preparative Chemoenzymatic Glycoside Syntheses

Reactions were conducted in 2 dram vials (total volume 4 mL) and contained acceptor (3 mM), donor (5 mM UDP-glucose or 3 mM 2-chloro-4-nitrophenyl-GlcNH2 with 0.1 mM UDP) in 50 mM Tris HCl, and 5 mM MgCl2, pH 8.0. Reactions were initiated via the addition of catalyst (OleD Loki, 2.6 μM) and incubated at 25 °C for up to 12 h with stirring and periodically monitored via ΔA410, TLC, and/or analytical HPLC (method A). Upon completion, reactions were quenched by the addition of 2 mL of MeOH and filtered using Amicon Ultra-15 centrifugal filter units (3 kDa cutoff; Millipore), and the filtrate was subsequently frozen and lyophilized. The corresponding concentrated crude reaction debris was resuspended in 1 mL of DMSO and centrifuged (18000g, 20 min, 4 °C), and the resulting crude supernatant was resolved by preparative HPLC (method B). Product-containing fractions were subsequently collected and lyophilized, and the corresponding products advanced for spectroscopic characterization.

Antibacterial, Antifungal, and Cancer Cell Line Viability Assays

Antibacterial (Staphylococcus aureus ATCC 6538, Micrococcus luteus NRRL B-287, Escherichia coli NRRL B-3708, Salmonella enterica ATCC 10708), antifungal (Saccharomyces cerevisiae ATCC 204508), and cell line cytotoxicity (non-small-cell lung A549) assays were accomplished in triplicate following our previously reported protocols.61,81

EtOH Damage Neuroprotection Assay

Assays to assess modulation of EtOH damage were accomplished following previously reported protocols.71,76,77

2-O-β-D-Glucosylspoxazomicin C (3a)

Using the general preparative scale methodology, spoxazomicin C (3; 3 mg, 0.015 mmol) and UDP-Glc (6 mg, 0.018 mmol) yielded 3a (1.2 mg, 3.3 μmol, 20% yield) and 3b (0.5 mg, 1.4 μmol, 10% yield) as white solids: 1H NMR (CD3OD, 500 MHz) δ 7.90 (dd, J = 7.9, 1.6 Hz, 1H), 7.49 (m, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 5.02 (d, J = 7.8 Hz, 1H), 4.16 (t, J = 5.4 Hz, 1H), 3.90 (dd, J = 12.2, 2.0 Hz, 1H), 3.67–3.81 (m, 5H), 3.53–3.59 (m, 1H), 3.38–3.51 (m, 3H); (+)-HRESIMS m/z 356.1340 [M + H]+ (calcd for C16H22NO8, 356.1340).

12-O-β-D-Glucosylspoxazomicin C (3b)

1H NMR (D2O, 500 MHz) δ 7.73 (d, J = 7.3 Hz, 1H), 7.50 (t, J = 8.8 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 5.07 (dd, J = 4.6, 2.7 Hz, 1H), 4.44–4.49 (m, 1H), 4.37 (m, 1H), 3.69–3.83 (m, 4H), 3.59–3.67 (m, 2H), 3.48 (m, 2H), 3.38 (d, J = 8.8 Hz, 1H); (+)-HRESIMS m/z 356.1334 [M + H]+ (calcd for C16H22NO8, 356.1340).

2-O-(2′-Amino-2′-deoxy-β-D-glucosyl)spoxazomicin C (3c)

Using the general preparative scale methodology, spoxazomicin C (3; 3 mg, 0.015 mmol) and (2-chloro-4-nitrophenyl)-2-amino-2-deoxy-β-D-glucoside (2; 5 mg, 0.015 mmol) yielded 3c (3.9 mg, 11 μmol, 71% yield) as a white solid: 1H NMR (CD3OD, 400 MHz) δ 7.77 (dd, J = 7.8, 1.6 Hz, 1H), 7.70–7.74 (m, 1H), 7.44 (t, J = 15.3 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 5.30 (d, J = 9.0 Hz, 1H), 4.07–4.16 (m, 1H), 3.82–3.90 (m, 4H), 3.70–3.81 (m, 2H), 3.66 (dd, J = 12.1, 5.9 Hz, 1H), 3.42–3.49 (m, 1H), 3.12–3.23 (m, 2H); (+)-HRESIMS m/z 355.1498 [M + H]+ (calcd for C16H23N2O7, 355.1500).

23-O-β-D-Glucosyloxachelin (5a)

Using the general preparative scale methodology, oxachelin (5; 5 mg, 0.007 mmol) and UDP-Glc (4 mg, 0.012 mmol) yielded 5a (1.5 mg, 1.9 μmol, 24% yield) as a white solid: 1H NMR (400 MHz, CD3OD) δ 8.22 (s, 1H), 7.70 (dd, J = 7.8, 1.6 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 6.85–7.02 (m, 2H), 5.10 (d, J = 7.8 Hz, 1H), 4.58–4.73 (m, 4H), 4.28–4.49 (m, 3H), 3.82–3.95 (m, 4H), 3.64–3.68 (m, 2H), 3.28–3.46 (m, 9H), 2.33–2.36 (m, 3H), 1.83–1.60 (m, 8H); (+)-HRESIMS m/z 798.3162 [M + H]+ (calcd for C33H48N7O16, 798.3152).

23-O-(2′-Amino-2′-deoxy-β-D-glucosyl)oxachelin (5b)

Using the general preparative scale methodology, oxachelin (5; 5 mg, 0.007 mmol) and (2-chloro-4-nitrophenyl)-2-amino-2-deoxy-β-D-glucoside (2; 3 mg, 0.009 mmol) yielded 5b (2.0 mg, 2.5 μmol, 32% yield) as a white solid: 1H NMR (400 MHz, CD3OD) δ 8.36 (d, J = 2.7 Hz, 1H), 8.21 (dd, J = 9.0, 2.7 Hz, 1H), 7.49 (d, J = 9.4 Hz, 1H), 6.96 (d, J = 8.6 Hz, 1H), 6.86–6.93 (m, 1H), 5.46 (d, J = 8.6 Hz, 1H), 5.06 (dd, J = 10.6, 7.8 Hz, 1H), 5.00 (m, 1H), 4.64–4.71 (m, 1H), 4.61 (dd, J = 8.0, 3.3 Hz, 1H), 4.36–4.44 (m, 4H), 3.82–3.92 (m, 3H), 3.39–3.77 (m, 12H), 3.10–3.13 (m, 1H), 2.34–2.37 (m, 2H), 1.96–2.04 (m, 3H), 1.81–1.83 (m, 2H), 1.60–1.66 (m, 3H); (+)-HRESIMS m/z 797.3308 [M + H]+ (calcd for C33H49N8O15, 797.3312).

N-(6-O-β-D-Glucosyl)salicyloyl-2-aminopropane-1,3-diol (6a)

Using the general preparative scale methodology, N-salicyloyl-2-aminopropane-1,3-diol (6; 6 mg, 0.028 mmol) and UDP-glucose (15 mg, 0.028 mmol) yielded 6a (2.0 mg, 5.4 μmol, 19% yield) as a white solid: 1H NMR (400 MHz, CD3OD) δ 7.88 (dd, J = 7.0, 1.6 Hz, 1H), 7.43–7.51 (m, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 5.01 (d, J = 7.4 Hz, 1H), 4.15 (q, J = 5.3 Hz, 1H), 3.88 (dd, J = 11.9, 1.8 Hz, 1H), 3.61–3.79 (m, 5H), 3.52–3.58 (m, 1H), 3.33–3.51 (m, 3H); HRESIMS m/z 374.1446 [M + H]+ (calcd for C16H24NO9, 374.1446).

N-[6-O-(2′-Amino-2′-deoxy-β-D-glucosyl)]salicyloyl-2-aminopropane-1,3-diol (6b)

Using the general preparative scale methodology, N-salicyloyl-2-aminopropane-1,3-diol (6; 6 mg, 0.028 mmol) and (2-chloro-4-nitrophenyl)-2-amino-2-deoxy-β-D-glucoside (2; 5 mg, 0.015 mmol) yielded 6b (0.8 mg, 2.2 μmol, 15% yield) as a white solid: 1H NMR (400 MHz, CD3OD) δ 7.65 (dd, J = 7.4, 1.6 Hz, 1H), 7.50 (td, J = 7.4, 1.6 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 7.20 (t, J = 7.2 Hz, 1H), 5.20 (d, J = 8.2 Hz, 1H), 4.16 (q, J = 5.7 Hz, 1H), 3.87–3.93 (m, 1H), 3.67–3.78 (m, 3H), 3.64–3.66 (m, 1H), 3.54–3.63 (m, 2H), 3.44–3.52 (m, 2H), 3.24 (dd, J = 10.6, 8.6 Hz, 1H); HRESIMS m/z 373.1607 [M + H]+ (calcd for C16H25N2O8, 373.1605).

(2R)-N-(6-O-β-D-Glucosyl)salicyloyl-2-aminopropan-1-ol (7a)

Using the general preparative scale methodology, (2R)-N-salicyloyl-2-aminopropan-1-ol (7; 6 mg, 0.031 mmol) and UDP-glucose (17 mg, 0.031 mmol) yielded 7a (1.6 mg, 4.5 μmol, 15% yield) as a white solid: 1H NMR (400 MHz, CD3OD) δ 7.81 (dd, J = 7.6, 1.4 Hz, 1H), 7.42–7.52 (m, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 5.00 (d, J = 7.4 Hz, 1H), 4.17 (dq, J = 12.1, 6.7 Hz, 1H), 3.79–3.96 (m, 1H), 3.70 (dd, J = 11.9, 5.3 Hz, 1H), 3.58–3.64 (m, 2H), 3.33–3.57 (m, 4H), 1.23 (d, J = 6.7 Hz, 3H); HRESIMS m/z 358.1497 [M + H]+ (calcd for C16H24NO8, 358.1496).

(2R)-N-[6-O-(2′-Amino-2′-deoxy-β-D-glucosyl)]salicyloyl-2-aminopropan-1-ol (7b)

Using the general preparative scale methodology, (2R)-N-salicyloyl-2-aminopropan-1-ol (7; 6 mg, 0.031 mmol) and (2-chloro-4-nitrophenyl)-2-amino-2-deoxy-β-D-glucoside (2; 10 mg, 0.031 mmol) yielded 7b (4.6 mg, 12.9 μmol, 42% yield) as a white solid: 1H NMR (400 MHz, CD3OD) δ 7.56 (dd, J = 7.8, 1.2 Hz, 1H), 7.49 (t, J = 8.2 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H), 5.17 (d, J = 8.6 Hz, 1H), 4.16 (dq, J = 12.3, 6.5 Hz, 1H), 3.90 (dd, J = 11.9, 1.4 Hz, 1H), 3.72–3.78 (m, 1H), 3.54–3.65 (m, 3H), 3.42–3.51 (m, 2H), 3.18 (dd, J = 10.2, 9.0 Hz, 1H), 1.20 (d, J = 6.7 Hz, 3H); HRESIMS m/z 357.1661 [M + H]+ (calcd for C16H25N2O7, 357.1656).

(2S)-N-(6-O-β-D-Glucosyl)salicyloyl-2-aminopropan-1-ol (8a)

Using the general preparative scale methodology, (2S)-N-salicyloyl-2-aminopropan-1-ol (8; 6 mg, 0.031 mmol) and UDP-glucose (17 mg, 0.031 mmol) yielded 8a (1.8 mg, 5.1 μmol, 16% yield) as a white solid: 1H NMR (400 MHz, CD3OD) δ 7.86 (dd, J = 7.8, 1.6 Hz, 1H), 7.46 (td, J = 8.2, 2.0 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.09–7.18 (m, 1H), 4.98 (d, J = 7.8 Hz, 1H), 4.18 (dq, J = 12.1, 6.7 Hz, 1H), 3.90 (dd, J = 11.9, 2.2 Hz, 1H), 3.70 (dd, J = 11.9, 5.7 Hz, 1H), 3.60 (d, J = 5.1 Hz, 2H), 3.43–3.56 (m, 3H), 3.39 (t, J = 9.0 Hz, 1H), 1.25 (d, J = 6.7 Hz, 3H); HRESIMS m/z 358.1500 [M + H]+ (calcd for C16H24NO8, 358.1496).

(2S)-N-[6-O-(2′-Amino-2′-deoxy-β-D-glucosyl)]salicyloyl-2-aminopropan-1-ol (8b)

Using the general preparative scale methodology, (2S)-N-salicyloyl-2-aminopropan-1-ol (8; 6 mg, 0.031 mmol) and (2-chloro-4-nitrophenyl)-2-amino-2-deoxy-β-D-glucoside (2; 10 mg, 0.031 mmol) yielded 8b (4.8 mg, 13.5 μmol, 44% yield) as a white solid: 1H NMR (400 MHz, CD3OD) δ 7.54 (dd, J = 7.6, 1.4 Hz, 1H), 7.45–7.52 (m, 1H), 7.38–7.42 (m, 1H), 7.19 (t, J = 7.4 Hz, 1H), 5.15 (d, J = 8.6 Hz, 1H), 4.17 (sxt, J = 6.3 Hz, 1H), 3.92 (dd, J = 11.9, 1.8 Hz, 1H), 3.75 (dd, J = 11.9, 5.3 Hz, 1H), 3.53–3.65 (m, 3H), 3.50 (d, J = 5.9 Hz, 2H), 3.21 (dd, J = 10.4, 8.8 Hz, 1H), 1.22 (d, J = 7.0 Hz, 3H); HRESIMS m/z 357.1667 [M + H]+ (calcd for C16H25N2O7, 357.1656).

2-O-β-D-Glucosylspoxazomicin D (4a)

To the solution of 1-bromo-α-D-glucose tetraacetate (60 mg, 0.15 mmol) in anhydrous CH3CN (5 mL) was added 4 (15 mg, 0.07 mmol) and 4 Å molecular sieves. After stirring the mixture for 30 min at room temperature, Ag2O (35 mg, 0.15 mmol) was added and the reaction allowed to continue for 24 h. The reaction was filtered through Celite and concentrated under vacuum, and the recovered crude material resuspended in 1 mL of MeOH, centrifuged, and resolved by preparative HPLC (method B). The product-containing fractions were subsequently collected and lyophilized to provide the desired product as an anomeric mixture (2:1 β:α), which was purified by HPLC (method A) to give the desired β-anomer (5 mg, 0.01 mmol). The β-anomer was dissolved in MeOH (1 mL), and global deprotection was accomplished via the addition of K2CO3 (5 mg, 0.04 mmol) followed by stirring at room temperature while monitoring the reaction via TLC and HPLC (method A). Upon completion (~6 h), the reaction was quenched by adding Amberlite 120 (H+) (4 equiv), stirred for 30 min, and filtered through Celite, and the crude reaction was dried under vacuum. The recovered crude material was subsequently dissolved in 1 mL of MeOH and resolved via preparative HPLC (method B). Product-containing fractions were subsequently collected and lyophilized to provide the desired 4a (white solid, 2 mg, 0.005 mmol, 8% yield in two steps): 1H NMR (CD3OD, 400 MHz) δ 7.94 (d, J = 7.4 Hz, 1H), 7.46–7.56 (m, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.14 (t, J = 9.8 Hz, 1H), 5.06 (d, J = 8.2 Hz, 1H), 4.63 (t, J = 4.3 Hz, 1H), 3.97 (dd, J = 4.3,11.0 Hz, 1H), 3.80–3.93 (m, 2H), 3.71 (dd, J = 5.3, 11.9 Hz, 1H), 3.65 (t, J = 8.6 Hz, 1H), 3.45–3.51 (m, 2H), 3.41 (t, J = 8.6 Hz, 1H); 13C NMR (CD3OD, 100 MHz) δ 173.7 (Cq-12), 165.9 (Cq-7), 155.9 (Cq-2), 133.0 (CH-4), 130.6 (CH-6), 122.4 (CH), 122.1 (CH), 116.0 (Cq-1), 102.2 (CH-1′), 77.0 (CH), 76.2 (CH), 73.2 (CH), 69.6 (CH), 61.9, 60.9, 55.5 ppm; HRESIMS m/z 387.1404 [M + H3O]+ (calcd for C16H23N2O9, 387.1398).

Supplementary Material

SI

Acknowledgments

This work was supported in part by NIH R37 AI52188, NIH T32 DA016176 (YZ), the University of Kentucky College of Pharmacy, the University of Kentucky Markey Cancer Center, and the National Center for Advancing Translational Sciences (UL1TR001998).

Footnotes

ORCID

Jon S. Thorson: 0000-0002-7148-0721

The authors declare the following competing financial interest(s): The authors report competing interests. JST is a co-founder of Centrose (Madison, WI).

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00949.

Chemical structures of compounds 10–20; HRMS/NMR spectra of compounds 3a–c, 4a, 5a,b, 6a,b, 7a,b, and 8a,b (PDF)

References

  • 1.Moloney MG. Trends Pharmacol Sci. 2016;37:689–701. doi: 10.1016/j.tips.2016.05.001. [DOI] [PubMed] [Google Scholar]
  • 2.Harvey AL, Edrada-Ebel R, Quinn RJ. Nat Rev Drug Discovery. 2015;14:111–129. doi: 10.1038/nrd4510. [DOI] [PubMed] [Google Scholar]
  • 3.Cragg GM, Grothaus PG, Newman DJ. J Nat Prod. 2014;77:703–723. doi: 10.1021/np5000796. [DOI] [PubMed] [Google Scholar]
  • 4.Cragg GM, Newman DJ. Biochim Biophys Acta, Gen Subj. 2013;1830:3670–3695. doi: 10.1016/j.bbagen.2013.02.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Li JW, Vederas JC. Science. 2009;325:161–165. doi: 10.1126/science.1168243. [DOI] [PubMed] [Google Scholar]
  • 6.Ma Z, Wang X, Wang X, Rodriguez RA, Moore CE, Gao S, Tan X, Ma Y, Rheingold AL, Baran PS, Chen C. Science. 2014;346:219–224. doi: 10.1126/science.1255677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Nicolaou KC, Hale CR, Nilewski C, Ioannidou HA. Chem Soc Rev. 2012;41:5185–5238. doi: 10.1039/c2cs35116a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Rodrigues T, Reker D, Schneider P, Schneider G. Nat Chem. 2016;8:531–541. doi: 10.1038/nchem.2479. [DOI] [PubMed] [Google Scholar]
  • 9.Garcia-Castro M, Zimmermann S, Sankar MG, Kumar K. Angew Chem, Int Ed. 2016;55:7586–7605. doi: 10.1002/anie.201508818. [DOI] [PubMed] [Google Scholar]
  • 10.Danishefsky S. Nat Prod Rep. 2010;27:1114–1116. doi: 10.1039/c003211p. [DOI] [PubMed] [Google Scholar]
  • 11.Buschleb M, Dorich S, Hanessian S, Tao D, Schenthal KB, Overman LE. Angew Chem, Int Ed. 2016;55:4156–4186. doi: 10.1002/anie.201507549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Zhang Y, Ye Q, Wang X, She QB, Thorson JS. Angew Chem, Int Ed. 2015;54:11219–11222. doi: 10.1002/anie.201505022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Ziemert N, Alanjary M, Weber T. Nat Prod Rep. 2016;33:988–1005. doi: 10.1039/c6np00025h. [DOI] [PubMed] [Google Scholar]
  • 14.Awan AR, Shaw WM, Ellis T. Adv Drug Delivery Rev. 2016;105:96. doi: 10.1016/j.addr.2016.04.010. [DOI] [PubMed] [Google Scholar]
  • 15.Walsh CT. Nat Chem Biol. 2015;11:620–624. doi: 10.1038/nchembio.1894. [DOI] [PubMed] [Google Scholar]
  • 16.Rutledge PJ, Challis GL. Nat Rev Microbiol. 2015;13:509–523. doi: 10.1038/nrmicro3496. [DOI] [PubMed] [Google Scholar]
  • 17.Gaudelli NM, Long DH, Townsend CA. Nature. 2015;520:383–387. doi: 10.1038/nature14100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Cimermancic P, Medema MH, Claesen J, Kurita K, Wieland Brown LC, Mavrommatis K, Pati A, Godfrey PA, Koehrsen M, Clardy J, Birren BW, Takano E, Sali A, Linington RG, Fischbach MA. Cell. 2014;158:412–421. doi: 10.1016/j.cell.2014.06.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Smanski MJ, Bhatia S, Zhao D, Park Y, Woodruff L, Giannoukos G, Ciulla D, Busby M, Calderon J, Nicol R, Gordon DB, Densmore D, Voigt CA. Nat Biotechnol. 2014;32:1241–1249. doi: 10.1038/nbt.3063. [DOI] [PubMed] [Google Scholar]
  • 20.Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, Geistlinger T, Henry R, Hepp M, Horning T, Iqbal T, Jiang H, Kizer L, Lieu B, Melis D, Moss N, Regentin R, Secrest S, Tsuruta H, Vazquez R, Westblade LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD. Nature. 2013;496:528–32. doi: 10.1038/nature12051. [DOI] [PubMed] [Google Scholar]
  • 21.Singh S, Phillips GN, Jr, Thorson JS. Nat Prod Rep. 2012;29:1201–1237. doi: 10.1039/c2np20039b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kim HJ, Ruszczycky MW, Choi SH, Liu YN, Liu HW. Nature. 2011;473:109–112. doi: 10.1038/nature09981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.King JR, Edgar S, Qiao K, Stephanopoulos G. F1000Research. 2016;5:397. doi: 10.12688/f1000research.7311.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Hubert CB, Barry SM. Biochem Soc Trans. 2016;44:738–744. doi: 10.1042/BST20160063. [DOI] [PubMed] [Google Scholar]
  • 25.Tibrewal N, Tang Y. Annu Rev Chem Biomol Eng. 2014;5:347–366. doi: 10.1146/annurev-chembioeng-060713-040008. [DOI] [PubMed] [Google Scholar]
  • 26.Podust LM, Sherman DH. Nat Prod Rep. 2012;29:1251–1266. doi: 10.1039/c2np20020a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Olano C, Mendez C, Salas JA. Nat Prod Rep. 2010;27:571–616. doi: 10.1039/b911956f. [DOI] [PubMed] [Google Scholar]
  • 28.Huber TD, Johnson BR, Zhang J, Thorson JS. Curr Opin Biotechnol. 2016;42:189–197. doi: 10.1016/j.copbio.2016.07.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Gantt RW, Peltier-Pain P, Thorson JS. Nat Prod Rep. 2011;28:1811–1853. doi: 10.1039/c1np00045d. [DOI] [PubMed] [Google Scholar]
  • 30.Cipolla L, Peri F. Mini-Rev Med Chem. 2011;11:39–54. doi: 10.2174/138955711793564060. [DOI] [PubMed] [Google Scholar]
  • 31.Elshahawi SI, Shaaban KA, Kharel MK, Thorson JS. Chem Soc Rev. 2015;44:7591–7697. doi: 10.1039/c4cs00426d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Goff RD, Thorson JS. MedChemComm. 2014;5:1036–1047. doi: 10.1039/C4MD00117F. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Calvaresi EC, Hergenrother PJ. Chem Sci. 2013;4:2319–2333. doi: 10.1039/C3SC22205E. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Thuan NH, Sohng JK. J Ind Microbiol Biotechnol. 2013;40:1329–1356. doi: 10.1007/s10295-013-1332-0. [DOI] [PubMed] [Google Scholar]
  • 35.Dwek RA. Chem Rev. 1996;96:683–720. doi: 10.1021/cr940283b. [DOI] [PubMed] [Google Scholar]
  • 36.Kapoor VK, Kaur A. Mini-Rev Med Chem. 2013;13:584–596. doi: 10.2174/1389557511313040010. [DOI] [PubMed] [Google Scholar]
  • 37.La Ferla B, Airoldi C, Zona C, Orsato A, Cardona F, Merlo S, Sironi E, D’Orazio G, Nicotra F. Nat Prod Rep. 2011;28:630–648. doi: 10.1039/c0np00055h. [DOI] [PubMed] [Google Scholar]
  • 38.Wilkinson B, Bachmann BO. Curr Opin Chem Biol. 2006;10:169–176. doi: 10.1016/j.cbpa.2006.02.006. [DOI] [PubMed] [Google Scholar]
  • 39.Doores KJ, Gamblin DP, Davis BG. Chem - Eur J. 2006;12:656–665. doi: 10.1002/chem.200500557. [DOI] [PubMed] [Google Scholar]
  • 40.De Bruyn F, Maertens J, Beauprez J, Soetaert W, De Mey M. Biotechnol Adv. 2015;33:288–302. doi: 10.1016/j.biotechadv.2015.02.005. [DOI] [PubMed] [Google Scholar]
  • 41.Schmolzer K, Gutmann A, Diricks M, Desmet T, Nidetzky B. Biotechnol Adv. 2016;34:88–111. doi: 10.1016/j.biotechadv.2015.11.003. [DOI] [PubMed] [Google Scholar]
  • 42.McArthur JB, Chen X. Biochem Soc Trans. 2016;44:129–142. doi: 10.1042/BST20150200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Yang Y, Zhang X, Yu B. Nat Prod Rep. 2015;32:1331–1355. doi: 10.1039/c5np00033e. [DOI] [PubMed] [Google Scholar]
  • 44.Fraser-Reid B. Top Curr Chem. 2011;301:xi–xiii. [PubMed] [Google Scholar]
  • 45.Crich D. Acc Chem Res. 2010;43:1144–1153. doi: 10.1021/ar100035r. [DOI] [PubMed] [Google Scholar]
  • 46.Boltje TJ, Buskas T, Boons GJ. Nat Chem. 2009;1:611–622. doi: 10.1038/nchem.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Galonic DP, Gin DY. Nature. 2007;446:1000–1007. doi: 10.1038/nature05813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Lairson LL, Wakarchuk WW, Withers SG. Chem Commun (Cambridge, U K) 2007:365–367. doi: 10.1039/b614636h. [DOI] [PubMed] [Google Scholar]
  • 49.Melancon CE, 3rd, Thibodeaux CJ, Liu HW. ACS Chem Biol. 2006;1:499–504. doi: 10.1021/cb600365q. [DOI] [PubMed] [Google Scholar]
  • 50.Lairson LL, Henrissat B, Davies GJ, Withers SG. Annu Rev Biochem. 2008;77:521–555. doi: 10.1146/annurev.biochem.76.061005.092322. [DOI] [PubMed] [Google Scholar]
  • 51.Lepenies B, Yin J, Seeberger PH. Curr Opin Chem Biol. 2010;14:404–411. doi: 10.1016/j.cbpa.2010.02.016. [DOI] [PubMed] [Google Scholar]
  • 52.Zhang C, Griffith BR, Fu Q, Albermann C, Fu X, Lee IK, Li L, Thorson JS. Science. 2006;313:1291–1294. doi: 10.1126/science.1130028. [DOI] [PubMed] [Google Scholar]
  • 53.Williams GJ, Zhang C, Thorson JS. Nat Chem Biol. 2007;3:657–662. doi: 10.1038/nchembio.2007.28. [DOI] [PubMed] [Google Scholar]
  • 54.Williams GJ, Goff RD, Zhang CS, Thorson JS. Chem Biol. 2008;15:393–401. doi: 10.1016/j.chembiol.2008.02.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Zhang J, Singh S, Hughes RR, Zhou M, Sunkara M, Morris AJ, Thorson JS. ChemBioChem. 2014;15:647–652. doi: 10.1002/cbic.201300779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Gantt RW, Peltier-Pain P, Singh S, Zhou M, Thorson JS. Proc Natl Acad Sci U S A. 2013;110:7648–7653. doi: 10.1073/pnas.1220220110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Gantt RW, Peltier-Pain P, Cournoyer WJ, Thorson JS. Nat Chem Biol. 2011;7:685–691. doi: 10.1038/nchembio.638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Chen Z, Zhang J, Singh S, Peltier-Pain P, Thorson JS, Hinds BJ. ACS Nano. 2014;8:8104–8112. doi: 10.1021/nn502181k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Peltier-Pain P, Marchillo K, Zhou M, Andes DR, Thorson JS. Org Lett. 2012;14:5086–5089. doi: 10.1021/ol3023374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Gantt RW, Thorson JS. Methods Enzymol. 2012;516:345–360. doi: 10.1016/B978-0-12-394291-3.00009-5. [DOI] [PubMed] [Google Scholar]
  • 61.Shaaban KA, Saunders MA, Zhang Y, Tran T, Elshahawi SIP, LV, Wang X, Zhang J, Copley GC, Sunkara M, Kharel MK, Morris AJ, Hower JC, Tremblay MS, Prendergast MA, Thorson JS. J Nat Prod. 2016 doi: 10.1021/acs.jnatprod.6b00948. [DOI] [Google Scholar]
  • 62.Inahashi Y, Iwatsuki M, Ishiyama A, Namatame M, Nishihara-Tsukashima A, Matsumoto A, Hirose T, Sunazuka T, Yamada H, Otoguro K, Takahashi Y, Omura S, Shiomi K. J Antibiot. 2011;64:303–307. doi: 10.1038/ja.2011.16. [DOI] [PubMed] [Google Scholar]
  • 63.Sontag B, Gerlitz M, Paululat T, Rasser HF, Grun-Wollny I, Hansske FG. J Antibiot. 2006;59:659–663. doi: 10.1038/ja.2006.88. [DOI] [PubMed] [Google Scholar]
  • 64.Egleton RD, Mitchell SA, Huber JD, Palian MM, Polt R, Davis TP. J Pharmacol Exp Ther. 2001;299:967–972. [PubMed] [Google Scholar]
  • 65.Fichna J, Mazur M, Grzywacz D, Kamysz W, Perlikowska R, Piekielna J, Sobczak M, Salaga M, Toth G, Janecka A, Chen C, Olczak J. Bioorg Med Chem Lett. 2013;23:6673–6676. doi: 10.1016/j.bmcl.2013.10.041. [DOI] [PubMed] [Google Scholar]
  • 66.Egleton RD, Mitchell SA, Huber JD, Janders J, Stropova D, Polt R, Yamamura HI, Hruby VJ, Davis TP. Brain Res. 2000;881:37–46. doi: 10.1016/s0006-8993(00)02794-3. [DOI] [PubMed] [Google Scholar]
  • 67.Scherrmann JM. Vasc Pharmacol. 2002;38:349–354. doi: 10.1016/s1537-1891(02)00202-1. [DOI] [PubMed] [Google Scholar]
  • 68.Moradi SV, Hussein WM, Varamini P, Simerska P, Toth I. Chemic Sci. 2016;7:2492–2500. doi: 10.1039/c5sc04392a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Tajuddin NF, Przybycien-Szymanska MM, Pak TR, Neafsey EJ, Collins MA. Alcohol. 2013;47:39–45. doi: 10.1016/j.alcohol.2012.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Crews FT, Nixon K. Alcohol Alcohol. 2009;44:115–127. doi: 10.1093/alcalc/agn079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Wang X, Reynolds AR, Elshahawi SI, Shaaban KA, Ponomareva LV, Saunders MA, Elgumati IS, Zhang Y, Copley GC, Hower JC, Sunkara M, Morris AJ, Kharel MK, Van Lanen SG, Prendergast MA, Thorson JS. Org Lett. 2015;17:2796–2799. doi: 10.1021/acs.orglett.5b01203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Zhou M, Hamza A, Zhan CG, Thorson JS. J Nat Prod. 2013;76:279–286. doi: 10.1021/np300890h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Gantt RW, Goff RD, Williams GJ, Thorson JS. Angew Chem, Int Ed. 2008;47:8889–8892. doi: 10.1002/anie.200803508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Gouliaras C, Lee D, Chan L, Taylor MS. J Am Chem Soc. 2011;133:13926–13929. doi: 10.1021/ja2062715. [DOI] [PubMed] [Google Scholar]
  • 75.Monch B, Gebert A, Emmerling F, Becker R, Nehls I. Carbohydr Res. 2012;352:186–190. doi: 10.1016/j.carres.2012.01.002. [DOI] [PubMed] [Google Scholar]
  • 76.Prendergast MA, Harris BR, Mullholland PJ, Blanchard JA, 2nd, Gibson DA, Holley RC, Littleton JM. Neuroscience. 2004;124:869–877. doi: 10.1016/j.neuroscience.2003.12.013. [DOI] [PubMed] [Google Scholar]
  • 77.Zimmer J, Kristensen BW, Jakobsen B, Noraberg J. Amino Acids. 2000;19:7–21. doi: 10.1007/s007260070029. [DOI] [PubMed] [Google Scholar]
  • 78.Zhou M, Thorson JS. Org Lett. 2011;13:2786–2788. doi: 10.1021/ol200977u. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 79.Zhou M, Hou Y, Hamza A, Pain C, Zhan CG, Bugni TS, Thorson JS. Org Lett. 2012;14:5424–5427. doi: 10.1021/ol3024924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 80.Williams GJ, Yang J, Zhang C, Thorson JS. ACS Chem Biol. 2011;6:95–100. doi: 10.1021/cb100267k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 81.Shaaban KA, Elshahawi SI, Wang X, Horn J, Kharel MK, Leggas M, Thorson JS. J Nat Prod. 2015;78:1723–1729. doi: 10.1021/acs.jnatprod.5b00429. [DOI] [PMC free article] [PubMed] [Google Scholar]

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