Post Polyketide Synthase Carbon-Carbon Bond Formation in Type-II

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Post Polyketide Synthase Carbon-Carbon Bond Formation in TypeII PKS Derived Natural Products from Streptomyces venezuelae Andrew William Robertson, Jeanna M MacLeod, Logan W MacIntyre, Stephanie M Forget, Steven R. Hall, Leah G Bennett, Hebelin Correa, Russell G. Kerr, Kerry B Goralski, and David L. Jakeman J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02823 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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The Journal of Organic Chemistry

Post Polyketide Synthase Carbon-Carbon Bond Formation in Type-II PKS Derived Natural Products from Streptomyces venezuelae Andrew W. Robertson,† Jeanna M. MacLeod,‡ Logan W. MacIntyre,† Stephanie. M. Forget,† Steven R. Hall,§ Leah G. Bennett,‡ Hebelin Correa, Russell G. Kerr, Kerry B. Goralski,†,§ and David L. Jakeman*,†,‡ †

‡

§

Department of Chemistry, College of Pharmacy, Department of Pharmacology, Dalhousie University, Halifax, NS, B3H 4R2, Canada

Department of Chemistry, University of Prince Edward Island, Charlottetown, PE, C1A 4P3, Canada

ABSTRACT: Polyketide synthase (PKS) derived natural products are biosynthesized by head-to-tail addition of acetate and malonate extender units resulting in linear extended-polyketide chains. Despite the well documented structural diversity associated with PKS derived natural products, C-C chain branching deviating from the usual linear pattern is relatively rare. Herein, type-II PKS angucyclic natural products containing a hemiaminal functionality were identified and proposed as the parent of a series of CC branched analogues. These C-C linked acetate or pyruvate branching units were located at the α-positions on the extended polyketide chains. Labeling studies utilizing [1-13C]-D-glucose provided mechanistic evidence that the C-C bond formation occurred as a result of a previously unidentified post-PKS processing, additional to the enzymes encoded within the biosynthetic gene cluster. Selected compounds were evaluated in cytotoxic or antimicrobial assays.

Introduction The Actinomyces genus Streptomyces has been a rich reservoir for bioactive natural products, representing one of the most prolific sources for clinically relevant molecules.1, 2 Many strains have been extensively screened for structurally unique lead compounds.3 The genome of Streptomyces venezuelae ISP5230 (ATCC 10712) was sequenced in 2011 and found to contain a number of putative natural product gene clusters, in addition to the two well-characterized and previously known clusters responsible for the biosynthesis of chloramphenicol and the jadomycins (Figure 1).4 Subsequently, the natural products gaburedin, venezuelin, foroxymithine, (+)isodauc-8-en-11-ol, venemycin, and, most recently, watasemycin and isopyochelin have been isolated from the strain.5-10 The jadomycins are cryptic type-II polyketide synthase (PKS) derived natural products produced under stress conditions.11 The jadomycins are members of the angucyclines, a large group of type-II PKS derived natural products, gaining their name from the angled structures of their polyaromatic scaffold (Figure 1).12 Interest in the jadomycins has arisen from their exhibited antibiotic,13 cytotoxic,14 and drug efflux transporter-evading properties,15 as well as their unique biosynthesis, involving incorporation of an amino acid present in the culture media into the polyaromatic core. Biosynthesis of the jadomycin polyketide backbone has been previously elucidated, terminating in the formation of dehydrorabelomycin.16 Thereafter, an enzyme catalyzed post PKS transformation occurs resulting in a 7-membered ringcontaining oxepinone intermediate (Figure 1).17 The enzymatic opening of the oxepinone produces an aldehyde that reacts

non-enzymatically with an amino acid present in the growth medium to ultimately furnish a cyclized E-ring (Figure 1a-b).13, 18, 19 This precursor-directed biosynthetic approach towards jadomycin congeners has included the incorporation of proteinogenic and non-proteinogenic amino acids.19-21 Evidence for the spontaneous cyclization and formation of the E-ring has been demonstrated through independent total synthesis of jadomycin analogues.22 The presence of a cyclized E-ring has been a feature of all previously described jadomycin congeners. In this manuscript, the isolation of a series of eight new jadomycin analogues 1, 1a, 2-7 (Figure 1) is described through culture with amino acids prohibitive of E-ring cyclization, 3and 4-(aminomethyl)benzoic acid (3AMBA and 4AMBA). Three of the isolated jadomycin analogues possessed reactive hemiaminal centers, susceptible to solvolysis with protic solvents, to compensate for the absence of intramolecular cyclization (1, 1a, and 5). We have studied the dynamic properties of the hemiaminal functionality. Three further congeners (3, 4 and 7) demonstrated a previously unreported post typeII PKS core carbon-carbon bond forming reaction that introduced either acetate (compounds 3 and 7) or pyruvate (compound 4). Biosynthesis of the jadomycin polyaromatic core is highly specific, with no C-C bifurcation of the scaffold observed in any of the isolated analogues to date. Herein, we have provided evidence to support a proposed post-PKS incorporation of the C-C branched units through [1-13C]-Dglucose labeling studies indicating that hemiaminal 1a is the likely precursor of compounds 2-4, and that these C-C deriva-

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tives are the result of the recruitment of additional enzymes

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to neutralize the reactive hemiaminal functionality.

Figure 1. Jadomycin congeners 1-7 isolated herein, and proposed formation; (a) representative E-ring cyclized jadomycins; (b) non-E-ring cyclized congeners and C-C branched congeners isolated and characterized from S. venezuelae ISP5230 cultures with 4(aminomethyl)benzoic acid (4AMBA) and 3-(aminomethyl)benzoic acid (3AMBA).

RESULTS Co-Amino Acid Supplementation. With the aim to elaborate our previous studies19, 21 on the incorporation of nonproteinogenic amino acids, and to further probe the structural diversity permissible from non-enzymatic amino acid incorporation, 3AMBA and 4AMBA, were selected to prohibit Ering cyclization given that their amino and carboxylate groups are separated by rigid benzyl moieties (Figure 1). We postulated that the amino groups of 3AMBA and 4AMBA would react with the aldehyde biosynthetic intermediate to form an imine but would preclude E-ring formation by preventing intramolecular cyclization. This could give rise to longer-lived imine intermediates allowing for alternate chemistry to occur. Initial cultures were carried out using established bacterial growth conditions with either 3AMBA or 4AMBA (60 mM).23 Streptomyces venezuelae ISP5230 was incapable of proliferation in the presence of either amino acid as the sole nitrogen

source, suggesting an inability of the bacteria to efficiently process the compounds as a nutrient source. To overcome this caveat, we implemented a modified procedure, supplementing with D-serine (15 mM) alongside either 3AMBA or 4AMBA. D-Serine was selected as it has been established that S. venezuelae cultures in the presence of serine supports cell growth and boosts jadomycin production.24, 25 S. venezuelae cultures with D-serine (15 mM) and either 3AMBA or 4AMBA (60 mM) promoted both organism growth and natural product production as observed by a darkening of the culture media characteristic for presence of colored natural products, including jadomycins. TLC analysis of the processed cultures identified several new colored compounds that did not correspond to the previously reported D-serine jadomycin analogue (jadomycin DS),25 implying preferential use of D-serine as a nutrient source. Growths were subsequently scaled up

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for isolation and characterization of the produced jadomycin family natural products. 1

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Figure 2. Characterization of compounds 1, and 1a, and their interconversion. (a) Structure of 1, illustrating NMR H- H COSY correlations 1 13 13 (bold lines), and H- C HMBC correlations (solid arrows), overlaid with C-NMR (176 MHz, CD3OD) showing the splitting pattern of the – 1 OCD3 moiety; (b) aromatic region of H-NMR spectrum of 1 recorded in CD3OD (700 MHz); (c) mixture of 1 and 1a, sample treated with D2O and recorded in CD3OD (700 MHz) illustrating interconversion. The small shift changes for H3’ and H4’ in 1 between (b) and (c) is

attributed to the mixed solvent system.

Isolation of Dynamic 3a-O-Methyl Jadomycin 4AMBA (1) and 3a-O-Methyl Jadomycin 3AMBA (5). A red compound appearing as the major natural product by TLC analysis from the 4AMBA culture extract was isolated by a series of chromatographic techniques and subsequently characterized by HRMS and NMR analysis (see supplemental information). 1H-NMR chemical shifts of the new compound were indicative of a glycosylated jadomycin-like compound present as a mixture of inseparable diastereomers arising from the stereochemistry at the 3aposition.19 These diastereomers maintain a dynamic equilibrium with one another owing to a ring opening event in which the B-ring of the jadomycin core can open and re-cyclize.18 High-resolution mass spectrometry (HRMS) analysis identified an m/z of 601.1878 corresponding to an [M – H]- of C33H31NO10. 1H NMR and 1H-1H COSY spectral analysis identified the intact jadomycin A-ring, D-ring, and L-digitoxose spin systems, and the presence of the incorporated 4AMBA for both diastereomers (Figure 2a). This confirmed that the coamino acid production protocol facilitated the incorporation

of the unnatural amino acid into the jadomycin core. Analysis of the HMBC spectrum completed characterization of the intact polyaromatic core, and confirmed the glycosylation site of the L-digitoxose moiety at the 12-position (Figure 2a). Next, the key structural features of the B-ring were examined to understand how ring formation was facilitated. Analysis of the HMBC spectrum identified correlations from H3a to C4, C13a, and C1′. AddiMonally, correlaMons were observed from H1′ to C3a and C13a conﬁrming the fully cyclized B-ring containing the fused 4AMBA. The remaining unaccounted-for mass from the HRMS analysis corresponded to CH3O, indicating methoxylation of the molecule. However, no methoxy signals were observed in the 1H NMR spectrum. The 1H and 13 C-NMR shifts of the 3a-position (δH = 5.61, δC = 92.7) suggested a similar environment to that of a typical oxazolone ring containing jadomycin, with the 3a-position adjacent to two heteroatoms (nitrogen and oxygen) and an aromatic carbon.19 The key correlation for structural elucidation was an HMBC correlation from H3a to a carbon with a chemical shift of 57.5 ppm. HSQC analysis did not reveal 1J1H-13C correlations to a carbon with that shift. Upon examination of the 13C-NMR

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spectrum, the C3a carbon signal was identified as a multiplet with a coupling constant of J = 21.5 Hz, resembling the 13C splitting pattern of CD3OD (Figure 2a). This splitting pattern indicated the methoxy moiety was −OCD3 and not -OCH3 as HRMS data had identified. The inconsistencies between the NMR and HRMS data could be rationalized by the fact that HRMS were collected using non-deuterated solvents. When HRMS was collected with CD3OD as the solvent the expected deuterated analogue m/z 603.2070 [M - H]- was found, corresponding to a molecular formula of C33H28D3NO10. We next set out to further probe the observation that substitution at the 3a-position was controlled by nucleophilic solvent addition. The compound was dissolved in ethanol and HRMS was recorded. An m/z of 614.2026 was found for [M – H]-, corresponding to a molecular formula of C34H33NO10 matching the expected m/z for the ethoxylated derivative. Due to the inability of the incorporated 4AMBA to undergo the intramolecular cyclization, we postulated that water could act as a nucleophile during fermentations to facilitate the B-ring closure (Figure 1c) resulting in the reactive intermediate jadomycin hydroxy-4AMBA (1a). The -OCD3 derivative 1 was dried in vacuo and brought up in D2O. On standing in D2O for several hours, a second set of peaks emerged in the NMR spectrum (Figure 2b-c), while the signals associated with analogue 1 correspondingly becoming less intense. Upon sequential drying and re-dissolving of the compound in D2O several times, the NMR spectra was significantly reduced of signals from 1, and the -OD derivative, jadomycin hydroxyl-4AMBA (1a), was characterized. All NMR correlations observed with analogue 1 were found, except for the HMBC correlation from H3a to -OCD3. HRMS was performed in H2O and identified an m/z of 610.1668 corresponding to [M + Na]+ giving a molecular formula of C32H29NO10, in agreement with the proposed structure. The corresponding jadomycin methoxy-3AMBA (5) derivative was isolated and characterized, and was found to possess the expected NMR correlations and mass spectral data analogous to those described for 1 (see SI). The nature of the C3a across compounds 1a, 1 and 5 are reflected in the 13C and 1H shifts at the 3a position and those in close proximity (Table 1). In the analogous methoxylated compounds (1 and 5), the chemical shifts are closely conserved, while hydroxylated 1a differs based on the electron donation capacity of the substituent. Table 1. Chemical shifts of C3a and adjacent protons effected by the nature of the C3a. Cmpd*

1a 1 5 2 6 3 7 4

δC or H C3a 86.6 92.7 91.6 164.1 163.9 65.6 64.8 61.9

H3a 5.88 5.61 5.58 5.03 5.06 5.12

*Major diastereomers only

H4 6.44 6.67 6.63 7.96 7.91 6.44 6.39 6.37

H6 6.39 6.85 6.82 6.94 7.20 6.66 6.61 6.67

H1'a 5.10 5.28 4.95 5.60 5.67 4.85 4.86 4.80

H1'b 5.92 5.51 5.75 6.10 6.22 5.75 5.74 5.77

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Isolation of Jadomycin 4AMBA Lactam (2) and Jadomycin 3AMBA Lactam (6). An orange compound with a similar Rf to 1 by TLC analysis was observed in the crude extract from the 4AMBA culture. The compound was purified (see SI) and subsequently characterized by HRMS and NMR analysis. The 1 H-NMR spectrum indicated a glycosylated jadomycin-like compound in which the chemical shifts H4 and H6 (δH4 = 7.96, δH6 = 6.94) of the A-ring were shifted downfield compared to that of 1 (δH4 = 6.67, δH6 = 6.85), likely owing to the presence of an adjacent carbonyl group (Figure 3a). Only one diastereomer was observed, which is unusual for jadomycins as they typically possess a 3a-stereocenter. The observation of an HMBC correlation between the H4 (δH = 7.57) and C3a (δC =164) supported a structure of jadomycin 4AMBA-lactam (2) with oxidation of the 3a position, consistent with reported data for a previously isolated lactam derivative (Figure 3a).26 HRMS analysis identified an m/z of 584.1640 corresponding to [M – H]- of C32H26NO10, consistent with the proposed structure of 2. 1H-NMR, HMBC, and 1H-1H COSY analysis confirmed the intact core jadomycin polyaromatic backbone and presence of the L-digitoxose glycosylation at the 12-position. Additionally, the 4AMBA spin system was present confirming the structure of 2 (Figure 3a). Purification and characterization of the corresponding jadomycin 3AMBA lactam (6) was accomplished (see SI), identifying this as a consistent oxidative event (see SI). Table 1 highlights the similar chemical shifts about the C3a between compounds 2 and 6, and the notable downfield shift in the H4 and H6 in comparison to the differentially substituted compounds. The oxidation is likely a spontaneous, based on the evidence for a series of structurally similar synthetic lactams.27 Isolation and Structural Characterization of Jadomycin Acetate-4AMBA (3), Jadomycin Acetate-3AMBA (7) and Jadomycin Pyruvate-4AMBA (4). From extracts of the 4AMBA culture, the presence of a series of polar purple unknown compounds were observed by TLC analysis. The compounds were isolated and purified (see supporting information). Initial 1H-NMR spectral analysis established the compounds as a mixture of diastereomers with a jadomycin-like core polyaromatic backbone. HRMS analysis indicated a molecular formula of C34H31NO11, identifying an m/z of 628.1818 (Figure S15) corresponding to [M – H]-. As was the case for 1, COSY NMR analysis identified the intact jadomycin A-ring, D-ring, and Ldigitoxose spin systems, as well as the incorporated 4AMBA side chain for both diastereomers (Figure 3a). HMBC analysis also identified the same jadomycin polyaromatic backbone connectivity identified for 1 (Figure 3a). Of particular interest was the observation in the 1H-NMR spectrum that H3a appeared as a doublet of doublets (J = 6.4, 8.2 Hz), showing COSY correlations to a pair of diastereotopic methylene protons with chemical shifts ranging between δH = 2.3-2.7. This unexpected 1H-1H coupling suggested a C-C bond at the 3a position. Analysis of the HSQC spectrum also revealed an upfield 13C-chemical shift of δC = 65 associated with the C3a position, while typically the C3a chemical shifts of most jadomycins appear between δC ~ 90-96,19 suggesting a significant difference in chemical environment. Observed HMBC correla-

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tions for 1 were also present for 3, with correlations from H1′ of the amino acid side chain to C13 and C3a, and from H3a to C1′, C13 and C4 observed (Figure 3a). These correlations indicated the presence of a fully cyclized heterocyclic B-ring incorporated with 4AMBA. The key correlations for structural elucidation were identified by 1H-13C HMBC analysis. H3a and

the adjacent methylene protons showed correlations to a carbon at δC = 178.1. This data along with the chemical shifts of the additional methylene protons (δH = 2.3-2.7) strongly suggested a carbonyl carbon associated with carboxylic acid functionality adjacent to the methylene group.

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Figure 3. Characterization data of compounds 2-4 and C-labelling studies of 3. (a) NMR COSY correlations (bold lines), and HMBC corre13 lations (solid arrows) of (left) 2, (middle) 3 and (right) 4; (b-e) overlaid NMR spectra of C-labelled (top) and unlabeled (bottom) (3) illus1 1 trating (b) full spectra; (c) carbonyl regions; (d) C3a with J splitting with C2; (e) C2 with J splitting with C3a. All NMR spectra of 3 were recorded as a mixture of the 3aR and 3aS diastereomers, as is consistent with other jadomycins.

These data are consistent with the proposed structure of 3 as the first example of a jadomycin derivative containing a C-C bond at the 3a position. The corresponding acetate-3AMBA derivative 7 was isolated and characterized using similar methodology (see SI), in which comparable chemical shifts were observed (Table 1). A fourth more polar purple spot was also identified by TLC analysis of crude extracts. NMR characterization identified all spin systems and most correlations as being the same as observed for 3, indicative of a highly similar analogue (Figure 3a). Analysis of the 13C-NMR identified a fifth carbonyl shift at

δC = 202 in addition to the four other carbonyl peaks associated with 3 (C1, C8, C13, and C6′). HRMS found an m/z of 656.1778 corresponding to a [M - H]- of C35H30NO12, (Figure S17) consistent with the presence of an extra CO group relative to 3. Analysis of the HMBC spectrum identified a correlation between H3a and δC = 202, positioning the CO unit in proximity to position 3a. The structure consistent with the NMR and HRMS data was jadomycin pyruvate-4AMBA (4), a C-C bond-containing jadomycin linked to a pyruvate moiety (Figure 3a). The jadomycin pyruvate-3AMBA derivative was not identified. Crude 3-AMBA culture materials were evaluat-

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ed by LC-MS to probe for the pyruvate-derived analogue, but no evidence of its formation was observed (Figure S72). 13

C-Labeling Studies: Origin of the Acetate Branching Unit. C-labeling experiments were devised to elucidate the biosynthetic mechanism of C-C acetate addition. The labeling patterns of the jadomycin core arising from either [1-13C] or [2-13C]-acetate12 assembly can be predicted from the known mechanism for head-to-tail PKS addition. We hypothesized that in the presence of [2-13C]-acetate jadomycins would be enriched at both adjacent C3a and C2 positions (Figure S7). 13

The resulting labelling pattern would be evident by 13C-NMR spectroscopy; wherein the signals for each of the two 13Cisotopes adjacent to one another would appear as doublets with a coupling constant of ~35-40 Hz.28 S. venezuelae ISP5230 was cultured in the presence of [1-13C] and [2-13C]acetate, however, the addition of labelled acetate into culture media led to inhibition of jadomycin production as observed by lack of pigmentation in cultures. To overcome this obstacle, we instead selected [1-13C]-D-glucose as the isotopic carbon source.29 Similar labeling strategies have been successfully employed for probing primary and secondary metabolism within the literature.30 Previously, we have demonstrated that glycolysis is the primary pathway for glucose catabolism in S. venezuelae ISP5230 during jadomycin biosynthesis26 enabling us to accurately predict the resulting labelled metabolites.31, 32 Glycolysis enables conversion of the isotopically labelled [1-13C]-D-glucose to one equivalent of [313 C]-pyruvate and one equivalent of unlabeled pyruvate (Figure S8). Pyruvate is then further processed to [2-13C]-acetylCoA by pyruvate dehydrogenase, producing the initiating substrates for jadomycin PKS biosynthesis. The culture media for jadomycin production contains D-glucose as the major carbon source, allowing for facile supplementation with the 13 C-isotopomer. Cultures with S. venezuelae ISP5230 were carried out as previously described,26 with unlabeled glucose (4 gL-1) supplemented with [1-13C]-D-glucose (100% isotope) (2 gL-1). Analysis of the 13C-NMR spectrum from purified material showed the presence of the 13C-label (Figure 3b-e) at all anticipated carbon positions for both diastereomers of 3. Signals with 13C-enrichment (C3a, C4-6, C7a, C8, C9, C11, C12a, C13a, the C1′′ anomeric carbon, the C5′′-methyl substituent of Ldigitoxose, and C2 of the acetate unit) were readily identified when compared to the unlabeled standard. No enhancement, as one would anticipate, was observed for the 4-AMBA resonances, and these resonances were used as reference to determine the labeling efficiency. The average labeling efficiency was calculated as described in the literature30 to be approximately 9% (see SI). HRMS analysis provided additional evidence for the 13C-enrichment with the increases in signal intensities associated with a series of isotopes being observed (Figure S16). The signal with the largest relative intensity corresponded to [M – H + 1]-, indicating the majority of the isotopomers were enriched in at least one position. Inspection of the region for C3a (Figure 3d) and C2 (Figure 3e) showed diagnostic 1J coupling between these adjacent carbon atoms; for position C3a, two sets of doublets with J = 35 Hz, corresponding to each diastereomer, were evident. Signals

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corresponding to the coupling partners of position C2 was not resolved sufficiently to measure the corresponding coupling constant due to signal overlap (Figure 3e), however, C-C splitting was evident. The enrichment of C2 is consistent with C-C bond formation that is a result of C-C branching post-PKS addition. Hemiaminal 1a undergoes nucleophilic substitution at the 3a position in protic solvents (vide supra) demonstrating that this is a reactive center. There are no candidate gene products in the jadomycin biosynthetic gene cluster that could catalyze the formation of the new C-C bonds observed in 3, 4 and 7. Thus, enzymes and substrates are presumably recruited from primary metabolism, or from another secondary metabolite pathway, to catalyze the carbon-carbon bond forming step. Scheme 1. Proposed mechanism of formation of 3. Non jadomycin biosynthetic gene cluster enzymes are needed to catalyze the carbon-carbon bond formation.

Proposed Pyruvate Branch Incorporation. Incorporation of the pyruvate functionality was explained in a similar fashion to that of the branched acetate unit of 3. This process is likely linked to gluconeogenesis, in which oxaloacetate is converted to phosphoenolpyruvate at the start of the pathway. During this process, an enolate intermediate (Figure 4a) is produced which we propose may be trapped producing 4 (Figure 4b).33 This branching is consistent with the proposed mechanism of formation for benzopyrenomycin proposed by Hertweck and coworkers.34 The 13C-labeling pattern matched that of compound 3. If the C-C branched pyruvate moiety was introduced through the decarboxylation of oxaloacetate followed by nucleophilic attack by the enolate, the C3a and C2-positions should be labelled in [1-13C]-D-glucose fermentations (Figure 4a). When examining the signal for C3a, the same splitting pattern, a doublet, observed for 3, was identified for 4, with a coupling constant of ~36 Hz in the 13C-NMR. The signal intensity was weaker in comparison to 3, but nevertheless this coupling pattern indicates the presence of 1J 13C-13C coupling of adjacent carbons. (Figure 4c). Labeling at C2 was not directly observed due to poor signal intensity attributed to exchange of the H2 methylene protons with deuterons from CD3OD owing to their α-position to the keto-group. This split and broadened the C2 signal in the 13C-NMR making it difficult to identify labeling.

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The Journal of Organic Chemistry cin analogues are effective cytotoxic agents against MCF7 breast cancer cells and largely retain their potency in MDR MCF7 breast cancer cells that overexpress ABCB1.35 In comparison, drugs that are known ABC-transporter substrates, such as doxorubicin (Dox, Figure 5A), etoposide, or mitoxantrone lose their cytotoxic potency.35

Figure 4. Proposed biosynthetic mechanism for the formation of 13 C-labeled 4. (a) Metabolism of oxaloacetate produced by [113 C]-D-glucose to phosphoenolpyruvate for use in gluconeogenesis and jadomycin production, solid circle indicates presence of 13 13 13 C-label; (b) observed C-labelling pattern of 4; (c) C-NMR of 1 C3a region illustrating J splitting with C2. All NMR spectra of 4 were recorded as a mixture of the 3aR and 3aS diastereomers, as is consistent with other jadomycins.

Biological Activity. Compounds 1 and 3 were selected for testing against the National Cancer Institute’s (NCI) 60 DTP human tumor cell line one-dose screen (Table S2-S3). Compound 1 exhibited better activity compared to 3, likely owing to the reactive hemiaminal center of 1, compared to the more stable C-C bond containing 3. Neither compound exhibited sufficient bioactivity to warrant further investigation by the NCI. Compounds 1, 3 and 7 were selected and screened for their antimicrobial bioactivity. Compounds 3 and 7 showed no appreciable activity against any of the microbes tested. Compound 1, was active against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus warneri and vancomycin-resistant Enterococcus faecium (VRE), exhibiting IC50 values of 12.5, 12.5 and 10 μgmL-1 respectively (Table S4). Cytotoxicity screening revealed a similar trend, with 3 and 7 showing no toxicity towards healthy human fibroblast and monkey kidney cells (Vero) while compound 1 was found to be toxic to both (Table S5).

All 3-AMBA jadomycin analogues demonstrated cytotoxicity towards the 231-CON cells in the low micromolar range (Figure 5B). Dox and jadomycin F (JdF, F, Figure 5A) were assayed as parallel controls. Their IC50 values in the 231-CON cells (Figure 5B) were comparable to our previous reports.35-37 With the exception of 5, the 3-AMBA jadomycin analogues were less potent cytotoxic agents compared to the positive control jadomycin F, which we previously identified as one of the most potent cytotoxic jadomycins.35, 36 The IC50 values of all 3-AMBA were higher in the 231-TXL cells, indicating a small loss of potency versus 231-CON cells. The observed foldresistance values for the 3-AMBA jadomycins were equivalent to that observed with jadomycin F (Figure 5C), and to the fold-resistance values reported previously when comparing jadomycin F cytotoxicity in control versus ABCB1overexpressing MCF7 or MDA-MB-231 breast cancer cell lines.35, 37 In contrast, the 231-TXL cells were significantly more resistant to the known ABCB1-transporter substrate, doxorubicin (38-fold)44 jadomycin analogues or jadomycin F (2.1 – 3.4-fold) (Figure 5C). Drugs effective in treating MDR cancers show equivalent small decreases in efficacy in vitro, such as the epothilone derivative ixabepilone, which demonstrated a 2.2-fold reduction in potency in ABCB1-overexpressing versus drug-sensitive colon carcinoma cells, versus a 28-fold reduction observed with paclitaxel.45 This suggests that the minor fold-decreases in potency observed with the 3-AMBA jadomycins in 231-TXL versus 231-CON cells, does not equate to their being inappropriate candidates for the treatment of such MDR cancers. These data suggest the anti-breast cancer activity of jadomycins is largely retained when the oxazolone ring of typical jadomycins, such as jadomycin F, is omitted, as in the 3-AMBA jadomycin analogues, albeit with decreased potency.

Compounds 5-7 were also screened for their anticancer activity against drug-sensitive and drug-resistant MDA-MB-231 triple negative breast cancer cells using a MTT cytotoxicity assay. Breast cancers are stratified for treatment based on the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Jadomycins have previously demonstrated equal cytotoxicity in MCF7 (ER+, PR+, HER2-), BT474 (ER-, PR+, HER2+), SKBR3 (ER-, PR-, HER2+), and MDA-MB-231 (ER-, PR-, HER2-) breast cancer cell lines.35-38 Metastatic breast cancer treatment is limited by the development of multidrug resistance (MDR) within the cancerous cells.39, 40 A major MDR mechanism is the overexpression of ABCB1, a broadspectrum multidrug efflux pump, which expels chemotherapeutics from within the cell and renders treatments ineffective.41 In breast cancer, the expression of ABCB1 is increased after certain therapies, including taxanes.42, 43 Many jadomy-

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units have been well documented for type-II PKS derived natural products.16, 48, 49 Examples of post-PKS C-C branching include but are not limited to the non-enzymatic pyruvate derivatization of urdamycin A,50 the proposed enzymatic dimerization of actinorhodin,51 the C-glycosylation seen in the urdamycins52 and marangucyclines,53 and lipidations catalyzed by prenyltransferases.54, 55 The enzymes responsible for these transformations are coded within the respective biosynthetic gene clusters.

Figure 5. (A) Jadomycin F (JdF) and doxorubicin (Dox); (B) IC50 values of 7a, 7b, 5, 6, versus positive controls Dox and JdF in 231CON and 231-TXL breast cancer cells. P ≤ 0.05, n ≥ 4 In 231-CON cells, the IC50 value is significantly different versus that of + all 3AMBA jadomycins; and ^ all other treatments. In 231-TXL cells, the IC50 value is significantly different versus that of $ 7a, 7b, and 6; and # all 3-AMBA jadomycins, as determined by one-way ANOVAs, followed by Bonferroni’s multiple comparison tests. *The IC50 value of the particular drug treatment is significantly higher in the 231-TXL versus 231-CON cells, as determined by unpaired t tests; (C) fold-resistance values of jadomycins and Dox, measuring the relative resistance of the 231-TXL cells to each drug treatment versus 231-CON cells, n ≥4. *P ≤ 0.05, the value is significantly lower than that of Dox, as determined by a one-way ANOVA, followed by Bonferroni’s multiple comparison test. No jadomycin fold-resistance value was significantly different from that of any other Jadomycin.

DISCUSSION Jadomycin acetate-4AMBA and 3AMBA (3 and 7) and jadomycin pyruvate-4AMBA (4) are examples of type-II PKS derived natural products containing carbon branching chains at αpositions relative to carbonyls on their extended polyketide chains. Alkylations at α-positions in PKS derived natural products are typically introduced by unusual substituted starter or extender units during PKS chain elongation, or can arise from methylene alkylation during or after PKS core biosynthesis.46, 47 Examples of chain branching with unique starter/extender

The jadomycin gene cluster has been extensively studied and contains no candidate genes encoding enzymes capable of these type of C-C bond formations. Recently, Hertweck and coworkers identified a new enzymatic polyketide-branching domain responsible for the introduction of a carbon branched acetate unit onto the type-I PKS natural product rhizoxin.46, 56, 57 This acetate unit is introduced by an enzyme mediated vinylogous-Michael addition to an unsaturated alkene during PKS chain elongation. Interestingly, this branched acetate is the same identified within 3, with a key difference being alkylation in rhizoxin is at a β-position.58 Hertweck has also previously shown branching within the biosynthesis of benzopyrenomycin, a type II derived PKS compound, by addition of the primary metabolite oxaloacetate to a biosynthetic intermediate.34 Due to the similarities between these branching chains, a similar mechanism of formation is plausible. There is no known enzyme present within jadomycin biosynthesis to facilitate this addition, thus derivatization was likely occurring as a post-core-PKS addition of primary metabolite building blocks by addition to the reactive hemiaminal 3a position by an enzyme from primary metabolism, or other biosynthetic gene clusters within the organism.46, 56, 57 Similar synthetic transformations also support the reactivity of hemiaminals, including the alkaloid cotarnine which has been chemically derivatized with barbituric acids,59, 60 acetylides,61 and malonate.62 Herein, however, the reactivity of the hemiaminal occurs within S. venezuelae ISP5230 and as a consequence is most likely enzyme catalyzed. In the case of 3, we propose acetate addition by a decarboxylative alkylation, invoking primary metabolites malonyl-CoA or malonate, that occurs post amino-acid condensation, reacting either with the imine species or the hemiaminal species (Scheme 1). Isolation of 3, 4 and 7 broadens the scope of this process and indicates the potential for the existence of other C-C derived jadomycins arising from the convergence of primary and secondary metabolic pathways or multiple secondary metabolite pathways. Investigations into the nature of the catalyst responsible for effecting the carbon-carbon bond formation will be reported in due course. CONCLUSIONS In summary, we have developed methodology involving supplementation of D-serine to facilitate incorporation of an amino acid into the jadomycin scaffold which S. venezuelae is incapable of utilizing as nitrogen source. We anticipate that this co-supplementation approach will be broadly applicable towards further expansion of the jadomycin library with diverse amine-containing precursors. This methodology led to the isolation and characterization of a series of new natural

8


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products including, jadomycin hydroxy-4AMBA (1a), containing a reactive hemiaminal center. This reactive core is the result of the inability for the amino acid to undergo the typical intramolecular cyclization associated with jadomycin biosynthesis, leaving the imine or the hemiaminal intermediates free to scavenge nucleophiles. The hemiaminal site was shown to readily undergo solvent dependent substitution resulting in the characterization of jadomycin methoxy4AMBA and 3AMBA (1 and 5, respectively). We have also reported a series of novel C-C branched jadomycins including jadomycin acetate-4AMBA and 3AMBA (3 and 7 respectively) and jadomycin pyruvate-4AMBA (4) that we believe are derived from this reactive hemiaminal. To the best of our knowledge, 3, 4 and 7 represent the first examples of this type of “branching” involving a reactive hemiaminal in a Type-II PKS derived natural product and represent the first series of structurally diverse C-C bond containing jadomycins. The structure activity relationship indicates that the hemiaminal containing compounds are broadly cytotoxic, likely owing to this reactive moiety. Potentially, C-C bond formation may be implemented by S. venezuelae as a self-resistance mechanism to the reactive moiety. Future research will attempt to delineate this process

9



Page 10 of 19

Table 2. NMR Characterization data for 1 (700 MHz, Methanol-d4) and 1a (700 MHz, deuterium oxide) position

δC, type

δH, (J in Hz)

3a

92.7, CH

5.61, s

3a-OCD3 3b 4 5 5-CH3 6 7 7a 7b 8 8a 9 10 11 12 12a 13 13a 1′a

57.5, CD3 132.3, C 120.4, CH 142.4, C 21.1, CH3 120.7, CH 155.0, C 113.7, C 115.4*, C 185.5, C 136.6, C 121.5, CH 136.4, CH 120.7, CH 156.1, C 120.7*, C 185.3, C 149.0, C 57.6, CH2 a: b:

2′ 3′ 4′ 5′ 6′ 1′′ 2′′a

141.6, C 127.9, CH 130.7, CH 138.3, C 174.8, C 96.4, CH 36.3, CH2 a: b:

3′′ 4′′ 5′′ 5′′-CH3

68.4, CH 74.1, CH 66.4, CH 18.2, CH3

1 3a Major HMBC 1′, 3a-CD3, 4, 7a, 7b, 13a

6.67, bd (1.4)

3a, 5-CH3, 6, 7a

2.35, s 6.85, s

4, 5, 6 4, 5-CH3, 7, 7a

7.81, obs. 7.69, m 7.52, d (8.4)

10, 11 9, 12 12a

5.28, d (15.6) 5.51, d (15.6)

3a, 3′, 13a 3a, 3′, 13a

7.07, d (8.0) 7.84, d (8.0)

1′, 3′, 5′

5.86, obs. 3′′, 5′′, 12 2.21, obs. 2.36, dd (15.2, 3.2) 4.10, m 3.29, obs. 5′′ 3.90, m 1.22, d (6.2) 4′′, 5′′

δC, type 92.7, CH 57.5, CD3 130.4, C 120.4, CH 141.6, C 21.1, CH3 120.7, CH 155.0, C 114.1, C 115.4*, C 183.7, C 136.5, C 121.4, CH 136.4, CH 121.3, CH 155.6, C 120.4*, C 183.3, C 153.6, C 59.2, CH2 141.4, C 128.4, CH 130.5, CH 138.2, C 174.9, C 96.4, CH 36.1, CH2 68.4, CH 74.0, CH 66.4, CH 18.2, CH3

1 3a Minor δH, (J in Hz) 5.59, s

6.69, bd (1.4) 2.34, s 6.85, s

7.81, obs. 7.69, m 7.49, d (8.4)

4.93, d (15.6) 5.87, d (15.6) 7.25, d (8.0) 7.77, d (8.0)

5.83, d (3.2) 2.14, dt (15.2, 3.2) 2.24, obs. 4.00, obs. 3.22, dd (10.0, 3.2) 3.81, m 1.17, d (6.2)

δC, type

1a δH, (J in Hz)

HMBC

86.6, CH

5.88, s

1′, 3b, 4, 7a, 13a

6.44, s

3a, 5-CH3, 6, 7a

1.99, s 6.39, s

4, 5, 6 5-CH3, 6, 7, 7a

7.51, obs. 7.56, obs. 7.17, bd

12 8a, 12, 12a 9, 12

5.10, d (17.1) 5.92, d (17.1)

3a, 13a, 2′, 3′ 3a, 13a, 2′, 3′

7.26, d (8.2) 7.71, d (8.2)

1′, 3′, 5′ 2′, 4′, 6′

5.39, bd 1.51, bm 1.92, bm 4.00, bm 3.37, dd (9.9, 2.9) 3.92, m 1.30, d (6.2)

3′′

131.7, C 120.2, CH 142.1, C 21.5, CH3 120.2, CH 152.7, C 112.8, C 113.0*, C 182.6, C 135.4, C 121.7, CH 137.2, CH 120.1, CH 154.6, C 119.4*, C 183.9, C 154.7, C 57.9, CH2 143.2, C 126.7, CH 130.8, CH 136.1, C 175.9, C 96.6, CH 35.1, CH2 67.9, CH 73.5, CH 66.5, CH 18.4, CH3

1′′ 5′′-CH3 1′′, 5′′-CH3 1′′, 4′′, 5′′

13

*Assignment by C NMR only, resonances may be interchangeable

10


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Table 3. NMR Characterization data for 2 and 3 (700 MHz, Methanol-d4) 3 3a Major

2 position δC, type 1 2 3a 3b 4 5 5-CH3 6 7 7a 7b 8 8a 9 10 11 12 12a 13 13a 1′ 2′ 3′ 4′ 5′ 6′ 1′′ 2′′ 3′′ 4′′ 5′′ 5′′-CH3

δH, (J in Hz)

HMBC

δC, type 178.1, C 43.0, CH2

a: b: 164.1, C 148.2, C 119.3, CH 143.9, C 21.3, CH3 125.7, CH 155.1, C 118.7, C 111.2*, C 182.3*, C 133.5*, C 121.5, CH 136.3, CH 121.2, CH 155.2, C Obs. 180.2*, C 148.2*, C 49.6, CH2 a: b: 141.2, C 128.2, CH 130.6, CH 138.2, C 174.7, C 96.4, CH 36.2, CH2 a: b: 68.5, CH 73.8, CH 66.3, CH 18.2, CH3

7.57, s

3a, 5-CH3

2.31, s 6.94, s

4, 5, 6 5-CH3, 7a

7.56, d (7.6) 7.51, t (7.9) 7.36, d (8.3)

11 12 9

5.60, d (15.4) 6.10, bs

2', 3'

7.06, d (8.1) 7.68, d (8.3)

1′, 3′, 5′ 2′, 3′, 4′, 6′

5.72, d (2.8) 2.13, dt (3.5, 15) 2.29, dd (obs.) 4.03, d (3.2) 3.19, dd (obs.) 3.78, m 1.11, d (6.1)

3′′, 5′′, 12

65.6, CH 134.8, C 118.8, CH 141.4, C 21.0, CH3 119.4, CH 154.4, C 114.1, C 112.4, C 181.6, C 137.6, C 121.3, CH 136.4, CH 120.7, CH 156.4, C 120.7, C 184.3, C 154.8, C 57.8, CH2 141.0, C 127.9, CH 130.5, CH 138.3, C 175.2, C 96.7, CH 36.3, CH2

1′′, 3′′, 4′′ 3′′, 5′′, 5′′-CH3 5′′-CH3 4′′, 5′′

68.1, CH 74.1, CH 66.4, CH 18.2, CH3

δH, (J in Hz)

HMBC

2.32, obs. 1, 3a, 3b 2.69, dd (14.0, 8.2) 1, 3a, 3b 5.03, dd (8.2, 6.4) 1, 2, 3b, 4, 7a 13a, 1′ 6.44, d (1.2)

3a, 5-CH3, 6, 7a

2.23, s 6.66, bs

4, 5, 6 4, 5-CH3, 7, 7a

7.85, obs. 7.71, t (8.1) 7.40, d (8.4)

8, 11 8a, 9, 12 9, 12, 13

4.85, d (15.8) 5.75, d (15.8)

3a, 13a, 2′, 3′ 3a, 13a, 2′, 3′

7.20, d (8.2) 7.87, d (8.2)

1′, 3′, 4′, 5′ 2′, 3′, 4′, 6′

5.89, d (3.4) 2.19, dt (15.0, 3.6) 2.28, dd (15.0, 2.3) 4.03, m 3.25, dd (10.0, 3.2) 3.95, m 1.22, d (6.2)

12, 3′′, 5′′ 1′′, 3′′, 4′′ 1′′, 3′′, 4′′ 5′′, 5′′-CH3 4′′, 5′′

δC, type

3 3a Minor δH, (J in Hz)

178.1, C 42.9, CH2 2.38, dd (14.0, 6.4) 2.71, dd (14.0, 8.2) 65.3, CH 5.02, dd (8.2, 6.4) 134.1, C 118.7, CH 6.42, d (1.2) 141.3, C 21.0, CH3 2.22, s 119.3, CH 6.66, bs 154.3, C 114.1, C 112.4, C 180.7, C 137.6, C 121.1, CH 7.84, dd (7.5, 0.8) 136.5, CH 7.72, t (8.1) 119.8, CH 7.50, d (8.4) 155.7, C 120.7, C 186.1, C 156.6, C 56.5, CH2 4.92, obs. 5.73, d (15.8) 140.4, C 127.7, CH 7.22, d (8.2) 130.6, CH 7.90, d (8.2) 138.4, C 175.0, C 96.3, CH 5.94, d (3.4) 36.3, CH2 2.20, dt (15.0, 3.4) 2.33, dd (15.0, 2.3) 68.3, CH 3.98, m 74.1, CH 3.23, dd (10.0, 3.2) 66.4, CH 3.87, m 18.2, CH 1.19, d (6.2)

13


11



Page 12 of 19

Table 4. NMR Characterization data for 4 (700 MHz, Methanol-d4) position δC, type 1 1a 2

169.7, C 202.4, C 43.9, CH2

3a 3b 4 5 5-CH3 6 7 7a 7b 8 8a 9 10 11 12 12a 13 13a 1′

61.9, CH 133.7, C 118.6, CH 141.5, C 21.0, CH3 119.6, CH 154.5, C 114.0, C 114.3, C 181.3, C 137.4, C 121.4, CH 136.4, CH 120.9, CH 156.2, C 120.0*, C 185.9, C 153.8, C 56.6, CH2 a: b: 141.0, C 127.9, CH 130.6, CH 138.6, C 175.1, C 96.3, CH 36.3, CH2 a: b: 68.2, CH 74.1, CH 66.4, CH 18.2, CH3

2′ 3′ 4′ 5′ 6′ 1′′ 2′′ 3′′ 4′′ 5′′ 5′′-CH3

a: b:

4 3a Major δH, (J in Hz) COSY

HMBC

4 3a Minor

2.90, bm 3.38, bm 5.12, bm

3a 3a 2

3a 3a 1a, 3b, 4, 7a, 13a, 1′

6.37, s

5-CH3, 6

3a, 5-CH3, 6, 7a

2.21, s 6.67, bs

4, 6 4, 5-CH3

4, 5, 6 4, 5-CH3, 7, 7a

7.85, obs. 10 7.71, obs. 9, 11 7.50, d (8.5) 10

8, 11 7a, 12 9

4.80, obs. 5.77, d (15.5)

3a, 13a, 2′, 3′ 3a, 13a, 2′, 3′

7.18, d (7.9) 4′ 7.84, obs. 3′

1′, 3′, 5′ 2′, 4′, 6′

5.94, d (3.0) 2.18, m 2.37, m 4.04, m 3.25, m 3.96, m 1.19, d (6.2)

3′′ 1′′, 3′′, 4′′ 1′′, 3′′, 4′′ 1′′ 5′′-CH3 1′′, 5′′-CH3 1′′, 4′′, 5′′

2′′ 1′′, 3′′ 1′′, 3′′ 2′′, 4′′ 3′′, 5′′ 4′′, 5′′-CH3 5′′

δC, type δH, (J in Hz) 169.7, C 202.6, C 43.9, CH2 2.90, bm 3.38, bm 62.2, CH 5.13, bm 134.5, C 118.7, CH 6.35, s 141.8, C 21.0, CH3 2.22, s 119.6, CH 6.66, bs 154.6, C 113.9, C 114.3, C 182.5, C 137.3, C 121.2, CH 7.85, obs. 136.6, CH 7.72, obs. 120.6, CH 7.52, d (8.5) 156.2, C 120.7*, C 184.0, C 156.2, C 58.4, CH2 4.80, obs. 58.4 5.77, d (15.5) 140.2, C 128.2, CH 7.22, d (7.9) 130.5, CH 7.81, obs. 138.5, C 175.0, C 96.5, CH 5.90, d (3.0) 36.3, CH2 2.18, m 2.37, m 68.4, CH 3.99, m 74.1, CH 3.25, m 66.4, CH 3.88, m 18.2, CH3 1.22, d (6.2)

13

*Assignment by C NMR only

12


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Table 5. NMR Characterization data for 5 and 6 (700 MHz, Methanol-d4)

position δC, type

δH, (J in Hz)

3a

91.6, CH

5.58, bs

3a-OCD3 3b 4 5 5-CH3 6 7 7a 7b 8 8a 9 10 11 12 12a 13 13a 1′

54.6, CD3 132.0, C 120.5, CH 141.5, C 21.02, CH3 120.67, CH 154.9, C 114.2, C 113.8, C* 183.5, C 136.5, C* 121.4, CH 136.6, CH 120.9, CH 155.9, C 121.5, C* 185.4, C 154.9, C 58.9, CH2

2′ 3′ 4′ 5′ 6′ 7′ 8′ 1′′ 2′′

138.6, C 130.08, CH 136.6, C 175.2, C 129.59, CH 129.2, CH 130.6, CH 96.6, CH 36.26, CH2

3′′ 4′′ 5′′ 5′′-CH3

68.29, CH 74.11, CH 66.43, CH 18.19, CH3 13

a: b:

a: b:

5 3a Major HMBC 1′, 3a-OCD3, 3b, 4, 6, 7a, 13a

6.63, bs

3a, 6

2.30, bs 6.82, bs

4, 5, 6 4, 7

7.83, d (7.2) 7.71, m 7.53, t (9.2)

8 12

5.75, d (15.5) 4.95, d (15.5)

2′, 3a, 8′ 2′, 3a, 8′

7.94, s

5′

7.82, d (obs.) 7.25, t (7.6) 4′ 7.31, d (7.6) 5.86, d (2.9) 2.19, m 2.41, dd (2.6, 15.1) 4.08, dd (3.1, 6.3) 3.27, dd (3.3, 9.9) 3.91, m 1.18, d (6.2) 4′′, 5′′

δC, type

5 3a Minor δH, (J in Hz)

δC, type

91.6, CH

5.56, bs

163.9, C

54.6, CD3 132.0, CH 120.5, CH 142.2, C 21.05, CH3 120.71, CH 154.9, C 113.76, C 113.75, C* 185.4, C 136.6, C* 121.5, CH 136.4, CH 120.8, CH 156.3, C 121.5, C* 185.4, C 154.9, C 57.0, CH2 138.2, C 130.13, CH 136.6, C 175.2, C 129.63, CH 129.1, CH 131.1, CH 96.5, CH 36.31, CH2 68.28, CH 74.08, CH 66.41, CH 18.20, CH3

6.62, bs 2.30, bs 6.81, bs

7.82, d (7.4) 7.70, m 7.53, t (9.2)

5.57, d (15.4) 5.15, d (15.4) 7.79, s

7.76, d (7.2) 7.21, t (7.4) 7.19, d (7.6) 5.86, d (2.9) 2.17, m 2.30, dd (3.1, 14.2) 4.00, dd (3.2, 6.3) 3.23, dd (3.1, 9.8) 3.87, m 1.22, d (6.2)

148.3, C* 121.9, CH 144.0, C 21.3, CH3 125.2, CH 156.7, C* 118.1 C 115.2, C* 186.9, C 129.1, C* 121.9, CH 136.6, CH 121.9, CH 154.7, C 120.7, C* 170.3, C 158.8, C* 48.9, CH2 135.7, C* 129.0, CH 138.7, C 174.6, C 129.4, CH 129.3, CH 131.0, CH 96.7, CH 36.3, CH2 68.5, CH 74.1, CH 66.4, CH 18.2, CH3

6 δH, (J in Hz)

HMBC

7.91, d (1.2)

3a, 6, 7a

2.47, s 7.20, d (1.3)

4, 5, 6 4, 7a

7.77, d (7.6) 7.67, t (8.4) 7.51, d (8.4)

8 12 9

5.67, d (15.4) 6.22, bs 7.68, s 5' 7.72, d (7.9) 7.24, t (7.7) 7.31, d (7.5) 5.82, d (3.0) 2.19, dt (3.6, 15.2) 2.45, dt (2.8, 14.7) 4.10, dd (3.3, 6.3) 3.27, dd (3.0, 9.9) 3.84, m 1.18, d (6.1)

4' 3", 5"

5′′ 4′′, 5′′


13



Page 14 of 19

Table 6. NMR Characterization data for 7a and 7b (700 MHz, Methanol-d4) 7a 3a Major position

δC, type

1 2

178.2, C 42.9, CH2

3a 3b 4 5 5-CH3 6 7 7a 7b 8 8a 9 10 11 12 12a 13 13a 1′

64.8, CH 134.3, C 118.8, CH 141.2, C 21.1, CH3 119.4, CH 154.4, C 114.2, C 112.5, C* 180.8, C 137.7, C 121.2, CH 136.6, CH 119.9, CH 155.9, C 120.9, C 186.2, C 156.9, C 56.4, CH2 a: b: 137.7, C 130.1, CH 139.6, C 175.2, C 129.8, CH 129.1, CH 130.7, CH 96.5, CH 36.4, CH2 a: b: 68.4, CH 74.1, CH 66.5, CH 18.3, CH3

2′ 3′ 4′ 5′ 6′ 7′ 8′ 1′′ 2′′ 3′′ 4′′ 5′′ 5′′-CH3

δH, (J in Hz) a: b:

COSY

HMBC

2.33, m 2b, 3a 2.70, dd (8.0, 6.0) 2a, 3a 5.06, t (6.7) 2a, 2b

1, 3a 1, 3a, 3b 2, 3b, 4, 7a, 13a

6.39, s

5-CH3, 6

3a, 5-CH3, 6, 7a

2.19, s 6.61, s

4, 6 4, 5-CH3

5, 6 4, 5-CH3, 7a, 7

7.83, m 7.70, t (8.3) 7.50, d (8.5)

10 9, 11 10

8, 11 8a, 12, 12a 10, 12, 13

5.74, d (15.1) 4.86, d (15.1)

1′a 1′b

2′, 3a, 8′, 13a 2′, 3a, 8′, 13a

8.01, s

7.84, m 7.26, t (7.6) 7.31, d (7.5) 5.92, d (3.1) 2.18, m 2.35, m 3.99, dd (2.8, 5.9) 3.23, dd (2.8, 9.8) 3.91, m 1.2, d (6.1)

1', 6'

7′ 6′, 8′ 7′ 2′′a 1′′, 2′′b, 3′′ 2′′a, 3′′ 2′′a, 2′′b, 4′′ 3′′, 5′′ 4′′, 5′′-CH3 5′′

5′, 8′ 4′ 3′ 3′′, 5′′, 12

5′′, 5′′-CH3 5′′-CH3 4′′, 5′′

δC, type 178.1, C 42.9, CH2 64.8, CH 134.8, C 118.8, CH 141.3, C 21.0, CH3 119.3, CH 154.3, C 114.0, C 114.3, C* 181.5, C 137.6, C 121.3, CH 136.4, CH 120.9, CH 156.5, C 121.0, C 184.4, C 155.1, C 57.5, CH2 138.1, C 129.9, CH 139.5, C 175.1, C 129.5, CH 128.9, CH 130.7, CH 96.9, CH 36.3, CH2 68.0, CH 74.1, CH 66.4, CH 18.2, CH3

7a 3a Minor δH, (J in Hz) HMBC 2.35, dd (7.9, 6.2) 2.67, dd (7.9, 6.2) 5.03, dd (6.6, 1.3)

3a 3a, 3b 1, 1′, 2, 3b, 4, 7a, 13a

6.39, s

3a, 5-CH3, 6, 7a

2.19, s 6.62, s

4, 5, 6 4, 5-CH3, 7a, 7

7.85, d (7.9) 7.70, t (8.3) 7.48, d (8.3)

8, 11 8a, 12 9, 12

5.78, d (15.1) 4.81, d (15.1)

2′, 3a, 13a 2′, 3a, 13a

7.94, s

1′, 5′, 6′, 8′

7.81, d (7.7) 7.25, t (7.7) 7.28, d (7.7) 5.88, d (3.4) 2.17, dt (3.8, 15.2) 2.40, dd (2.5, 15.2) 4.03, d (3.1) 3.24, dd (2.4, 6.7) 3.97, t (5.8) 1.22, d (6.6)

5′ 4′, 8′ 3′ 5′′, 12

5′′, 5′′-CH3 4′′, 5′′

13

*Assignment by C NMR only

14


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EXPERIMENTAL SECTION General Experimental Procedures All reagents were purchased from commercial sources and used without further purification unless otherwise stated. Solvents used for all reactions and chromatographic methods were HPLC grade. Flash chromatography was performed using a Biotage SP1™ unit using pre-packed normal phase silica columns from SiliCycle®. Glass-backed thin layer chromatography (TLC) plates (SiliCycle®) layered with 250 μm silica were used to assess purity of compounds, determine the Rf values, and purify compounds by preparative TLC. Preparative TLC using 20 × 20 cm plates was performed using the appropriate solvent mixture to develop the plates, which were then allowed to dry, then re-developed and dried again. This process was repeated until good separation was observed (2-4 cycles). Bands of interest were scraped off the glass backing and eluted with 100% methanol. Size-exclusion chromatography was accomplished using Sephadex™ LH-20 (GE Healthcare) resin. Compounds were characterized by liquid chromatography tandemmass spectrometry (LC-MS/MS), high resolution mass spectrometry (HRMS), and 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy. Low resolution LC-MS/MS spectra were obtained on an Applied Biosystems hybrid triple quadrupole linear ion trap (2000Qtrap) mass spectrometer using an electrospray ionization (ESI) source. This was coupled with an Agilent 1100 high performance liquid chromatography (HPLC) instrument with a Phenomenex Kinetex 2.6 μm Hilic column (150 mm × 2.10 mm). Samples were prepared in methanol and 5 μL aliquots were injected onto the column. Elution of compounds was accomplished using an isocratic gradient of (7:3) CH3CN : 2 mM ammonium acetate in water (pH 5.5) with a flow rate of 120 μLmin-1 for 10 min. For all jadomycins, the instrument was used in positive mode (ESI+). Enhanced product ionization (EPI) was performed with a capillary voltage of +4500 kV, declustering potential +80 V, and curtain gas 10 arbitrary units. EPI scans were conducted over a range of 300-900 m/z scanning for [M+H]+ and the appropriate jadomycin fragmentation. Scans were conducted using two steps, 300.0 amu to 320 amu (0.005 s) and 300.0 amu to 900.0 amu (0.150 s). Spectra were analyzed using Analyst software version 1.4.1 (Applied Biosystems). HRMS traces of all jadomycins were recorded on a Bruker Daltonics MicroTOF Focus Mass Spectrometer using either and ESI+ or ESI- source depending on the compound. NMR spectra of all jadomycins (1a, 1-7b) were recorded using a Bruker AV-III 700 MHz Spectrometer (1H: 700 MHz, 13C: 176 MHz) equipped with an ATMA 5 mm TCI cryoprobe located at the Canadian National Research Council Institute for Marine Biosciences (NRC-IMB) in Halifax, Nova Scotia. All spectra were recorded in MeOD-d4 or D2O. Appropriate solvents used in each case can be found with the accompanying supplemental NMR-spectra. Chemical shifts (δ) were given in ppm, and calibrated to residual solvent peaks (MeOD: 3.31 ppm; D2O: 4.79 ppm). Structural characterization and signal assignments were accomplished using 1H-NMR chemical shifts and multiplicities, and 13C-NMR chemical shifts. In addition, 1H-1H correlated spectroscopy (COSY), 1H-13C heteronuclear single quantum coherence (HQSC) NMR, and 1H-13C heteronuclear multiple bond correlation (HMBC) NMR experiments were used to support assignments where appropriate. Ultra-violet-visible (UV-vis) spectroscopy was carried out on a SpectraMax Plus Microplate Reader (Molecular Devices), and analyzed using SoftMax® Pro Version 4.8 Software. Samples were dissolved in methanol (or water for compound 1a) placed in a quartz cuvette (1 cm path length) and scanned over a range of 280-700 nm using 1 nm intervals. Two separate dilutions were used (concentrations listed with characterization data) to calculate a series of extinction coefficients (ε) from several maximal absorbance wavelengths (λmax). HPLC analyses of jadomycin analogues 1a, and 2-7b were performed on a Hewlett Packard Series 1050 instrument with an Agilent Zorbax 5 μm Rx-C18 column (4.6 × 150 mm). Elution of the compounds was monitored at an absorbance of 254 nm using an isocratic gradient of 9:1 (A:B) over 0.5 min followed by an increasing linear gradient from 9:1 (A:B) to 4:6 (A:B) over 7.5 min, followed by an isocratic gradient of 4:6 (A:B) for an additional 2 min. This was then followed by a decreasing linear gradient from 4:6 (A:B) to 9:1 (A:B) over 1 min, ending with an isocratic gradient of 9:1 (A:B) over 4 min (total time 15 min; flow rate of 1 ml/min).

Buffer A was an aqueous buffer comprised of 12 mM Bu4NBr, 10 mM KH2PO4, and 5% HPLC grade CH3CN (pH 4.0) and B was HPLC grade CH3CN. Preparatory scale HPLC was performed using a C-18 reversed phased column (Ultrasphere ODS, 5 μm particle size, 10 mm x 25 cm) with degassed methanol (A) and water (B) using the following method: a linear gradient from 5:95 A:B to 95:5: A:B over 30 min, followed by a hold at 95:5 A:B for 30 min at 5 mL min−1.

Bacteria Growth Conditions Refer to SI for media recipes. MYM or MSM broth (250 mL) were prepared in 1 L glass Erlenmeyer flasks. MYM agar (125 mL) was prepared in 250 mL glass Erlenmeyer flasks. Agar solutions were supplemented with 50 μgmL-1 apramycin sulfate before being poured into standard petri dishes while molten. All media was adjusted to pH 7 or 7.5 with 5 M NaOH or 5 M HCl as required. All solutions were autoclaved at 120 °C for 20 minutes prior to use. Streptomyces venezuelae ISP5230 (ATCC 10712) VS1099 was maintained on MYM agar plates supplemented with apramycin for 1-3 weeks for use in jadomycin production fermentations. For long term storage, spore solutions were stored in 20% glycerol at -70°C. Jadomycin fermentations were carried out using modified conditions for jadomycin production previously established in the Jakeman laboratory.23 A 1 × 1 cm lawn of S. venezuelae ISP5230 VS1099 was used to inoculate 250 mL MYM media (4 × 250 mL in 4-1 L flasks per growth). Growths were incubated at 30°C with agitation (250 RPM) for 16-24 hours. The cell suspension was centrifuged at 3750 RPM (4°C) for 30-45 minutes. Supernatant was removed and the cell pellet was washed with 100 mL MSM containing no amino acid. The washing step was repeated twice more to ensure removal of all nutrient rich MYM. The cell pellet was re-suspended in 100 mL MSM without amino acid. Autoclaved MSM media containing 4(aminomethyl)benzoic acid or 3-(aminomethyl)benzoic acid (60 mM), and D-serine (15 mM) in 4-1 L flasks (4 × 250 mL) were supplemented with separately sterilized glucose (33 mM) and phosphate (50 μM) before being inoculated with the pre-growth S. venezuelae ISP5230 VS1099 cell suspension to an initial OD600 of 0.6. Growths were immediately ethanol shocked with 100% ethanol (3% v/v) and incubated at 30°C with agitation (250 RPM) for 48 hours. After 24 hours, the pH of the media was readjusted to a pH of 7.5 by the drop wise addition of 5 M NaOH. Bacterial growths were monitored by absorbance at 600 nm (OD600), colored natural product production was monitored by absorbance of clear growth solution at 526 nm. Labeled fermentations were carried out following the exact same method for the unlabeled fermentations (see above) on a 1 L scale except the media was comprised of unlabelled glucose (4 gL-1) and [1-13C]-Dglucose (100% isotope) (2 gL-1). Bacterial growths were monitored by absorbance at 600 nm (OD600) and at 526 nm, as above.

Culture Extraction Bacterial cells were removed via suction filtration through Whatman No. 5 filter paper, followed by 0.45 μm then 0.22 μm Millipore Durapore® membrane filters. The clear media was passed through a reversed-phase SiliCycle® phenyl column (70 g) and washed with distilled water until flow through was colorless to remove all water-soluble material. Remaining material was eluted with 100% methanol and dried in vacuo yielding crude extract. The unlabeled 4-AMBA culture yielded ~340 mgL-1 crude, the 13C-labeled 4-AMBA culture yielded ~132 mgL-1, and the unlabeled 3AMBA culture yielded ~280 mgL-1 crude.

Purification and Characterization Jadomycin methoxy-4AMBA (1) The ~340 mgL-1 crude material was brought up in minimal H2O (100 mL) and extracted with EtOAc (3 × 250 mL). The organic layers were combined and dried in vacuo yielding a crude solid purple mixture (99 mgL-1). Crude ethyl acetate extract (99 mg) was dissolved in 40 mL of ethyl acetate and loaded onto two 80 g silica columns (20 mL/column) preconditioned with DCM. Material was eluted with a flow rate of 45 mL/min collecting 9 mL fractions. Purification was accomplished using a gradient system comprised of solvent A (DCM), and solvent B (methanol). To start, an initial isocratic gradient step using solvent A (1 CV) was performed. This was followed by a linear increasing gradient from 0% to 20% B over 10 CV, followed by a linear increasing gradient from 20% to 80% B

15



over 5 CV. Finally, an isocratic gradient of 80% B over 5 CV was performed. Fractions were checked by TLC for the presence of the compound of interest. Fractions containing 1 were combined and dried yielding 21 mg of crude 1. Preparatory TLC was then performed using a 9:1 (DCM:MeOH) solvent system yielding 5.2 mg. Final purification was accomplished by semi-preparatory HPLC. Material was eluted using the previously described and dried yielding 1 as a red solid (2.5 mg) as a mixture of diastereomers (Mj/Mn = 100/80) by NMR. 13 C-labeled jadomycin methoxy-4AMBA (1) Crude phenyl column extract (132 mg) from a 1 L D-glucose-1-13C labeled fermentation, was brought up in minimal methanol (~4 mL) and loaded onto preparatory TLC plates. Preparatory TLC was performed using a 9:1 (DCM:MeOH) solvent system developing the plates 2× to ensure appropriate separation. The compounds of interest were scraped from the plate and eluted from the silica with 100% methanol. Material was dried in vacuo yielding a red solid (1, 1.8 mg). 13 C-Jadomycin hydroxy-4AMBA (1a) The compound of interest (1a) was purified by preparative TLC as previously described, yielding 2.7 mg of a crude red solid. Final purification was accomplished by semi-preparatory HPLC. Material was eluted using the previously described method and dried yielding 1a as a red solid (1.8 mg). For all subsequent NMR and HRMS analyses, material was dissolved in H2O or D2O. NMR analysis was inconclusive in determining if the compound exists as a single diastereomer or an unresolved mixture of diastereomers. Jadomycin 4AMBA-lactam (2) A crude mixture containing 2 (65 mg) was dissolved in minimal methanol and loaded onto preparative normal phase silica TLC plates (250 μm) and developed with DCM:MeOH (9:1). The orange band of interested was scraped from the plate and eluted with MeOH to give 6.7 mg of crude 2. Further purification was accomplished through two successive rounds of semi-preparatory HPLC, yielding 2 as an orange solid (1.2 mg). Jadomycin acetate-4AMBA (3) Crude ethyl acetate extract (99 mg) was dissolved in 40 mL of ethyl acetate and loaded onto two 80 g silica columns (20 mL/column) preconditioned with DCM. Material was eluted with a flow rate of 45 mL/min collecting 9 mL fractions. Purification was accomplished using a gradient system comprised of solvent A (DCM), and solvent B (methanol). To start, an initial isocratic gradient step using solvent A (1 CV) was performed. This was followed by a linear increasing gradient from 0% to 20% B over 10 CV, followed by a linear increasing gradient from 20% to 80% B over 5 CV. Finally, an isocratic gradient of 80% B over 5 CV was performed. Fractions were checked by TLC for the presence of the compound of interest. Fractions containing the compound were combined and dried yielding 28 mg of crude 3. Preparatory TLC was then performed using a 9:1 (DCM:MeOH) solvent system yielding 21 mg. Final purification was accomplished by semi-preparatory HPLC, yielding 3 as a purple solid (11 mg) as a mixture of diastereomers (Mj/Mn = 100/85) by NMR. 13 C-Jadomycin acetate-4AMBA (3) The compound of interest (3) was purified by preparative TLC as previously described, yielding 2.5 mg of a crude purple solid. Final purification was accomplished by semi-preparatory HPLC, yielding 3 as a purple solid (1.6 mg) as a mixture of diastereomers (Mj/Mn = 100/85) by NMR. 13 C-Jadomycin pyruvate-4AMBA (4) The compound of interest (4) was purified by preparative TLC as previously described, yielding 3.7 mg of a crude purple solid. Final purification was accomplished by semi-preparatory HPLC, yielding 4 as a purple solid (1.7 mg) as a mixture of diastereomers (Mj/Mn = 100/95) by NMR. 3AMBA Jadomycins The 280 mg of crude natural product extract material was fractionated over a prepacked C18 column (12 g, 70 mL). The material was reconstituted in minimal methanol, applied to the column and air-dried, then eluted with a stepwise gradient of the following solvents (100 mL), in order: water, 1:1 water:MeOH, MeOH, and ethyl acetate. The solvents were removed in vacuo. The crudely fractionated material was analysed by TLC before proceeding. The C18 fractionated material was purified by

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preparative TLC, using 20 × 20 cm glass backed, normal phase silica TLC plates (250 μm thickness). The material was re-suspended in minimal methanol. The C18 aqueous fraction (58 mg), enriched with 7a and 7b, was spotted onto four silica plates and the C18 1:1 water:MeOH fraction, enriched with 5 and 6, (15 mg) was spotted onto three silica plates. All plates were developed twice in 5:95 MeOH:CH2Cl2 and twice more in 10:90 MeOH:CH2Cl2. The developed plates were air-dried. The jadomycins of interest were removed by scraping the silica from the plate. The compounds were eluted from the silica using methanol, which was subsequently removed in vacuo. Following preparative TLC, 6.8 mg of violet material (7a), 6.1 mg of violet material (7b), 2.6 mg of red material (5), and 1.6 mg of orange material (6) were isolated. Jadomycins were finally purified for characterization by size exclusion chromatography using LH20 in methanol (10 g in 35 mL). Samples were solvated in minimal methanol to apply to column and were eluted at 1 mLmin-1. Following the LH20 separation, 3.8 mg of violet Jad 3-AMBA acetate major (7a), 2.7 mg of violet Jad 3-AMBA acetate minor (7b), 1.4 mg of red Jad 3-AMBA methoxy (5), and 0.9 mg of orange Jad 3-AMBA lactam (6) were isolated. Jadomycin 4-AMBA 3a-O-Methyl (1): red amorphous solid (yield 2.5 mgL-1); (Mj/Mn = 100/80) by NMR; TLC Rf: 0.50 (9:1 CH2Cl2: MeOH); 1H NMR (MeOD, 700 MHz) and 13C{1H} NMR (MeOD, 176 MHz) see Table 2. HRMS (ESI-) m/z: [M-H]- Calcd for C33H30NO10 600.1875; found 600.1880, (Figure S9). Jadomycin hydroxy 4-AMBA (1a): red amorphous solid (yield 1.8 mgL-1); 1 H NMR (D2O, 700 MHz) and 13C{1H} NMR (D2O, 176 MHz) see Table 2. UV-Vis (7.66 × 10-4 and 9.57 × 10-5 M, H2O): λmax (ε) = 287 (15000), 394 (3280), 516 (2500). HRMS (ESI+) m/z: [M+Na]+ Calcd for C32H29NNaO10 610.1684; found 610.1668, (Figure S14). Jadomycin 4-AMBA lactam (2): orange amorphous solid (yield 1.2 mgL1 ); TLC Rf: 0.48 (9:1 CH2Cl2: MeOH); 1H NMR (MeOD, 700 MHz) and 13C{1H} NMR (MeOD, 176 MHz) see Table 3. HRMS (ESI+) m/z: [M+H]+ Calcd for C32H26NO10 586.1708; found 586.1708, (Figure S13). Jadomycin acetate 4-AMBA (3): purple solid (yield 11 mgL-1); (Mj/Mn = 100/85) by NMR; TLC Rf: 0.24 and 0.36 (9:1 CH2Cl2: MeOH); HPLC Rt = 7.5 min; 1H NMR (MeOD, 700 MHz) and 13C{1H} NMR (MeOD, 176 MHz) see Table 3. UV-Vis (6.35 × 10-4 and 7.94 × 10-5 M, MeOH): λmax (ε) = 316 (12500), 381 (2630), 551 (1840); LRMS (ESI-): MS/MS (628) found 628 [M−H]-, 304 [M−H− C16H20O7]-; HRMS (ESI-) m/z: [M-H]- Calcd for C34H31NO11 628.1824; found 628.1818, (Figure S15). Jadomycin acetate 4-AMBA (4): purple solid (yield 3.7 mgL-1); (Mj/Mn = 100/95) by NMR; TLC Rf: 0.04 (9:1 CH2Cl2: MeOH); HPLC Rt = 7.5 min; 1H NMR (MeOD, 700 MHz) and 13C{1H} NMR (MeOD, 176 MHz) see Table 4. UV-Vis (6.47 × 10-4 and 8.08 × 10-5 M, MeOH): λmax (ε) = 316 (10400), 386 (2200), 547 (1600); HRMS (ESI-) m/z: [M-H]- Calcd for C35H30NO12 656.1773; found 656.1778. Jadomycin 3-AMBA 3a-O-Methyl (5): burgundy amorphous solid (yield 1.4 mgL-1); TLC Rf: 0.80 (9:1 CH2Cl2: MeOH); HPLC Rt = 8.99 min (Figure S3); 1H NMR (MeOD, 700 MHz) and 13C{1H} NMR (MeOD, 176 MHz) see Table 5; UV-Vis (2.5 × 10-4 M, 1.0 × 10-3 M, MeOH), λmax (ε) [(287 (13313), 324 (3824), 389 (2696), 526 (1923)]. HRMS (ESI-) m/z: [M-H]- Calcd for C33H30NO10 600.1875; found 600.1853, (Figure S18). Jadomycin 3-AMBA Lactam (6): orange amorphous solid (yield 0.9 mgL1 ); TLC Rf: 0.82 (9:1 CH2Cl2: MeOH); HPLC Rt = 10.26 min (Figure S5); 1H NMR (MeOD, 700 MHz) and 13C{1H} NMR (MeOD, 176 MHz) see Table 5; UV-Vis (2.5 × 10-4 M, 1.0 × 10-3 M, MeOH), λmax (ε) [(264 (8954)]. HRMS (ESI-) m/z: [M-H]- Calcd for C32H26NO10 584.1562; found 584.1542, (Figure S20). Jadomycin 3-AMBA Acetate 3a-Major (7a): violet amorphous solid (yield 3.8 mgL-1); TLC Rf: 0.26 (9:1 CH2Cl2: MeOH); HPLC Rt = 8.75 min (Figure S4); 1H NMR (MeOD, 700 MHz) and 13C{1H} NMR (MeOD, 176 MHz) see Table 6; UV-Vis (2.0 × 10-4 M, 8.0 × 10-4 M, MeOH), λmax (ε) [(297 (9813), 325 (9252), 552 (1329)]. HRMS (ESI-) m/z: [M-H]- Calcd for C34H30NO11 628.1824; found 628.1824, (Figure S21). Jadomycin 3-AMBA Acetate 3a-Minor (7b): violet amorphous solid (yield 2.7 mgL-1); TLC Rf: 0.37 (9:1 CH2Cl2: MeOH); HPLC Rt = 8.78 min (Figure S6); 1H NMR (MeOD, 700 MHz) and 13C{1H} NMR (MeOD, 176 MHz) see Table 6; UV-Vis (2.0 × 10-4 M, 8.0 × 10-4 M, MeOH), λmax (ε) [(298

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(3981), 325 (5464), 381 (900), 552 (724)]. HRMS (ESI-) m/z: [M-H]- Calcd for C34H30NO11 628.1824; found 628.1847, (Figure S22). Labeling Efficiency. Labeling efficiency of 3 for the 13C-enriched fermentation was calculated as reported in the literature.30, 63 13C-NMR of labeled and unlabeled compounds were recorded under identical experimental conditions. The 13C-signals from both the labeled and unlabeled 13 C-NMR spectra were integrated with respect to C6′ (integraMon value = 1), the carbonyl of the 4-amino(methyl)benzoic acid side chain, this served as an appropriate probe as the incorporated amino acid did not possess an isotopic label. This process was repeated for each set of peaks corresponding to each diastereomer of 3. Labeled and unlabeled spectra were compared by the ratio (r) for each peak, where: ( ) = ( ) The total percentage 13C at each site was calculated by multiplying r for that carbon by the factor (f) required to scale the average r for unenriched carbons to 1.108%. The factor (f) was calculated using C1′-C6′, as these carbons were known to be unenriched. The f values were determined to be 0.99 and 1.08 for the major and minor diastereomers of 3 respectively. Total percent 13C at each position was calculated according to the equation63: 13 = The percent enrichment was calculated according to the equation30: ℎ =

( − ) ×

Some diastereomeric carbon shifts were not resolved sufficiently to accurately integrate values for each diastereomer. In these cases, 13C-values were calculated from the sum of signals and the average of the calculated f values. Due to the expected conversion of D-glucose to one equivalent of both labeled and unlabeled pyruvate, the potential labeling efficiency was halved. Since glucose used for fermentations consisted of a 2:1 ratio of D-glucose to [1-13C]-D-glucose (33% D-[1-13C]-glucose), a theoretical labeling efficiency of ~16-17% was expected. It was found, on average, labeled carbons within the polyaromatic backbone experienced a labeling efficiency of 9.6% ± 0.4. A full table of all enrichment percentages at each site can be found in the supplemental information (Table S1).

domonas aeruginosa ATCC 14210, Proteus vulgaris ATCC 12454, and Candida albicans ATCC 14035. Compounds were tested in three replicates against each organism. Compounds were serially diluted to generate a range of concentrations in a final well volume concentration of 2% DMSO. Each plate contained eight uninoculated positive controls (media with 2% DMSO), eight untreated negative controls (Media with 2% DMSO + organism), and one column containing a concentration range of a control antibiotic (vancomycin for MRSA and S. warneri, rifampicin for VRE, gentamycin for P. aeruginosa, ciprofloxacin for P. vulgaris, or nystatin for C. albicans). The optical density of the plate at 600 nm was recorded at 0 h and 22 h (after incubation of the plates at 37°C). The percentages of microorganism survival relative to vehicle control wells were calculated and the IC50 was determined.

ASSOCIATED CONTENT Supporting Information 13 Media recipes, supporting figures, C-labeling efficiencies, HRMS spectra, cytotoxicity data, antimicrobial activity, NMR-spectra and additional data are described. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Tel: +1 902 494 7159; [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources We thank NSERC, CIHR, and CHRP for financial support of this research.

Bioassay Methods

ACKNOWLEDGMENT

MDA-MB-231 (231-CON) were used to create polyclonal, ATP-binding cassette (ABC) B1 transporter-overexpressing, paclitaxel-resistant MDAMB-231 (231-TXL) cells in-house using slowly increasing concentrations of paclitaxel over seven months until the cells could survive a final concentration of 470 nM,37 the same paclitaxel concentration used in our previously described MCF7-TXL cells.35 All MDA-MB-231 cells were cultured in phenol red-free Dulbecco's modified Eagle's medium supplemented with 10% FBS, 100 IU/mL penicillin, 250 µg/mL streptomycin, and 1 mM sodium pyruvate (standard assay medium), with the 231-TXL cells maintained with 470 nM paclitaxel. The cells were split, and growth medium changed every 3-4 days up to a maximum of 35 passages. Cells were maintained in a humidified, 95% air/5% CO2 atmosphere at 37°C. MTT assays were used to evaluate 5, 6, 7a, 7b (0.5 – 100 µM), jadomycin F (0.1 – 25 µM), and doxorubicin (0.008 – 100 µM) in 231-CON and 231-TXL breast cancer cells according to previously described methods.36 The fold-resistance values for each drug were obtained by dividing their half-maximal inhibitory concentration (IC50) values in the drug-resistant 231-TXL cells by their mean IC50 value determined in the 231-CON cells. All MTT data are presented as the mean value of at least four separate replicate trials with each trial’s values displayed in scatter plots. An unpaired t test was performed for dual comparisons in experiments with one independent variable. A one-way analysis of variance (ANOVA) was performed for multiple comparisons in experiments with one independent variable. A Bonferroni’s multiple comparison test was used for post-hoc analysis of the significant ANOVA. A difference between mean values between groups was considered significant if P ≤ 0.05. All microbroth antibiotic susceptibility testing was carried out in 96well plates in accordance with Clinical Laboratory Standards Institute testing standards (2003, M7-A6) using the following pathogens: methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), S. warneri ATCC 17917, vancomycin-resistant Enterococcus faecium EF 379 (VRE), Pseu-

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Post Polyketide Synthase Carbon-Carbon Bond Formation in Type-II

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