Discovery, Isolation, and Structure Elucidation of Dretamycin - Journal

Jun 16, 2014 - ... 2014 The American Chemical Society and American Society of Pharmacognosy. *Tel: 1-908-654-3915. E-mail: [email protected]...
3 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/jnp

Discovery, Isolation, and Structure Elucidation of Dretamycin Kenneth E. Wilson,*,†,‡ Scott K. Smith,†,§ Rosemarie Kelly,†,⊥ Prakash Masurekar,†,∥ Deming Xu,†,¶ Craig A. Parish,† Hao Wang,† Debbie L. Zink,†,● Julian E. Davies,□ and Terry Roemer† †

Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, United States Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4

□

S Supporting Information *

ABSTRACT: The Candida albicans fitness test is a whole cell screening platform that utilizes a mixed-pool of C. albicans mutants, each of which carries a heterozygous deletion of a particular gene. In the presence of an antifungal inhibitor, a subset of these mutants exhibits a growth phenotype of hypersensitivity or hyposensitivity. Collectively these mutants reflect aspects of the mechanism of action of the compound in question. In the course of screening natural products a culture of Streptomyces sp. MS-1-4 was discovered to produce a compound, dretamycin, which yielded a fitness profile exhibiting significant hypersensitivity of the DRE2 heterozygote and hyposensitivity of the DIP5 heterozygote. Herein we report the production, isolation, and structure elucidation of dretamycin. he Candida albicans fitness screening platform was developed with the goal of increasing the overall efficiency of antifungal antibiotics discovery by targeting specific processes in the fungal pathogen essential for survival.1,2 Since C. albicans is a diploid organism, deletion of one allele on C. albicans usually confers no phenotypic defect to the resulting heterozygous mutant. However, when partially inhibited by an antifungal compound, some of these mutant strains become preferentially sensitive or resistant. In many cases the profile of these hypersensitive and hyposensitive heterozygotes captures functional aspects of inhibitory activity shedding light on the mode of action and even the target. A collection of C. albicans heterozygous mutants was constructed in which each strain was uniquely barcoded, thereby allowing parallel screening of all strains in coculture. Growth properties of the coculture could be determined by the relative abundance of each strain by DNA microarray to which each barcode was hybridized.3 The format of this assay was particularly useful in screening crude natural product extracts and led to the identification of multiple new antifungal compounds, one of which was dretamycin.2 During the course of screening 1800 crude fermentation broths and 200 pure compounds from fungal and bacterial sources, a single extract (ECC619) resulted in hypersensitivity of the C. albicans DRE2 heterozygote. The DRE2 gene encodes an essential Fe/S cluster protein involved in oxidative stressinduced cell death.4 ECC619 also caused hyposensitivity of the DIP5 heterozygote. The DIP5 gene encodes a permease, controlling the transport of dicarboxylic L-amino acids.5,6 Extract EEC619 was derived from a fermentation of Streptomyces sp. MS-1-4, isolated from a moss from British Columbia. The compound accounting for the fitness activity of EEC619 was isolated and identified as N-hydroxy-3-carbamyl-5carboxy-2-pyrroline (1). This report describes the detection, production, and structure elucidation of 1, which we give here the name dretamycin. Dretamycin was isolated and charac-

T

© 2014 American Chemical Society and American Society of Pharmacognosy

terized chemically and biologically as the potassium salt 1a (Figure 1).

Figure 1. Structure of dretamycin (1) and dretamycin, potassium salt (1a).

■

RESULTS AND DISCUSSION Candida Fitness Test of ECC619. A fermentation broth of Streptomyces sp. MS-1-4 was found to be active against Saccharomyces cerevisiae and C. albicans but not bacteria (data not shown). Broth extract (ECC619) was assayed in the fitness test (Figure 2). Two significantly hypersensitive genes were identified, DRE2 (orf19.2825) and RVB2 (orf19.6539), neither of which has been characterized in C. albicans. However, their Saccharomyces homologues are involved in oxidative stressinduced cell death and in regulation of gene expression as part of the chromatin remodeling complex, respectively.4,7 The fitness test profile also identified a number of hyposensitive genes whose functions were not clearly related (Figure 2). One of them, DIP5 (orf19.2942, represented by two independently constructed strains) encodes a permease involved in transport of dicarboxylic L-amino acids.5,6 Its hyposensitivity was interpreted as a growth advantage rendered by the loss of one allele of the gene that is involved in the uptake of ECC619, suggesting that the active compound is an analogue of an amino Received: December 7, 2013 Published: June 16, 2014 1280

dx.doi.org/10.1021/np401019t | J. Nat. Prod. 2014, 77, 1280−1286

Journal of Natural Products

Article

1 was eluted from the resin with 1 M acetic acid. To remove the acetic acid, the Dowex 1 × 4 rich cut was adsorbed at pH 2 onto Dowex 50 × 2 resin (hydrogen cycle). Dretamycin was eluted from this resin by adjusting the pH of a stirred, waterslurry of the resin to pH 8.2. (For long-term storage the Dowex 50 × 2 active fraction was diluted with methanol to a final aqueous concentration of 30% and stored at −80 °C in a liquid state). Purification of the Dowex 50 × 2 rich cut was carried out by preparative HPLC on a Phenomenex Aqua C18 column, eluting with 10 mM potassium formate at pH 4.0. Final desalting by adsorption on Dowex 50 × 4 (hydrogen cycle) and slurry elution at pH 8.2 with potassium hydroxide, followed by lyophilization, afforded pure dretamycin, potassium salt (1a, 38% overall yield). (Concentrating aqueous solutions of 1a at pH 7 or lower resulted in significant decomposition of 1 based on HPLC and 1H NMR data, suggesting that the solution instability of 1 at neutral and acidic pH is due to second-order rate kinetics.) Purified dretamycin potassium salt (1a) reproduced the fitness test profile of the original extract ECC619 (Figure 2). Structure Determination of Dretamycin. From the isolation studies compound 1a was suspected to be a small, hydrophilic molecule with both acidic and basic functionality. The compound was found to be soluble only in water. Compound 1a exhibited a UV chromophore at 253 nm. 13C NMR and HSQC NMR data of 1a in D2O indicated the presence of six carbons: C (180.4 ppm, 177.1 ppm, 82.7 ppm), CH (158.3 ppm, 58.3 ppm), and CH2 (26.4 ppm). 1H NMR and COSY NMR data of 1a indicated the presence of four nonexchangeable protons and a three-proton spin system. HMBC NMR data led to partial structure 2, accounting for the six observed carbons and the four nonexchangeable protons. On the basis of chemical shift, it was considered likely that the carbon at 158.3 ppm bears a nitrogen substituent (Figure 3).

Figure 2. C. albicans fitness test profiles of extract ECC619, dretamycin, 6-diazo-5-oxo-L-norleucine (DON), trans-azetidine-2,4dicarboxylic acid (AzDC) and L-azetidine-2-carboxylic acid (AzC). The fitness test and the heat-map display are described elsewhere.1,2 The experimental conditions are listed on top of the heat map, with gene designations and S. cerevisiae homologues on the right.

acid. Since ECC619 was the only antifungal extract that induced hypersensitivity of DRE2, we hypothesized that ECC619 contained a novel antifungal amino acid analogue that warranted isolation. Fermentation and Isolation of 1. Fermentation of Streptomyces sp. MS-1-4 under submerged, aerated conditions produced dretamycin at a titer of 180 mg/L. The isolation of 1 from harvested fermentation broth was monitored by agar diffusion assay using a wild-type Candida albicans and a DRE2heterozygous deletion strain. Compound 1 typically exhibited a hazy zone of inhibition on the wild-type strain and a larger clearer zone on the heterozygote. Initial studies indicated that 1 was a water-soluble, amphoteric compound that was not retained on polystyrene-divinylbenzene reverse phase resins under any pH conditions. The isolation of 1 was complicated by two factors. (i) Instability - the antifungal activity of an acetone extract of harvested broth prepared during initial isolation studies, although stable at 5 °C for several days, was lost after the extract had been frozen and stored at −80 °C. Furthermore, the activity was observed to be more stable at alkaline pH than acidic pH. (ii) A high concentration of 3-(Nmorpholino)-propanesulfonic acid buffer (MOPS, 20 g/L), which has amphoteric properties similar to those of 1, was a component of the fermentation production medium. Compound 1 was present uniquely in the clarified fermentation broth. The clarified broth was filtered through a bed of Mitsubishi SP207 resin at pH 3.5 to remove lipophilic impurities and then adsorbed at pH 5 onto Dowex 1 × 4 resin (acetate cycle) to remove the MOPS media buffer. Compound

Figure 3. Partial 1H and 13C chemical shift assignments of 1 in D2O at 25 °C with COSY and selected HMBC correlations.

ESI-HRMS studies of 1a did not identify a likely molecular ion or fragment ion that was consistent with partial structure 2. In order to determine the carbon/nitrogen ratio, 1a was submitted for elemental combustion analysis. An initial analysis of the pure sample was carried out with the standard protocol of drying to constant weight before combustion, resulting in a C/N atom ratio of 6/1 and the presence of only trace sulfur (C6.0 H6.45 N1.03 S0.021). Later when chemical instability issues of 1a were better appreciated, the elemental analysis of 1a was repeated without first drying to constant weight. The results indicated a C/N atom ratio of 6/2 (C6.0 H9.6 N1.9). It was concluded that 1a has a C/N ratio of 6/2 but loses nitrogen upon drying to constant weight. Accordingly, assuming the presence of 6 carbons from the 13C NMR data, 1a must also contain 2 nitrogen atoms. Two plausible structures for 1, 1281

dx.doi.org/10.1021/np401019t | J. Nat. Prod. 2014, 77, 1280−1286

Journal of Natural Products

Article

(0.23 mg) of 8.5%. Attempts were made to improve the reaction conditions and recovery of 6. Diazomethane treatment of 1 at higher temperatures yielded more complex product mixtures. Compound 6 decomposed in acetonitrile−0.1% aqueous formic acid but was stable in methanol−0.1% aqueous formic acid, suggesting that 6 was reactive with acetonitrile. ESI-HRMS data established the dimethyl analogue 6 to have a molecular ion M+ at 200.07698 m/z, corresponding to the molecular formula C8H12N2O4. 1H and COSY45 NMR data of 6 in DMSO-d6 showed that the three-proton spin system and the single olefinic proton of partial structure 3 had been preserved: C4H2(2.30 ppm, 2.41 ppm)-C5H (3.44 ppm); C2H (8.20 ppm). In addition two new methyl singlets at 3.58 and 3.27 ppm were present. The HSQC spectrum of 6 revealed all protonated carbons except C4. Carbons C4, C6, and C7 were identified indirectly from HMBC data. Based on HMBC, NOESY-1D and 15N-HMBC data, the methyl singlet at 3.58 ppm (δc 52 ppm) is part of a methyl ester at C5 in 3 and the methyl singlet at 3.27 ppm (δc 40 ppm) is attached to N-1 in 3. Together these fragments have an atom count of C8H10N2O3, two protons and one oxygen atom less than the molecular formula of 6 determined by HRMS. In light of partial structure 3 for dretamycin and the fact that 6 is a dimethyl derivative of dretamycin, the two protons must be assigned to a carboxamide group at C3 and the oxygen atom must be attached to N1 (Figure 6a). The two NH protons of the C3 carboxamide

consistent with the NMR and elemental analysis data, are shown below (Figure 4).

Figure 4. Proposed structures of 1 based upon NMR and elemental analysis data.

In order to obtain confirmation of the overall structure and to differentiate between the enamine (3) and imine (4) isomers, compound 1a was hydrogenated over PtO2 in acetic acid/water. Examination of the crude reaction product by 1H NMR and COSY NMR in D2O indicated a mixture of predominantly three reduction products (5a, 5b, 5c), all with similar proton spin systems, in the relative ratio of 54/35/11. 13 C NMR and DEPT NMR data on the crude mixture indicated that the major component (5a) contains two methine carbons (59.0 ppm, 43.0 ppm) and two methylene carbons (45.7 ppm, 30.6 ppm). The four carbons are connected as a 6-proton spin system based on COSY and HSQC data. HMBC NMR data indicated the presence of two carbonyl carbons in 5a (171.8 ppm, 177.0 ppm) not evident in the 13C NMR spectrum. On the basis of correlations from those carbons to protons in the 6proton spin system, the structure of the major product (5a) was determined to be that shown in Figure 5.

Figure 6. 1H and 13C chemical shift assignments of 6 in DMSO-d6 at 25 °C: (a) selected HMBC correlations; (b) NOESY-1D and 15NHMBC correlations.

1

group were not observed in the 1H NMR or COSY NMR of 6, presumably because of exchange of these protons with protons from residual water in the sample/DMSO-d6. The presence of the N-methyl-N-oxo functionality in 6 was supported by 15N-HMBC data, which showed a correlation from the 15N1 signal at 146.4 ppm to H2 at 8.20 ppm (Figure 6b). The 15N chemical shift of N1 is consistent with known 15N chemical shift data for N-oxides.8,9 The expected NOESY-1D correlations were observed between for H4a/H4b, H5/H4a, and H2/NMe. However, no NOESY-1D correlation was seen for H5/NMe. Therefore, the configuration of the NMe bond of 6 was assigned trans with respect to H5 since a cis configuration would be expected to have shown a NOESY correlation (MM2 energy minimization modeling: H5-NMe dihedral angle = 30°). From the structure of 6 it could be concluded that the structure of dretamycin was N-hydroxy-3-carbamyl-5-carboxy2-pyrroline (1, MW 172, C6H8N2O4). With this knowledge reexamination of earlier MS data on 1 identified the presence of an ion at 173 m/z [M + H]+ by positive ion ESI-MS and an ion at 171 m/z [M − H]− by negative ion ESI-MS. ESI-HRMS data of the 173 m/z ion confirmed the molecular formula.

13

Figure 5. H and C chemical shift assignments of hydrogenation product 5a in D2O at 25 °C with COSY and selected HMBC correlations.

The six-proton spin system of the hydrogenation product 5a is only consistent with dretamycin potassium salt (1a) having the enamine structure 3 and not the imine structure 4. Minor hydrogenation products 5b and 5c are likely isomers of 5a. Interpretation of the ESI-MS data of mixture 5a−c was equivocal. No attempt was made to separate and further characterize the hydrogenation products. Final elucidation of the structure of 1a was obtained from NMR studies of the dimethyl derivative 6, prepared by the treatment of dretamycin free acid 1 with diazomethane in MeOH−water at −80 °C for 10 min followed by quenching excess diazomethane at −80 °C with formic acid. Based upon HPLC data of the quenched reaction the dimethyl analogue 6 (27% yield) was the most predominant of four products formed under these conditions. Compound 6 was purified by preparative HPLC in a disappointing overall isolated yield 1282

dx.doi.org/10.1021/np401019t | J. Nat. Prod. 2014, 77, 1280−1286

Journal of Natural Products

Article

prevent osmotic loss of intracellular water to the environment and subsequent loss of cellular viability, the intracellular concentration of an osmo-protectant such as proline is increased through active transport from the environment or from up-regulation of cellular synthesis. For example when S. griseus was grown both in minimal media and in minimal media containing 0.5 M NaCl, the respective concentrations of free intracellular proline were 3.1 mM and 151 mM, respectively. The proline levels were similar in studies where proline was an ingredient of the media, namely 5.8 mM and 162 mM, respectively. In the latter case half of the intracellular high-level proline was from uptake and half was from cellular synthesis.15 MOPS buffer was an ingredient of the production medium for Streptomyces sp. MS-1-4 at a concentration high enough (20 g/ L, 96 mM) to likely induce an osmo-protective response, leading to high concentrations of intracellular proline. Without an understanding of the biosynthetic origin of dretamycin it is impossible to speculate on the relationship between dretamycin and proline production in the fermentation of Streptomyces sp. MS-1-4. The chemical instability of dretamycin was particularly problematic during its isolation. Vinylogous hydroxamic acids are reported to be unstable, existing in solution in equilibrium with a nitrone isomeric form.16 The nitrone moiety can react through 1,3-dipolar cycloaddition either (i) with itself or (ii) with the vinylogous hydroxamic acid. With respect to dretamycin both processes are shown in Scheme 1, the nitrone

With regard to the absolute stereochemistry of 1, growth of the DIP5 heterozygous mutant in the Candida fitness test was stimulated by dretamycin. DIP5 encodes a permease which mediates the transport of dicarboxy-L-amino acids into cells.2,5,6 Dretamycin is a structural analogue of a dicarboxy-L-amino acid; therefore, it was suspected that the absolute stereochemistry of 1 is L (S-C5). (The enhanced growth of the DIP5 heterozygote relative to the diploid strain can be explained by a reduced uptake of antifungal dretamycin into the heterozygote.) Chemical approaches to confirm the L configuration were unsuccessful. Specifically, preparation of a larger sample of dimethyl analogue 6 was attempted since 6 was suspected to be intrinsically more stable than dretamycin and more conducive to crystallization. Unfortunately scale-up of the diazomethane reaction and isolation of pure 6 produced disappointing results because of the multiple reaction products, the sensitivity of the yield of 6 to reaction conditions, and the lack of a robust method to purify 6. An attempt was also made to prepare the N-(O-4-bromobenzyl)-4-bromobenzyl ester of hydrogenation product 5a since exciton coupling of the dibromobenzyl groups in the CD spectrum of such a derivative would establish the absolute stereochemistry of C5. Crude hydrogenation product of dretamycin (mainly 5a, 5b, 5c) was converted to the tetraethylammonium salt and was treated with excess 4bromobenzyl bromide in DMF at 37 °C. Based on HPLC analysis a complex product mixture was obtained (not unexpectedly) with no major component dominating the mixture; resolution of the mixture was not pursued. Purified dretamycin potassium salt (1a) reproduced the fitness test profile of original extract EEC619 and specifically the respective hyper- and hyposensitivity of the DRE2 and DIP5 strains, indicating that 1a was the sole active component in the original extract to which the Candida fitness profile could be attributed (Figure 2). The hypersensitivity of the DRE2 mutant has been shown to be responsible for the antifungal activity of 1a while the hyposensitivity of the DIP5 mutant has identified this dicarboxylic-L-acid amino permease as responsible for the uptake of 1a into C. albicans.2 Dretamycin exhibited weak antifungal activity against Candida spp. (seven strains) with minimum inhibitory concentrations (MICs) ranging from 8 μg/mL to >32 μg/mL. MICs of la against S. cerevisiae and Aspergillus fumigatus were >32 μg/mL (data not shown). The structure of dretamycin was determined to be Nhydroxy-3-carbamyl-5-carboxy-2-pyrroline (1). There is insufficient experimental evidence to confidently assign the absolute stereochemistry of 1; however, the active transport of 1 into Candida using the dicarboxylic acid-L-amino-acid permease DIP5 infers that the absolute stereochemistry of 1 is L (S-C5). Dretamycin is a 4,5-dehydro-proline analogue containing an unusual vinylogous hydroxamic acid fragment. No close analogues of dretamycin have been reported in the literature. trans-4-Carboxy-L-proline is a natural product isolated from Afzelia bella seed and from algae.10,11 The ethyl ester of 4ethoxycarbonyl-4,5-dehydro-proline has been reported as a synthetic compound.12 Dretamycin is produced by aerobic fermentation of Streptomyces sp. MS-1-4 at a titer of 180 mg/L (1.04 mM). The high titer is remarkable and may be related to the fact that dretamycin is an analogue of proline. Proline plays an important role in osmo-regulation in bacteria and plants.13,14 Organisms experience osmotic stress when the concentration of solutes in the aqueous environment becomes too high. To

Scheme 1. Plausible Pathways to Explain the Chemical Instability of Dretamycin (1) and 6

isomer 7 leading to symmetric adduct 8 or adduct 9. The second-order nature of both processes explains why the instability of dretamycin increases with concentration. The observed loss of nitrogen upon predrying a sample of dretamycin for elemental analysis can be explained by the isomerization of 1 to nitrone 10, with subsequent loss of ammonia to afford ketene 11 (Scheme 1). It was mentioned earlier that dimethyl analogue 6 was unstable in aqueous acetonitrile solutions containing 0.1% formic acid. A plausible explanation for the instability is the condensation of 6 and acetonitrile to form the 1,2,4oxadiazoline 12, shown in Scheme 1.17 Dretamycin potassium salt (1a) did not afford interpretable ESI-MS data. Because of this the preparation and structural analysis of dihydro-dretamycin 5a and dimethyl analogue 6 were necessary to complete the structure elucidation of dretamycin. The syntheses of these derivatives were inefficient and unaesthetic, leading to mixtures of products. An aspect of 1283

dx.doi.org/10.1021/np401019t | J. Nat. Prod. 2014, 77, 1280−1286

Journal of Natural Products

■

the 1H spectrum of dimethyl analogue 6 not mentioned above was that the spectrum showed evidence of multiple conformers of 6 in DMSO-d6. Specifically, downfield from the dominant methyl singlet at δ 3.58 ppm (OMe in 6) were three additional minor methyl singlets at δ 3.628 ppm (OMea), 3.640 ppm (OMeb) and 3.683 ppm (OMec, Figure 7). The proton signals

Article

EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were recorded on a Beckman DU-70 spectrophotometer. IR spectra were recorded on a PerkinElmer Spectrum 1 spectrophotometer. NMR data were acquired on a Varian VnmrS 600 spectrometer with an indirect detection probe. MS data were collected on a Thermo Finnigan LTQ FTMS with ESI ionization. HPLC assays of solutions of 1a and of 6 were performed using a Phenomenex Aqua C18 column (4.6 mm × 75 mm) with an eluent of 20 mM potassium formate, pH 4.0 at 0.50 mL/min and 25 °C, monitoring eluent at 205 and 260 nm. Dretamycin eluted with a retention time of 2.15 min (0.86 column volume retention). Compounds 1a and 6 were stored as solutions in MeOH−water (7:3) at −80 °C (liquid state). The concentration of 1a in solution was initially determined by quantitative 1H NMR of 1a in D2O containing 2.86 μmol of CH3CN as internal standard. Dilutions of that solution were used to determine the UV chromophore of la (253 nm, log ε = 3.97). HPLC assay standards of 1a were prepared based on this log ε value. As an approximation the log extinction coefficient of the UV chromophore of 6 (270 nm) was also assumed to be 3.97. MM2 energy minimization modeling was done using ChemDraw 3D Ultra Ver. 4. Production of Dretamycin (1). (i) Seed Stage. The surface of 10 agar plates of ISP Medium 4 (Becton Dickinson & Co.) was inoculated with culture Streptomyces sp. MS-1-4. The plates were incubated for 7 days at 30 °C. ISP Medium 4 was prepared using (g/L in distilled water) agar (20), soluble starch (10), K2HPO4 (1.0), MgSO4·7H2O (1.0), NaCl (1.0), (NH4)2SO4 (2.0), CaCO3 (2.0), FeSO4·7H2O (0.0001), MnCl2·4H2O (0.0001), ZnSO4·7H2O (0.0001). (ii) Production Stage. Erlenmeyer flasks (250 mL) containing 30 mL of production medium were each inoculated with two loops of growth from the seed stage plates. The flasks were incubated on a gyratory shaker (220 rpm) at 30 °C for 10 days. Production medium was prepared using (g/L in distilled water) glucose (10), glycerol (15), soy peptone (15), malt extract (5), yeast extract (5), Tween 80 (1), MOPS (20), NaCl (3), pH adjusted to 7.0. Isolation of Dretamycin. Harvested fermentation broth (2.34 L, pH 7.6) was centrifuged (8000 rpm, 5 °C) to pellet the mycelium. The clarified centrifugate (2.32 L, 442 mg 1) was adjusted to pH 3.5 with acetic acid and HCl and passed through a resin bed of Mitsubishi SP207 resin (200 mL). The resin was washed with 500 mL of water. The combined aqueous material (2.8 L) was adjusted to pH 5.0 with potassium hydroxide and adsorbed onto a column of Dowex 1 × 4 resin (700 mL, 200−400 mesh, acetate cycle). The resin was washed with 1.8L of water and eluted with 1.0 M aqueous acetic acid. The dretamycin rich cut (2.70 L, 284 mg 1) was adjusted to pH 2.0 with concentrated HCl and adsorbed onto a bed of Dowex 50 × 2 resin (240 mL, 200−400 mesh, hydrogen cycle). The resin was washed with 1.8 L of water and transferred to a beaker. The resin was stirred in 100 mL of water, and the slurry was adjusted carefully to pH 8.2 with aqueous KOH solution (45 wt %). The resin slurry was transferred to a column. The column was drained to collect the dretamycin-rich aqueous solution, and the resin was washed with 360 mL of water. The two aqueous solutions were combined as the desalted partially purified 1 (550 mL, 252 mg). For long-term storage in a liquid state this fraction was diluted to 2.7 L with MeOH and stored at −80 °C. Aliquots of this

Figure 7. 1H NMR spectrum of the OMe signals of 6 and tabulated 13 C, COSY45, and HMBC Data.

of OMeb and OMec were unresolved in the proton domain in the COSY, HSQC, and HMBC spectra. HSQC data indicated that the cross-peak of the protons of OMeb and OMec correlated to a carbon with a chemical shift (52 ppm) the same as that of the carbon associated with the dominant OMe at 3.58 ppm. HMBC correlations analogous to the correlation between OMe (3.58 ppm) and H2 in 6 were observed for the three minor singlets. Finally, the presence of exchange correlations among the four OMe singlets in the COSY NMR spectrum of 6 established that the singlets arise from interconverting conformers of 6, likely from energetically preferred rotational orientations of the carbomethoxy group at C5 due to the adjacent, geminally disubstituted nitrogen moiety with mixed charge properties (Figure 7). In summary, the Candida fitness assay, which utilizes a large collection of cocultured C. albicans heterozygous deletion mutants, detected an antifungal activity in crude methanol extract EEC619 of Streptomyces sp. MS-1-4 that inhibited the growth of the DRE2 heterozygote but stimulated the growth of the DIP5 heterozygote. Since it is known that in S. cerevisiae DRE2 is associated with oxidative, stress-induced, cell death and DIP5 encodes a dicarboxy-L-amino acid permease, it was suspected that the antifungal activity in the extract was due to a compound that targets the DRE2 gene product of C. albicans and is transported into the cell via a dicarboxy-L-amino acid permease. Detramycin (1, N-hydroxy-3-carbamyl-5-carboxy-2pyrroline), was isolated from the extract and found to reproduce the Candida fitness profile. As predicted from the DIF5 activity of the original Candida fitness profile of the crude extract, 1 was a dicarboxy-amino acid analogue. Dretamycin was unstable in aqueous solutions below pH 7 presumably due to isomerization to nitrone 7 followed by 1,3-cycloaddition to itself or 1. Recognition of the N-methyl-N-oxo functionality in dimethyl analogue 6 was pivotal in the structure elucidation of 1. Dretamycin potassium salt has weak activity against clinical strains of Candida spp. and A. fumigatus. 1284

dx.doi.org/10.1021/np401019t | J. Nat. Prod. 2014, 77, 1280−1286

Journal of Natural Products

Article

methanolic solution were used for final prep HPLC purification. Typically a 100 mL aliquot (9.0 mg 1) was concentrated to 4 mL. After adjusting the pH of the concentrate from 9.0 to 2.1 with HCl, the concentrate was injected onto a Phenomenex Aqua C18 column (19 mm × 300 mm). The column was eluted at room temperature with 10 mM potassium formate pH 4.0 at 8.0 mL/min. Fraction purity was judged by comparing the analytical HPLC profiles at 205 and 260 nm. Fractions containing pure 1 were pooled and adsorbed onto BioRad AG50 × 2 resin (1.0 mL, 200−400 mesh, hydrogen cycle). After washing with water the resin was slurried in 3 mL of water and adjusted to pH 8.2 with concentrated KOH solution. Removal of the spent resin afforded an aqueous solution of pure dretamycin, potassium salt 1a (6 mg, 2.4 mL, pH 8.3). Dretamycin, potassium (1a): white, amorphous solid; UV (H2O) λmax (log ε) 253 (3.97); FTIR (neat) νmax 3379 (br, s), 2925, 1635 (s), 1404; 1H NMR (D2O, 600 MHz) δ 7.96 (1H, s, H2), 3.84 (1H, dd, J = 7, 4, H5); 2.78 (1H, dd, J = 16, 4, H4a); 2.67 (1H, dd, J = 16, 7, H4b); 13C NMR (D2O, 150 MHz) δ 180.4 (C, C7); 177.1 (C, C6), 158.3 br (C, C2), 82.7 (C, C3), 58.3 (CH, C5); 26.4 (CH2, C4); ESI-FTMS m/z 173.05568 [M + H]+ (calcd for C6H8N2O4 + H, 173.05623); Elemental Analysis (1a not predried to constant weight) C 24.46%, H 3.30%, N 9.02% [calcd for C6H7.8N2O4K0.2. (H2O)6.8: C 23.84%, H 2.60%, N 9.27%]. Elemental Analysis (1a predried to constant weight) C 47.5%, H 4.26%, N 9.50%, S 0.58%, empiric formula C6H6.4.N1.0S0.021. Hydrogenation of Dretamycin. A solution of 1.1 mg of 1a in 0.5 mL water adjusted to pH 6 was hydrogenated at 5 °C using prereduced PtO2 (1.1 mg) in 4 mL of glacial acetic acid. After 30 min the catalyst was removed by centrifugation and the liquid was evaporated to dryness. The residue was taken up in 0.40 mL water and 2.0 mL of MeOH and evaporated to dryness, affording a 54:35:11 mixture of 5a, 5b, and 5c. Dihydro-dretamycin 5a. 1H and 13C NMR data (D2O) see Figure 5. Preparation of N-Methyl-N-oxo-3-carbamyl-5-methoxycarbonyl-2-pyrroline (6). Dretamycin potassium salt 1a (2.6 mg in 2.6 mL 7:3 MeOH/water) was passed through Amberlite CG50 resin (1 mL, 100−200 mesh, hydrogen cycle) in 7:3 MeOH/water, followed by 3.0 mL of 7:3 MeOH/water wash. The combined spent and wash (pH 4.8) was adjusted to pH 2.8 with 2 N HCl. The solution was adjusted to 75% MeOH by addition of 1.0 mL MeOH and then cooled to −80 °C. The solution was stirred and treated with excess diazomethane (in ethyl ether). After 10 min stirring at −80 °C, the reaction was quenched with formic acid. The solution was warmed and concentrated to 3 mL. HPLC analysis of the crude reaction concentrate indicated the presence of four products all of which eluted later than 1a (tR = 2.15 min, UVmax = 252 nm): A (3.86 min, 270 nm), B (5.12 min, 255 nm), C (7.68 min, br, 255 nm), and D (9.34 min, 270 nm, 6). The total product yield was 61% and the yield of 6 was 27% (0.715 mg). Compound 6 was purified by preparative HPLC (Phenomenex Aqua C18, 4.6 mm × 150 mm, 20 mM K formate pH 4.0) followed by desalting on Mitsubishi CHP20P resin (0.5 mL), eluting with MeOH. The MeOH eluate contained 0.319 mg of pure 6. Evaporation of the MeOH eluate in preparation of the NMR sample resulted in 30% decomposition of 6 based on HPLC analysis: 0.23 mg 6 (8.5% overall yield). N-Methyl-N-oxo-3-carbamyl-5-methoxycarbonyl-2-pyrroline (6). UV (7:3 MeOH/H2O) λmax (log ε) 270 (3.97

assumed); 1H, 13C, 15 NMR (DMSO-d6) see Figure 6; ESIFTMS m/z 201.0875 [M + H]+ (calcd for C8H12N2O4 + H, 201.0875).

■

ASSOCIATED CONTENT

S Supporting Information *

Annotated 1D and 2D NMR spectra of 1a, 5a and 6, (limited sample and instability prevented obtaining a 13C NMR spectrum of 6). This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 1-908-654-3915. E-mail: [email protected]. Present Addresses ‡

Kenneth E. Wilson (120 S. Scotch Plains Avenue, Westfield, NJ 07090. E-mail: [email protected]). § Scott K. Smith (Agilent Technologies, 5301 Stevens Creek Blvd., Santa Clara CA 950051. E-mail: scott.smith@agilent). ⊥ Rosemarie Kelly (Novartis Pharmaceuticals Corp., One Health Plaza, E. Hanover, NJ 07936. E-mail: kelly. [email protected]). ∥ Prakash Masurekar (35 Christie Dr., Warren, NJ 07059. Email: [email protected]). ¶ Deming Xu (AppTec Co. Ltd., 288 Fute Zhong Road, Shanghai 200131, P.R. China. E-mail: xu_deming@hotmail. com). ● Debbie L. Link (20 Woodhollow Dr., Manalapan, NJ 07726. E-mail: [email protected]). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Roemer, T.; Jiang, B.; Davison, J.; Ketela, T.; Veillette, K.; Breton, A.; Tandia, F.; Linteau, A.; Sillaots, S.; Marta, C.; Martel, N.; Veronneau, S.; Lemieux, S.; Kauffman, S.; Becker, J.; Storms, R.; Boone, C.; Bussey, H. Mol. Microbiol. 2003, 50, 167−181. (2) Roemer, T.; Xu, D.; Singh, S. B.; Parish, C. A.; Harris, G.; Wang, H.; Davies, J. E.; Bill, G. F. Chem. Biol. 2011, 18, 148−164. (3) Shoemaker, D. D.; Lashkari, D. A.; Morris, D.; Mittmann, M.; Davis, R. W. Nat. Genet. 1966, 14, 450−456. (4) Vernis, L.; Facca, C.; Delagoutte, E.; Soler, N.; Chanet, R.; Guiard, B.; Faye, G.; Baldacci, G. PLoS One 2009, 4, e4376. (5) Regenberg, B.; Holmberg, S.; Olsen, L. D.; Kielland-Brandt, M. C. Curr. Genet. 1998, 33, 171−177. (6) Regenberg, B.; Düring-Olsen, L.; Kielland-Brandt, M. C.; Holmberg, S. Curr. Genet. 1999, 36, 317−328. (7) Gribun, A.; Cheung, K. L. Y.; Huen, J.; Ortega, J.; Houry, W. A. J. Mol. Biol. 2008, 376, 1320−1333. (8) Farley, K. A.; Bowman, P. B.; Brumfield, J. C.; Crow, F. W.; Duholke, W. K.; Guido, J. E.; Robins, R. H.; Sims, S. M.; Smith, R. F.; Thamann, T. J.; Vonderwell, B. S.; Martin, G. E. Magn. Reson. Chem. 1998, 36, S11−S16. (9) Martin, G. E.; Hadden, C. E.; Blinn, J. R.; Sharaf, M. H. M.; Tackie, A. N.; Schiff, P. L., Jr. Magn. Reson. Chem. 1999, 37, 1−6. (10) Impellizzeri, G.; Piatelli, M.; Sciuto, S.; Fattorusso, E. Phytochemistry 1977, 16, 1601−1602. (11) Welter, A.; Marlier, M.; Dardenne, G. Phytochemistry 1978, 17, 131−134. (12) Kraus, G. A.; Nagy, J. O. Tetrahedron 1985, 41, 3537−3545. (13) Csonka, L. N. Microbiol. Rev. 1989, 53, 121−147. (14) Delauney, A. J.; Verma, D. P. S. Plant J. 1993, 4, 215−223. (15) Killham, K.; Firestone, M. K. Appl. Environ. Microbiol. 1984, 48, 239−241. 1285

dx.doi.org/10.1021/np401019t | J. Nat. Prod. 2014, 77, 1280−1286

Journal of Natural Products

Article

(16) Wright, S. W.; Harris, R. R.; Kerr, J. S.; Green, A. M.; Pinto, D. J.; Bruin, E. M.; Collins, R. J.; Dorow, R. L.; Mantegna, L. R.; Sherk, S. R.; Covington, M. B.; Nurnberg, S. A.; Welch, P. K.; Nelson, M. J.; Magolda, R. L. J. Med. Chem. 1992, 35, 4061−4068. (17) Plate, R.; Hermkens, H. H.; Smits, J. M. M.; Nivard, R. J. F.; Ottenheijm, H. C. J. J. Org. Chem. 1987, 52, 1047−1051.

1286

dx.doi.org/10.1021/np401019t | J. Nat. Prod. 2014, 77, 1280−1286