Puromycins B–E, Naturally Occurring Amino-Nucleosides Produced

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Puromycins B−E, Naturally Occurring Amino-Nucleosides Produced by the Himalayan Isolate Streptomyces sp. PU-14G Muhammad Abbas,†,‡,§ Sherif I. Elshahawi,†,‡,∥ Xiachang Wang,†,‡,⊥ Larissa V. Ponomareva,†,‡ Imran Sajid,§ Khaled A. Shaaban,*,†,‡ and Jon S. Thorson*,†,‡

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Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States ‡ Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States § Department of Microbiology and Molecular Genetics, University of the Punjab, Quid-i-Azam Campus, Lahore 54590, Pakistan ∥ Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, California 92618, United States ⊥ Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China S Supporting Information *

ABSTRACT: The isolation and structure elucidation of four new naturally occurring amino-nucleoside [puromycins B−E (1− 4)] metabolites from a Himalayan isolate (Streptomyces sp. PU-14-G, isolated from the Bara Gali region of northern Pakistan) is reported. Consistent with prior reports, comparative antimicrobial assays revealed the need for the free 2″-amine for anti-Grampositive bacteria and antimycobacterial activity. Similarly, comparative cancer cell line cytotoxicity assays highlighted the importance of the puromycin-free 2″-amine and the impact of 3′-nucleoside substitution. These studies extend the repertoire of known naturally occurring puromycins and their corresponding SAR. Notably, 1 represents the first reported naturally occurring bacterial puromycin-related metabolite with a 3′-N-amino acid substitution that differs from the 3′-N-tyrosinyl of classical puromycin-type natural products. This discovery suggests the biosynthesis of 1 in Streptomyces sp. PU-14G may invoke a uniquely permissive amino-nucleoside synthetase and/or multiple synthetases and sets the stage for further studies to elucidate, and potentially exploit, new biocatalysts for puromycin chemoenzymatic diversification.

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Lake Saif ul Malook; 34°52′37′ N, 73°41′40′ E; altitude 10 577 ft) and Bara Gali (located in the Ayubia National Park; 34°6′0′ N, 73°21′0′ E; altitude 7710 ft) in Northern Pakistan. Bioactivity screens (antibacterial and cancer cell line cytotoxicity) and HPLC-MS comparative profiling with known metabolites in AntiBase2 implicated the Bara Gali isolate Streptomyces sp. PU-14G as capable of unique metabolic potential. In this report, we describe the isolation, structure elucidation, and biological activity of four new Streptomyces

ctinomycetes continue to serve as an abundant source of novel bioactive probes and/or leads, particularly in the areas of anti-infective and anticancer discovery, where the use of microbes isolated from untapped environments reduces the rates of rediscovery.1−3 As one of the youngest and tallest mountain ranges in the world, the Kashmir, Kohistan, Kaghan, Deosai, and Chilas valleys of the Greater Himalaya Range in Pakistan represent unexplored biodiverse environments for microbial natural products discovery. As a part of an ongoing microbial natural product and biocatalyst discovery initiative program,4−17 we examined the actinomycete diversity (and their corresponding secondary metabolic potential) within soil samples collected from the Himalayan Kaghan Valley (near © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 24, 2018

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DOI: 10.1021/acs.jnatprod.8b00720 J. Nat. Prod. XXXX, XXX, XXX−XXX

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puromycin-related metabolite that bears a 3′-amino acid substitution that differs from the 3′-tyrosinyl of classical puromycin-type natural products, 1 highlights Streptomyces sp. PU-14G as a potential source for new biocatalyst discovery.

sp. PU-14G-derived naturally occurring amino-nucleosides, puromycins B−E (1−4, Figures 1 and 2). Three additional

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RESULTS AND DISCUSSION Streptomyces sp. PU-14G was fermented in liquid A medium8−13 for both small-scale (50 mL) screening and large-scale (10 L) production of metabolites. HPLC-MS screening of the crude extract revealed three predominant metabolities that shared similar UV signatures (220 and 277 nm) and molecular weights of 513, 472, and 449 Da, among the range of microbial metabolites. Scale-up fermentation of Streptomyces sp. PU-14G, followed by solid-phase (XAD) and mycelial extraction, revealed predominant bioactivity and metabolites to reside within the XAD extract (Supporting Information, Figure S6). Subsequent purification of compounds from the XAD extract using various chromatographic techniques led to the isolation of four new amino-nucleosides: puromycins B (1; 9.8 mg), C (2; 2.53 g), D (3; 2.6 mg), and E (4; 1.8 mg), along with three other known compounds: 5′methylthioinosine (6; 28.6 mg), nocardamine (7; 91.8 mg), and ferrioxamine E (8; 15.4 mg); see Experimental Section and Supporting Information for details (Figure 1 and Supporting Information, Figures S1 and S6). The chemical structures of new metabolites were determined by cumulative 1D and 2D NMR spectroscopy, high-resolution mass spectrometry (HRMS), and chemical methods. Structure Elucidation. The physicochemical properties of compounds 1−4 are summarized in the Experimental Section and Supporting Information, Table S1. Compound 1 was isolated as a white powder using standard chromatographic techniques (Supporting Information, Figure S6). The molecular formula of 1 was deduced as C20H31N7O5 on the basis of (+)- and (−)-HRESIMS and NMR. The proton NMR data of 1 in DMSO-d6 (Table 2) revealed an array of aliphatic proton signatures and two additional aromatic proton signals at δ 8.44 (s) and 8.23 (s). In addition, two proton doublets, observed at δ 8.09 (d, J = 7.7 Hz) and 7.99 (d, J = 8.2 Hz), suggested the presence of two substituted amides (−CONH−). The 13C NMR/HSQC spectra of 1 (Table 1) displayed 20 signals corresponding to five methyl, two methylene, eight methine, and five carbon groups. In addition, 13C NMR highlighted the presence of two acid/amide carbonyls (δC 172.7 and 169.2) and a sugar anomeric carbon (δC 89.4) in 1. Two singlet proton signals [δ 8.44 (δC 137.9) and 8.23 (δC 151.7)] in conjunction with three carbon signals [δC 154.1, 149.6, and 119.6] further implicated the presence of adenine. Cumulative analyses of the 1H,1H-COSY/NOESY/HMBC spectra (Figures 1 and 2) also confirmed the presence of leucine and 3′amino-3′-deoxy-β-ribose in compound 1. Key 3J/2J HMBC correlations [anomeric proton signal (H-1′, δ 6.00) to C-4 (δ 149.6) and C-8 (δ 137.9), 3′-NH (δ 8.09) to CH-3′ (δC 50.3) and C-1″ (δC 172.7); Figure 1] confirmed the adenine N9sugar and corresponding sugar 3′-amino-leucine connectivities, the acetylation of the latter for which was also established via 2J HMBC correlations [from 2″-NH (δ 7.99) to CH-2″ (δC 51.0) and to the 2″-carbonyl (δC 169.2) and from the 2″NHCOCH3 methyl (δ 1.83, singlet) to the 2″-carbonyl signal (δC 169.2); Figure 1 and Tables 1 and 2]. An AntiBase 20172 query using the corresponding adenosyl N-9-amino-pentose 3′amino-3′-deoxy-β-ribose substructure revealed four reported bacterial metabolites: puromycin (5; produced by Streptomyces

Figure 1. 1H,1H-COSY and selected HMBC correlations of puromycins B−E (1−4) and puromycin (5, commercial standard).

Figure 2. TOCSY and selected NOESY couplings of puromycins B− E (1−4) and puromycin (5, commercial standard).

known compounds [5′-methylthioinisine (6),18 nocardamine (7),9,19,20 and ferrioxamine E (8)9,21,22] were also isolated from the same culture extract (Supporting Information, Figures S1 and S2). As the first reported naturally occurring B

DOI: 10.1021/acs.jnatprod.8b00720 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C (100 MHz) NMR Spectroscopic Data for Compounds 1−5 in DMSO-d6 (δ in ppm)a puromycin C (2)

puromycin D (3)

puromycin E (4)

puromycin (5)

position

puromycin B (1) δC, type

position

δC, type

δC, type

δC, type

δC, type

2 4 5 6 6-N(CH3)2 8 1′ 2′ 3′ 4′ 5′ 1″ 2″ 2″-NHCOCH3 2″-NHCOCH3 3″ 4″ 5″ 6″

151.7, CH 149.6, C 119.6, C 154.1, C 38.1, CH3 137.9, CH 89.4, CH 73.1, CH 50.3, CH 83.2, CH 60.8, CH2 172.7, C 51.0, CH 169.2, C 22.5, CH3 41.2, CH2 24.3, CH 23.0, CH3 21.7, CH3

2 4 5 6 6-N(CH3)2 8 1′ 2′ 3′ 4′ 5′ 1″ 2″ 2″-NHCOCH3 2″-NHCOCH3 3″ 4″ 5″/9″ 6″/8″ 7″ 7″-OCH3

151.9, CH 149.7, C 119.7, C 154.3, C 37.9, CH3 138.0, CH 89.6, CH 73.2, CH 49.9, CH 83.8, CH 61.2, CH2 173.9, C 72.5, CH

151.8, CH 149.6, C 119.6, C 154.2, C 38.0, CH3 137.9, CH 89.3, CH 73.0, CH 50.2, CH 83.2, CH 60.9, CH2 171.8, C 54.1, CH 169.0, C 22.5, CH3 37.3, CH2 129.7, C 130.2, CH 113.4, CH 157.7, C 54.9, CH3

151.9, CH 149.6, C 119.6, C 154.3, C 37.9, CH3 137.7, CH 89.4, CH 73.1, CH 50.2, CH 82.4, CH 60.6, CH2 169.7, C 22.7, CH3

149.3, CH 148.6, C 119.5, C 151.9, C ND 138.7, CH 89.5, CH 73.4, CH 50.4, CH 82.7, CH 60.2, CH2 168.4, C 53.7, CH

39.5, CH2 130.5, C 130.4, CH 113.5, CH 157.7, C 55.0, CH3

36.1, CH2 126.8, C 130.6, CH 113.9, CH 158.4, C 55.1, CH3

a

See Supporting Information for NMR spectra. ND = not detected. Assignments were supported by 2D HSQC and HMBC experiments.

Table 2. 1H NMR Spectroscopic Data for Compounds 1−5 in DMSO-d6 (δ in ppm)a puromycin B (1)b position

δH, mult (J in [Hz])

position

2 6-N(CH3)2 8 1′ 2′ 2′-OH 3′ 3′-NH 4′ 5′

2 6-N(CH3)2 8 1′ 2′ 2′-OH 3′ 3′-NH 4′ 5′

5′−OH 2″ 2″-NH 2″-NHCOCH3 3″ 4″ 5″

8.23, s 3.45, brs 8.44, s 6.00, d (2.8) 4.47, brm 5.95, brs 4.45−4.36, m 8.09, d (7.7) 4.00, brm 3.69, dd (12.2, 2.0) 3.49, dd (12.3, 3.8) 5.95, brs 4.45−4.36, m 7.99, d (8.2) 1.83, s 1.44, t (7.2) 1.57, m 0.86, d (6.5)

6″

0.83, d (6.6)

5″/9″ 6″/8″ 7″-OCH3

5′-OH 2″ 2″-OH 2″-NH 2″-NH2 2″-NHCOCH3 3″

puromycin C (2)b

puromycin D (3)b

puromycin E (4)c

puromycin (5)b

δH, mult (J in [Hz])

δH, mult (J in [Hz])

δH, mult (J in [Hz])

δH, mult (J in [Hz])

8.24, s 3.41, brs 8.46, s 6.02, brs 4.52−4.45, m 6.27, brs 4.52−4.45, m 7.74, brs 4.03, brm 3.75, d (11.1) 3.62−354, m 5.18, brs 4.13, m 5.69, d (5.3)

8.23, 3.40, 8.44, 5.97, 4.47, 6.03, 4.42, 8.16, 3.91, 3.64, 3.45, 5.18, 4.56,

8.22, 3.38, 8.46, 5.97, 4.43, 6.03, 4.43, 8.04, 4.00, 3.72, 3.52, 5.23, 1.87,

8.34, s 3.40. brs 8.60, s 6.01, d (2.3) 4.48, brm 4.53−4.13, brs 4.43, m 8.65, d (7.6) 3.89, m 3.64, d (11.7) 3.37, dd (12.4, 3.2) 4.53−4.13, brs 4.03, m

s brs s brd (1.6) brm brs m d (7.4) brm d (12.0) brm brs m

s brs s d (2.4) brm brs m d (7.5) m d (11.1) dd (12.5, 3.0) brs s

8.09, d (8.4) 8.35, brs 2.97, 2.66, 7.16, 6.83, 3.70,

d (13.3) dd (13.4, 9.1) d (7.8) d (7.8) s

1.75, 2.88, 2.68, 7.17, 6.82, 3.71,

s dd (13.5, 4.8) dd (13.3, 9.7) d (7.7) d (7.6) s

2.97, m 7.18, d (8.2) 6.90, d (8.0) 3.73, s

a

See Supporting Information for NMR spectra. Assignments were supported by 2D HSQC and HMBC experiments. b400 MHz. c500 MHz.

alboniger)23 and A201A (9), A201C (10), A201D (11), and A201E (12) (uniquely glycosylated analogues from Streptomyces capreolus; Supporting Information, Figure S3).24,25 Comparison of the 1H and 13C NMR chemical shifts of 1 to that of a commercially available puromycin (5) standard revealed a common 3′-amino-nucleoside core with structural divergence at the 3′-N-moiety (3′-N-tyrosinyl in 5 versus an Nacetyl-leucyl in 1; Tables 1 and 2). All of the remaining HMBC

correlations (Figure 1) and NMR data (Tables 1 and 2) are in full agreement with structure 1. The relative configurations of 1 stereocenters were indirectly established through NOESY correlations (Figure 2) and by comparison of the NMR shifts/ coupling constants and optical rotation to those of 5 (Experimental Section and Tables 1 and 2). The absolute configuration of the 1-appended amino acid was determined to be L-leucine by Marfey’s method26 (see Experimental Section C

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Streptomyces alboniger)23 and other closely related metabolites A201A (9), A201C (10), A201D (11), and A201E (12) (derived from Streptomyces capreolus, Supporting Information, Figure S3).24,25 Puromycin (5) is an aminoacyl-tRNA mimetic and is a potent inhibitor of translation, where the free 5-2″amine has been noted as important for bioactivity.28 To further extend puromycin structure−activity relationship studies, the determined bioactivities (antibacterial, antifungal, and cancer cell line cytotoxicity) of 1−4 and 6 were compared to that of 5 (Figure 3 and Supporting Information, Table S2, Figures S4

and Supporting Information, Figure S19). In summary, cumulative analyses of 1D (1H, 13C) and 2D (HSQC, 1 1 H, H−COSY, TOCSY, HMBC, and NOESY) NMR and chemical degradative methods established the structure of 1 (Figures 1 and 2), and, as a new puromycin analogue, 1 was subsequently named puromycin B. The physicochemical properties of compound 2 (2.53 g) were similar to those of 1 and 5. The molecular formula of compound 2 was deduced as C22H28N6O6 based on the molecular ion peak observed at m/z 473.2145 [M + H]+ (calcd 473.2143 for C22H29N6O6) in the HRESIMS spectrum. The proton NMR spectrum of 2 was nearly identical to that of puromycin (5), with the exception of a loss of the broad singlet at δ 8.35 (2″-NH2, 2H) in 5 and the presence of a new doublet at δ 5.69 (2″-OH, 1H) (Table 2) in compound 2. Likewise, the 13C NMR spectrum of 2 differed from that of 5 via an observed downfield shift in the 5 CH-2″ resonance (5, δ 53.7; 2, δ 72.5), consistent with a replacement of the 2″-NH2 of 5 with a 2″-OH in 2 (Table 1). The relative configurations of 2 stereocenters were indirectly established through NOESY correlations (Figure 2) and by comparison of the NMR shifts/ coupling constants and optical rotation to those of 5 (Tables 1 and 2). An AntiBase 20172 query revealed 2 as a new naturally occurring puromycin analogue, and, as such, 2 was subsequently named puromycin C. We note that, while the synthesis and partial spectroscopic characterization of 2 have been previously reported,27 the complete physicochemical and NMR spectroscopic data of 2 (Figures 1 and 2 and Tables 1 and 2) are reported herein for the first time. Likewise, compounds 3 and 4 were obtained as white solids and displayed similar physicochemical properties to 1, 2, and 5. The molecular formula of 3 was deduced as C24H31N7O6, consistent with a 5-acetylated variant. Consistent with this, the 1 H NMR spectrum of 3 highlighted an exchange of the 5 2″NH2 signature (δ 8.35, 2H) with a corresponding doublet at δ 8.09 (2″-NH, 1H, J = 8.4 Hz) and the presence of an additional methyl singlet at δ 1.75. Similarly, the 13C NMR spectrum of 3 displayed the requisite acetyl carbonyl (δ 169.0) and methyl (δ 22.5) carbon signals. The attachment of the acetyl moiety in compound 3 at the 2″-NH position was established by 3J HMBC cross-peaks observed from H-2″ to CO (δ 169.0) and 2J HMBC correlation from 2″-NH (δ 8.09) to C-2″ (δ 54.1) and CO (δ 169.0). In contrast, the 4 deduced molecular formula of C14H20N6O4 and corresponding 1H, 13C, and 2D NMR were consistent with replacement the 3′-Ntyrosinyl moiety in compound 5 with a 3′-N-acetyl residue in 4. All of the remaining HMBC correlations (Figure 1) and NMR data (Tables 1 and 2) were in full agreement with structures 3 and 4 (Figures 1 and 2). The relative configurations of compound 3 and 4 stereocenters were indirectly established through NOESY correlations (Figure 2) and by comparison of the NMR shifts/coupling constants and optical rotation to those of 5 (Tables 1 and 2). As additional new naturally occurring puromycin analogues, 3 and 4 were subsequently named puromycins D and E. While the syntheses of 3 and 4 have been previously reported,23,28−30 we report their full physicochemical and NMR spectroscopic data assignments herein for the first time. Discussion. Including the new naturally occurring puromycins B−E (1−4) reported herein, the 3′-amino-3′deoxy-adenosine-derived nucleosides make up nine of the 32 naturally occurring adenine-based glycosides.2,31 These include the amino-nucleoside antibiotic puromycin (5, produced by

Figure 3. Viability of A549 (non-small-cell lung) and PC3 (prostate) human cancer cell lines at 100 μM treatment with compounds 1−6 after 48 h.

and S5). Consistent with prior reports,32 these comparative assays revealed the parental puromycin prototype 5 as the only member tested to display anti-Gram-positive bacteria and antimycobacterial activity [MICs 4−30 μM (2−14 μg/mL), Supporting Information, Table S2] and also as the most cytotoxic against A549 (non-small-cell lung) and PC3 (prostate) human cancer cell lines (IC50 300 nM; Figure 3, Supporting Information, Figures S4 and S5). While 1−4 were also found to be ∼2 orders of magnitude less cytotoxic (Supporting Information, Figure S4) than 5, single-dose studies (Figure 3) suggest puromycin 3′-amino substitutions to have some influence on potency, as observed for 2 (2″hydroxy-5), 4 (3′-N-acetyl-substituted), and 3 (2″-N-acetyl-5) when compared to 5. With the exception of 5, all other puromycin-related metabolites tested [≤120 μM (62−36 μg/ mL)] lacked appreciable anti-Gram negative bacteria (Escherichia coli and Salmonella enterica) or antifungal (Saccharomyces cerevisiae) activity (Supporting Information, Table S2). Notably, 1 represents the first reported naturally occurring puromycin-related bacterial metabolite with a 3′-N-amino acid substitution that differs from the 3′-N-tyrosinyl of classical puromycin-type natural products. This discovery suggests the biosynthesis of puromycin-based metabolites in Streptomyces sp. PU-14G may invoke a uniquely permissive aminonucleoside synthetase and/or multiple synthetases. While genomic, genetic, and preliminary biosynthetic studies have identified Pur6 as the requisite tyrosinyl-aminonucleoside synthetase responsible for 3′-N-tyrosinyl conjugation en route to puromycin in S. alboniger,33−35 systematic in vitro Pur6 substrate specificity studies are lacking. Similar genomic and genetic analyses have yet to expose a pur6 homologue in S. capreolus NRRL 3817 (the producer of the A201-based metabolites that share the common 3′-N-tyrosinyl-containing puromycin core).36,37 The putative availability of new Streptomyces sp. PU-14G-based biocatalysts to enable rapid D

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England Biolabs, Ipswich, MA, USA) following manufacturer’s recommendations. The plasmids were purified using QIAprep Spin Miniprep plasmid purification kit (Qiagen) and sequenced using plasmid forward and reverse primers. Three different clones were sequenced for verification. The insert 16S rRNA sequence (1377 bp) displayed 99% identity (BLAST search) to the 16S rRNA gene sequence of Streptomyces f locculus strain NBRC 13041 and has been deposited in the NCBI nucleotide database with the accession number KY484996. Fermentation, Extraction, Isolation, and Purification. The terrestrial Streptomyces sp. PU-14G was cultivated on M2-agar plates at 28 °C for 3 days. To prepare the seed culture, small pieces of the agar with the fully grown strain were used to inoculate two 250 mL baffled flasks, each containing 50 mL of A medium [glucose (10.0 g), yeast extract (5.0 g), soluble starch (20.0 g), peptone (5.0 g), NaCl (4.0 g), K2HPO4 (0.5 g), MgSO4·7H2O (0.5 g), and CaCO3 (2 g) dissolved in 1 L of H2O (pH 7.0) and sterilized by autoclaving for 33 min at 121 °C] and grown at 28 °C with shaking (210 rpm) for 2 days. An aliquot of seed culture (1 mL) was subsequently used to inoculate 100 250 mL baffled flasks, each containing 100 mL of A medium. Fermentation was continued at 28 °C with shaking (210 rpm) for 8 days. The obtained yellowish-gray culture broth was centrifuged and filtered over Celite. The supernatant was mixed with XAD-16 (4%) resin overnight, followed by filtration. The resin was washed with water (3 × 1200 mL) and then extracted with methanol (5 × 800 mL). The methanol extract was subsequently evaporated in vacuo at 38 °C to afford 11.86 g of reddish-brown, oily crude extract. The biomass (mycelium) was extracted with methanol (4 × 800 mL) and subsequently evaporated in vacuo at 38 °C to yield 54.1 g of yellow, oily crude extract. Both extracts revealed different sets of metabolites based upon HPLC and TLC analyses wherein the targeted bioactive metabolites were found to be mostly present in the XAD extract (11.86 g). Thus, this extract was subjected to the following workup and isolation procedure. As highlighted in Figure S6, the XAD extract (11.86 g) was subjected to silica gel column chromatography (column 5 × 50 cm, 150 g) and fractionated with a gradient of CH2Cl2/0−100% CH3OH followed by HPLC and TLC analysis. This resulted in the generation of 27 fractions [1.5 L 0% CH3OH → fractions F1 (55.7 mg) and F2 (9.6 mg), 750 mL each; 1.0 L 3% CH3OH → fractions F3 (306.1 mg) and F4 (51.2 mg), 500 mL each; 1.2 L 5% CH3OH → fractions F5 (10.4 mg), F6 (67.8 mg), and F7 (171.8 mg), 400 mL each; 1.2 L 7% CH3OH → fractions F8 (1.36 g), F9 (1.1 g), and F10 (498.8 mg), 400 mL each; 1.2 L 10% CH3OH → fractions F11 (133.3 mg), F12 (91.7 mg), and F13 (53.5 mg), 400 mL each; 1.2 L 15% CH3OH → fractions F14 (48.3 mg), F15 (93.8 mg), and F16 (240.7 mg), 400 mL each; 500 mL 20% CH3OH → fractions F17 (342.8 mg) and F18 (153.6 mg), 250 mL each; 500 mL 30% CH3OH → fractions F19 (319.9 mg) and F20 (186.7 mg), 250 mL each; 500 mL 40% CH3OH → fraction F21 (603.1 mg); 500 mL 50% CH3OH → fractions F22 (286.7 mg) and F23 (219.9 mg), 250 mL each; 500 mL 60% CH3OH → fraction F24 (503.3 mg); 500 mL 80% CH3OH → fraction F25 (604.3 mg); 1.0 L 100% CH3OH → fractions F26 (1.1 g) and F27 (1.07 g), 500 mL each]. Fractions F8−10 (2.96 g) were combined based on the HPLC and TLC similarity, followed by purification using Sephadex LH-20 (MeOH, 2.5 × 50 cm) to afford puromycin C (2; 2.46 g) in pure form as a white solid. Similarly, fractions F11−13 (278.5 mg) were combined and purified using preparative TLC (CH2Cl2/5% MeOH, 2.5 × 50 cm) followed by Sephadex LH-20 (CH2Cl2/40% MeOH, 1.0 × 40 cm) and semipreparative HPLC to yield puromycins B (1; 9.8 mg), C (2; 65.8 mg), D (3; 2.6 mg), and E (4; 1.8 mg) in pure form as white solids. In the same manner, fractions F17−20 were combined based on their HPLC/MS and TLC similarities. Further purification of the combined fractions using Sephadex LH-20 (MeOH, 2.5 × 50 cm) and semipreparative HPLC afforded 5′-methylthioinosine (6; 28.6 mg, white powder), nocardamine (7; 91.8 mg, white solid), and ferrioxamine E (8; 15.4 mg, red solid) in pure forms. Remaining fractions (F1−F7, F14−F16, and F21−F27) and the mycelium extract lacked related puromycin

one-pot puromycin semisynthetic modification may facilitate ongoing efforts to generate and exploit innovative puromycinbased analogues for targeting alternative proteins (e.g., aminopeptidases),38 PET imaging,39 ribosome engineering,40 and/or real-time proteome profiling studies.41−43

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation measurements were recorded on a Jasco DIP-370 digital polarimeter (Jasco, Easton, MD, USA). UV spectra were recorded on an Ultrospec 8000 spectrometer (GE, Pittsburgh, USA). All NMR data was recorded at 500 or 400 MHz for 1H and 100 MHz for 13C with Varian Inova NMR spectrometers (Agilent, Santa Clara, CA, USA), where δ-values were referenced to respective solvent signals [DMSO-d6, δH 2.50 ppm, δC 39.51 ppm]. HPLC-MS was conducted with an Agilent 6120 Quadrupole MSD mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent 1200 Series Quaternary LC system and an Eclipse XDB-C18 column (150 × 4.6 mm, 5 μm; solvent A: H2O−0.1% formic acid, solvent B: CH3CN−0.1% formic acid; flow rate: 0.4 mL min−1; 0−4 min, 10% B; 4−22 min, 10−100% B; 22−27 min, 100% B; 27−29 min, 100−10% B; 29−35 min, 10% B). HRESIMS spectra were recorded on an AB SCIEX Triple TOF 5600 system. HPLC analyses were performed on an Agilent 1260 system equipped with a photodiode array detector and a Phenomenex C18 column (Phenomenex, Torrance, CA; 250 × 4.6 mm, 5 μm; solvent A: H2O−0.1% TFA, solvent B: CH3CN; flow rate: 1.0 mL min−1; 0−30 min, 5−100% B; 30−35 min, 100% B; 35−36 min, 100−5% B; 36−40 min, 5% B). Semipreparative HPLC was accomplished using a Phenomenex C18 column (10 × 250 mm, 5 μm) on a Varian (Palo Alto, CA, USA) ProStar model 210 equipped with a photodiode array detector and a gradient elution profile (solvent A: 0.05% TFA−H2O, solvent B: CH3CN; flow rate: 5.0 mL min−1; 0−2 min, 25% B; 2−15 min, 25−100% B; 15−17 min, 100% B; 17−18 min, 100−25% B; 18−19 min, 25% B). Silica gel (230−400 mesh) for column chromatography was purchased from Silicycle (Quebec City, Canada). Thin layer chromatography was conducted using Polygram SIL G/UV254 (Macherey-Nagel & Co., Dueren, Germany). C18-functionalized silica gel (40−63 μm) was purchased from Material Harvest Ltd. (Cambridge, UK). Amberlite XAD16N resin (20−60 mesh) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Size exclusion chromatography was performed on Sephadex LH-20 (25−100 μm; GE Healthcare, Piscataway, NJ, USA). Puromycin (5) standard was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Staphylococcus aureus, Bacillus subtilis, Salmonella enterica, Mycobacterium aurum, and Saccharomyces cerevisiae strains and PC3 and A549 cells were obtained from ATCC (Manassas, VA, USA); Micrococcus luteus and Escherichia coli were obtained from NRRL (Peoria, IL, USA). All solvents used were of ACS grade and purchased from the Pharmco-AAPER (Brookfield, CT, USA). All other reagents used were reagent grade and purchased from Sigma-Aldrich. Isolation of Streptomyces sp. PU-14G and Its Taxonomy. A soil sample containing PU-14G was collected from the Himalayan Mountain Range (Bara Gali, Pakistan; 34°6′0′ N, 73°21′0′ E; altitude 7710 ft). Streptomyces sp. PU-14G was isolated following previously reported methods.44−46 A full colony isolated on M2 agar (glucose, 4.0 g; yeast extract, 4.0 g; malt extract, 10.0 g; CaCO3, 2.0 g; agar, 18.0 g) was used to inoculate M2 broth. After 3 days of incubation at 28 °C the cell pellet was collected, and genomic DNA was prepared using an UltraClean microbial DNA isolation kit (Mo Bio Laboratories, CA, USA). The partial 16S rRNA gene fragment was amplified using universal primers (27F, 5′-AGAGTTTGATCMTGGCTCAG-3′; 1492R, 5′-GGTTACCTTGTTACGACTT3′)47 and Advantage GC2 polymerase (Clontech, Mountain View, CA, USA). QIAquick gel extraction kit (Qiagen, Valencia, CA, USA) was used to gel-purify the amplified product. The product was cloned into pGEM T-easy vector (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The corresponding ligated plasmid construct was transformed into NEB 5-alpha competent E. coli (New E

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protocols.4,5,9,14,49 Antibacterial and antifungal MIC values were obtained after 16−48 h of incubation (Supporting Information, Table S2). Kanamycin and ampicillin (S. aureus, M. luteus, B. subtilis, M. aurum, S. enterica, and E. coli), amphotericin B (S. cerevisiae), and actinomycin D (A549 and PC3) were used as positive controls (Figure 3 and Supporting Information, Table S2 and Figures S4 and S5).

analogues based on HPLC/MS and TLC analyses (Supporting Information, Figure S6). Puromycin B (1): white solid, UV absorbing (254 nm, TLC); Rf 0.41 (CH2Cl2−MeOH, 90:10); pale yellow with anisaldehyde/H2SO4 spraying reagent; [α]25D −29.0 (c 1.0, MeOH); UV/vis (MeOH) λmax (log ε) 219 (4.35), 277 (4.30) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 2; 13C NMR (DMSO-d6, 100 MHz), see Table 1; (+)-APCI-MS m/z 450 [M + H]+; (−)-APCI-MS m/z 448 [M − H]−; (+)-HRESIMS m/z 450.2460 [M + H]+ (calcd for C20H32N7O5, 450.2459); (−)-HRESIMS m/z 448.2302 [M − H]− (calcd for C20H30N7O5, 448.2313). Puromycin C (2): white solid, UV absorbing (254 nm, TLC); Rf 0.44 (CH2Cl2−MeOH, 90:10); pale yellow with anisaldehyde/H2SO4 spraying reagent; [α]25D −49.0 (c 1.0, MeOH); UV/vis (MeOH) λmax (log ε) 218 (4.28), 277 (4.16) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 2; 13C NMR (DMSO-d6, 100 MHz), see Table 1; (+)-APCI-MS m/z 473 [M + H]+; (−)-APCI-MS m/z 471 [M − H]−; (+)-HRESIMS m/z 473.2145 [M + H]+ (calcd for C22H29N6O6, 473.2143); (−)-HRESIMS m/z 471.1984 [M − H]− (calcd for C22H27N6O6, 471.1997). Puromycin D (3): white solid, UV absorbing (254 nm, TLC); Rf 0.44 (CH2Cl2−MeOH, 90:10); pale yellow with anisaldehyde/H2SO4 spraying reagent; [α]25D −45.0 (c 1.0, MeOH); UV/vis (MeOH) λmax (log ε) 219 (4.36), 277 (4.21) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 2; 13C NMR (DMSO-d6, 100 MHz), see Table 1; (+)-APCI-MS m/z 514 [M + H]+; (−)-APCI-MS m/z 512 [M − H]−; (+)-HRESIMS m/z 514.2409 [M + H]+ (calcd for C24H32N7O6, 514.2409); (−)-HRESIMS m/z 512.2252 [M − H]− (calcd for C24H30N7O6, 512.2263). Puromycin E (4): white solid, UV absorbing (254 nm, TLC); Rf 0.32 (CH2Cl2−MeOH, 90:10); pale yellow with anisaldehyde− H2SO4 spraying reagent; [α]25D −53.0 (c 1.0, MeOH); UV/vis (MeOH) λmax (log ε) 214 (4.31), 277 (4.24) nm; 1H NMR (DMSOd6, 500 MHz), see Table 2; 13C NMR (DMSO-d6, 100 MHz), see Table 1; (+)-APCI-MS m/z 337 [M + H]+; (−)-APCI-MS m/z 371 [M + Cl−]−; (+)-HRESIMS m/z 337.1620 [M + H]+ (calcd for C14H21N6O4, 337.1619); (−)-HRESIMS m/z 335.1465 [M − H]− (calcd for C14H19N6O4, 335.1473), 371.1229 [M + Cl−]− (calcd for C14H20N6ClO4, 371.1240). Determination of Amino Acid Configuration. The absolute configuration of the amino acid residue in compound 1 was determined following Marfey’s method.26 Specifically, compound 1 (1.0 mg) was hydrolyzed in 6 N HCl (1 mL) at 110 °C for 10 h under magnetic stirring (250 rpm). After drying under nitrogen, the corresponding hydrolysate was dissolved in 50 μL of H2O, to which a solution of 1% Marfey’s [Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (FDAA)] reagent in acetone (200 μL) was subsequently added, followed by 1 M NaHCO3 (50 μL). The reaction was heated to 40 °C for 1 h, cooled to room temperature, and acidified with 2 N HCl (25 μL). The reaction mixture was diluted with MeOH (0.5 mL), followed by centrifugation, and analyzed by HPLC (Phenomenex C18 column, 250 × 4.6 mm, 5 μm; solvent A: 50 mM TEAP buffer, solvent B: CH3CN; flow rate: 1.0 mL min−1; 0−55 min, 10−55% B; 55−56 min, 55−100% B; 56−60 min, 100% B; 60−61 min, 100−10% B; 61−65 min, 10% B; 430 nm). Derivatized standards were prepared from the authentic L-Leu and D-Leu (50 μL of a 50 mM stock) following an identical procedure.10,26,48 The retention times for the standard Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (FDAA; tR = 32.0 min) derivatives of authentic L-Leu and D-Leu were 36.5 and 42.3 min, respectively. HPLC analysis of the Marfey’s (FDAA) derivatives of the acid hydrolysate of 1 showed a peak corresponding to L-Leu (36.4 min) (Supporting Information, Figure S19). Antibacterial, Antifungal, and Cancer Cell Line Viability Assays. Antibacterial (Staphylococcus aureus ATCC 6538, Micrococcus luteus NRRL B-287, Bacillus subtilis ATCC 6633, Mycobacterium Aurum ATCC 23366, Escherichia coli NRRL B-3708, Salmonella enterica ATCC 10708), antifungal (Saccharomyces cerevisiae ATCC 204508), and mammalian cell line cytotoxicity [A549 (non-small-cell lung) and PC3 (prostate) human cancer cell lines] assays were accomplished in triplicate following our previously reported

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00720.

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Chemical structures of compounds 6−12, workup isolation scheme, antimicrobial activities, cytotoxicity figures, and full spectroscopic data (HPLC/UV, HPLC/ MS, HRMS, and NMR) of compounds 1−7 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Sherif I. Elshahawi: 0000-0003-1879-1552 Xiachang Wang: 0000-0002-1106-1904 Jon S. Thorson: 0000-0002-7148-0721 Notes

The authors declare the following competing financial interest(s): J.S.T. is a co-founder of Centrose (Madison, WI).

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ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant R24 OD21479 (J.S.T.), the University of Kentucky College of Pharmacy, the University of Kentucky Markey Cancer Center, and the National Center for Advancing Translational Sciences (UL1TR001998). M.A. would like to thank the Pakistan Higher Education Commission (grant IRSIP 31 BMS 21) and the University of the Punjab Department of Microbiology and Molecular Genetics for support.

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REFERENCES

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