Structure Elucidation and Antimalarial Activity of Apicidin F: An

Note pubs.acs.org/jnp

Structure Elucidation and Antimalarial Activity of Apicidin F: An Apicidin-like Compound Produced by Fusarium fujikuroi Katharina Walburga von Bargen,† Eva-Maria Niehaus,‡ Klaus Bergander,⊥ Reto Brun,§ Bettina Tudzynski,‡ and Hans-Ulrich Humpf*,† †

Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany Institute of Molecular Biology and Biotechnology of Fungi, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48143 Münster, Germany § Swiss Tropical and Public Health Institute, P.O. Box, CH-4002 Basel, Switzerland ⊥ Organic Chemistry Institute, Westfälische Wilhelms-Universität Münster, Corrensstraße 40, 48149 Münster, Germany ‡

S Supporting Information *

ABSTRACT: Apicidins are cyclic tetrapeptides with histone deacetylase inhibitory activity. Since their discovery in 1996 a multitude of studies concerning the activity against protozoa and certain cancer cell lines of natural and synthetic apicidin analogues have been published. Until now, the only published natural sources of apicidin are the fungus Fusarium pallidoroseum, later known as F. semitectum and two unspecified Fusarium strains. The biosynthetic origin of apicidins could be associated with a gene cluster, and a biosynthetic pathway has been proposed. Recently, our group was able to identify for the first time an apicidin-like gene cluster in F. f ujikuroi that apparently does not lead to the production of any known apicidin analogue. By overexpressing the pathway-specific transcription factor we were able to identify a new apicidin-like compound. The present study provides the complete structure elucidation of the new compound, named apicidin F. Activity evaluation against Plasmodium falciparum showed good in vitro activity with an IC50 value of 0.67 μM.

A

1-methylpyrrolidine. Furthermore, exchange of tryptophan with other aromatic or heteroaromatic substituents resulted in a decrease or loss of HDAC inhibition10 except for naphthylalanine, which appeared to show higher inhibition.12 These studies suggest that differences in the amino acid substituent of apicidin are of great interest, as they might lead to increased HDAC-inhibitory activity or selectivity and therefore to more potent pharmaceutical agents. Besides the activity against several protozoa,1 apicidin was shown to inhibit cell proliferation in several human cancer cell lines. For instance, it induces apoptosis in HL-60- or Bcr-Ablpositive leukemic cells through the activation of caspase-3 and -9 cascades13,14 or in Neuro-2a neuroblastoma cells due to endoplasmatic reticulum stress and mitochondrial dysfunction15 and also suppresses the growth of human breast and ovarian cancer cells.16,17 Apicidins are thus a quite promising group of pharmaceutically active compounds. To our knowledge, the production of apicidins was only described in F. pallidoroseum, which is synonymic to F. semitectum and in the two unspecified strains Fusarium sp. KCTC16676 and KCTC16677.18,19 Recently, Jin et al. identified the apicidin gene cluster in F. semitectum and proposed a biosynthetic pathway. Based on modifications of the gene cluster, new analogues were identified.20 Recent genome sequencing of the rice pathogen F. f ujikuroi revealed a similar gene cluster that is not present in any other so far sequenced Fusarium species. Overexpression of the pathway-specific

picidin is a nonribosomal cyclic tetrapeptide that consists of the four amino acids N-methoxy-L-tryptophan, L-isoleucine, D-pipecolic acid, and L-2-amino-8-oxodecanoic acid (Aoda) (Figure 1A). It was first isolated in 1996 from Fusarium pallidoroseum (later known as F. semitectum) as a new agent showing histone deacetylase (HDAC)-inhibiting activity in apicomplexan parasites such as Plasmodium berghei.1,2 Apicidin is structurally related to other known cyclic tetrapeptidic HDAC inhibitors such as chlamydocin3 and HC-toxin.4 However in contrast to apicidin, both latter substances have an α-ketoepoxide moiety in their Aoda side chain. Removal of this epoxide from HC-toxin results in a loss of the biological activity.5 Thus, the HDAC-inhibiting activity of apicidin with the missing epoxide is not easily explained.5,6 Structure−activity relationship studies were performed to identify a more active derivative.6−12 Several synthetic and naturally occurring apicidins were evaluated for their potential to inhibit HDACs derived from human HeLa cells, Eimeria tenella, and Plasmodium falciparum. The cyclic structure of apicidin, the C8-keto group of the Aoda substituent, and the tryptophan substituent appeared to be essential for the activity.6,9,10,12 Variations in the Aoda and tryptophan moieties resulted in distinct differences in the HDAC-inhibiting activity. For example, an additional epoxide in the Aoda chain (comparable to HC-toxin or chlamydocin) increased the inhibition of HDAC from HeLa cells about 10-fold compared to apicidin,9 whereas reduction of the keto group resulted in a loss of activity.6 An approximate 5-fold increase was observed for a substance where the N-methoxy group attached to the indole ring was replaced by © 2013 American Chemical Society and American Society of Pharmacognosy

Received: July 25, 2013 Published: November 6, 2013 2136

dx.doi.org/10.1021/np4006053 | J. Nat. Prod. 2013, 76, 2136−2140

Journal of Natural Products

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Figure 1. (A) Structures of the known natural apicidin analogues.6,20,23 (B) Structure of the new apicidin F.

transcription factor-encoding gene led to increased accumulation of a metabolite with the molecular formula C35H43N5O7, which is similar to that of apicidin (C34H49N5O6).21 The differing molecular masses between apicidin and the new compound suggested a different amino acid composition. The current work describes the complete structure elucidation and first results for antimalarial activity of this new apicidin-like metabolite. The compound was named apicidin F and is the eighth naturally occurring apicidin-like substance (Figure 1). For detailed structure elucidation apicidin F was isolated and analyzed by NMR and MS. The NMR data confirmed the presence of the three amino acids N-methoxytryptophan, pipecolic acid, and phenylalanine that were proposed after acid hydrolysis.21 The fourth amino acid was identified as 2-aminooctanedioic acid based on the NMR data (Table 1). Due to the overlap of the alkyl signals, HRMS/MS was used to completely confirm the structure of the fourth amino acid. Commercially available 2-aminooctanedioic acid was used as a reference compound, and the fragmentation pattern and retention time were compared with those of the acid hydrolysate of apicidin F (see Figure S1 in the Supporting Information). Thus, by a combination of NMR and MS measurements it was possible to fully discover the amino acid composition of the isolated compound. For complete structure elucidation two questions remained to be answered: What is the configuration of the amino acids, and what is their sequence in the peptide? In a study by Singh et al. a combined strategy of NMR analysis and peptide hydrolysis followed by amino acid derivatization was used for the structure elucidation of apicidins.2 Based on these data, the following strategy for the structure elucidation was applied. Apicidin F was hydrolyzed in 6 M hydrochloric acid with the addition of 5% thioglycolic acid in order to obtain all amino acids, including tryptophan that derives from N-methoxytryptophan. Additionally, apicidin was hydrolyzed to prove the applicability of the method. The peptide hydrolysates were derivatized with the chiral Marfey’s reagent to obtain diastereomers of the amino acids that can be separated on a C8 column. For comparison, available standards of the D- and L-configured amino acids were also derivatized. With this method, the stereochemistry of isoleucine, N-methoxytryptophan, and pipecolic acid of the apicidin standard could be confirmed, demonstrating that this method is valid for stereochemistry determination of apicidin F. Comparison of the retention times revealed that apicidin F consists of L-tryptophan (like apicidin), D-pipecolic acid (like apicidin), L-phenylalanine, and L-2-aminooctanedioic acid (see Figure S2 in the Supporting Information). The last question concerning the sequence of the amino acids could unfortunately not be elucidated using NMR analysis as described for the elucidation of other apicidins,2,6 as in a ROESY experiment measured in pyridine no CH/NH ROE signals of the different amino acids could be observed. The use of deuterated dichloromethane for those experiments as also described for

Table 1. NMR Spectroscopic Data (600 MHz, CD3OD and C5D5N) for Apicidin F CD3OD position

δC, type

C5D5N δH (J in Hz)

δC, type

δH (J in Hz)

N-Methoxytryptophan NH 1 2 3

10.00, d (5.0) 175.4, C 59.4, CH 26.6, CH2

4.32, t (7.3) 3.25, d (8.0) 3.28, d (8.7)

4 107.7, C 4a 124.9, C 5 119.7, CH 7.60, d (8.0) 6 120.8, CH 7.10, t (7.5) 7 123.6, CH 7.22, d (7.7) 8 109.3, CH 7.40, d (8.2) 8a 133.8, C 10 123.0, CH 7.06, s OCH3 66.2, CH3 4.03, s 2-Aminooctanedioic Acid NH 1 175.8, C 2 56.4, CH 4.37−4.33, m 3 30.6, CH2 1.55−1.52, m 1.75−1.70, m 4 26.8, CH2 1.67−1.62, m 5 29.7, CH2 1.39−1.31, m 6 25.8, CH2 1.60−1.56, m 7 34.8, CH2 2.28, t (7.4) 8 177.6, C Pipecolic Acid 1 173.4, C 2 52.1, CH 5.20, d (5.9) 3 25.2, CH2 2.00, d (14.8) 1.47−1.40, m 4 20.6, CH2 2.09−2.03, m 1.52−1.48, m 5 26.3, CH2 1.39−1.31, m 1.18−1.15, m 6 45.1, CH2 2.97, td (13.7, 2.5) 3.84, d (13.4) Phenylalanine NH 1 174.9, C 2 51.4, CH 5.30, t (7.7) 3 37.8, CH2 3.08, dd (13.6, 7.5) 3.24−3.21, m 4 138.4, C 5/9 130.3, CH 7.32−7.27, m 6/8 129.5, CH 7.32−7.27, m 7 127.8, CH 7.27−7.22, m 2137

174.1 61.8 26.1 108.5 124.4 119.7 120.5 123.6 109.2 133.2 123.2 66.0

4.51−4.56, m 3.80−3.72, m 4.21−4.13, m

7.74, d (8.0) 7.18, d (6.8) 7.37−7.30, m 7.54, d (8.3) 7.50−7.42, m 3.93, s 7.34, d (7.8)

174.5 55.3 30.6 26.5 29.4 25.6 34.6 177.0 172.3 51.4 24.8 20.2 25.9 44.6

4.78, dd (16.9, 8.0) 2.02−1.98, m 1.68−1.60, m 1.51−1.40, m 1.29−1.24, m 1.72−1.66 2.40, t (7.4)

5.47, d (5.8) 2.04−2.00, m 1.35−1.31, m 2.35−2.24, m 1.35−1.31, m 1.29−1.24, m 1.51−1.40 3.26, t (13.5) 4.32, d (13.3) 8.56, d (10.1)

173.3 50.8 37.8 138.7 130.1 129.1 127.2

5.85, dd (17.9, 8.0) 3.55, dd (14.0, 7.4) 3.40, dd (13.8, 7.4) 7.50−7.42, m 7.37−7.30, m 7.28−7.25, m



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Figure 2. (A) Tri- and dipeptidic compounds resulting from partial hydrolysis of apicidin F analyzed by HPLC-HRMS. Shown is the TIC from m/z 50 to 700. (B) Possible sequences of apicidin F compared to di- and tripeptides produced by hydrolysis. For the fragmentation of the potential tripeptides 3−6, see Figure 3. (C) Two possible structures of apicidin F.

apicidins2,22 was not applicable either, as apicidin F could not be dissolved in dichloromethane. In a structural study of apicidins, Kranz et al. described conformational differences of apicidin in different solvents and described only weak αCH/NH ROE signals,22 which might explain the missing ROE signals of apicidin F. To elucidate the sequence of the amino acids despite the insufficient NMR data, apicidin F was treated using short-time hydrolysis to generate linear di- and tripeptides, which were analyzed by HRMS (Figure 2). The tripeptide [M + H]+ ions with m/z 503.2484, 539.2515, and 448.2452 gave no hint of the possible sequence, as they were in accordance with all of them (Figure 2B), but the resulting dipeptide [M + H]+ ions excluded four of the six theoretical possibilities for the amino acid sequence (Figure 2B). The ion at m/z 301.1747 results from pipecolic acid linked to 2-aminooctanedioic acid, m/z 392.1826 is in accordance with 2-aminooctanedioic acid linked to N-hydroxytryptophan (the methyl group was cleaved), m/z 368.1605 fits N-hydroxytryptophan connected with phenylalanine, and m/z 277.1552 corresponds to pipecolic acid linked to phenylalanine (Figure 2B). On the basis of these data, two of the six hypothetical structures of apicidin F (1) and 2 remain possible (Figure 2C). Additionally, CID fragmentation of the generated di- and tripeptides was performed, and the fragment ions were compared to the expected theoretical fragmentation of the two hypothetical structures. As expected, we observed backbone fragmentation of the peptides. By these fragmentation experiments it was possible to confirm one of the two remaining hypothetical structures (1). Two tripeptides with m/z 503.25 and 448.25 derived from the hypothetical structures 1 and 2, respectively, with their fragmentation pattern are shown in Figure 3A and B, 3−6. The fragment ions m/z 392.1815 and 221.0923 are y2 and y1 fragments resulting of structure 3; the b2 and a2 fragments with m/z 283.1654 and 255.1705 can also be found with high intensities. The fragment m/z 175.0866 might be the a1 fragment of structure 4 but can also be formed from structure 3. The fragmentation of m/z 448.2452 shows one main fragment of m/z 259.1443, which fits the b2 fragment of structure 5. The a2 and y2 fragments with m/z 231.1494 and 301.1761 can also be found, in

contrast to possible fragments of structure 6, which do not appear. The data clearly show that the tripeptides 3 and 5 (Figure 3) derived from 1 (Figure 2C) are the only structures for which the corresponding backbone fragments can be found. This confirms the structure of apicidin F as depicted in Figure 1B. It has an Lphenylalanine moiety instead of L-isoleucine and L-2-aminooctanedioic acid instead of Aoda incorporated into the tetrapeptide. Neither amino acid has been reported in naturally occurring apicidins (Figure 1A). For a first characterization of the biological activity of apicidin F, a P. falciparum assay was performed. The determined IC50 value of 0.67 μM (average of two replicates) is about 3-fold higher compared to the IC50 value of apicidin, which is about 0.2 μM.1 Additionally to the antimalarial effects, structure−activity relationship studies of apicidin-derived compounds already showed that differences in the structure might result in increased HDAC inhibitory effects or selectivity especially in HeLa cells or E. tenella.6,10,12,23 Therefore, future activity tests are useful to characterize the activity spectrum of apicidin F, as it already showed promising first results in inhibiting P. falciparum.

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EXPERIMENTAL SECTION

General Experimental Procedures. The 1H, 13C, and 2D NMR spectra were recorded on a 600 MHz NMR spectrometer (Varian, Palo Alto, CA, USA). The signals are reported in parts per million and are referenced to the solvent CD3OD or C5D5N. For structure elucidation, additionally to the 1D NMR experiments, 2D NMR experiments such as H,H-correlated spectroscopy (H,H-COSY), heteronuclear multiplequantum correlation (HMQC), heteronuclear multiple bond correlation (HMBC), and rotating Overhauser enhancement spectroscopy (ROESY) were performed. The pulse programs were taken from the software library. HPLC-HRMS was carried out on a HPLC system (Accela LC with Accela pump 60057-60010 and Accela autosampler 60057-60020, Thermo Scientific, Dreieich, Germany) coupled to a Fourier transform mass spectrometer with a heated ESI source (LTQ Orbitrap XL, Thermo Scientific, Dreieich, Germany). Chemicals. All solvents and reagents were purchased from SigmaAldrich (Deisenhofen, Germany), VWR (Darmstadt, Germany), or Merck Schuchardt (Hohenbrunn, Germany) in gradient or analytical 2138



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Figure 3. (A) MS2 fragmentation (CID 35.0%) of the tripeptide with m/z 503.25 compared to the backbone fragmentation of possible tripeptide structures. (B) MS2 fragmentation (CID 35.0%) of the tripeptide with m/z 448.25 compared to the backbone fragmentation of possible tripeptide structures. 2 μg of the peptide (apicidin or apicidin F) was hydrolyzed with 50 μL of the degassed acid mixture in a deactivated brown autosampler glass vial that was flushed with nitrogen for 10 min and closed at 110 °C for 8 h. Afterward, the hydrolysate was evaporated to dryness at 110 °C under a stream of nitrogen. During this procedure, the N-methoxy group was cleaved, but the remaining tryptophan was stable and could be used for the derivatization. The dried residue was dissolved in 50 μL of water. HPLC-HRMS of the Apicidin F Hydrolysate. The analysis was performed as previously described.21 Additionally, the 2-aminooctanedioic acid was analyzed using the same method, and MS2 spectra were generated using collision-induced dissociation (35%). Partial Hydrolysis of Apicidin F. A 5 μg amount of apicidin F was hydrolyzed with 50 μL of 6 M HCl in a closed autosampler glass, which was flooded with nitrogen for 30 min at 100 °C. The HCl was removed under a stream of nitrogen at 100 °C. The hydrolysate was dissolved in 50 μL of water and used for HPLC-HRMS analysis. HPLC-HRMS of the Partial Hydrolyzed Apicidin F. The data were acquired in the scan mode from m/z 50 to 700 with ionization in the positive ionization mode. The used parameters were as follows: capillary temperature 275 °C, vaporizer temperature 350 °C, sheath gas flow 40 units, auxiliary gas flow 20 units, source voltage 3.5 kV, tube lens 119 V. The software Xcalibur 2.07 SP1 (Thermo Scientific, Dreieich, Germany) was used for data acquisition and analysis. MS2 spectra were generated using collision-induced dissociation (35%). The HPLC column used was a 150 mm × 2.00 mm i.d., 5 μm, Gemini C18 with a 4 mm × 2 mm Gemini NX C18 guard column (Phenomenex, Aschaffenburg, Germany). Solvent A was 1% formic acid in methanol (v/v); solvent B, 1% formic acid in water (v/v). The gradient was at 40 °C from 5% A to 100% A in 35 min, followed by column flushing at 100% A for 5 min and equilibration to the starting conditions for 5 min. The flow rate was 250 μL/min, and the injection volume was 10 μL. Marfey’s Derivatization. The derivatization of the amino acids with Marfey’s reagent was carried out as similarly described in Jamindar and Gutheil.29 The reference amino acids described under reagents were derivatized separately and desalted on a Strata-X 33 μm polymeric reversed phase (30 mg/1 mL) (Phenomenex, Aschaffenburg, Germany) following the described procedure. A 15 μL portion of the hydrolysate of apicidin and apicidin F was used for Marfey’s derivatization. For quenching 5−10 μL of 1 M HCl was added until the color turned from red to yellow. The complete derivatization solution was used for the desalting. After the desalting step all eluates were dried under a stream of

grade. Water for extraction and chromatography was purified with a Milli-Q Gradient A10 system (Millipore, Schwalbach, Germany). Apicidin (AppliChem, Darmstadt, Germany), (S)-2-(Boc-amino)octanedioic acid, (R)-2-(Boc-amino)octanedioic acid, (R)-N-Fmocpiperidine-2-carboxylic acid, L-isoleucine, DL-isoleucine, L-phenylalanine, DL-phenylalanine, L-tryptophan, DL-tryptophan (Sigma-Aldrich, Deisenhofen, Germany), and L-pipecolic acid (Bachem, Bubendorf, Switzerland) were used as reference materials for analyses. Fungal Material. The fungus Fusarium f ujikuroi IMI58289 (Commonwealth Mycological Institute, Kew, UK) was used for the isolation. Growth conditions under laboratory conditions were as described in a previous publication: preculture occurred for 3 days in a 300 mL Erlenmeyer flask with 100 mL of Darken medium; then 500 μL of the preculture was used for inoculation of 100 mL of ICI medium containing 60 mM glutamine as nitrogen source (growth for 6 days). Both were incubated on a rotary shaker at 180 rpm at 28 °C in the dark.21 Deactivation of Autosampler Vials. The deactivation was carried out as previously described by Kleigrewe et al.24 Isolation. The isolation of apicidin F was carried out as described in a previous publication.21 Deprotection of Fmoc- or BOC-Protected Amino Acids. A 5 mg amount of (S)- and (R)-2-(Boc-amino)octanedioic acid were deprotected as described by Han et al.,25 then dried at 100 °C under a stream of nitrogen, dissolved in water, and directly used for further analysis. An 80 mg sample of (R)-N-Fmoc-piperidine-2-carboxylic acid was dissolved in 50% morpholine in dichloromethane (v/v) and stored at room temperature for 3 h based on the method by Fields.26 The solvent was removed at 40 °C under a stream of nitrogen, and the residue dissolved in 3 mL of 0.1 M HCl. The cleaved amino acid was purified on Strata-X-C 33 μm Polymeric Strong Cation 200 mg/3 mL cartridges (Phenomenex, Aschaffenburg, Germany) using a protocol based on Fábián et al.27 The column was first activated with 2 mL of MeOH and 2 mL of H2O; then the sample was applied and washed with 2 mL of H2O. The amino acid was eluted with 2 mL of 7 N NH3 and afterward dried at 40 °C under a stream of nitrogen, dissolved in H2O, and used for Marfey’s derivatization. Total Hydrolysis of Apicidin F. To preserve the N-methoxytryptophan from degradation during hydrolysis, a method by Gehrke and Takeda28 that was used already for other apicidins was modified to the present laboratory equipment. A 6 M HCl solution containing 5% thioglycolic acid (v/v) was degassed with nitrogen for 10 min. About 2139



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nitrogen at 40 °C and dissolved in 20% acetonitrile in water with 0.1% formic acid (v/v/v) to end up with a 10-fold concentration. HPLC-HRMS of Derivatized Amino Acids. The data were acquired in the scan mode at m/z 100−700 in the positive ionization mode. The used parameters were the same as described above. The HPLC column used was a 150 mm × 2.00 mm i.d., 3 μm, Hyperclone BDS C8 with a guard column of the same material (Phenomenex, Aschaffenburg, Germany). Solvent A was 0.1% formic acid in acetonitrile (v/v); solvent B was 0.1% formic acid (v/v). The gradient at 40 °C was 25% A for 2 min followed by a gradient to 45% A in 12 min. After holding 45% A for 1 min, the column was equilibrated to the starting conditions for 10 min. The flow rate was 200 μL/min, and the injection volume was set to 10 μL. Plasmodium falciparum Assay. The in vitro activity against P. falciparum NF54 strain (sensitive to all known drugs) was performed as described earlier.30

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Crumley, T. M.; Cannova, C.; Schmatz, D. M.; Wyvratt, M. J.; Fisher, M. H.; Meinke, P. T. Bioorg. Med. Chem. Lett. 2001, 11, 113−117. (11) Meinke, P. T.; Colletti, S. L.; Ayer, M. B.; Darkin-Rattray, S. J.; Myers, R. W.; Schmatz, D. M.; Wyvratt, M. J.; Fisher, M. H. Tetrahedron Lett. 2000, 41, 7831−7835. (12) Olsen, C. A.; Ghadiri, M. R. J. Med. Chem. 2009, 52, 7836−7846. (13) Kwon, S. H.; Ahn, S. H.; Kim, Y. K.; Bae, G.-U.; Yoon, J. W.; Hong, S.; Lee, H. Y.; Lee, Y.-W.; Lee, H.-W.; Han, J.-W. J. Biol. Chem. 2002, 277, 2073−2080. (14) Cheong, J.-W.; Chong, S. Y.; Kim, J. Y.; Eom, J. I.; Jeung, H. K.; Maeng, H. Y.; Lee, S. T.; Min, Y. H. Clin. Cancer Res. 2003, 9, 5018− 5027. (15) Choi, J. H.; Lee, J. Y.; Choi, A. Y.; Hwang, K. Y.; Choe, W.; Yoon, K.-S.; Ha, J.; Yeo, E. J.; Kang, I. Apoptosis 2012, 17, 1340−1358. (16) Ueda, T.; Takai, N.; Nishida, M.; Nasu, K.; Narahara, H. Int. J. Mol. Med. 2007, 19, 301−308. (17) Im, J. Y.; Park, H.; Kang, K. W.; Choi, W. S.; Kim, H. S. Chem.-Biol. Interact. 2008, 172, 235−244. (18) Jin, J.; Baek, S.-R.; Lee, K.-R.; Lee, J.; Yun, S.-H.; Kang, S.; Lee, Y.W. Plant. Pathol. J. 2008, 24, 417−422. (19) Park, J.-S; Lee, K.-R.; Kim, J.-C.; Lim, S.-H.; Seo, J.-A.; Lee, Y.-W. Appl. Environ. Microbiol. 1999, 65, 126. (20) Jin, J.-M.; Lee, S.; Lee, J.; Baek, S.-R.; Kim, J.-C.; Yun, S.-H.; Park, S.-Y.; Kang, S.; Lee, Y.-W. Mol. Microbiol. 2010, 76, 456−466. (21) Wiemann, P.; Sieber, C. M. K.; von Bargen, K. W.; Studt, L.; Niehaus, E.-M.; Espino, J. J.; Huß, K.; Michielse, C. B.; Albermann, S.; Wagner, D.; Bergner, S. V.; Connolly, L. R.; Fischer, A.; Reuter, G.; Kleigrewe, K.; Bald, T.; Wingfield, B. D.; Ophir, R.; Freeman, S.; Hippler, M.; Smith, K. M.; Brown, D. W.; Proctor, R. H.; Münsterkötter, M.; Freitag, M.; Humpf, H.-U.; Güldener, U.; Tudzynski, B. PLoS Pathog. 9 (6): e1003475, doi 10.1371/journal.ppat.1003475. (22) Kranz, M.; Murray, P. J.; Taylor, S.; Upton, R. J.; Clegg, W.; Elsegood, M. R. J. J. Pept. Sci. 2006, 12, 383−388. (23) Singh, S. B.; Zink, D. L.; Liesch, J. M.; Dombrowski, A. W.; Darkin-Rattray, S. J.; Schmatz, D. M.; Goetz, M. A. Org. Lett. 2001, 3, 2815−2818. (24) Kleigrewe, K.; Aydin, F.; Hogrefe, K.; Piecuch, P.; Bergander, K.; Würthwein, E. U.; Humpf, H.-U. J. Agric. Food Chem. 2012, 60, 5497− 5505. (25) Han, G.; Tamaki, M.; Hruby, V. J. J. Pept. Res. 2001, 58, 338−341. (26) Fields, G. B. Methods Mol. Biol. 1994, 35, 17−27. (27) Fábián, V.; Morvai, M.; Pintér-Szakács, M.; Molnár-Perl, I. J. Chromatogr. A 1991, 553, 87−92. (28) Gehrke, C. W.; Takeda, H. J. Chromatogr. 1973, 76, 77−89. (29) Jamindar, D.; Gutheil, W. G. Anal. Biochem. 2010, 396, 1−7. (30) Orhan, I.; Şener, B.; Kaiser, M.; Brun, R.; Tasdemir, D. Mar. Drugs 2010, 8, 47−58.

ASSOCIATED CONTENT

S Supporting Information *

Identification of 2-aminooctanedioic acid by HPLC-HRMS/MS, results of the Marfey’s derivatization, and NMR spectra of apicidin F. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 251 8333391. Fax: +49 251 8333396. E-mail: humpf@ wwu.de. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was funded by the Deutsche Forschungsgemeinschaft (DFG) HU 730/9-1 and TU 101/16-2. REFERENCES

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