Profiling of Acarviostatin Family Secondary Metabolites Secreted by

Aug 23, 2008 - Tianjin 300071, People's Republic of China, College for Basic Medical Science, Tianjin Medical University,. Tianjin 300070, People's Re...
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Anal. Chem. 2008, 80, 7554–7561

Profiling of Acarviostatin Family Secondary Metabolites Secreted by Streptomyces coelicoflavus ZG0656 Using Ultraperformance Liquid Chromatography Coupled with Electrospray Ionization Mass Spectrometry Peng Geng,†,‡ Xiansheng Meng,§ Gang Bai,*,†,| and Guoan Luo| Department of Microbiology, College of Life Sciences, and College of Pharmaceutical Sciences, Nankai University, Tianjin 300071, People’s Republic of China, College for Basic Medical Science, Tianjin Medical University, Tianjin 300070, People’s Republic of China, and College of Pharmaceutical Sciences, Liaoning University of Traditional Chinese Medicine, Dalian 116600, People’s Republic of China Profiling of acarviostatin family secondary metabolites secreted by Streptomyces coelicoflavus ZG0656 was performed by means of a rapid and facile procedure using ultraperformance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC/ESIMS). The acarviostatins were separated on a C18 UPLC column with a series of acetonitrile-aqueous ammonia gradients. The target homologues were detected using the multiple reaction monitoring mode, and the chemical structures were confirmed by analyzing the diagnostic fragment ions in their MS/MS spectra. All six known reference acarviostatins (I03, II03, II13, II23, III03, IV03) were thus identified. In addition, at least 74 acarviostatin homologues, including 65 novel compounds, were characterized. Some of the features of the novel structures included having up to five acarviosine moieties, an acarviosine moiety at the reducing terminus, or an incomplete acarviosine moiety at the nonreducing terminus. This type of investigation may be useful for researchers who study secondary metabolomics in microorganisms and plants, especially those who perform metabolic profiling of aminooligosaccharides and other natural products with similar structures. Saccharide hydrolase inhibitors, such as amylase and glucodase inhibitors, are well-known as treatments and prophylactics for diabetes, hyperlipoproteinemia, hyperlipidemia, obesity, or other secondary symptoms caused by these diseases.1 In the course of previous screening for novel R-amylase inhibitors, we discovered a series of compounds secreted by Streptomyces coelicoflavus ZG0656, termed acarviostatins. This family of secondary metabolites consists of acarviosine-containing aminooligosaccharides, which show remarkable inhibitory activity against * To whom correspondence should be addressed. Tel/Fax: 86-22-23508371. E-mail: [email protected]. † College of Life Sciences, Nankai University. ‡ Tianjin Medical University. § Liaoning University of Traditional Chinese Medicine. | College of Pharmaceutical Sciences, Nankai University. (1) Si, D.; Zhong, D.; Chen, X. Anal. Chem. 2001, 73, 3808–3815.

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porcine pancreatic R-amylase.2 The chemical structures of six major acarviostatins (I03, II03, II13, II23, III03, IV03) have been described elsewhere.3,4 These mixed noncompetitive inhibitors of R-amylase have a repeating pseudotrisaccharide core formed by an acarviosine unit and a D-glucopyranose group through an R-(1f4) quinovosidic bond. Acarviosine is composed of a cyclohexitol unit (hydroxymethylconduritol residue) and a 4-amino4,6-dideoxy-D-glucopyranose unit (4-amino-4-deoxy-D-quinovopyranose residue). These aminooligosaccharides are therefore named acarviostatins followed by a Roman numeral and two numbers. Acarvios originates from the acarviosine core; the Roman numeral I, II, III, or IV represents one, two, three, or four pseudotrisaccharide residues, respectively; the middle digit represents the number of glucose units at the nonreducing end; and the last digit represents the number of glucose units at the reducing end (Table 1). Similar to other known secondary metabolites, many acarviostatin homologues are secreted by S. coelicoflavus ZG0656. Because the content and activity of different secondary metabolites often differ vastly, metabolic profiling in microbial culture at an early stage is crucial to further the drug development processes of pharmacodynamics, pharmacokinetics, and quality control. Metabolic profiling is also known as metabolomics or metabonomics. This “omics” has exploded onto the scientific scene in the past few years, and is the latest up-and-coming “omics” science.5 Ultraperformance liquid chromatography (UPLC)6,7 is a promising separation technique for metabolomics. The reduced particle size (1.4-1.7 µm) of the packing material offers increased separation due to narrower chromatographic peaks over normal particle size (3.5-5 µm) high-performance liquid chromatography (2) Bai, G.; Geng, P.; Zhang, L.; Gao, Z.; Zhang, Q. CN Patent 200710058410.1, 2007. (3) Geng, P.; Bai, G. Carbohydr. Res. 2008, 343, 470–476. (4) Geng, P.; Qiu, F.; Zhu, Y.; Bai, G. Carbohydr. Res. 2008, 343, 882–892. (5) Robertson, D. G. Toxicol. Sci. 2005, 85, 809–822. (6) Shen, Y. F.; Zhang, R.; Moore, R. J.; Kim, J.; Metz, T. O.; Hixson, K. K.; Zhao, R.; Livesay, E. A.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2005, 77, 3090–3100. (7) Wilson, I. D.; Nicholson, J. K.; Castro-Perez, J.; Granger, J. H.; Johnson, K. A.; Smith, B. W.; Plumb, R. S. J. Proteome Res. 2005, 4, 591–598. 10.1021/ac801117s CCC: $40.75  2008 American Chemical Society Published on Web 08/23/2008

Table 1. Structures and Properties of Reference Acarviostatins

name a

I03 II03b II13 II23 III03c IV03d

m

n

q

format

MW

tR (min)

[M + H]+ (m/z)

0 0 1 2 0 0

1 2 2 2 3 4

2 2 2 2 2 2

C37H63NO28 C56H94N2O40 C62H104N2O45 C68H114N2O50 C75H125N3O52 C94H156N4O64

969 1434 1596 1758 1899 2364

10.85 9.69 9.34 9.18 6.21 5.71

970 1435 1597 1759 950.5e 1183e

fragment ions from quinovosidic bond cleavages (m/z) 304(B2) 304(B2), 466(B3), 628(B4), 304(B2), 304(B2),

769(B5), 1132(Y7) 931(B6), 1132(Y7) 1093(B7), 1132(Y7) 769(B5), 1234(B8), 1132(Y7), 1597(Y10) 769(B5), 1234(B8), 850(B11)e, 1132(Y7), 1597(Y10), 1031.5(Y13)e

a Acarviostatin I series compounds were separated with 1.5:98.5 (v/v) acetonitrile-aqueous ammonia. b Acarviostatin II series compounds were separated with 3:97 (v/v) acetonitrile-aqueous ammonia. c Acarviostatin III series compounds were separated with 5:95 (v/v) acetonitrile-aqueous ammonia. d Acarviostatin IV series compounds were separated with 6.5:93.5 (v/v) acetonitrile-aqueous ammonia. e Doubly charged ions.

(HPLC), resulting in increased peak capacity, lower ion suppression, and a potentially better signal-to-noise (S/N) ratio for observed components. When analyzing complex mixtures with liquid chromatography/mass spectrometry (LC/MS), as often is the case in metabolomics investigations, use of UPLC can be advantageous over regular microbore HPLC in that more components can be detected.8 On the other hand, MS has established itself as a useful tool for metabolomics analysis for its capability to measure compounds present at very low levels and at the same time provide structural information.9,10 In addition, electrospray ionization (ESI) MS and MS/MS techniques are becoming the method of choice for analysis of oligosaccharides.4 The use of UPLC/ESI-MS for metabolomics is becoming a pretty welldeveloped area now, and lots of excellent studies have been reported in recent years.11-17 Since acarviostatins have shown acceptable separation on UPLC systems and high sensitivities in positive ESI-MS/MS analysis, we chose UPLC coupled with ESIMS/MS to screen for desired acarviostatin family secondary metabolites secreted by S. coelicoflavus ZG0656. This paper is the first to describe a rapid and sensitive UPLC/ ESI-MS/MS method based on analysis of the six known reference acarviostatins and, subsequently, introduces the secondary metabolic profiling of S. coelicoflavus ZG0656, including some novel types of acarviostatin homologues, by using this same technique. The chemical profiling results provide a blueprint of the secondary (8) Plumb, R.; Castro-Perez, J.; Granger, J.; Beattie, I.; Joncour, K.; Wright, A. Rapid Commun. Mass Spectrom. 2004, 18, 2331–2337. (9) Want, E. J.; Cravatt, B. F.; Siuzdak, G. Chembiochem 2005, 6, 1941–1951. (10) Villas-Boas, S. G.; Mas, S.; Akesson, M.; Smedsgaard, J.; Nielsen, J. Mass Spectrom. Rev. 2005, 24, 613–646. (11) Yin, P.; Zhao, X.; Li, Q.; Wang, J.; Li, J.; Xu, G. J. Proteome Res. 2006, 5, 2135–2143. (12) Plumb, R. S.; Granger, J. H.; Stumpf, C. L.; Johnson, K. A.; Smith, B. W.; Gaulitz, S.; Wilson, I. D.; Castro-Perez, J. Analyst 2005, 130, 844–849. (13) Plumb, R. S.; Johnson, K. A.; Rainville, P.; Shockcor, J. P.; Williams, R.; Granger, J. H.; Wilson, I. D. Rapid Commun. Mass Spectrom. 2006, 20, 2800–2806. (14) Gika, H. G.; Theodoridis, G. A.; Wilson, I. D. J. Chromatogr., A 2008, 1189, 314–322. (15) Nordstrom, A.; O’Maille, G.; Qin, C.; Siuzdak, G. Anal. Chem. 2006, 78, 3289–3295. (16) Patterson, A. D.; Li, H.; Eichler, G. S.; Krausz, K. W.; Weinstein, J. N.; Fornacejr, A. J.; Gonzalez, F. J.; Idle, J. R. Anal. Chem. 2008, 80, 665–674. (17) Chan, E. C.; Yap, S. L.; Lau, A. J.; Leow, P. C.; Toh, D. F.; Koh, H. L. Rapid Commun. Mass Spectrom. 2007, 21, 519–528.

metabolites that make up the acarviostatin family of aminooligosaccharides secreted by S. coelicoflavus ZG0656. This investigation on secondary metabolomics will benefit researchers who study the secondary metabolic pathways of S. coelicoflavus ZG0656 and will allow us to learn more about the producer of acarviostatins. EXPERIMENTAL SECTION Microorganism. S. coelicoflavus strain ZG0656 was collected from soil at the Nankai University campus, Tianjin, China, in 2005, and was identified by the Department of Microbiology, Nankai University. A voucher specimen (CGMCC 2097) was deposited in the China General Microbiological Culture Collection Center, Institute of Microbiology, Academia Sinica. Reference Compounds. The reference substances (acarviostatins I03, II03, II13, II23, III03, and IV03) were isolated and purified from the culture filtrate of S. coelicoflavus ZG0656. Their chemical structures have been established by chemical and spectroscopic methods including acid hydrolysis, ESI-FTMS experiments, and 2D NMR techniques (Table 1).3,4 Analytical Sample Preparation. The acarviostatin-containing complex, AIB656, was extracted from the culture filtrate of S. coelicoflavus ZG0656 as described by Geng et al.3,4 A 5-µL aliquot of AIB656 solution (∼100 µg/mL in the LC mobile phase, filtered through a 0.45-µm membrane) was directly injected into the LC/ MS system. UPLC. All separations were performed with a Waters Acquity UPLC system (Waters Co.), operated on a 50 × 2.1 mm (1.7 µm) Waters Beh C18 column (Waters Co.) for 20 min. The mobile phase was acetonitrile-1.5 mM aqueous ammonia with a flow rate of 0.2 mL/min. Using 1.5:98.5 (v/v), acarviostatin I series compounds were separated; using 3:97 (v/v), acarviostatin II series compounds were separated; using 5:95 (v/v), acarviostatin III series compounds were separated; and using 6.5:93.5 (v/v), acarviostatin IV and V series compounds were separated. Mass Spectrometry. Mass spectrometric analysis was performed on a Q-TOF premier mass spectrometer (Waters Co.) equipped with an ESI source and a mass range up to m/z 2000. The positive ion mode was employed, and the spray voltage was set at 4.5 kV. The capillary voltage was fixed at 5.0 kV, and the temperature was maintained at 220 °C. The solvent was nebulized using N2 as both the sheath gas, at a flow rate of 0.80 L min-1, Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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Figure 1. Positive ESI-MS/MS fragmentation and MS/MS spectrum of [M + H]+ of acarviostatin II23 at m/z 1759.

and the auxiliary gas, at a flow rate of 0.08 L min-1. Multistage MS experiments were performed using helium as the collision gas, and the collision energy was set at 25-40 V. RESULTS AND DISCUSSION ESI-MS Analysis of Reference Acarviostatins. Direct injection of six known acarviostatins onto the ESI source gave positive full-scan mass spectra of each. Acarviostatins I03, II03, II13, and II23 each generated a strong [M + H]+ signal at m/z 970, 1435, 1597, and 1759, respectively. However, acarviostatins III03 and IV03 showed a strong [M + 2H]2+ signal at m/z 951 and 1183, respectively. Since the ESI mode is known to produce multiply charged ions, it is reasonable that acarviostatins III03 and IV03, which contain multiple basic secondary amine groups in their chemical structures, exhibited [M + 2H]2+ signals. Furthermore, the difference of 0.5 amu between the isotopic peaks confirmed that these signals corresponded to the doubly charged ions. ESI-MS/MS Analysis on Fragmentation Patterns of Reference Acarviostatins. Both the [M + H]+ and [M + 2H]2+ signals for the six known acarviostatins were able to yield MS/MS spectra. These spectra play key roles in structure elucidation procedures. Nomenclature for the fragmentation of oligosaccharides as obtained by FAB-MS/MS has been suggested by Domon and Costello.18 This nomenclature has been adopted here, and the formation of Bi and Yj fragment ions is characteristic of glycosidic bond dissociation of protonated oligosaccharides. However, acarviostatin molecules contain cyclohexitol, a pseudosaccharide unit. The cyclohexitol-nitrogen bond is defined as a pseudoglycosidic bond, and the cleavage of the pseudoglycosidic bond also yields Bi and Yj fragment ions. All six acarviostatins yielded similar ESI-MS/MS fragmentation patterns (Figure 2B and Table 1). A typical example of structure elucidation using a MS/MS spectrum is as follows. The positive full-scan ESI-MS of acarviostatin II23 showed a strong [M + H]+ signal at m/z 1759. The ESI-MS/MS spectrum of [M + H]+ is (18) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397–409.

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shown in Figure 1. The fragment ion at m/z 1741 corresponded to the neutral loss of one water molecule. The product ions at m/z 1579 (B10), 1417 (B9), and 1255 (B8) matched with the ordinal loss of one to three glucose units attached to the reducing end. The ions at m/z 1597 (Y10) and 1435 (Y9) agreed with the ordinal loss of one and two glucose units attached to the nonreducing end. The cleavages occurred on every C-1-oxygen bond, which could differentiate a monosaccharide unit on the “left side” from one on the “right side”. The most abundant fragment ions at m/z 1132 (Y7), 1093 (B7), and 628 (B4) were produced by the cleavages of the quinovosidic bonds between the quinovopyranose unit and glucose unit, which indicated relatively weaker bonds compared to other ordinary glycosidic bonds in the molecule. The ions at m/z 1277 (Y8), 812 (Y5), and 948 (B6) resulted from the dissociations of the pseudoglycosidic bonds within the acarviosine moieties. The ions at m/z 970 (Y6) and 790 (B5) were due to the cleavage of the glycosidic bond between two pseudotrisaccharide residues. These features of the MS/MS spectrum indicated the structure to be of acarviostatin II23, as outlined in Figure 1. The results from the MS/MS spectra provided the most useful information for sequence determination of unknown acarviostatins: (1) All glycosidic bonds dissociated easily, including the pseudoglycosidic bond within the acarviosine moiety. The quinovosidic bond achieved cleavage much more readily than ordinary glucosidic bonds. As a result, the fragment ions produced by quinovosidic bond cleavages yielded the most abundant signals, giving key information in the structure determination of unknown acarviostatins. (2) Most of the abundant fragment ions originated from single bond cleavages. Product ions derived from more than one bond cleavage did exist in the MS/MS spectra for every acarviostatin, but their intensities were relatively low. (3) Each abundant ion in ESI positive-ion mode possessed at least one nitrogen-containing moiety. Since secondary amine residues exhibit fairly strong basicity, ESI positive-ion detections

Figure 2. UPLC/ESI-MS/MS traces for six reference acarviostatins I03, II03, II13, II23, III03, and IV03. (A) Chromatograms; (B) ESIMS/MS spectra of [M + H]+ or [M + 2H]2+.

are usually enhanced by the presence of amine-containing molecules or fragments. (4) The fragments with relatively high molecular weights showed a propensity for forming doubly protonated ions. However, the ones with relatively low molecular weights normally formed single protonated ions. Although each secondary amine residue could be protonated in theory, it was difficult to discover multiprotonated ions from fragments containing more than two amine residues. UPLC/ESI-MS/MS Analysis on Combined Separation and Determination of Reference Acarviostatins. When screening for desired components in the crude extract by ESI-MS/MS, hyphenation with UPLC provided not only the qualitative retention times (tR) but also the purified eluate for acquiring convincing MS/MS spectra. In pre-experiments for the separation of acarviostatins using UPLC, we found that increasing the number of acarviosine moieties resulted in a much longer retention time for an acarviostatin analogue. It was too difficult to separate all types of acarviostatin analogues using uniform conditions. As a result, we used different mobile phases, as described in the Experimental Section, to separate the various acarviostatins. Figure 2 shows the UPLC/ESI-MS/MS traces of reference acarviostatins (10 µg/ mL, 5-µL aliquots), revealing excellent chromatographic separa-

tion. The respective MS/MS spectra were similar to those obtained by direct flow injection analysis. Therefore, the present UPLC/ESI-MS/MS method provides two independent types of parameters, retention time and mass spectrometric information, for the identification of both known and novel acarviostatins in a crude extract. UPLC/ESI-MS/MS Screening for Desired Acarviostatins in AIB656. The existence of a desired acarviostatin was determined based on the tR of each chromatographic peak and the corresponding MS/MS spectra by UPLC/ESI-MS/MS analysis. The summary of fragmentation patterns for acarviostatins was used to predict the m/z values corresponding to peaks derived from quinovosidic bond cleavages. As a result, these monitored product ions became key evidence for structure confirmation. Many acarviostatins are positional isomers and have the same molecular weights and m/z values. That is to say, they have the same number of glucose units in the whole molecule, but they align differently (e.g., acarviostatins II01 and II10 in Figure 3A and B). Therefore, the selected ion monitoring chromatograms of each individual [M + H]+ or [M + 2H]2+ ion exhibited multiple ion peaks. These peaks could usually be distinguished by the multiple reaction monitoring (MRM) mode and be further evaluated by ion pattern analyses. For example, Figure 3A and B show the tR of MRM chromatograms and the corresponding MS/MS spectra for two identified components with the same molecular weight of 1110. The fragmentation profile in the MS/MS spectrum for the eluate at tR 8.27 min, especially the most abundant signals at m/z 304, 769, and 808 originating from the dissociation of quinovosidic bonds, is in accordance with the compound acarviostatin II01. Whereas the diagnostic fragment ions at m/z 466, 646, and 931 in the MS/MS spectrum for the isomer at tR 7.50 min, compared to that of acarviostatin II01, showed that one glucose unit from the reducing end had been transferred to the nonreducing end, which corresponds to acarviostatin II10. On the calculation of the content of analytes and their S/N ratios for the chromatographic peaks, the MRM technique could reach a minimum detection limit of one acarviostatin molecule at ∼50 pg/aliquots. However, to obtain a MS/MS spectrum satisfactory enough to judge structure would need at least ∼500 pg/aliquots acarviostatin. As described above, we identified 80 desired acarviostatin analogues from AIB656, the crude extract from the culture filtrate of S. coelicoflavus ZG0656. The information for these components is listed in Tables 2-4 in detail. The UPLC data indicated that the retention times of acarviostatins on the column were affected by two factors. First, an additional acarviosine moiety brought a predominantly longer retention time. Second, the effect of an additional glucose unit depended on its position: in general, one at the reducing end led to a slightly longer retention time; whereas one at the nonreducing end gave a somewhat shorter retention time. Compounds listed in Table 2 are ordinary acarviostatin analogues. They contain 1-5 pseudotrisaccharide units, 0-5 glucose units at the nonreducing end, and 0-3 glucose units at the reducing end. Among them, 43 compounds are novel oligomers and 15 compounds are known ones. This is the first report of a series of natural product compounds containing up to five acarviosine moieties. Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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Figure 3. Positive ESI-MS/MS fragmentation and MS/MS spectra of [M + H]+ of four similar acarviostatins II series compounds (separated with 3:97 (v/v) acetonitrile-aqueous ammonia on UPLC).

Two special cases should be mentioned. The first one was that some acarviostatin analogues directly ended with a quinovopyranose (4-amino-4,6-dideoxy-D-glucopyranose) unit at the reducing terminus. Using standard nomenclature, the number (-1) was given to the last digit in their names due to the absence of one glucose group in the pseudotrisaccharide core. As described above, the quinovosidic bond between the quinovopyranose unit and glucose unit easily dissociates. In fact, this is due to the weak bond between the anomeric C-1 atom and the hydroxy oxygen atom in quinovopyranose. As a result, the hydroxyl group at the reducing terminus in the compounds ending with a quinovopyranose was usually lost in the ESI-MS spectrum. This is the diagnostic characteristic of this series of acarviostatin analogues. An example is acarviosta7558

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tin II2(-1), shown in Figure 3C. Despite the fact that it has the same molecular weight of 1110 as acarviostatin II01 (Figure 3A) and II10 (Figure 3B), we only found a [M - OH]+ signal at m/z 1093 in the MS/MS spectrum. Further, the diagnostic fragment ions at m/z 466 and 628 resulted from the dissociation of the quinovosidic bond, which is in accordance with the structure of acarviostatin II2(-1). All of the 11 compounds in this series of acarviostatin analogues are novel structures and are listed in Table 3. This is the first report of a series of natural product compounds directly ending with an acarviosine moiety at the reducing terminus. The other peculiar observation was that some acarviostatin analogues ended with a quinovopyranose (4-amino-4,6-dideoxyD-glucopyranose) unit at the nonreducing terminus. Using

Table 2. Ordinary Acarviostatins Identified from the Culture of S. coelicoflavus ZG0656 by UPLC/ESI-MS/MS format

MW

tR (min)

[M + H]+ (m/z)

I01 I02f I03f I12f I13 I20f I21f I22 I23 I30 I31f I32 I33 I42 I43 I53 II00b,f II01f II02f II03f II10 II11 II12 II13f II20 II21 II22 II23f II30 II31 II32 II40 II41 III00c III01 III02 III03f III10 III11 III12 III13 III20 III21 III22 III23 III31 IV00d IV01 IV03f IV12 IV13 IV20 IV21 IV22 V02d

C25H43NO18 C31H53NO23 C37H63NO28 C37H63NO28 C43H73NO33 C31H53NO23 C37H63NO28 C43H73NO33 C49H83NO38 C37H63NO28 C43H73NO33 C49H83NO38 C55H93NO43 C55H93NO43 C61H103NO48 C67H113NO53 C38H64N2O25 C44H74N2O30 C50H84N2O35 C56H94N2O40 C44H74N2O30 C50H84N2O35 C56H94N2O40 C62H104N2O45 C50H84N2O35 C56H94N2O40 C62H104N2O45 C68H114N2O50 C56H94N2O40 C62H104N2O45 C68H114N2O50 C62H104N2O45 C68H114N2O50 C57H95N3O37 C63H105N3O42 C69H115N3O47 C75H125N3O52 C63H105N3O42 C69H115N3O47 C75H125N3O52 C81H135N3O57 C69H115N3O47 C75H125N3O52 C81H135N3O57 C87H145N3O62 C81H135N3O57 C76H126N4O49 C82H136N4O54 C94H156N4O64 C94H156N4O64 C100H166N4O69 C88H146N4O59 C94H156N4O64 C100H166N4O69 C107H177N5O71

645 807 969 969 1131 807 969 1131 1293 969 1131 1293 1455 1455 1617 1779 948 1110 1272 1434 1110 1272 1434 1596 1272 1434 1596 1758 1434 1596 1758 1596 1758 1413 1575 1737 1899 1575 1737 1899 2061 1737 1899 2061 2223 2061 1878 2040 2364 2364 2526 2202 2364 2526 2667

10.58 10.27 10.85 11.45 12.64 7.83 12.06 13.25 13.80 8.42 11.36 12.58 13.15 10.72 11.29 9.76 6.89 8.27 8.88 9.69 7.50 8.61 8.79 9.34 7.93 8.42 8.65 9.18 6.95 8.73 8.83 6.30 8.49 5.31 5.45 5.90 6.21 4.49 5.23 5.63 5.99 4.45 5.18 5.51 5.80 5.07 4.58 4.82 5.71 5.25 5.40 3.67 4.02 4.91 11.37

646 808 970 970 1132 808 970 1132 1294 970 1132 1294 1456 1456 1618 1780 949 1111 1273 1435 1111 1273 1435 1597 1273 1435 1597 1759 1435 1597 1759 1597 1759 1414 1576 1738 950.5e 1576 1738 950.5e 1031.5e 1738 950.5e 1031.5e 1112.5e 1031.5e 940e 1021e 1183e 1183e 1264e 1102e 1183e 1264e 1334.5e

V03

C113H187N5O76

2829

12.85

1415.5e

V12

C113H187N5O76

2829

10.74

1415.5e

V21

C113H187N5O76

2829

9.16

1415.5e

name a,f

fragment ions from quinovosidic bond cleavages (m/z) 304(B2) 304(B2) 304(B2) 466(B3) 466(B3) 628(B4) 628(B4) 628(B4) 628(B4) 790(B5) 790(B5) 790(B5) 790(B5) 952(B6) 952(B6) 1114(B7) 304(B2), 769(B5), 646(Y4) 304(B2), 769(B5), 808(Y5) 304(B2), 769(B5), 970(Y6) 304(B2), 769(B5), 1132(Y7) 466(B3), 931(B6), 646(Y4) 466(B3), 931(B6), 808(Y5) 466(B3), 931(B6), 970(Y6) 466(B3), 931(B6), 1132(Y7) 628(B4), 1093(B7) 646(Y4) 628(B4), 1093(B7), 808(Y5) 628(B4), 1093(B7), 970(Y6) 628(B4), 1093(B7), 1132(Y7) 790(B5), 1255(B8), 646(Y4) 790(B5), 1255(B8), 808(Y5) 790(B5), 1255(B8), 970(Y6) 952(B6), 1417(B9), 646(Y4) 952(B6), 1417(B9), 808(Y5) 304(B2), 769(B5), 1234(B8), 646(Y4), 1111(Y7) 304(B2), 769(B5), 1234(B8), 808(Y5), 1273(Y8) 304(B2), 769(B5), 1234(B8), 970(Y6), 1435(Y9) 304(B2), 769(B5), 1234(B8), 1132(Y7), 1597(Y10) 466(B3), 931(B6), 1396(B9), 646(Y4), 1111(Y7) 466(B3), 931(B6), 1396(B9), 808(Y5), 1273(Y8) 466(B3), 931(B6), 1396(B9), 970(Y6), 1435(Y9) 466(B3), 931(B6), 1396(B9), 1132(Y7), 1597(Y10) 628(B4), 1093(B7), 1558(B10), 646(Y4), 1111(Y7) 628(B4), 1093(B7), 1558(B10), 808(Y5), 1273(Y8) 628(B4), 1093(B7), 1558(B10), 970(Y6), 1435(Y9) 628(B4), 1093(B7), 1558(B10), 1132(Y7), 1597(Y10) 790(B5), 1255(B8), 1720(B11), 808(Y5), 1273(Y8) 304(B2), 769(B5), 1234(B8), 850(B11)e, 646(Y4), 1111(Y7), 1576(Y10) 304(B2), 769(B5), 1234(B8), 850(B11)e, 808(Y5), 1273(Y8), 869.5(Y11)e 304(B2), 769(B5), 1234(B8), 850(B11)e, 1132(Y7), 1597(Y10), 1031.5(Y13)e 466(B3), 931(B6), 1396(B9), 1861(B12), 970(Y6), 1435(Y9), 950.5(Y12)e 466(B3), 931(B6), 1396(B9), 1861(B12), 1132(Y7), 1597(Y10), 1031.5(Y13)e 628(B4), 1093(B7), 1558(B10), 1012(B13)e, 646(Y4), 1111(Y7), 1576(Y10) 628(B4), 1093(B7), 1558(B10), 1012(B13)e, 808(Y5), 1273(Y8), 869.5(Y11)e 628(B4), 1093(B7), 1558(B10), 1012(B13)e, 970(Y6), 1435(Y9), 950.5(Y12)e 304(B2), 769(B5), 1234(B8), 850(B11)e, 1082.5(B14)e, 970(Y6), 1435(Y9), 950.5(Y12)e, 1183(Y15)e 304(B2), 769(B5), 1234(B8), 850(B11)e, 1082.5(B14)e, 1132(Y7), 1597(Y10), 1031.5(Y13)e, 1264(Y16)e 466(B3), 931(B6), 1396(B9), 1861(B12), 1163.5(B15)e, 970(Y6), 1435(Y9), 950.5(Y12)e, 1183(Y15)e 628(B4), 1093(B7), 1558(B10), 1012(B13)e, 1244.5(B16)e, 808(Y5), 1273(Y8), 869.5(Y11)e, 1102(Y14)e

a Acarviostatin I series compounds were separated with 1.5:98.5 (v/v) acetonitrile-aqueous ammonia. b Acarviostatin II series compounds were separated with 3:97 (v/v) acetonitrile-aqueous ammonia. c Acarviostatin III series compounds were separated with 5:95 (v/v) acetonitrile-aqueous ammonia. d Acarviostatin IV and V series compounds were separated with 6.5:93.5 (v/v) acetonitrile-aqueous ammonia. e Doubly charged ions. f Known compounds.

standard nomenclature, the number (-1) was given to the first digit in their names due to the absence of one cyclohexitol (hydroxymethylconduritol) group in the pseudotrisaccharide core. An example is acarviostatin II(-1)2, which is shown in

Figure 3D. In the MS/MS spectrum, the most abundant signals at m/z 611 and 970 were due to the cleavages of quinovosidic bonds, which corresponds to the structure of acarviostatin II(-1)2. All of the 11 compounds in this series of acarviostatin Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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Table 3. Acarviostatins with an Acarviosine Moiety at the Reducing Terminus Identified from the Culture of S. coelicoflavus ZG0656 by UPLC/ESI-MS/MS name a

I2(-1) I3(-1) II0(-1)b II1(-1) II2(-1) II3(-1) II4(-1) III0(-1)c III1(-1) III2(-1) IV2(-1)d

format

MW

tR (min)

[M - OH]+ (m/z)

fragment ions from quinovosidic bond cleavages (m/z)

C25H43NO18 C31H53NO23 C32H54N2O20 C38H64N2O25 C44H74N2O30 C50H84N2O35 C56H94N2O40 C51H85N3O32 C57H95N3O37 C63H105N3O42 C82H136N4O54

645 807 786 948 1110 1272 1434 1251 1413 1575 2040

10.44 8.05 4.74 4.70 4.59 4.43 4.15 4.01 3.20 3.14 4.67

628 790 769 931 1093 1255 1417 1234 1396 1558 1012e

628(B4) 790(B5) 304(B2), 466(Y3) 466(B3,Y3) 628(B4), 466(Y3) 790(B5), 466(Y3) 952(B6), 466(Y3) 304(B2), 769(B5), 466(Y3), 931(Y6) 466(B3), 931(B6), 466(Y3), 931(Y6) 628(B4), 1093(B7), 466(Y3), 931(Y6) 628(B4), 1093(B7), 1558(B10), 466(Y3), 931(Y6), 1396(Y9)

a Acarviostatin I series compounds were separated with 1.5:98.5 (v/v) acetonitrile-aqueous ammonia. b Acarviostatin II series compounds were separated with 3:97 (v/v) acetonitrile-aqueous ammonia. c Acarviostatin III series compounds were separated with 5:95 (v/v) acetonitrile-aqueous ammonia. d Acarviostatin IV series compounds were separated with 6.5:93.5 (v/v) acetonitrile-aqueous ammonia. e Doubly charged ions.

Table 4. Acarviostatins with an Incomplete Acarviosine Moiety at the Nonreducing Terminus Identified from the Culture of S. coelicoflavus ZG0656 by UPLC/ESI-MS/MS name a

II(-1)(-1) II(-1)0 II(-1)1 II(-1)2 II(-1)3 III(-1)(-1)b III(-1)0 III(-1)1 III(-1)2 III(-1)3 IV(-1)3c

format

MW

tR (min)

[M + H]+ (m/z)

C25H44N2O16 C31H54N2O21 C37H64N2O26 C43H74N2O31 C49H84N2O36 C44H75N3O28 C50H85N3O33 C56H95N3O38 C62H105N3O43 C68H115N3O48 C87H146N4O60

628 790 952 1114 1276 1093 1255 1417 1579 1741 2206

3.11 3.72 4.00 4.65 5.13 4.05 3.48 4.94 5.09 5.32 3.81

611d 791 953 1115 1277 1076d 1256 1418 1580 1742 1104e

fragment ions from quinovosidic bond cleavages (m/z) 466(Y3) 611(B4), 611(B4), 611(B4), 611(B4), 611(B4), 611(B4), 611(B4), 611(B4), 611(B4), 611(B4),

646(Y4) 808(Y5) 970(Y6) 1132(Y7) 466(Y3), 931(Y6) 1076(B7), 646(Y4), 1111(Y7) 1076(B7), 808(Y5), 1273(Y8) 1076(B7), 970(Y6), 1435(Y9) 1076(B7), 1132(Y7), 1597(Y10) 1076(B7), 771(B10)e, 1132(Y7), 1597(Y10), 1031.5(Y13)e

a Acarviostatin II series compounds were separated with 3:97 (v/v) acetonitrile-aqueous ammonia. b Acarviostatin III series compounds were separated with 5:95 (v/v) acetonitrile-aqueous ammonia. c Acarviostatin IV series compounds were separated with 6.5:93.5 (v/v) acetonitrile-aqueous ammonia. d [M - OH]+. e Doubly charged ions.

analogues are novel structures and are listed in Table 4. This is the first report of a series of natural product compounds ending with an incomplete acarviosine moiety at the nonreducing terminus. Biosynthesis of Acarviostatins. In this section, we will mainly discuss the proposed acarviostatin biosynthetic pathway and the conceivable key enzymes, according to the above screening results and correlative reports for similar compounds. Since acarviostatins belong to the group of acarviosinecontaining aminooligosaccharides, the most important references are reports on acarbose biosynthesis. The acb-gene cluster in Actinoplanes sp. SE50/110 is putatively responsible for the entire procedure of acarbose production.19 It covers ∼35 kb of chromosomal DNA and encompasses 25 ORFs, including acbA to Z. The acarviosine moiety is assembled by a series of intracellular Acb proteins first and subsequently transferred as a whole unit. Then different maltooligosaccharides are randomly added to the acarviosine moiety via catalysis by the extracellular AcbD protein.20 Because the basic structural units of acarviostatins are identical to those of acarbose, their correlative biosynthetic pathways would be expected to be similar. However, the arrangements of the structural units in acarviostatins are quite different from those of (19) Stratmann, A.; Mahmud, T.; Lee, S.; Distler, J.; Floss, H. G.; Piepersberg, W. J. Biol. Chem. 1999, 274, 10889–10896. (20) Hemker, M.; Stratmann, A.; Goeke, K.; Schro ¨der, W.; Lenz, J.; Piepersberg, W.; Pape, H. J. Bacteriol. 2001, 183, 4484–4492.

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acarbose, which suggests their prominent contrast. In acarviostatins, the pseudotrisaccharide unit, composed of one acarviosine moiety and one glucose moiety, forms the repeating structural core. Obviously, the pseudotrisaccharide unit should act as a whole module in acarviostatin producing transfer procedures. Thus, there is most likely one enzyme responsible for these transfer reactions. This hypothetical “pseudotrisaccharide-transferase (TSTase)” might play a key role in acarviostatin biosynthesis as does the AcbD protein in acarbose production. The TSTase would be expected to have maximum affinity to trisaccharide moieties, which not only leads to the reappearing pseudotrisaccharide core in acarviostatins but also contributes to the main products of ZG0656, such as acarviostatins I03, II03, III03, and IV03, which all end with a maltotriose at the reducing terminus. On the other hand, the TSTase might have lower affinity to saccharides with more or less monosaccharides, which results in the minor products of ZG0656 as described in this article. The diverse saccharides likely originate from intracellular middle metabolites or extracellular hydrolysates from starch or amylose. CONCLUSION Extensive inspection of the positive ESI-MS/MS spectra for six reference acarviostatin family compounds resulted in the interpretation of their fragmentation patterns, which could be applied for structure determination of other acarviostatin analogues. In addition, a satisfactory UPLC separation of acarviostatins

was achieved on a C18 column with acetonitrile-aqueous ammonia as the mobile phase. Therefore, the established UPLC/ESI-MS/ MS method provided two independent types of parameters, retention time and mass spectrometric information, for the identification of both known and novel acarviostatins in a crude extract. Using MRM chromatograms and the corresponding MS/MS spectra, we identified the presence of 80 acarviostatin family analogues from the culture of S. coelicoflavus ZG0656. Among them, 65 compounds were novel oligomers, whereas 15 compounds were known. Some of the features of the novel acarviostatins included having up to five acarviosine moieties, an acarviosine moiety at the reducing terminus, or an incomplete acarviosine

moiety at the nonreducing terminus. The results of the current investigation demonstrated that the UPLC/ESI-MS/MS technique provides a rapid and sensitive approach for the profiling of acarviostatin family secondary metabolites secreted by S. coelicoflavus ZG0656. ACKNOWLEDGMENT This work was supported in part by the National Key Basic Research and Development Program of China (973 Program) (2007CB914803). Received for review June 2, 2008. Accepted July 22, 2008. AC801117S

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