Structural elucidation studies of polyketide tetrasubstituted δ-lactones by gas chromatography/tandem mass spectrometry and electrospray mass spectrometry

Weissman, Kira J.; Kearney, Gordon C.; Leadlay, Peter F.; Staunton, James

doi:10.1002/(sici)1097-0231(19991115)13:21<2103::aid-rcm760>3.0.co;2-y

Cited by 9 publications

(3 citation statements)

References 29 publications

(25 reference statements)

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“…Another possible way to generate the ion at m / z 285 is via ring opening of the lactone moiety followed by the loss of a water molecule from the ester bond (path 2, Scheme ). Such process was observed in previous studies by Weissman et al 31. They investigated the fragmentation pattern for β‐hydroxy lactones using EI and ESI ionization sources.…”

Section: Resultssupporting

confidence: 60%

Fragmentation study of simvastatin and lovastatin using electrospray ionization tandem mass spectrometry

Wang

Zhao

2001

J. Mass Spectrom.

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The fragmentation mechanism of simvastatin and lovastatin was investigated using both triple quadrupole and ion trap mass spectrometers. The elimination of the ester side-chain followed by dehydration and dissociation of the lactone moiety were observed as the main fragmentation pathways for both compounds. Another major fragmentation process was a C==C double-bond facilitated rearrangement. Our tandem mass spectrometric (MS/MS) data suggested that the beta-hydroxy group was involved in the fragmentation by interacting with the carboxyl group generated from the ring opening of the lactone. As a result, a facile neutral loss of 60 Da (CH(3)COOH or a combination of CH(2)==C==O and H(2)O) was detected. MS/MS studies of the structural analogs also provided evidence that the dehydration of the beta-hydroxy lactone generated preferentially the beta,gamma-unsaturated lactones.

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Section: Resultssupporting

confidence: 60%

Fragmentation study of simvastatin and lovastatin using electrospray ionization tandem mass spectrometry

Wang

Zhao

2001

J. Mass Spectrom.

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“…GC − MS Analysis of Triketide Lactone Diastereomers. The synthetic reference samples of each of the four triketide lactone diastereomers 4a − 4d can be readily separated and analyzed by capillary GC−MS, with sensitivity down to 1 nmol or better using chemical ionization (CI) and extracted ion monitoring (XIM) of the parent [M + H] + ion at m / z 245 for the corresponding TMS derivatives . At this scale, each enzyme incubation can be carried out in a convenient volume of 500 μL, using 10−20 nmol of [KS][AT] didomain in combination with 100−150 nmol of each of the other PKS domains.…”

Section: Discussionmentioning

confidence: 99%

Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

Castonguay

Chen

et al. 2007

J. Am. Chem. Soc.

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Abstract6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted system has been developed involving combinations of ketosynthase (KS) -acyl transferase (AT) didomains with acyl-carrier protein (ACP) and KR domains from different DEBS modules. Incubations with (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone 4 by either base-or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-mass spectrometry technique has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct comparison with synthetic standards of each of the four possible diasteromers 4a-4d. Using the [KS][AT] didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6 gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-δ-lactone (4a). The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5][ACP5] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACPbound 2-methyl-3-ketoacyl triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried only an unepimerized D-2-methyl group. The results confirm the predicted stereospecificity of the individual KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl thioester intermediate. The methodology should be applicable to the study of any combination of heterologous [KS][AT] and [KR] domains.Modular polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of polyketides metabolites that exhibit a wide range of important biological properties, including antibacterial, antifungal, anticancer, and immunosuppressive activity. 1 They catalyze repetitive Claisen-like condensations between methylmalonyl or malonyl thioester building blocks and the growing polyketide acyl thioesters. Using an assembly line of active sites, each module is responsible for one cycle of polyketide chain elongation and *To whom correspondence should be addressed. E-mail: David_Cane@brown.edu. (TE) domain, located at the C-terminus of the furthest downstream module, catalyzes release and concomitant cyclization of the mature, full-length macrocyclic polyketide. NIH Public AccessThe 6-deoxyerythronolide synthase (DEBS) from Saccharopolyspora erythraea, which is by far the most thoroughly studied modular PKS, is responsib...

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“…The major novel compound 3 (Fig. 3A) displayed chromatographic and mass spectrum (MS) fragmentation characteristics 12 appropriate for a pentaketide lactone with a hydroxy group, rather than a keto group at C3. This hydroxy group derives from the action of the KR domain in module 4 of the PKS and corresponds to the C15 position of the full spinosyn structure.…”

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confidence: 99%

Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthaseElectronic supplementary information (ESI) available: Further details of the construction of pCJR308, the fermentation of BIOT-0966 and the isolation of pentaketide lactone, 3, and figures showing the13C NMR and 1H COSY spectra of 3. See http://www.rsc.org/suppdata/ob/b3/b310740j/

Martin¹,

Timoney

Sheridan³

et al. 2003

Org. Biomol. Chem.

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A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed.

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Structural elucidation studies of polyketide tetrasubstituted δ-lactones by gas chromatography/tandem mass spectrometry and electrospray mass spectrometry

Cited by 9 publications

References 29 publications

Fragmentation study of simvastatin and lovastatin using electrospray ionization tandem mass spectrometry

Fragmentation study of simvastatin and lovastatin using electrospray ionization tandem mass spectrometry

Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

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