Structural elucidation studies of erythromycins by electrospray tandem mass spectrometry

Abstract

Erythromycin A (EryA) was studied by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for the structural elucidation of novel erythromycins developed by biological synthetic methods. Skimmer dissociation along with sequential mass spectrometry studies (up to MS5) have been employed in this study. In the low-resolution MS/MS analysis of the polyketides, there are several fragment ions that are easily assigned to various neutral losses. These have all been confirmed by accurate-mass measurements. There is also a series of peaks due to ring opening and fragmentation that can only be assigned by high-resolution MSn analysis. Further experiments were performed in deuterated media (D2O/CD3OD 50%) which, along with the high-resolution MSn of erythromycin analogues, has enabled us to identify some of the steps in the ring fragmentation, particularly the loss of the polyketide starter acid. This is an essential step for determining structural alterations in the novel polyketides, but further labelling experiments and studies on more erythromycin analogues are required before the complete fragmentation pathway can be confirmed.

Characterization of erythromycin analogs by collisional activated dissociation and infrared multiphoton dissociation in a quadrupole ion trap

“…Protonated SP1 (with a C-9 hydroxyl instead of a carbonyl) and SP4 (in which the C-9 oxygen is involved in binding to the trimethylbenzyl protecting group) exhibit virtually no water loss in either the CAD or IRMPD spectra, corroborating the C-6/C-9 oxonium formation mechanism proposed by Gates et al [4], in which the carbonyl oxygen at C-9 was involved in dehydration. The dissociation spectrum of protonated SP3 displays a small signal corresponding to water loss; the presence of a new hydroxyl group at C-3 likely accounts for this fragmentation pathway.…”

“…Based on the 18 O-labeling, it was found that the first loss of water occured from the C-9 carbonyl group, indicating that the carbonyl oxygen was protonated, not the more basic amine group in the desosamine moiety. They also confirmed via MS n experiments that the first macrolide ring cleavage involved the C-13 position and adjacent oxygen, with the net loss identified as a substituted ketone moiety [4]. Volmer and Hui incubated erythromycin in aqueous solutions at non-physiological pH [6], and subsequently used solid phase microextraction/high performance liquid chromatography/ESI-MS to determine the decomposition products of protonated erythromycin in a triple quadrupole mass spectrometer.…”

“…The clinical testing of these erythromycin analogs for bacteriostatic potential, possible human toxicity, and pharmacokinetics requires effective analytical techniques. Mass spectrometry has been utilized in several recent studies aimed at identifying the structures and fragmentation pathways of macrolide antibiotics [3][4][5][6][7][8][9]. For example, Gross and co-workers used fast atom bombardment (FAB) to create both protonated and alkali metal cationized species of erythromycin A, megalomicin A, desmycosin, megalalosamine, and tylosin, followed by CAD for structural characterization [3].…”

“…They noted that the protonated macrolides gave fragmentation patterns that lacked mid-range fragment ions, whereas the alkali metal cationized species gave a greater variety of fragment ions. Gates et al used isotopic labeling to determine the origin of the initial water loss from protonated erythromycin [4,5]. For these studies, electrospray ionization (ESI) was used to introduce samples into either a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (for accurate mass measurements), a quadrupole-time-offlight (Q-TOF) instrument, or a quadrupole ion trap for MS/MS experiments.…”

Structural elucidation studies on 14- and 16-membered macrolide aglycones by accurate-mass electrospray sequential mass spectrometry

“…We therefore also performed fragmentation analysis on acetate starter EB (Ac-EB) (Structure 3), to investigate whether fragments containing the starter acid could be pinpointed on the fragmentation pathway. Previous MS-MS studies [11,12] had achieved this using erythromycin A analogs with different starter acids incorporated.…”