Structural elucidation of biologically active neomycin N-octyl derivatives in a regioisomeric mixture by means of liquid chromatography/ion trap time-of-flight mass spectrometry Martin Giera * , Jon S. B. de Vlieger, Henk Lingeman, Hubertus Irth and Wilfried M. A. Niessen BioMolecular Analysis Group, Department of Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Received 30 January 2010; Revised 3 March 2010; Accepted 3 March 2010 Structural elucidation of six regioisomers of mono-N-octyl derivatized neomycin is achieved using MS n (up to n ¼ 4) on an ion trap time-of-flight (IT-TOF) instrument equipped with electrospray ionization. The mixture of six derivatized neomycin analogues was generated by reductive amination in a shotgun synthetic approach. In parallel to the liquid chromatography/mass spectrometry (LC/ MS) detection, the antibacterial activity of the neomycin regioisomers was tested by post-column addition of buffer and bacterial inocula, subsequent microfractionation of the resulting mixture, incubation, and finally a chemiluminescence-based bioactivity measurement based on the pro- duction of bacterial ATP. The MS-based high-resolution screening approach described can be applied in medicinal chemistry to help in designing and producing new antibiotic substances, which is particularly challenging due to the high functionality of most antibiotic substances, therefore requiring advanced (hyphenated) separation and detection techniques for compound mixtures. Copyright # 2010 John Wiley & Sons, Ltd. In the past few years, we have been developing hyphenated and mass spectrometry (MS)-based analytical technologies enabling high-resolution biological activity screening of (complex) mixtures and simultaneous identification of active sample constituents. 1–3 Such a hyphenated continuous-flow system usually consists of an initial chromatographic separation step, followed by the on-line addition of all necessary biochemical reagents. MS can then be used to monitor either the column effluent (analyte detection) or additionally the biochemical reaction. One of the disadvan- tages of such a continuous-flow approach is that it is only applicable to relatively fast biological reactions. Addition- ally, reaction constituents may hinder efficient MS detection. In order to keep the benefits of high-resolution screening assays, but enabling longer reaction times in the bioassay, we have recently described a microfractionation system. 4 Such an approach has the additional advantage that the bioactivity screening and the mass spectral identification step are essentially decoupled (parallel assay), which allows the use of chemicals in the bioassay which are not at all amenable to MS detection. This is especially true if the bioactivity screening would involve bacteria in order to screen for new antibacterial agents. The screening for antibacterial substances is usually carried out by incubating a small number of colony- forming units (CFU) in the presence or absence of test substances in a suitable broth medium. 5 After incubating the bacterial inocula under suitable conditions for 18 h or longer, the growth is monitored visually, or by the use of a UV spectrophotometer. The concentration of a substance which just does not lead to a detectable bacterial growth is the so- called minimal inhibitory concentration, or MIC. Another more sensitive readout possibility for MIC determinations is the detection of ATP, which is formed by metabolically active bacteria and is directly proportional to the number of bacteria found under standardized conditions. 6 In the ongoing struggle to find new antibiotics, complex mixtures like natural extracts are still the most prominent source for antibiotic substances 7,8 or lead structures which are synthetically modified. 9 The high numbers of functional groups and stereocenters of most antibiotics make it very difficult to synthetically address selected groups of an antibiotic scaffold without rebuilding the backbone with modified building blocks. These and other difficulties in producing novel antibiotic derivatives make it necessary to find new strategies where separation sciences, MS, micro- biology, and medicinal chemistry are combined in a single platform to overcome bottlenecks of today’s drug discovery processes. 10 The model compound used in this study is the aminogly- coside antibiotic neomycin, which is an RNA targeting substance. 11 Recently, it has been shown that neomycin lipid RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2010; 24: 1439–1446 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4534 *Correspondence to: M. Giera, BioMolecular Analysis Group, Department of Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. E-mail: mgiera@few.vu.nl Copyright # 2010 John Wiley & Sons, Ltd.