5.2 Preliminary MS Experiments
Durham University Chemistry Department operates an analytical mass spectrometry service, utilising a QToF Premier (Waters Corp., UK) with ESI source to obtain routine accurate mass data for samples. In the case of complex samples, the instrument can be utilised in combination with an Acquity UPLC (Waters Corp., UK) running a seven minute linear gradient of either H2O:MeCN or H2O:MeOH. Initially, it was considered that this validated accurate mass system of moderate sensitivity may be suitable for the study of small molecule intrinsic lipidation, focussing on direct observation of acylated small molecules.
In order to test the instrumentation system described, several test samples were required.
Given the complexity of natural biological cell membranes, a model system was deemed more appropriate for initial experiments in order to simplify the resulting data. Use of a model eliminates factors which may cause confusion during data interpretation, including the presence of enzymes, the variety of acyl chain types present within the membrane, and the potential loss of material through solvent extraction steps. Liposomes, Fig. 5.1, also known as large unilamellar vesicles (LUV) were selected for study due to their robustness, simple preparation, the abundance of available research, and their widely accepted similarity to natural membranes.241 Additionally, since liposomes are single bilayer structures prepared from solution phase phospholipids, their lipid composition can be tuned as desired. In order to assimilate biological conditions, liposomes utilised within this research were prepared via the extrusion method at a diameter of 100 nm and in 10 mm ammonium bicarbonate buffer at pH 7.4.
Figure 5.1 A liposome formed of a bilayer of phospholipids. Head groups are shown in blue and acyl chains in green.
Liposomes were prepared at a known concentration containing, for simplicity, only one type of phospholipid, DOPC. DOPC contains oleoyl (18:1) acyl chains at both the sn-1 and the sn-2 position of the phospholipid backbone. These chains, highlighted purple in Fig. 5.2, are one of the most common acyl chains known to be present in natural cell membranes. In addition,
Chapter 5. Small Molecule Intrinsic Lipidation 99
DOPC contains the PC phosphate head group, highlighted blue in Fig. 5.2, the primary phospholipid head group present in eukaryotic membranes. A eukaryotic model membrane system is deemed appropriate due to the therapeutic relevance of small molecule intrinsic lipidation.
Me3N
OOP O O
O O
O O
(CH2)7CH3 (CH2)7CH3
Figure 5.2 Chemical structure of the membrane phospholipid DOPC. Key structural features highlighted: (i) oleoyl chains in purple; (ii) glycerol backbone in green; (iii) phosphate in red; (iv) choline head group in blue.
In order to accurately mimic a natural biological system, the ideal small molecule:lipid molar ratio utilised within this research would be in the range of 1:10000-100000. Unfortunately, samples containing lipid in this excess would require significant dilution in preparation for analysis by mass spectrometry to prevent overloading the chromatography column. This required dilution would therefore reduce the concentration of the modified small molecule injected into the instrument to below the limit of detection. Since visualisation of modified small molecule is key to proving the existence of small molecule intrinsic lipidation, a 1:10 small molecule:lipid molar ratio was utilised. Although the proportion of small molecule is high, this ratio provides the best balance between upper concentration capacity of the instrument and the limit of detection.
Fig. 5.3 details the small molecules selected for the initial study of small molecule intrinsic lipidation. Six cationic amphiphilic small molecules were selected, each containing an amine nucleophile in order to facilitate nucleophilic attack on the phospholipid acyl chains. The small molecules were selected due to their predicted reactivity towards intrinsic lipidation, based on previous research monitoring lysolipid levels in their presence.77 Further, the small molecules selected cover a range of aromatic moieties and exhibit significant structural variation, in order to ensure the greatest likelihood of observing reactivity. Simultaneously, three cationic amphiphilic commercial pharmaceuticals were selected for study: (i) propranolol 1; (ii) procaine 2; (iii) tetracaine 3. The three pharmaceutical containing samples were at this time prepared by undergraduate student Sanna Appleby.76
100 5.2. Preliminary MS Experiments
Figure 5.3 Cationic amphiphilic small molecules studied during preliminary screening of small molecule intrinsic lipidation by LCMS on a QToF Premier (Waters Corp., UK).
In accordance with previous research conducted into small molecule intrinsic lipidation, small molecule:phospholipid mixtures were incubated under physiological conditions of pH 7.4 and 37◦C.77 After 72 hours, samples were centrifuged and vortexed in order to combine and mix the contents. A portion of the sample was diluted in 1:1 H2O:MeCN to a concentration of small molecule approximated to be 0.1 mg mL−1. Samples were then analysed on a QToF Premier (Waters Corp., UK) with Acquity C18 BEH stationary phase, using a five minute linear gradient from H2O (0.1% formic acid):MeCN (99:1) to H2O (0.1% formic acid):MeCN (1:99), followed by a four minute organic wash. Analyses were then repeated with a five minute linear gradient from H2O (0.1% formic acid):MeOH (99:1) to H2O (0.1% formic acid):MeOH (1:99), followed by a four minute organic wash. Instrument parameters applied to samples were those used as standard for all accurate mass samples analysed by Durham University mass spectrometry service, summarised in the Experimental section of this thesis.
Fig. 5.4 shows one example of the data obtained following analysis of phospholipid:small molecule samples under the standard analytical conditions employed by Durham University mass spectrometry service. Several problems are evident upon analysis of the data, including poor chromatographic resolution and insufficient sensitivity. Particularly problematic is the presence of considerable levels of background interference. Given the low levels of desired modified small molecule product expected, combined with poor ionisation resulting from their increased hydrophobicity, background interference may suppress the signal and prevent visualisation. Overall, it is clear that the instrumentation and conditions utilised for routine analysis of samples by Durham University mass spectrometry service is insufficient for the study of small molecule intrinsic lipidation.
Chapter 5. Small Molecule Intrinsic Lipidation 101
Figure 5.4 Example TIC chromatogram of small molecule incubated for 72 hours with phospholipid, and analysed by LCMS on a QToF Premier (Waters Corp., UK) under non-optimised conditions. No distinct peaks attributed to acylated small molecule or lysolipid species are observed. Analysis was conducted on a BEH C18column (Waters Corp., UK) with the ten minute gradient of mobile phase A (H2O containing 0.1 % formic acid) and mobile phase B (MeOH) shown in grey.