process progressed via the relevant dipalmitoyled propranolol 41 and dioleoylated propranolol 42 intermediates, ultimately resulting in formation of the desired N -acylated products. How-ever, problems were encountered during product purification due to the modified properties of palmitic and oleic acid, byproducts of the ester hydrolysis step, compared to acetic acid.
Palmitic and oleic acid are amphiphilic in nature, resulting in micelle formation in aqueous solution. N -palmitoyl propranolol 12 and N -oleoyl propranolol 13 are also amphiphilic and likely to form micelles. Within the polar mobile phase required for flash column purification of N -acylated propranolol derivatives, mixed micelles are predicted to form. These mixed micelles containing fatty acid and N -acylated propranolol prevent separation and purification of the two species. A solution to this problem is presented in Scheme 5.4.248 Prior to column chromatography the fatty acid is converted into a methyl ester, reducing hydrophilicity of the compound and preventing mixed micelle formation. Desired products N -palmitoyl propranolol 12 and N -oleoyl propranolol 13 can then be separated from the relevant methyl ester by flash column chromatography.
Scheme 5.4 Synthetic method employed to methylate excess fatty acids, facilitating pu-rification of N -palmitoyl propranolol 12 and N -oleoyl propranolol 13. Palm represents the palmitoyl group (CH2)14CH3 and oleo represents the oleoyl group (CH2)7CHCH(CH2)7CH3.
5.4 Optimisation of ESI Parameters
Armed with synthetic acylated small molecule standards, analytical conditions for the study of small molecule intrinsic lipidation can be developed. ESI was selected as the ionisation technique, due to its reputation for effective ionisation with minimal in-source fragmentation.105 Optimising ESI instrument parameters allows for efficient ionisation of the hydrophobic acylated small molecules, given their diminished ionisation potential following conversion of an amine in the original small molecule into an amide. The presence of a hydrophobic acyl chain connected via a relatively weak amide bond within the acylated small molecules, means conditions for effective ionisation must be carefully balanced with minimal fragmentation of the desired molecular ion.
Chapter 5. Small Molecule Intrinsic Lipidation 105
Ionisation parameters were optimised by direct infusion of a 0.5 µg mL−1 solution of N -palmitoyl procaine 35 in 1:1 H2O:MeCN into the mass spectrometer at a flow rate of 5 µL/ min.
The mass spectrum was collected over one minute for each set of conditions, and the resulting data compared in order to determine optimal ionisation parameters. Data quality was determined based upon ion intensity, signal to noise, and level of in-source fragmentation.
Ionisation parameters determined to play major role in analyte ionisation for ESI on a Synapt G2-S (Waters Corp., UK) mass spectrometer are:
• Desolvation gas temperature
• Desolvation gas flow rate
• Source temperature
• Capillary voltage
• Sampling cone voltage
• Source offset voltage
Nitrogen is the desolvation gas utilised within the ESI source throughout the study of small molecule intrinsic lipidation by mass spectrometry. It is selected due to its availability at the necessary purity, inert nature, and stability at high temperatures. The desolvation gas promotes the desolvation of solvated ions released in an aerosol from the capillary needle into gas phase ions. Desolvation gas temperature and flow rate are two vital parameters which can be used to control the desolvation process and modify analyte ionisation. Increased desolvation temperature and increased gas flow rate provide more energy to the gas particles, promoting ion desolvation. However, an increase in desolvation gas energy can also increase collisions between ions and the inert gas, resulting in energy transfer to the ion which is then distributed among all the available degrees of freedom causing bond dissociation. As such, modification to desolvation gas flow rate and temperature were expected to influence ionisation of N -palmitoyl procaine 35. Surprisingly, alterations to the desolvation gas resulted in minimal change to the ionisation profile of N -palmitoyl procaine 35. Optimised values of 350 ◦C and 600 L h−1 were selected for desolvation temperature and flow rate respectively, determined by published research into small molecule ESI.249 Similarly, the source temperature was maintained at 150
◦C in accordance with both literature data and compatibility with other instrument users.40,77
106 5.4. Optimisation of ESI Parameters
5.4.1 Capillary Voltage
Capillary voltage is an instrument parameter which describes the voltage applied at the tip of an ESI capillary needle.81 Voltage application creates a potential difference between the needle and the rest of the mass spectrometer. As a result, selection of correct capillary voltage is vital to ensure charge transfer and motion of ESI droplets formed at the needle tip.
Typical positive ESI capillary voltages are in the range of 0.5 to 4.0 kV, however the optimum value is highly dependent upon analyte properties and sample matrix, including solvent.
Application of a capillary voltage that is too low prevents development of a Taylor cone at the capillary tip, resulting in formation of large droplets with little horizontal directionality.
As a result, droplet desolvation becomes more challenging and sensitivity is reduced, resulting in diminished ionisation. Application of a higher capillary voltage is necessary for certain solvent systems such as those lacking acidic additives or with a high aqueous proportion, due to its corresponding increases in ionisation and sensitivity. However, application of too high a capillary voltage for a given analyte also causes ionisation issues. Major problems include loss of the Taylor cone resulting in rim emission and poor droplet formation, and discharge reducing ionisation stability.250
To investigate the effect of capillary voltage upon the ionisation of acylated small molecules a 0.5 µg mL−1 solution of N -palmitoyl procaine 35 in 1:1 H2O:MeCN was direct infused into the mass spectrometer. Data was collected over a one minute period with all parameters kept consistent apart from the capillary voltage, which underwent incremental increases for each analysis. Fig. 5.6 presents the ionisation profile of N -palmitoyl procaine 35 at a selection of these capillary voltages. Below 1.0 kV the ion intensity of N -palmitoyl procaine 35 is greatly reduced, as indicated by the profile at a capillary voltage of 0.5 kV. The reduction in ionisation results in a corresponding signal to noise decrease, making the capillary voltage inappropriate for the study of intrinsic lipidation. High capillary voltages of 2.0 kV and 2.5 kV exhibit increased ion intensity, however in-source fragmentation to ion m/z 193.0736 of molecular formula C10H11NO3 is observed in tandem. Between 1.0 kV and 1.5 kV capillary voltage modification has minimum impact upon ion abundance or fragmentation of N -palmitoyl procaine 35. Considering these observations, and stability issues associated with a high capillary voltages, 1.0 kV was deemed most appropriate for the ionisation of acylated small molecules.250
Chapter 5. Small Molecule Intrinsic Lipidation 107
Figure 5.6 Ion profiles of N -palmitoyl procaine 35 upon application of differing capillary voltages: (a) 0.5 kV; (b) 1.0 kV; (c) 1.5 kV; (d) 2.0 kV; (e) 2.5 kV.
5.4.2 Sampling Cone Voltage
The sampling cone is an orifice within the ESI source which lies perpendicular to the plume of droplets produced at the capillary needle tip. A voltage in the region of 10 - 100 V is applied to the sampling cone in order to draw ions from the atmospheric pressure region and into the intermediate vacuum region of the mass spectrometer.81 Voltage application aids in ion transmission, exclusion of unwanted neutral species and declustering of heavily hydrated ions. Selection of an appropriate sampling cone voltage is highly dependent upon analyte type and charge state, with ions of increased mass requiring application of a higher cone voltage. Two key factors are taken into account when tuning the sampling cone voltage for a particular analyte. Firstly, the voltage must be sufficiently high in order to pull the maximum number of ions into the mass spectrometer. Maximising ion transmission results in an increase in instrument sensitivity and aids detection of low abundance or poorly ionising species. However, the increased ion acceleration and transmission associated with a high sampling cone voltage also promotes collisions with molecules of desolvation gas. Inelastic collisions result in increased ion internal energy, causing breakage of weak internal bonds.
108 5.4. Optimisation of ESI Parameters
This in-source fragmentation process has the undesirable side effect of reducing abundance of the molecular ion, however it can be manipulated for use in MSMS analyses.
0 200 400 600
Figure 5.7 Ion profiles of N -palmitoyl procaine 35 upon application of differing sampling35 cone voltages: (a) 30 V; (b) 40 V; (c) 50 V; (d) 60 V; (e) 70 V.
Retaining an intact acylated small molecule molecular ion is vital for detection of small molecule intrinsic lipidation. Prevention of in-source fragmentation at the weak amide bond is vital when selecting an appropriate sampling cone voltage. Mass spectra of N -palmitoyl procaine 35 were collected over one minute whilst increasing the sampling cone voltage in 10 V increments. Fig. 5.7 presents the ionisation profiles of N -palmitoyl procaine 35 over the region of interest. Across values 30 V to 70 V, the sampling cone voltage promotes transmission of the molecular ion m/z 475.3928 with minimal evidence of in-source fragmentation. The maximum molecular ion intensity is reached at a sampling cone voltage of 50 V, therefore this is considered the optimum value for further study.
5.4.3 Source Offset Voltage
Following transmission through the sampling cone into the intermediate vacuum region of the mass spectrometer, ions must continue into the high vacuum region.81 Ions are pulled down a potential gradient set by the source offset voltage into the StepWave ion transfer,
Chapter 5. Small Molecule Intrinsic Lipidation 109
facilitating entry to the mass analyser region of the mass spectrometer. High source offset voltages increase ion transmission into the mass spectrometer, increasing ion abundance and sensitivity. However, as with a high sampling cone voltage, the increased ion acceleration promotes collisions with gas phase molecules resulting in ion fragmentation.
0 200 400 600
Figure 5.8 Ion profiles of N -palmitoyl procaine 35 upon application of differing source35 offset voltages: (a) 20 V; (b) 30 V; (c) 40 V.
Minimisation of in-source fragmentation at the weak amide bond of N -palmitoyl procaine 35 is a key consideration throughout tuning of the ESI source offset voltage. Ionisation profiles obtained over one minute from the direct infusion of N -palmitoyl procaine 35 are shown in Fig. 5.8. The source offset voltage was modified over the range of 20 V to 40 V in increments of 10 V. In-source fragmentation of N -palmitoyl procaine 35 was not noted to contribute significantly to observed ion profiles across the voltage range tested. Optimum source offset voltage is selected as 30 V based upon its ability to produce the maximum observed ion intensity by facilitating ion transmission.
5.4.4 Summary of Optimised ESI Parameters
Table 5.1 summarises the optimised ESI instrument parameters for the study of N -palmitoyl procaine 35. However, suitability of the parameters for studying acylated small molecules in general, and thus small molecule intrinsic lipidation, must be determined. Utilising the optimised instrument parameters, the five other synthetic acylated small molecule standards were direct infused into the mass spectrometer. These were N -oleoyl procaine 36, N -palmitoyl tetracaine 37, N -oleoyl tetracaine 38, N -palmitoyl propranolol 12 and N -oleoyl propranolol 13. Mass spectra for each acylated small molecule standard under optimised condition are
110 5.4. Optimisation of ESI Parameters
shown in Fig. 5.9. Molecular [M+H]+ ions are observed with good signal to noise for each desired analyte, with additional sodium adduct formation and dimerisation noted for N -palmitoyl propranolol 12 and N -oleoyl propranolol 13 due to preparation methodology. Low level in-source fragmentation is observed for all species, along with in-source double bond oxidation for N -oleoyl procaine 36. However, observed issues are minimal and successful ionisation of all acylated small molecules cements the optimised parameters as those to be utilised for the study of small molecule intrinsic lipidation.
Parameter Optimised Value Capillary Voltage (kV) 1.0 Source Temperature (◦C) 150.0 Sampling Cone Voltage (V) 50.0
Source Offset Voltage (V) 30.0 Desolvation Temperature (◦C) 350.0
Cone Gas Flow (L/Hr) 60.0 Desolvation Gas Flow (L/Hr) 600.0
Nebuliser Gas Flow (Bar) 6.0
Table 5.1 Optimised positive mode electrospray parameters for mass spectrometry analysis of acylated small molecules.
Intensity (arb. units) [M+H]+(b) [Tetracaine]+
Intensity (arb. units) [M+H]+ [Tetracaine]+
Figure 5.9 Ionisation profiles of synthetically prepared acylated small molecules under optimised instrument parameters given in Table 5.1: (a) N oleoyl procaine 36; (b) N -palmitoyl tetracaine 37; (c) N -oleoyl tetracaine 38; (d) N --palmitoyl propranolol 12; (e) N-oleoyl propranolol 13 .
Chapter 5. Small Molecule Intrinsic Lipidation 111