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Reaction kinetics for both solution phase and biological systems are known to be greatly altered by environmental temperature. Increased temperature provides reactive molecules with higher energy, aiding their ability to overcome the activation energy barrier to reaction participation. Further, higher energy levels increase molecular motion, facilitating the collisions between reactive species required for solution phase reactivity. Influence of temperature upon propranolol 1 intrinsic lipidation is predicted to be more complex than upon standard solution phase reaction kinetics. Increased molecular energy within the phospholipid bilayer favours an increasingly fluid and mobile membrane system, with heightened hydration levels. It is unclear how increased membrane fluidity will impact the proximity between the phospholipid ester linkage and the alcohol of membrane bound propranolol 1, however penetration is predicted to be similar assuming no modification to membrane phase or propranolol 1 ionisation. Proximity between reactive moieties is required for propranolol 1 intrinsic lipidation to successfully proceed via transesterification. Further, within a heavily hydrated membrane, hydrolysis of both O-acylated propranolol and membrane phospholipids is expected to be promoted.

Favoured hydrolysis compared to transesterification or intramolecular O to N migration would diminish formation of acylated propranolol derivatives. Determining how these factors are balanced, and the resulting impact upon propranolol 1 intrinsic lipidation requires study of the reaction at temperatures above and below physiological conditions of 37^◦C.

Examination of propranolol 1 intrinsic lipidation at varying temperature was conducted by combining propranolol 1 with a phospholipid membrane at 4^◦C, 37^◦C, and 57^◦C. Reaction mixture analysis was conducted under optimised LCMS conditions at time points of 24, 72 and 144 hours. Time points beyond this range were not investigated during the course of this study due to challenges associated with reaction mixture stability, and instrument detection limitations. Eukaryotic DOPC, viral DOPC:DOPS (4:1), and prokaryotic DOPE:DOPG (3:1) membranes were tested in order to investigate the effect of temperature upon reaction rate in the presence of different phosphate head groups associated with different membrane models.

POPC and OPPC were also tested in order to determine whether preferential sn-1 acyl chain transfer observed at 37 ^◦C is maintained despite changes in temperature.

The dependence of propranolol 1 intrinsic lipidation at different temperatures can be examined by considering total acylated product formation. Total acylated product is defined as the sum of O-palmitoyl propranolol 62, O-oleoyl propranolol 47, N -palmitoyl propranolol 12, and

Chapter 6. Propranolol Intrinsic Lipidation in vitro 185

N-oleoyl propranolol 13. Total acylated product formation within each membrane type studied following 72 hours is presented in Fig. 6.43 at 4 ^◦C, 37 ^◦C and 57^◦C. Increased acylated product formation, indicative of the extent of propranolol 1 intrinsic lipidation, is evident for each membrane type upon temperature increase. Reactivity is promoted by increased molecular energy available at higher temperatures, aiding in overcoming the activation energy barrier for the preliminary transesterification step required for propranolol 1 intrinsic lipidation.

Increased molecular mobility attributed to higher temperatures is not noted to reduce reactivity by disrupting the proximity of reactive moieties. One notable exception to these observations is within the POPC membrane, Fig. 6.43 (b), where 55 % conversion to acylated product is observed at 37 ^◦C, compared to 45 % at 57 ^◦C. At 37^◦C substantial differences in peak area of acylated products were observed between each of the triplicate samples analysed.

Identification of anomalous results therefore proves challenging, resulting in increased error and reduced confidence in the data at this time point. Consequently, the value of 55 % attributed to total acylated product within a POPC membrane at 37^◦C may be higher than the true value. Alternatively, propranolol 1 intrinsic lipidation within a POPC membrane may be favoured at 37^◦C compared to 57^◦C. Unfavourable disruption to key reactivity factors such as proximity of reactive moieties or membrane binding orientation may occur within the POPC membrane at 57 ^◦C, resulting in reduced propranolol 1 intrinsic lipidation.²⁶⁷

0 20 40 60

Figure 6.43 Total acylated product formed at varying temperatures (41 ^◦C, 37^◦C and 57

◦C) within 72 hours at pH 7.4, in the presence of five membrane systems: (a) DOPC (red circles), DOPC:DOPS 4:1 (blue squares) and DOPE:DOPG 3:1 (green triangles) ; (b) POPC (purple cicles) and OPPC (black squares).

Total acylated product formation can also be utilised to determine the extent of propranolol 1 intrinsic lipidation with varying temperature at time points of 24 and 144 hours, summarised in Table 6.22 and Table 6.23 respectively. Increased total acylated product, indicative of increased propranolol 1 intrinsic lipidation, is observed at higher temperatures across all membrane types. Promotion of reactivity at higher temperatures at 24 hours and 144 hours,

186 6.4. A Kinetic Model for Propranolol Intrinsic Lipidation

attributed to increased ability to overcome the activation energy barrier of transesterification, supports the observations made following analysis at 72 hours.

Total Acylated Product (%)

Membrane Type 4 ^◦C 37 ^◦C 57 ^◦C

DOPC 0.2 4.6 20.6

DOPC:DOPC (4:1) 0.0 0.9 5.2

DOPE:DOPG (3:1) 0.0 1.6 12.4

POPC 0.0 6.0 28.7

OPPC 0.0 9.3 23.6

Table 6.22 Proportion of total acylated product formed after 24 hour incubation at varying temperatures (4 ^◦C, 37^◦C, and 57 ^◦C) within five membrane types.

Total Acylated Product (%)

Membrane Type 4 ^◦C 37 ^◦C 57 ^◦C

DOPC 0.2 10.2 56.8

DOPC:DOPC (4:1) 0.1 1.0 20.9

DOPE:DOPG (3:1) 0.1 2.8 22.2

POPC 0.7 1.6 47.5

OPPC 0.5 3.7 65.4

Table 6.23 Proportion of total acylated product formed after 144 hour incubation at varying temperatures (4 ^◦C, 37^◦C, and 57 ^◦C) within five membrane types.

Preferential reactivity at temperatures greater than 37^◦C can be determined by comparison of the proportion of total acylated product formed by propranolol 1 intrinsic lipidation. To this end, eukaryotic membrane model DOPC, viral model DOPC:DOPS (4:1), and prokaryotic model DOPE:DOPG (3:1) were examined at 4^◦C and 57^◦C. Under physiological conditions the observed order of reactivity towards propranolol 1 intrinsic lipidation, reported in Section 6.3.3, is eukaryotic, followed by prokaryotic, and finally viral. Presented in Fig. 6.43, this observed reactivity trend is retained at 4^◦C and 57^◦C following 72 hours incubation. Further, the trend is also noted following analysis of samples incubated at 4^◦C and 57^◦C at 24 and 144 hours, as shown in Table 6.22 and Table 6.23. Conservation of this reactivity trend occurs despite temperature variation, and the associated modifications to membrane phase and fluidity. These membrane modifications may be expected to level subtle differences between the three membrane systems, such as domain formation and propranolol 1 binding orientation. However, given retention of the reactivity trend upon temperature variation, sufficient differences between the three membrane types as related to propranolol 1 intrinsic lipidation, must remain.

Chapter 6. Propranolol Intrinsic Lipidation in vitro 187

Investigation of the impact of temperature change upon the preferential sn-1 acyl chain transfer observed at 37^◦C, was also conducted. Investigation was facilitated by comparison of POPC and OPPC membranes, phospholipids comprised of identical acyl chain moieties in opposing backbone positions. Fig. 6.44 presents total palmitoylated product and total oleoylated product observed for each membrane type following 72 hour incubation at 4 ^◦C, 37 ^◦C, and 57 ^◦C. Preferential sn-1 transfer, palmitoyl for POPC and oleoyl for OPPC, is observed for both membrane compositions at 4 ^◦C and 57 ^◦C. This preference is noted despite the low abundance of palmitoylated and oleoylated products formed by propranolol 1 intrinsic lipidation at 4^◦C. Preferential sn-1 transfer at 4^◦C and 57^◦C after 72 hours is attributed to increased proximity between the alcohol of membrane bound propranolol 1 and the phospholipid sn-1 ester linkage, compared to the sn-2 linkage.

0 20 40 60

Figure 6.44 Total palmitoylated product (green circles) and total oleoylated product (red1 squares) formed by propranolol 1 intrinsic lipidation after 72 hours at varying temperatures (4^◦C, 37 ^◦C and 57 ^◦C) in the presence of: (a) POPC membrane; (b) OPPC membrane.

Preferential sn-1 acyl chain transfer persists at 57^◦C within both POPC and OPPC membranes at the additional time points of 24 and 144 hours, as highlighted in Table 6.24. However, more complex results are observed upon comparison of total palmitoylated product and total oleoylated product formed at 4^◦C after 24 and 144 hours. Following a 24 hour incubation at 4

◦C, neither palmitoylated propranolol nor oleoylated propranolol are observed analytically for POPC or OPPC membranes, Table 6.25. Lack of product observation prevents determination of preferential acyl chain transfer, and is attributed to reduced intrinsic lipidation reactivity at low temperatures combined with altered product distribution. Insufficient molecular energy to overcome reaction activation energy, combined with reduced molecular mobility, can explain observed reactivity limits. Analysis of samples incubated at 4^◦C for 144 hours with a POPC membrane suggest retention of preferential sn-1 acyl chain transfer. However, this preferential sn-1 transfer is not reflected within an OPPC membrane following 144 hours at 4^◦C, Table 6.25.

By contrast, increased sn-2 transfer results in slightly higher levels of palmitoylated propranolol

188 6.4. A Kinetic Model for Propranolol Intrinsic Lipidation

compared to oleoylated propranolol. Observed differences in preferential acyl chain transfer may be attributed to the low levels of propranolol 1 intrinsic lipidation at 4^◦C, resulting in product abundance at the limit of instrument detection and thus increasing the associated error. Alternatively, preferential sn-1 transfer may be replaced by either preferential sn-2 transfer or a distinction based upon acyl chain chemistry. A change in transfer preference at low temperature is possible within the confines of the less fluid membrane present at 4

◦C, particularly considering the increased activation energy attributed to palmitoyl chain migration in Section 6.3.2.

Membrane Type Time (hours) Palmitoylated Product (%) Oleoylated Product (%)

POPC 24 18.1 10.6

OPPC 24 9.5 14.1

POPC 144 29.9 17.7

OPPC 144 28.8 36.5

Table 6.24 Proportion of total palmitoylated product and total oleoylated product formed at 57 ^◦C within POPC and OPPC membranes after 24 and 144 hours.

Membrane Type Time (hours) Palmitoylated Product (%) Oleoylated Product (%)

POPC 24 0.0 0.0

OPPC 24 0.0 0.0

POPC 144 0.6 0.1

OPPC 144 0.3 0.2

Table 6.25 Proportion of total palmitoylated product and total oleoylated product formed at 4^◦C within POPC and OPPC membranes after 24 and 144 hours.

Analysis has been conducted to determine the influence of temperature upon propranolol 1 intrinsic lipidation rate and reactivity preferences. Temperature has also been determined to influence the rate of intramolecular O to N migration within acylated propranolol species.

Combining these two phenomena, it is possible to determine the influence of temperature upon the relative product distribution of O-acylated and N -acylated propranolol formed by intrinsic lipidation. Lowering environmental temperature from 37 ^◦C to 4 ^◦C results in formation of O-oleoyl propranolol 47 as sole intrinsic lipidation product within DOPC, DOPC:DOPS (4:1) and DOPE:DOPG (3:1) membranes over the time period studied, Fig. 6.45. Lack of N-oleoyl propranolol 13 formation is attributed to a diminished O to N migration rate at 4^◦C.

O-oleoyl propranolol 47 formation at 4 ^◦C is first observed at 24 hours within the eukaryotic DOPC membrane, and at 72 hours within the viral and prokaryotic systems. Following initial formation, a slow increase in O-oleoyl propranolol 47 is observed within DOPC:DOPS (4:1)

Chapter 6. Propranolol Intrinsic Lipidation in vitro 189

and DOPE:DOPG (3:1) membrane models, supporting a low rate of initial transesterification and minimal O to N migration.

0 50 100 150

0.0 0.1 0.2 0.3

Time (h)

%TotalPropranololContent

1

Figure 6.45 Total oleoylated product equal to O-oleoyl propranolol 47 only, formed over time at 4 ^◦C and pH 7.4 in the presence of DOPC (red circles), DOPC:DOPS 4:1 (blue squares) and DOPE:DOPG 3:1 (green triangles) membrane systems.

Study of acylated product formation at 4^◦C within POPC and OPPC membranes, Fig. 6.46, can also offer insight into the kinetics of propranolol 1 intrinsic lipidation. Acylated product formation is observed within both membrane systems from 72 hours, however unlike other membrane systems studied at 4^◦C, both O-acylated propranolol and N -acylated propranolol species contribute. N -palmitoyl propranolol 12 is noted as the sole N -acylated species in both POPC and OPPC membrane systems. Increased rate of O to N migration for O-palmitoyl propranolol 62 compared to O-oleoyl propranolol 47 could explain this observation. However, previously determined rate constants for migration (k1) of 0.7 × 10⁻³h⁻¹ and 2.6 × 10⁻³h⁻¹ for O-palmitoyl propranolol 62 and O-oleoyl propranolol 47 respectively do not support this theory. Alternatively, the presence of N -palmitoyl propranolol 12 as sole N -acylated product may suggest preferential transesterification of the palmitoyl moiety compared to the oleoyl moiety at 4 ^◦C. Despite low product abundance, it has been determined that N -palmitoyl propranolol 12 formation within POPC and OPPC systems proceeds at 4 ^◦C as observed under physiological conditions. An initial lag phase of 45 to 50 hours, highlighted by the vertical line in Fig. 6.46 (b), increased compared to 4 to 6 hours at 37 ^◦C, is followed by an increase in N -palmitoyl propranolol 12 concentration. Within the OPPC membrane the increase in N -palmitoyl propranolol 12 concentration occurs more steadily, as shown by the dashed line in Fig. 6.46 (b). By contrast, in the presence of the POPC membrane the increase seems initially fast but then plateaus, suggestive of rate variation. Simultaneously, production of O-palmitoyl propranolol 62 and O-oleoyl propranolol 47 is observed to progress linearly within POPC and OPPC membranes at 4 ^◦C, as shown by the solid green lines in Fig. 6.46.

190 6.4. A Kinetic Model for Propranolol Intrinsic Lipidation

Data extrapolation suggests transesterification commences at time point zero, with the lack of observed product at 24 hours attributed to the low abundance of acylated product, less that 0.05 % (<1 ng mL⁻¹).

Figure 6.46 Total O-acylated product (experimental data as green circles and trend fitted1 with a solid green line), and total N -acylated product (experimental data as red squares and trend fitted with a dashed red line), formed from propranolol 1 at 4^◦C in the presence of: (a) POPC membrane; (b) OPPC membrane. The box indicates the proportion of O-acylated product at 24 hours, below the limit of instument detection. The vertical line indicates end of N-acylated derivative lag phase.

Increasing environmental temperature from 37 ^◦C to 57 ^◦C is also predicted to influence the distribution of O-acylated and N -acylated products of propranolol 1 intrinsic lipidation.

Similar trends in product formation at 57 ^◦C are observed for membranes containing a PC phosphate head group, as shown in Fig. 6.47. Unlike at 4 ^◦C and 37 ^◦C, N -acylated propranolol production at 57^◦C does not exhibit a lag phase on the scale of the time period studied. Data extrapolation shown by the red dashed lines in Fig. 6.47, suggests N -acylated propranolol formation commences within minutes of time point zero, suggesting no requirement for prior O-acylated propranolol accumulation. N -acylated product concentration proceeds to increase linearly within DOPC and OPPC systems, Fig. 6.47 (a) and (c), with slope gradient providing a means of formation rate comparison. At 57^◦C the rate of N -acylated propranolol formation by intramolecular O to N migration is increased compared to at 37^◦C, as shown in Table 6.26. O-acylated propranolol formation within PC membranes at 57^◦C also differs from the logarithmic curve observed under physiological conditions. As shown in Fig. 6.47, an initial increase in O-acylated propranolol formation over 24 hours at 57 ^◦C is followed by a steady decrease in concentration at later time points of 72 and 144 hours. Decreasing concentration is indicative of a faster rate of O-acylated propranolol decomposition by O to N migration compared to O-acylated propranolol formation by transesterification.

Chapter 6. Propranolol Intrinsic Lipidation in vitro 191

Figure 6.47 Total O-acylated product (experimental data as green circles and trend fitted1 with a solid green line), and total N -acylated product (experimental data as red squares and trend fitted with a red dashed line), formed at 57^◦C in the presence of: (a) DOPC; (b) POPC;

(c) OPPC.

Membrane Type Rate at 37^◦C (h⁻¹) Rate at 57^◦C (h⁻¹)

DOPC 0.13 0.36

OPPC 0.17 0.19

Table 6.26 Comparison of the rate of N -acyl propranolol formation within two membrane types at 37^◦C and 57 ^◦C.

Study of N -oleoyl propranolol 13 production at 57 ^◦C within viral DOPC:DOPS (4:1) and prokaryotic DOPE:DOPG (3:1) membranes reveals similar results to those observed for PC membranes, Fig. 6.48. N -oleoyl propranolol 13 formation commences within minutes of time point zero in the prokaryotic membrane, indicating a diminished lag phase compared to at physiological temperatures. Viral membrane DOPC:DOPS (4:1) exhibits a 5 hour lag phase prior to N -oleoyl propranolol 13 production, consistent with the reduced membrane reactivity. N -oleoyl propranolol 13 concentration increases linearly up to 144 hours within the viral membrane model, Fig. 6.48 (a), whereas concentration increases and then plateaus in the presence of DOPE:DOPG (3:1), shown in Fig. 6.48 (b). O-oleoyl propranolol 47 concentration at 57 ^◦C does not exhibit a significant decrease over time within viral and prokaryotic membranes, in contrast to PC membranes. A logarithmic curve is observed for O-oleoyl propranolol 47 production within the viral membrane at 57^◦C. Analogous to observations under physiological conditions, this trend suggests similar reaction rates for