Structure-activity relationships of rationally designed AMACR 1A inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.04.024

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the ‘racemisation’ reaction and the difficulties in the separation of epimeric prod

Full text of DOI:10.1016/j.bioorg.2018.04.024

Accepted Manuscript
Structure-activity relationships of rationally designed AMACR 1A inhibitors
Maksims Yevglevskis, Guat L. Lee, Amit Nathubhai, Yoana D. Petrova, Tony
D. James, Michael D. Threadgill, Timothy J. Woodman, Matthew D. Lloyd
PII:
S0045-2068(18)30167-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.04.024
YBIOO 2347
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
21 February 2018
20 April 2018
24 April 2018
Please cite this article as: M. Yevglevskis, G.L. Lee, A. Nathubhai, Y.D. Petrova, T.D. James, M.D. Threadgill, T.J.
Woodman, M.D. Lloyd, Structure-activity relationships of rationally designed AMACR 1A inhibitors, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.04.024
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Structure-activity relationships of rationally
designed AMACR 1A inhibitors
Maksims Yevglevskis,a Guat L. Lee,a Amit Nathubhai,a Yoana D. Petrova,a Tony
D. James,b Michael D. Threadgill,a Timothy J. Woodmana and Matthew D.
Lloyda*
aDrug & Target Discovery, Department of Pharmacy & Pharmacology,
University of Bath, Claverton Down, Bath BA2 7AY, UK.
bDepartment of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY,
UK.
*Drug & Target Discovery, Department of Pharmacy & Pharmacology,
University
of
Bath,
Claverton
Down,
Bath
BA2
7AY,
U.K.;
M.D.Lloyd@bath.ac.uk
Keywords: α-Methylacyl-CoA racemase (AMACR; P504S); drug lipophilicity;
enzyme inhibitors; rational drug design; Structure-activity relationships.
1

Abstract
α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target
for prostate and other cancers. Assaying enzyme activity is difficult due to the
reversibility of the ‘racemisation’ reaction and the difficulties in the separating
epimeric products; consequently few inhibitors have been described and no
structure-activity relationship study has been performed. This paper describes
the first structure-activity relationship study, in which a series of 23 known and
potential rational AMACR inhibitors were evaluated. AMACR was potently
inhibited (IC50 = 400 - 750 nM) by ibuprofenoyl-CoA and derivatives. Potency
was positively correlated with inhibitor lipophilicity. AMACR was also inhibited
by straight-chain and branched-chain acyl-CoA esters, with potency positively
correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold
more potent inhibitors than decanoyl-CoA, demonstrating the importance of the
2-methyl group for effective inhibition. Elimination substrates and compounds
with modified acyl-CoA cores were also investigated, and shown to be potent
inhibitors. These results are the first to demonstrate structure-activity
relationships of rational AMACR inhibitors and that potency can be predicted by
acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric
assay for thorough inhibitor characterisation.
Abbreviations
used:
AMACR,
α-methylacyl-CoA
racemase;
trifluoride;
CDI,
carbonyldiimidazole;
DAST,
(Diethylamino)sulfur
DCC,
Dicyclohexylcarbodiimide; DMAP, (dimethylamino)pyridine; MCR, 2-methylacyl-
CoA racemase from M. tuberculosis; SAR, Structure-activity relationships; THF,
tetrahydrofuran; TMSCl, chlorotrimethylsilane.
2

Introduction
Branched-chain fatty acids (e.g. phytanic acid pristanic acids) are common
components of the human diet, and derivatives of such compounds are used as
drug molecules e.g. Ibuprofen [1, 2]. Degradation of branched-chain fatty acids
occurs as the acyl-CoA ester, and the acyl-CoA oxidases and other enzymes
involved in β-oxidation have an absolute requirement for S-2-methylacyl-CoAs
[3-5]. However, R-2-methylacyl-CoAs are produced from dietary and
endogenous fatty acids and these cannot be immediately degraded by β-
oxidation. The enzyme α-methylacyl-CoA racemase [1, 2] (AMACR; P504S;
E.C. 5.1.99.4) catalyses conversion of R-2-methylacyl-CoAs to a near 1:1
epimeric mixture [6, 7] by a deprotonation / reprotonation reaction [7, 8],
probably via an enolate intermediate [9] (this reaction is referred to as
“racemization” [10]). The resulting S-2-methylacyl-CoAs are degraded by β-
oxidation whilst the 2R epimers are further processed to the 2S epimers by
AMACR [1, 2]. AMACR also plays a key role in the in vivo pharmacological
activation of R-Ibuprofen to S-Ibuprofen, via the corresponding acyl-CoA esters
[1, 2, 11]. The S-Ibuprofen resulting from this pathway exerts its anti-
inflammatory effect by inhibiting cyclooxygenase-1 and -2 [12].
AMACR protein levels are increased in prostate [13, 14] and several other
cancers [1, 15-18]. Catalytic activity of AMACR is increased by 4 to 10-fold in
prostate cancer cells [19, 20], with the AMACR 1A splice variant [1, 2, 21-24]
(possessing “racemase” activity [7, 10]) showing the most significant increase in
expression [19, 20]. Reducing AMACR 1A levels using siRNA or shRNA
approaches [19, 25, 26] has been shown to reduce proliferation of prostate
cancer cells via a pathway which is synergistic with the use of an androgen
3

receptor antagonist, studies which have validated AMACR 1A as a
chemotherapeutic target. Some advanced prostate cancer cell lines revert from
castrate-resistant (a.k.a. androgen-independent) growth to androgen-dependent
growth upon knockdown of AMACR 1A [26]. Consequently, AMACR has
attracted considerable interest as a prostate cancer biomarker [1, 2, 27] and
drug target [25, 28-31]. However, the lack of a convenient assay to measure
AMACR activity [32, 33] has severely hampered the development of AMACR
inhibitors as new chemotherapeutic drugs against cancers that over-express
AMACR, and consequently only a few rationally designed inhibitors of AMACR
[28-30, 34] or MCR [31, 35] (M. tuberculosis homologue) have been reported.
No systematic study of AMACR inhibitor SAR has been undertaken [32, 33].
Recently, we reported a versatile continuous assay for AMACR based on
the utilisation of our novel substrate 1 that can eliminate 2,4-dinitrophenolate 2,
which can be monitored by absorbance at 354 nm, and unsaturated product 3
(Scheme 1). This new assay [32] was used to examine the potency of two
known acyl-CoA inhibitors (N-dodecyl-N-methylcarbamoyl-CoA 4 [29] and
ibuprofenoyl-CoA 5 [6]; Figure 1) and selected known non-specific protein
modification agents [25]. This paper reports the first systematic examination of
SAR for rationally designed acyl-CoA inhibitors of AMACR. Compounds
investigated (Figure 1) include those with aromatic side-chains, (5 - 11);
Straight-chain acyl-CoA esters (12 – 17); Branched-chain substrates (18 – 21)
and product 22. Analogues of known inhibitors with modified 2-methylacyl-CoA
moieties (4, 23 - 26) were also examined. The results reveal a correlation
between potency and lipophilicity of the inhibitors, consistent with observations
on MCR inhibitors [35], the homologous enzyme from M. tuberculosis.
4

Results and discussion
AMACR is a promising novel cancer drug target, but therapeutic
development in this field has been slow due to the lack of a robust enzyme
assay. Thus, the majority of studies reporting AMACR inhibitors have largely
focussed on rationally designed drugs [28-30, 32, 33]. In most cases, only one
or a few examples of each inhibitor type has been evaluated, and no
systematic SAR study has been performed. Initial SAR studies have been
carried out on reversible [31] and irreversible [35] inhibitors of MCR (the M.
tuberculosis homologue). In addition, different research groups have used
different assays during their studies, making it difficult to compare results. In
this study, the SAR of rational AMACR inhibitors were explored using a series
of acyl-CoA esters (Figure 1). These included compounds previously tested as
substrates (6 – 11 [6, 11]; 12 – 17 [36]; 18 – 20, 22 [7, 10]). Most of these
compounds have not been tested as inhibitors with the exception of 5 [28, 32,
37, 38] and 13, 15-17 (which were previously reported to be inactive [37, 38]).
Compound 21 was included as an epimer of 20, and has not been previously
reported as
a
substrate or inhibitor (although the 3-fluoro-2-
methyltetradecanoyl-CoA analogues are potent inhibitors [28]). Compound 24
is a synthetic intermediate to 25, and has not been previously tested as a
substrate or inhibitor. Compounds 22, 25 and 26 are intermediates in the
subsequent β-oxidation pathway [39], and have not been previously tested as
substrates or inhibitors. Analogues of compounds 23, and 25 with different
side-chains have been previously tested as inhibitors of AMACR or MCR [9,
30]. Compound 4 was previously reported as the most potent AMACR inhibitor
[29, 32], and is included as an acyl-CoA core analogue.
5

Chemical synthesis of acyl-CoA inhibitors
(2S,3S)-3-Fluoro-2-methyldecanoyl-CoA 21 was synthesised by an
analogous route to (2R,3R)-3-fluoro-2-methyldecanoyl-CoA 20 [10, 28], using
an Evans’ auxiliary strategy (Scheme 2). Aldol-like reaction of deprotonated 27
with octanal gave the (2S,3R)-3-hydroxy-2-methyl intermediate 28. From here,
alcohol 28 was activated and replaced with fluoride with inversion of
configuration, using DAST to give 3-fluoro-2-methyl derivative 29. The reaction
is thought to go with inversion of stereochemical configuration (by analogy with
the work of Carnell et al. [28]). Removal of the Evans’ auxiliary from
intermediate 29 provided the carboxylic acid 30 under mild conditions that
involved in situ generation of lithium hydroperoxide. Intermediate 30 was
subsequently converted to the CoA thioester 21 using the standard synthetic
method with N,N’-carbonyldiimidazole [6, 10, 11, 32, 33, 40]. This compound
was stable in solution in the absence of AMACR, showing that the relative
geometry of the α-proton and fluorine atom was syn- (anti- epimers rapidly
eliminate fluoride, presumably by an E2 mechanism [10]).
2-Methylenedecanoyl-CoA 23 was synthesised by an adaptation of the
method reported by Morgenroth et al. [30] (Scheme 3). Meldrum’s acid 33 was
acylated with octanoic acid 32 using DCC activation; the intermediate ketone
was reduced to the octyl-Meldrum’s acid 31 with sodium triacetoxyborohydride
generated in situ. Subsequent reaction of 31 with Eschenmoser’s salt gave the
2-methylene ester 34. Base-hydrolysis furnished the 2-methylene acid 35,
which was coupled with CoA-SH by a mixed anhydride approach to give 2-
methylenedecanoyl-CoA 23.
6

2-Methyl-3-oxodecanoyl-CoA 25 was synthesised by the method of Reen et
al. [41] from 36 (Scheme 4). The ketone in 37 was protected as the cyclic acetal
36. Hydrolysis of the ester group in 36 gave the corresponding acid 38, which
was then coupled with CoA [6, 10, 11, 32, 33, 40] to give 24. Acidolysis of the
acetal protection provided 25.
2S,3R-3-Hydroxy-2-methyldecanoyl-CoA 26 was synthesised (Scheme 5)
from the acyl-Evan’s auxiliary 28 (Scheme 2, vide supra) by hydrolysis with
lithium hydroperoxide to give acid 39, which was converted to the CoA ester 26
using the standard procedure [6, 10, 11, 32, 33, 40].
Evaluation of inhibitors
The selected AMACR inhibitors were evaluated using the colorimetric assay
[32]. Incubation of active human AMACR 1A with substrate 1 results in
production of 2,4-dinitrophenolate 2 and unsaturated product 3 (Scheme 1).
Hence, the potency of inhibitors can be determined based on measuring the
absorbance of 2 at 354 nm. Inhibitory potency was assessed using dose-
response curves to determine IC50 values (Figure 1).
As expected, ibuprofenoyl-CoA 5 and its derivatives 6 – 9 were inhibitors of
the enzyme, with most having IC50 values of ca. 500 nM (Figure 1). Variation of
the structure of the side-chain in these inhibitors appeared to make little
difference to inhibitory activity (as judged by IC50 values), although
fenoprofenoyl-CoA 6 appeared to be slightly more potent than the other
examples and naproxenoyl-CoA 9 appeared to be slightly less so. All these
compounds are known substrates of AMACR [6] and are predicted to behave as
competitive inhibitors. Ibuprofenoyl-CoA 5 has been previously confirmed to be
7

a competitive inhibitor of AMACR, with Ki = 60 nM [32], consistent with
observations of other workers on the human and rat enzymes [28, 37, 38]. The
mandelic acid derivatives R- and S-2-hydroxy-2-phenylacetyl-CoA 10 and 11
were also modest inhibitors (Figure 1), binding approximately ten times less
strongly than compounds 5 – 9. Compounds 10 and 11 are not substrates of
AMACR, since enzyme catalysed α-proton exchange does not occur [11]. This
result with 10 and 11 also demonstrates that inhibitors can possess a 2-
hydroxy- group in addition to the previously reported 2-trifluoromethyl- [28] and
2-chloro- [29] groups in place of the 2-methyl group. A wide range of aromatic
inhibitor side-chains can therefore be accomodated by the enzyme, consistent
with predictions made based on the MCR crystal structures [8] and biochemical
data [6, 11].
Acyl-CoA esters 12 - 17, possessing alkyl side-chains were also assessed as
inhibitors (Figure 1). The potency of inhibition for acyl-CoA esters with side-
chains of four carbons or fewer (15 – 17) is very weak, with low levels of
inhibition (15-30%) observed even at very high inhibitor concentrations (100
µM). Inhibition increased as alkyl chain-length increased. This behaviour is
consistent with that of straight-chain acyl-CoA esters acting as substrates [36],
where increased levels of α-proton exchange are observed with increasing
chain length. Our results showing inhibition of AMACR by straight-chain acyl-
CoA esters contrasts with the early observations of Schmitz et al., who reported
that these compounds were not inhibitors of the native human and rat enzymes
[37, 38].
Inclusion of a 2-methyl group on the inhibitor increased potency by about 3-
fold (compounds 18 and 19 compared to 12), again consistent with the finding
8

that 2-methylacyl-CoA esters are much more efficient substrates than their
straight-chain equivalents [36]. R-2-Methyldecanoyl-CoA 18 appeared to be a
slightly more potent inhibitor than S-2-methyldecanoyl-CoA 19, and this
probably reflects the physiological role of AMACR in the conversion of R-2-
methylacyl-CoAs to their S-2-methylacyl-CoA epimers [1, 2].
2-Methyldecanoyl-CoA derivatives with more acidic α-protons are better
inhibitors than their parent compounds (Figure 1). R,R-3-Fluoro-2-
methyldecanoyl-CoA 20 was about 5-fold more potent than was R-2-
methyldecanoyl-CoA 18. A similar trend was observed with S,S-3-fluoro-2-
methyldecanoyl-CoA 21 and S-2-methyldecanoyl-CoA 19, although both of
these compounds were slightly less potent than their R-epimers. The product of
the reaction, E-2-methyldec-2-enoyl-CoA 22, is also a potent inhibitor. It is
therefore difficult to determine if the observed IC50 values for 20 or 21 reflect the
conversion of these substrates [10], product inhibition by 22 or both. These
observations contrast with early studies [37, 38], which suggest that 22 was not
an inhibitor of AMACR.
2-Methyl-3-oxodecanoyl-CoA 25 was also a good inhibitor of AMACR. The α-
proton of this compound is relatively acidic and 25 undergoes rapid non-
enzymatic α-proton exchange with solvent via an enolate intermediate. It was
therefore not possible to analyse the influence of 2-methyl group
stereochemical configuration. The precursor 24 was a much poorer inhibitor
than 25, presumably due to reduced acidity of the α-proton. It is also possible
that the additional steric bulk at carbon-3 contributes to the lower potency of 24
compared to 25, as the 5-membered ring of 24 will be twisted out of plane
relative to the aliphatic side-chain. However, AMACR is known to be able to
9

accept substrates with diverse side-chain structures [6, 28, 36-38] and it is
notable that ibuprofenoyl-CoA 5 and derivatives 6 - 9 (which have aromatic
rings at the equivalent position) are potent inhibitors (Figure 1).
2S,3R-3-Hydroxy-2-methyldecanoyl-CoA 26 is also a relatively potent
inhibitor. Incubation of 26 with active AMACR did not result in an elimination
reaction, as judged by the lack of a peak at δ 1.75 ppm [10] from the 2-methyl
group of the anticipated product 22 (Figure 2), probably because hydroxide is a
relatively poor leaving group (water pKa = ~7), compared to fluoride (HF pKa =
1
3.2) [42]. Similarly, H NMR analysis of the reaction products showed that 26
did not undergo α-proton exchange, and hence 26 is not a substrate of AMACR.
It is notable that 22, 24 and 26 are intermediates in the branched-chain acyl-
CoA β-oxidation pathway. One may speculate that these compounds could
provide some regulation of AMACR by negative feedback control, and hence
control entry of R-2-methylacyl-CoA esters into the β-oxidation pathway.
Whether or not this is physiologically significant will depend on rates of flux
through the β-oxidation pathway and whether intermediates 22, 24 and 26 are
sequestered away from AMACR.
Acyl-CoAs which mimic the planar enolate intermediate are good inhibitors of
AMACR (Figure 1). E-2-Methyldec-2-enoyl-CoA 22 and 2-methylenedecanoyl-
CoA 23 bind strongly due to having a planar sp2-hybridized α-carbon. This
result is consistent with a previous study in which 2-methyleneacyl-CoAs were
shown to be good competitive inhibitors [2, 30]. The enolate analogue N-
dodecyl-N-methylcarbamoyl-CoA 4 is the best rationally designed inhibitor (IC50
= 0.4 nM) reported to date [29, 32]. Inhibition by 4 is ca. 2000 more potent
than by 18 and 19 (Figure 1). This high potency of 4 appears to largely result
10

from the mimicking of the enolate intermediate by the carbamoyl moiety. It is
also notable that the determined IC50 value for 4 in this study (0.4 nM) is
significantly lower than that previously determined by Carnell et al. (98 nM),
when assayed against HEK-derived human AMACR using ibuprofenoyl-CoA 5
as substrate [29]. It appears that the colorimetric assay consistently determines
higher levels of compound potency than other assays (e.g. Ki = 60 nM [32] vs.
56 µM [28, 38] for an ibuprofenoyl-CoA epimeric mixture) The reasons for this
discrepancy is not entirely clear, but higher apparent potency may be a
consequence of using a substrate undergoing an irreversible reaction to
measure activity (and hence avoiding the error introduced by the presence of
the reverse reaction). Alternatively, this may be related to the extent of
substrate or inhibitor micelle formation under the different assay conditions.
The determined IC50 value (0.4 nM) is around half the calculated ‘active’
enzyme concentration in the assay [32] (based on comparison of kcat/Km values
for the E. coli and HEK cell derived enzymes [29]), and hence 4 could be
behaving as a tight-binding inhibitor. Compound 4 behaves as a rapidly
reversible competitive inhibitor of AMACR, with a Hill coefficient of ~0.7 [32].
These observations are consistent with the zone A inhibitor behaviour described
by Straus and Goldstein [43, 44], i.e. the enzyme active site concentration is
<0.1  the apparent Ki value (0.65 nM [32]). This rapidly reversible inhibition is
significantly different behaviour to that observed for similar compounds (gem-
carbamoyl inhibitors and N-decyl-carbamoyl-CoA) with the highly homologous
bacterial enzyme MCR, where time-dependent inactivation was observed [35].
The reasons for this difference in behaviour are not entirely obvious.
11

Influence of the side-chain lipophilicity on inhibitor potency
AMACR is able to catalyse the ‘racemisation’ of substrates with structurally
diverse side-chains [1, 2]. The accommodation of these diverse structures is
thought to be a result of non-specific binding of the side-chain by hydrophobic
interactions to a methionine-rich surface [8]. Consistent with this, the MCR gem-
[31] and gem- carbamate [35] inhibitors show increased potency for compounds
with more hydrophobic alkyl side-chains. Consequently, we were interested to
investigate whether inhibitor potency was related to the lipophilicity of the
inhibitor side-chain. A plot of determined IC50 values vs. calculated LogP values
for the acyl-CoA inhibitor (Figure 3) showed that high LogP values tend to
produce low IC50 values. Systematic trends were observed within those
compounds containing aromatic side-chains (5 - 11), with potency positively
correlating with lipophilicity. It is also notable that 10 and 11, possessing a
single phenyl group side-chain are significantly less potent than compounds
containing more lipophilic side-chains. This consistent behaviour tends to
suggest that side-chain lipophilicity is driving potency, with the 2-hydroxy group
of 10 and 11 making a smaller contribution. Systematic trends were also
observed for inhibitors possessing alkyl side-chains (12 - 17), showing that
lipophilicity is also an important determinant of potency for this series.
The enolate analogue 4 has a potency increased by ~875-fold compared to
that predicted based on LogP values (measured IC50 = ~0.4 nM vs. ~350 nM
predicted for miLogP = 2.61) (Supplementary Information, Figure S1), showing
the effectiveness of the carbamate moiety in promoting inhibition. Acyl-CoA
esters do not comply with Lipinski guidelines and hence AMACR inhibitors are
delivered as their acid pro-drugs [28, 30, 32, 34] which are converted to the
12

acyl-CoA in vivo. Although 4 has very high potency, delivery as the pro-drug will
be challenging because carbamates readily decarboxylate to the corresponding
amine.
Conclusions
This is the first systematic SAR study of rationally designed AMACR inhibitors.
The study illustrates that extremely diverse side-chain structures which can be
accommodated. A minimal level of side-chain lipophilicity is required for efficient
binding. For compounds with aromatic side-chains, a single aromatic ring
results in modest inhibition whilst more than one aromatic ring or an aromatic
ring with alkyl substituents results in much more potent inhibition. Similarly, a
minimum of a 6-carbon alkyl chain appears to be required for reasonably
efficient inhibitor binding, with increased potency resulting from addition of
further –CH2– groups. Our results allow investigation of the contribution to
potency made by the individual structural elements of these inhibitors.
AMACR has attracted much attention as both a novel drug target and cancer
marker since its involvement in prostate cancer was reported [14, 19]. However,
exploitation of this discovery has been extremely limited, largely due to the
absence of a suitable assay with which to test inhibitor potency [33]. This study
shows that our novel colorimetric assay [32] allows quick and accurate
measurement of drug potency and detailed kinetic characterisation of inhibitors.
The systematic investigation of novel inhibitor SAR and therapeutic
development is now possible.
Materials and methods
Sources of materials
13

Chemicals were purchased from the Sigma-Aldrich Chemical Co. or Fisher
Scientific Ltd., unless otherwise stated and were used without further
purification. Reduced coenzyme A, tri-lithium salt was purchased from
Calbiochem. Acyl-CoA esters 4 [33], 5-9 [6], 10 and 11 [11] were synthesised
as previously described. Acyl-CoA esters 12 – 17 were purchased from Larodan
Lipids. Substrates 18-20 and product 22 were synthesised as described [7, 10].
Human recombinant AMACR 1A was expressed and purified and substrate 1
synthesised as previously described [32].
General experimental procedures
Thin layer chromatography was performed on Merck silica aluminium plates 60
(F254) and UV light, potassium permanganate or phosphomolybdic acid were
used for visualisation. Column chromatography was performed using Fisher
silica gel (particle size 35-70 micron). Purifications of acyl-CoA esters were
performed by solid phase extraction using Oasis HLB 6cc (200 mg) extraction
cartridges. Phosphate buffer was prepared from monobasic sodium phosphate
and NaOH at the required proportions. Citric acid buffer was prepared from
citric acid and NaOH at the required proportion for 0.8 M pH 4.0 buffer. The pH
of aqueous solutions was measured using a Corning 240 pH meter and Corning
general purpose combination electrode. The pH meter was calibrated using
Fisher Chemicals standard buffer solutions (pH 4.0 - phthalate, 7.0 - phosphate,
and 10.0 - borate) at either pH 7.0 and 10.0 or 7.0 and 4.0. Calibration and
measurements were carried out at ambient room temperature. IR spectra were
recorded on Perkin-Elmer RXI FTIR spectrometer instrument. NMR spectra
were recorded on Bruker Avance III 400.04 MHz or 500.13 MHz spectrometers
14

in D2O, (CD3)2SO or CDCl3 and the solvent was used as an internal standard.
Shifts are given in ppm and J values reported to  0.1 Hz. Multiplicities of NMR
signals are described as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet. Stock concentrations of acyl-CoA esters for assays were determined
using 1H NMR. Mass spectra were recorded by ESI TOF. High resolution mass
spectra were recorded in ES mode. Melting points were determined using a
Gallenkamp melting point apparatus and are uncorrected. Syntheses were
carried out at ambient temperature, unless otherwise specified. Solutions in
organic solvents were dried over anhydrous MgSO4 and the solvents were
evaporated under reduced pressure. Aqueous solutions for biological
experiments were prepared in Nanopure water of 18.2 MΩ.cm-1 quality and
were pH-adjusted with aq. HCl or NaOH.
(4S)-4-Benzyl-3-[(2S,3R)-3-hydroxy-2-methyldecanoyl]-1,3-oxazolidin-2-
one (28)
(S)-(+)-4-Benzyl-3-propanoyl-2-oxazolidinone 27 (3.0 g, 12.9 mmol) in CH2Cl2
(30.0 mL) was cooled to -78°C. Dibutylboron triflate (1.0 M in CH2Cl2, 13.0 mL,
12.9 mmol) and Pri2NEt (2.3 mL, 12.9 mmol) were added and the mixture was
stirred for 30 min before octanal (1.4 mL, 9.2 mmol) in CH2Cl2 (9.0 mL) was
added dropwise. The mixture was stirred at -78°C for further 30 min and then at
room temperature for 2 h. aq. Sodium phosphate buffer at pH 7.0 (100 mM, 100
mL) was added slowly to the reaction mixture. The organic layer was washed
[aq. HCl (1.0 M), aq. NaHCO3 (saturated), brine] and dried. Column
chromatography (Petroleum ether / EtOAc 10:1  6:1) gave 28 (2.53 g, 76%)
21
as a colourless oil. [α]D +51.4 (c 0.74 in CHCl3); IR max 3517 (OH), 1780
15

1
(C=O), 1692 (C=O) cm-1; H NMR (500.13 MHz; CDCl3) δH 7.26-7.07 (5 H, m,
Ar-H), 4.65-4.55 (1 H, m, 4-H), 4.16-4.05 (2 H, m, 5-H), 3.89-3.80 (1 H, m, 3’-H),
3.68 (1 H, qd J = 7.0, 3.0 Hz, 2’-H), 3.14 (1 H, dd, J = 13.0, 3.0 Hz, CHHAr),
2.92 (1 H, s, OH), 2.70 (1 H, dd, J = 13.0, 9.0 Hz, CHHAr), 1.50-1.10 (15 H, m,
13
6 × CH2 and CH3CH), 0.79 (3 H, t, J = 6.5 Hz, 10’-H3); C NMR (125.77 MHz,
CDCl3) δC177.23 (1’-C), 152.87 (2-C), 134.92 (Ar-C), 129.24 (Ar-C), 128.73 (Ar-
C), 127.18 (Ar-C), 71.35 (3’-C), 65.96 (5-C), 54.91 (4-C), 42.02 (2’-C), 37.53
(CHHAr), 33.77 (CH2), 31.62 (CH2), 29.35 (CH2), 29.05 (CH2), 25.84 (CH2),
22.45 (CH2), 13.90 (CH3CH), 10.31 (10’-C); ESI-MS m/z 384.2134 [M + Na]+
(C21H31NNaO4 requires 384.2151).
(4S)-4-Benzyl-3-[(2R,3S)-3-fluoro-2-methyldecanoyl]-1,3-oxazolidin-2-one
(29)
(Diethylamino)sulfur trifluoride (0.5 mL, 3.9 mmol) in CH2Cl2 (10.0 mL) was
added dropwise to 28 (1.4 g, 3.9 mmol) in CH2Cl2 (20.0 mL) at -78C and the
mixture was stirred for 2 h at this temperature. It was stirred for a further 2 h at
room temperature, before being quenched with water (50 mL). The organic
layer was washed (saturated aq. NaHCO3, brine). Column chromatography
21
(Petroleum ether / EtOAc 30:1 gave 29 (490 mg, 35%) as a colourless oil. [α]D
+49.2 (c 0.63 in CHCl3); IR max 1782 (C=O), 1700 (C=O) cm-1; 1H NMR (500.13
MHz; CDCl3) δH 7.27-7.06 (5 H, m, Ar-H), 4.76-4.57 (2 H, m, 4-H and 3’-H),
4.15-3.96 (3 H, m, 5-H and 2’-H), 3.17 (1 H, dd, J = 13.5, 3.5 Hz, CHHAr), 2.72
(1 H, dd, J = 13.5, 9.5 Hz, CHHAr), 1.70-1.14 (12 H, m, 6 × CH2), 1.10 (3 H, d, J
13
= 7.0 Hz, CH3CH), 0.80 (3 H, t, J = 6.5 Hz, 10’-H3); C NMR (125.77 MHz,
CDCl3) δC 174.37 (d, J = 2.8 Hz, 1’-C), 153.12 (2-C), 135.25 (Ar-C), 129.47 (Ar-
16

C), 128.93 (Ar-C), 127.37 (Ar-C), 94.89 (d, J = 169.8 Hz, 3’-C), 66.19 (5-C),
55.38 (4-C), 42.03 (d, J = 20.9 Hz, 2’-C), 37.85 (CHHAr), 32.04 (d, J = 20.9 Hz,
4’-CH2), 31.79 (CH2), 29.37 (CH2), 29.16 (CH2), 24.57 (d, J = 2.8 Hz, 5’-CH2),
22.64 (CH2), 14.10 (10’-C) and 13.62 (d, J = 8.5 Hz, 2’-CH3); δF (470 MHz,
CDCl3) -179.67; ESI-MS m/z 386.2126 [M + Na]+ (C21H30FNNaO3 requires
386.2107).
(2R,3S)-3-Fluoro-2-methyldecanoic acid (30)
H2O2 aq. [30% (w/w), 0.7 mL] and LiOH (62 mg, 2.6 mmol) were added to 29
(472 mg, 1.3 mmol) in THF (14 mL) at 0C. The mixture was stirred at ambient
temperature for 20 h before being quenched with sat. aq. sodium sulfite (14
mL). The THF was evaporated and the mixture was acidified with aq. HCl (1.0
M) to pH 1.0 and extracted with CH2Cl2 (100 mL). The organic phase was
washed with water (75 mL) and brine (75 mL). The combined organic layers
were dried and the solvent was evaporated. Column chromatography
(petroleum ether / EtOAc 5:1) gave 30 (245 mg, 92%) as a white solid. mp 64-
21
1
65°C; [α]D -7.8 (c 0.51 in CHCl3); IR max 2925 (OH), 1693 (C=O) cm-1; H
NMR (400.04 MHz; CDCl3) δH 10.89 (1 H, br s, OH), 4.58-4.50 (1 H, m, 3-H),
2.58-2.52 (1 H, m, 2-H), 1.75-1.23 (12 H, m, 6 × CH2), 1.20 (3 H, d, J = 7.2 Hz,
13
CH3CH), 0.88 (3 H, t, J = 6.8 Hz, 10-H3); C NMR (100.60 MHz, CDCl3) δC
180.02 (d, J = 5.6 Hz, C=O), 94.28 (d, J = 172.2 Hz, 3-C), 44.37 (d, J = 22.0 Hz,
2-C), 31.74 (d, J = 21.1 Hz, 4-CH2), 31.74 (CH2), 29.30 (CH2), 29.12 (CH2),
24.82 (d, J = 3.0 Hz, 5-CH2), 22.61 (CH2), 14.05 (10-C) and 12.56 (d, J = 6.6
19
Hz, 2-CH3); F NMR (470 MHz, CDCl3) δF -181.94; ESI-MS m/z 203.1449 [M -
H]- (C11H20FO2 requires 203.1447).
17

(2S,3S)-3-Fluoro-2-methyldecanoyl-CoA (21)
Carbonyldiimidazole (48 mg, 0.29 mmol) was added to (2R,3S)-3-fluoro-2-
methyldecanoic acid 30 (30.0 mg, 0.15 mmol) in anhydrous CH2Cl2 (2 mL) and
the mixture was stirred at ambient temperature for 1 h. CH2Cl2 (3 mL) was
added to the mixture, which was with water (5 × 2 mL) and brine (2 mL) and
dried. The solvent was evaporated to obtain the crude acyl-imidazole
intermediate. Aq. NaHCO3 (1.0 mL, 0.10 M) and tri-lithium CoA-SH (17.0 mg,
0.02 mmol) was added to the crude intermediate in THF (1.0 mL) and the
mixture was stirred at ambient temperature for 18 h. The THF was evaporated
and the residue was acidified to ca. pH 3 by addition of aq. HCl (1.0 M HCl).
The mixture was diluted with water (2.0 mL) and washed with EtOAc (3 × 3 mL).
The crude aqueous solution was freeze-dried and purified with solid-phase
1
extraction to give 21 (13.6 mg) as a white solid. H NMR (500 MHz, D2O) δH
8.63 (1 H, s, adenosine CH), 8.38 (1 H, s, adenosine CH), 6.16 (1 H, d, J = 6.0
Hz, adenosine CH), 4.30-4.10 (2 H, m, adenosine CH2), 3.97 (1 H, s,
adenosine CH), 3.85-3.72 (1 H, m, CoA(OCHH)), 3.58-3.47 (1 H, m,
CoA(OCHH)), 3.39 (2 H, t, J = 6.5 Hz, CoA(CH2)), 3.29 (2 H, t, J = 6.0 Hz,
CoA(CH2)), 3.08-2.90 (3 H, m, CoA(SCH2) and CHCH3), 2.36 (2 H, t, J = 6.5
Hz, CoA(CH2)), 1.68-1.44 (2 H, m, CHH and CHH), 1.39-1.12 (10 H, m, 5 ×
CH2), 1.07 (3 H, d, J = 7.0 Hz, CHCH3), 0.87 (3 H, s, CoA(CH3)), 0.81-0.70 (6
19
H, m, CH2CH3 and CoA(CH3)); F NMR (470 MHz) δF -181.11; ESI-MS m/z
475.6220 [M – 2 H]2- (C32H53FN7O17P3S requires 475.6208).
2,2-Dimethyl-5-octyl-1,3-dioxane-4,6-dione (31)
18

4-Dimethylaminopyridine (1.296 g, 10.6 mmol), N,N’-dicyclohexylcarbodiimide
(1.0 M in CH2Cl2, 11.1 mL, 11.1 mmol) and octanoic acid 32 (1.6 mL, 10 mmol,
1.0 eq.) were added to Meldrum’s acid 33 (1.455 g, 10.1 mmol) in dry CH2Cl2
(100 mL). The mixture was stirred at ambient temperature for 40 h. The
precipitate was removed by filtration and the filtrate was washed with aq.
KHSO4 (1.0 M, twice), water and brine, then dried. AcOH (6.0 mL) was added to
the filtrate. To this solution, NaBH4 (802 mg, 21.2 mmol) was added in portions
during 1 h and the mixture was stirred for an additional 20 h. The evaporation
residue was dissolved in Et2O (100 mL) and washed with water (twice) and
brine (100 mL). Drying and evaporation gave 31 (2.30 g, 89 %) as a white solid:
1
mp. 64-65°C (lit. [45] mp. 65-67C); H NMR (400.04 MHz, CDCl3): δH 3.49 (1
H, t, J = 4.9 Hz, dioxane 5-H), 2.14-2.04 (m, 2 H, octyl 1-H2), 1.77 (3 H, s, 2-
CH3), 1.75 (3 H, s, 2-CH3), 1.48-1.38 (2 H, m, octyl 3-H2), 1.37-1.19 (10 H, m,
13
octyl 4,5,6,7-H8), 0.86 (3 H, t, J = 7.0 Hz, octyl 8-H3); C NMR (125.77 MHz,
CDCl3): δC 165.6, 104.7, 46.1, 31.8, 29.5, 29.2, 29.1, 28.4, 26.9, 26.6, 26.5,
22.6, 14.1; IR (KBr disc) nmax 1752 (C=O) cm-1; ESI-MS m/z 279.1577 [M + Na]+
C14H24NaO4 requires 279.1572; 257.1732 [M + H]+ (C14H25O4 requires
257.1753).
Methyl 2-methylenedecanoate (34)
Compound 31 (2.10 g, 8.19 mmol) was dissolved in anhydrous MeOH (26 mL).
Eschenmoser's salt (3.79 g, 20.5 mmol, 2.5 eq.) was added and the mixture
was heated at reflux for 40 h. The solvent was evaporated. The residue, in Et2O
(100 mL), was washed with aq. KHSO4 (1.0 M), water and brine and was dried.
Evaporation and column chromatography (petroleum ether / EtOAc 10:1) gave
19

34 (1.10 g, 74 %) as a colourless oil (lit.[46] oil): IR (neat) nmax 1725 (C=O) cm-1;
1H NMR (400.04 MHz, CDCl3): δH 6.12-6.10 (1 H, m, =CHH), 5.52-5.49 (1 H, m,
=CHH), 3.73 (3 H, s, OCH3), 2.31-2.23 (2 H, m, 3-H2), 1.49-1.37 (2 H, m, 4-H2),
13
1.34-1.19 (10 H, m, 5,6,7,8,9-H10), 0.86 (3 H, t, J = 7.1 Hz, 10-H3); C NMR
(125.77 MHz, CDCl3): δC 167.8, 140.8, 124.4, 51.7 (two carbons), 31.8, 29.3,
29.2, 29.2, 28.3, 22.6, 14.0.
2-Methylenedecanoic acid (35)
Methyl ester 34 (870 mg, 4.39 mmol) was stirred at 50 °C for 2 h with aq. NaOH
(1.0 M, 15 mL, 15 mmol) in EtOH (57 mL), then cooled to ambient temperature
and acidified to pH ca. 3. The volatile solvents were evaporated. The residue, in
Et2O (50 mL), was washed with water (twice) and brine and was dried.
Evaporation and column chromatography (petroleum ether / EtOAc 3:1) gave
35 (650 mg, 80 %) as a colourless oil (lit. [47] oil): IR (neat) nmax 1696 (C=O)
cm-1;1H NMR (400.04 MHz, CDCl3): δH 12.05 (1 H, br s), 6.34-6.23 (1 H, m,
=CHH), 5.70-5.59 (1 H, m, =CHH), 2.32-2.25 (2 H, m, 3-H2), 1.54-1.40 (2 H, m,
13
4-H2), 1.36-1.19 (10 H, m, 5,6,7,8,9-H10), 0.87 (3 H, t, J = 7.0 Hz, 10-H3); C
NMR (125.77 MHz, CDCl3): δC 173.0, 140.2, 126.9, 31.8, 31.4, 29.3, 29.2, 29.2,
28.3, 22.6, 14.1; ESI-MS m/z 207.1349 [M + Na]+ C11H20NaO2 requires
207.1361); 183.1396 [M]- (C11H19O2 requires 183.1385).
2-Methylenedecanoyl-CoA (23)
Ethyl chloroformate (17 µL, 19 mg, 0.18 mmol) was added to 35 (33 mg, 0.18
mmol) and NEt3 (25 µL, 18 mg, 0.18 mmol) in anhydrous THF (2.0 mL) and the
mixture was stirred at ambient temperature for 1 h. CoA-SH tri-lithium salt [(28
mg, 0.04 mmol) in aq. KHCO3 (2.5%), 2.0 mL] was added and the mixture was
20

stirred at ambient temperature for 16 h. The mixture was acidified to pH ca. 3
with aq. HCl (1.0 M) and the THF was evaporated. The solution was washed
with EtOAc (5 × 3 mL) and the crude product was purified by SPE to give 23
1
(7.0 mg) as a colourless solid: H NMR (500.13 MHz, D2O): δH 8.47 (1 H, s),
8.16 (1 H, s), 6.07 (1 H, d, J = 7.0 Hz), 5.96 (1 H, s), 5.55 (1 H, s), 4.19-4.11 (1
H, m), 3.75 (1 H, dd, J = 9.8, 5.1 Hz), 3.46 (1 H, dd, J = 9.8, 4.8 Hz), 3.36 (2 H,
t, J = 6.5 Hz), 3.31-3.25 (2 H, m), 2.95 (2 H, t, J = 6.2 Hz), 2.33 (2 H, t, J = 6.6
Hz), 2.16 (2 H, t, J = 7.3 Hz), 1.30-1.22 (2 H, m), 1.18-1.06 (10 H, m), 0.79 (3 H,
s), 0.75 (3 H, t, J = 6.8 Hz), 0.65 (3 H, s); ESI-MS m/z 465.6152 [M – 2 H]2-
(C32H52N7O17P3S requires 465.6177).
Methyl 2-(2-heptyl-1,3-dioxolan-2-yl)propanoate (36)
Ethane-1,2-diol (5.27 g, 84.8 mmol) was added to 37 (606 mg, 2.83 mmol) in
dry CH2Cl2 (26 mL). Me3SiCl (1.84 g, 2.15 mL, 17.0 mmol) was added and the
mixture was stirred at ambient temperature for 3 d. Further ethane-1,2-diol (5.27
g) and Me3SiCl (1.84 g) were added and the mixture was stirred for a further 3
d. Water (25 mL) was added and the mixture was extracted with CH2Cl2 (3 × 20
mL). The combined organic layers were washed with water (5 × 70 mL) and
brine (70 mL) and were dried. Evaporation and column chromatography
(petroleum ether / EtOAc 10:1) gave 36 (600 mg, 82%) as a colourless oil: IR
1
(neat) nmax 1740 (C=O) cm-1; H NMR (400.04 MHz, CDCl3) δH 4.04-3.90 (4 H,
m, OCH2CH2O), 3.67 (3 H, s, OCH3), 2.83 (1 H, q, J = 7.2 Hz, CHCH3), 1.81-
1.62 (2 H, m, heptyl 1-H2), 1.42-1.20 (10 H, m, heptyl 2,3,4,5,6-H10), 1.18 (3 H,
d, J = 7.2 Hz, propanoate 3-H3), 0.86 (3 H, t, J = 7.1 Hz, heptyl 7-H3); 13C NMR
(125.77 MHz, CDCl3): δC 173.9, 111.3, 65.5, 65.4, 51.7, 46.7, 35.0, 31.7, 29.7,
21

29.2, 22.8, 22.6, 14.0, 12.5; ESI-MS m/z 281.1761 [M + Na]+ (C14H26NaO4
requires 281.1729), 259.1883 [M + H]+ (C14H27O4 requires 259.1909).
2-(2-Heptyl-1,3-dioxolan-2-yl)propanoic acid (38)
Aq. NaOH (1.0 M, 6.7 mL, 6.7 mmol) was stirred with 36 (345 mg, 1.34 mmol) in
MeOH (30 mL) at ambient temperature for 2 h, then at 65°C for 2 h. The
mixture was cooled to ambient temperature and citric acid buffer (0.8 M, pH 4.0,
15 mL) was added. The mixture was extracted with CH2Cl2 (3 × 30 mL). The
combined organic layers were washed with water and brine and dried.
Evaporation and column chromatography (petroleum ether / EtOAc 2:1) gave
38 (130 mg, 40 %) as a colourless oil. IR (neat) nmax 1709 (C=O) cm-1; 1H NMR
(400.04 MHz, CDCl3): δH 10.78 (1 H, br s, OH), 4.10-3.94 (4 H, m, OCH2CH2O),
2.83 (1 H, q, J = 7.2 Hz, CHCH3), 1.80-1.67 (2 H, m heptyl 1-H2), 1.43-1.20 (10
H, m, heptyl 2,3,4,5,6-H10), 1.23 (3 H, d, J = 7.2 Hz, CHCH3), 0.87 (3 H, t, J =
7.1 Hz, heptyl 7-H3); 13C NMR (125.77 MHz, CDCl3): δC 176.9, 111.4, 65.4,
65.4, 46.6, 34.7, 31.7, 29.6, 29.2, 22.8, 22.6, 14.0, 12.2; ESI-MS m/z 267.1549
[M + Na]+ (C13H24NaO4 requires 267.1572), 245.1729 [M + H]+ (C13H25O4
requires 245.1753).
2-(2-Heptyl-1,3-dioxolan-2-yl)propanoyl-CoA (24)
Using the same method as for 21, 24 was prepared from 38 (31 mg, 0.13 mmol,
1.0 eq.) by sequential treatment with N,N’-carbonyldiimidazole (41 mg, 0.25
mmol, 2.0 eq.) and CoA-SH tri-lithium salt (29 mg, 0.04 mmol, 0.3 eq.) to give
1
24 (10 mg) as a colourless solid: H NMR (500.13 MHz, D2O) δH 8.57 (1 H, s),
8.30 (1 H, s), 6.13 (1 H, d, J = 6.1 Hz), 4.21-4.14 (1 H, m), 3.99-3.88 (4 H, m),
22

3.78 (1 H, dd, J = 9.6, 4.5 Hz), 3.50 (1 H, dd, J = 9.7, 4.5 Hz), 3.39 (2 H, t, J =
6.6 Hz), 3.32-3.22 (2 H, m), 3.12 (1 H, 2 x q, J = 7.0 Hz; both epimers), 2.93 (2
H, t, J = 6.2 Hz), 2.36 (2 H, t, J = 6.6 Hz), 1.66-1.53 (2 H, m), 1.26-1.11 (8 H,
m), 1.08 (3 H, d, J = 7.0 Hz, CHCH3), 0.85 (3 H, s), 0.76 (3 H, t, J = 7.1 Hz,
CH2CH3), 0.72 (3 H, s); ESI-MS m/z 506.6219 [M + Na]2+ (C34H55N7NaO19P3S
requires 506.6192), 495.6301 [M + H]2+ (C34H56N7O19P3S requires 495.6282).
2R,S-2-Methyl-3-oxodecanoyl-CoA (25)
Compound 24 (7 mg, 7.1 µmol) was dissolved in water (0.8 mL) and acetone
(1.0 mL). Aq. HCl (1.0 M, 0.2 mL) was added and the reaction mixture was
stirred overnight. The acetone was evaporated and the residue freeze-dried to
give 25 (6 mg) as a colourless solid: 1H NMR (500.13 MHz, D2O): δH 8.49 (1 H,
s), 8.33 (1 H, s), 6.10 (1 H, d, J = 5.8 Hz), 4.20-4.10 (2 H, m), 3.99 (1 H, q, J =
7.0 Hz), 3.76 (1 H, dd, J = 9.7, 5.0 Hz), 3.38-3.28 (2 H, m), 3.22 (2 H, t, J = 6.3
Hz), 2.97-2.88 (2 H, m), 2.57-2.44 (2 H, m), 2.30 (2 H, t, J = 6.6 Hz), 1.41-1.32
(2 H, m), 1.16 (3 H, d, J = 7.0 Hz), 1.12-1.04 (8 H, m), 0.80 (3 H, s), 0.69 (3 H,
s), 0.68 (3 H, t, J = 7.0 Hz); ESI-MS m/z 473.6174 [M – 2 H]2- (C32H52N7O18P3S
requires 473.6151).
2S,3R-3-Hydroxy-2-methyldecanoyl-CoA (26)
2S,3R-3-Hydroxy-2-methyldecanoic acid 39 was synthesised [48] from the
Evan’s auxiliary protected acid 28 by hydrolysis with NaOH and H2O2. Following
the procedure used for 21, 2S,3R-3-hydroxy-2-methyldecanoyl-CoA 26 was
prepared from 2S,3R-3-hydroxy-2-methyldecanoic acid 39 (40 mg, 0.20 mmol,
1.0 eq.), CDI (64 mg, 0.40 mmol, 2.0 eq.) and CoA-SH tri-lithium salt (78 mg,
23

1
0.10 mmol, 0.5 eq.) to give 26 (12 mg) as a colourless solid: H NMR (500.13
MHz, D2O) δH 8.48 (1 H, s), 8.20 (1 H, s), 6.09 (1 H, d, J = 6.5 Hz), 4.22-4.10 (1
H, m), 3.81-3.70 (1 H, m), 3.48 (1 H, dd, J = 9.8, 4.8 Hz), 3.37 (2 H, t, J = 6.6
Hz), 3.25 (2 H, t, J = 6.3 Hz), 2.97-2.87 (2 H, m), 2.74-2.68 (1 H, m), 2.34 (2 H,
t, J = 6.5 Hz), 1.39-1.29 (2 H, m), 1.21-1.09 (10 H, m), 1.06 (3 H, d, J = 6.9 Hz,
CHCH3), 0.81 (3 H, s), 0.74 (3 H, t, J = 7.1 Hz, CH2CH3), 0.68 (3 H, s); ESI-MS
m/z [M – 2 H]2- 474.6217 (C32H54N7O18P3S requires 474.6229).
Evaluation of inhibition of AMACR by test compounds
Colorimetric assays were performed as previously described [32]. Dose
response curves were used to determine IC50 values for inhibitors. Enzyme (4 x
stock, 150 µL) and inhibitor at the appropriate concentration (4 x stock, 150 µL)
were incubated together in 96 well plates at ambient room temperature for 10
min. The sample was divided into three repeats of 100 µL before addition of
substrate (2 x stock, 3 x 100 µL; final concentration of 40 μM in the assay) and
monitored at 354 nm. Each 200 µL assay contained ca. 8 µg of total AMACR
protein (0.85 µM, assuming a molecular weight of 47,146.8 Da. with one active
site per monomer [7]). Final concentrations of inhibitor in the assay were 100,
33.3, 11.1, 3.7, 1.23, 0.411, 0.137 and 0.045 µM unless otherwise stated.
Positive controls contained enzyme and substrate 1 only and negative controls
buffer and substrate. Rates in ΔAbsorbance.min-1 were determined using Excel
and converted to nmol.min.-1mg-1 using the 2,4-dinitrophenoxide 2 extinction
coefficient (15,300 M-1 cm-1) [32] with the path-length (0.588 cm) determined by
the plate-reader. IC50 values were determined using reaction rate, with the data
fitted to a 4-parameter logistic using SigmaPlot 13 using Log10 inhibitor
concentration (in μM). In some cases 2-3% (v/v) DMSO was included in assays;
24

no significant change in enzyme activity was observed with DMSO
concentrations of up to 8% (v/v) [32]. Half-volume 96 well plates were used for
some inhibitors; identical IC50 values were obtained for standard inhibitors using
both types of microtitre plate. IC50 values [32] for N-dodecyl-N-
methylcarbamoyl-CoA
4
and ibuprofenoyl-CoA
5
were determined
contemporaneously with the inhibitors described in this study, with the same
batch of enzyme.
Computational analysis of potency of inhibition
Lipophilicity of acyl-CoA esters was assessed by calculation of miLogP values
using the molecular properties calculator (http://www.molinspiration.com/cgi-
bin/properties). miLogP values were calculated using molecular smiles obtained
from Chemdraw Professional 15. IC50 values (in nM) were plotted against the
obtained LogP value using SigmaPlot 13.
Acknowledgements
This work was funded by Prostate Cancer UK (S10-03 and PG14-009), a
University of Bath Overseas Research Studentship and a Biochemical Society
Summer Vacation studentship (2015). The authors are members of the Cancer
Research @ Bath (CR@B) network.
Disclosure statement
MJ, GLL, MDT, TJW and MDL are named inventors on patent applications on
the use and application of the colorimetric assay. The other authors report no
25

conflicts of interest. The authors alone are responsible for the content and
writing of this article.
Appendix A Supplementary Information
Supplementary Information contains dose-response curves and other biological
evaluation of inhibitors and spectroscopic characterisation data for compounds.
Supplementary Information can be found in the online version at:
References
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