Fluoromethylated derivatives of carnitine biosynthesis intermediates- synthesis and applications

Article abstract of DOI:10.1039/c3cc47581f

A convenient method for the synthesis of fluoromethylated carnitine biosynthesis intermediates, i.e. fluorinated derivatives of γ- butyrobetaine and trimethyllysine, is described. The fluoromethylated probes were useful in both in vitro and cell based ass

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Fluoromethylated derivatives of carnitine biosynthesis intermediates –
synthesis and applications
Anna M. Rydzika, Ivanhoe K. H. Leunga, Armin Thalhammera, Grazyna T. Kochanb§, Timothy D. W.
Claridgea, Christopher J. Schofielda*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A convenient method for the synthesis of fluoromethylated
carnitine biosynthesis intermediates, i.e. fluorinated
derivatives of γꢀbutyrobetaine and trimethyllysine, is
10 described. The fluoromethylated probes were useful in both
in vitro and in cell based assays employing 19F NMR and LCꢀ
MS analyses.
O
O
OH
A
+
+
TMLH
NMe3
NMe3
HO
HO
NH2
NH2
3-hydroxytrimethyllysine (TML-OH)
succinate
CO2
2OG
O2
trimethyllysine (TML)
2
(
)
1
(
)
O
O
OH
HTMLA
TMABA-DH
+
+
BBOX
NMe3
NMe3
HO
HO
-butyrobetaine (GBB)
succinate
CO2
(R)-carnitine (CAR)
2OG
O2
3
)
4
(
(
)
Carnitine, an essential metabolite in humans and other
eukaryotes, is vital for the transport of long chain fatty acid into
15 mitochondria1. In animals carnitine is biosynthetised from
trimethyllysine (TML) in four enzymeꢀcatalysed steps2ꢀ4. The
first and last steps in the pathway are catalysed by trimethyllysine
hydroxylase (TMLH) and γꢀbutyrobetaine hydroxylase (BBOX),
both of which belong to the 2ꢀoxoglutarate (2OG) dependent
20 oxygenase superfamily. TMLH and BBOX catalyse
hydroxylation of Nεꢀtrimethyllysine (TML, (1)) and γꢀ
butyrobetaine (GBB, (3)) respectively (Scheme 1A)5,6. The
inhibition of human carnitine biosynthesis reduces fatty acid
metabolism and has been targeted for the treatment of
25 cardiovascular disease7,8. With respect to efforts aimed at
understanding biological roles of 2OG dependent oxygenases in
carnitine metabolism9,10, we are interested in developing reagents
that enable assays for carnitine biosynthesis enzyme activities,
both in vitro11 and in more biologically representative systems.
30 Given that the monofluoromethyl group is similar in size to the
methyl group, we envisioned fluoromethylated GBB (9) and
TML (11) analogues as potentially useful tool compounds for
monitoring carnitine biosynthesis.
B
C
F
1. 1 eq. 1,2,3,4-
tetramethylbenzene
1 eq. Tf2O, Et2O
1. 2 eq CsF
PEG200/MeCN
80°C 1.5h
O
S
S
Cl
F
S+
0°C, 1h
2. 2.05 eg NBS/ H2O
2. xs. aq. NaBF4
0-10°C, 1h
-
6
)
(
5
BF4
(
)
7
)
(
1. (7), Cs2CO3
MeCN, 60°C
O
O
O
N+
F
N
HO
HO
O
2. LiOH
THF/H2O
9
) GBBNF
8
)
(
(
(
O
1. (7), Cs2CO3
MeCN, 60°C
N+
F
N
HO
2. LiOH
THF/H2O
3. HCl/Et2O
NHBoc
10
NH2
11
(
)
) TMLNF
50 Scheme 1 Synthesis of fluorinated analogues of carnitine biosynthesis
metabolites. A – The carnitine biosynthesis pathway in mammals. B –
Prepartaion of fluoromethylating agent (7). C – Synthesis of fluorinated
derivative of γꢀbutyrobetaine (GBBNF, (9)) and trimethylysine (TMLNF,
(11)). NBS – Nꢀbromosuccinimide; Tf2O – trifluoromethanesulfonic
55 anhydride.
that reacts under mild conditions17 with various nucleophiles,
including amines17,18. The reported preparation of (7) required the
use of liquefied CH2FCl, which can be inconvenient. We
therefore employed a route where chloromethylphenyl sulphide
60 (5) was converted to the corresponding fluoride by reaction with
caesium fluoride19 and then directly oxidised with aqueous N-
bromosuccinimide to yield fluoromethylsulphinyl benzene (6) – a
known intermediate in synthesis of fluoromethylating reagent (7)
(Scheme 1B). We then used (7) to efficiently prepare NꢀCH2F
65 modified derivatives of GBB (3) and TML (1) in 2 or 3 steps,
respectively, starting from appropriately protected N,Nꢀ
dimethylated precursors (Scheme 1C).
Organofluorine compounds have substantial applications in
35 chemistry and chemical biology; their applications include the
modification of physicochemical and conformational properties
of small molecules, and uses in radiomedicine as tracers12 or
labels for NMR studies13,14 19F NMR is emerging as a valuable
.
tool for studying biochemical processes in vitro and in vivo15. The
40 principle advantage of 19F NMR is that the absence of
endogenous fluorinated species in most living organisms
eliminates problem of background interference and signal
overlap. However, the use of 19F NMR is often limited by the
availability of appropriately labeled fluorinated compounds.
The fluoromethylated GBB analogue (GBBNF, (9)) was
evaluated in reaction with purified recombinant human BBOX
70 (hBBOX) in vitro. GBBNF (9) was found to be hBBOX substrate
as demonstrated by 1H NMR analyses (Fig. S1) undergoing
hydroxylation at the C3 position to give fluoromethylated
45 Various approaches for the incorporation of fluorine into small
molecules are available, but reagents for electrophilic
monofluoromethylation are limited16. Prakash et al. reported an
air and moisture stable electrophilic fluoromethylation reagent (7)
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Figure 2 Applications of GBBNF (9). A – Conversion of GBBNF to
CARNF by psBBOX can be followed by 19F NMR. B – 19F NMR based
35 assay enables determination of doseꢀresponse curves for potential
inhibitors, exemplified with the hBBOX inhibitor Mildronate. C – SDSꢀ
PAGE gel shows lysate samples from cells harvested at various times
after IPTG induction (lanes 4ꢀ8). Molecular weight marker (lane 1),
BBOX sample (lane 2) and uninduced cells harvested after 24h (lane 3)
40 are shown. BBOX activity in cell lysates can be quantified by 19F NMR.
Figure 1 Reactions catalysed by BBOX. GBBNF is a BBOX substrate
and undergoes hydroxylation in position analogous to GBB
hydroxylation. Time dependent product formation reveals that GBBNF is
a better substrate than GBBF.
a
5
Table 1 Kinetic properties of fluorinated GBB analogues
GBB (3) GBBF (12) GBBNF (9)
while in case of GBBF (12), the ratio of uncoupled:coupled
turnover was around two (Table 1, Fig. S5). Note that, as for
GBB (3), GBBF (12) displays substantial substrate inhibition
(apparently Ki values in micromolar range – see Table 1).
45 Interestingly, the extent of substrate inhibition by GBBNF (9)
was much less than for GBB (3) or GBBF (12) (Figure S6). The
mechanism of substrate inhibition of hBBOX is unknown, but is
of interest as it may be involved in regulating carnitine
biosynthesis in cells.
Hydroxylation
0.123
0.027
0.083
rate [ꢁM/s]
Succinate
formation
0.162
0.063
0.097
Ratio of succinate formation to
hydroxylation
1.1
2
1.3
KM [ꢁM]
4.2
0.83
24.5
19.8
0.14
135
16.6
0.30
ꢀ
Kinetic
kcat [1/s]
parameters
Ki [ꢁM]
carnitine analogue (CARNF) (14) (Fig. 1C; for NMR assignment
of the product see Supporting Information – Fig. S2ꢀS4), in an
analogous manner to hBBOX catalysed GBB hydroxylation (Fig.
50 hBBOX catalysed GBBNF (9) hydroxylation can be followed by
19F NMR, because the fluorine shift of the product is distinctively
different from a shift of the substrate (Fig. 2A). In these
compounds the 19F resonance appears with a 1:1:1 triplet fine
structure, not as a singlet. This structure arises from coupling of
55 the fluorine to the adjacent quadrupolar 14N nucleus (I= 1, 99.6%
abundance) and is apparent because the highly symmetrical
tetrahedral environment of the 14N centre suppresses rapid
quadrupolar relaxation that, in less symmetrical environments,
leads to loss of coupling fine structure. 19F NMR can also be
60 employed for IC50 measurements (Fig. 2B). We investigated
hBBOX inhibition by Mildronate7, which is an inhibitor and
competitive substrate for hBBOX9, 10. The obtained IC50 value
10 1A). We have reported the synthesis of another fluorinated
analogue of GBB (3S)ꢀfluoroꢀGBB (GBBF, (12)), Cꢀ3
–
hydroxylation of which leads to fluoride release concomitant with
ketone (13) formation (Fig. 1B)11. In NMR assays we did not
observed fluoride release in reaction of GBBNF (9) with hBBOX,
15 indicating that the fluoromethylated quaternary ammonium group
is stable under the assay conditions. Comparison of the initial
rates of hydroxylation of GBB (3), GBBNF (9) and GBBF (12)
by hBBOX revealed that GBBNF (9) is a better substrate than
GBBF (12) (Table 1, Fig. 1). The initial hydroxylation rate of
20 GBBNF (9) is ~65% of the initial hydroxylation rate of GBB (3),
while GBBF (12) is hydroxylated at ~20% of the initial rate of
GBB (3). The KM and kcat values also reveal that the properties of
GBBNF (9) as a substrate are closer to those of GBB (3) than
GBBF (12) (Table 1). Some 2OG dependent oxygenases catalyse
25 turnover of 2OG independent of substrate transformation
(uncoupled 2OG turnover)5 which can manifest with a poor
substrate20. We examined levels of uncoupled turnover when
GBB (3) and its fluorinated analogues were used, by comparing
rates of succinate formation to that of hydroxylation. The results
30 showed GBBNF (9) to be similar to GBB (3) (ratio of succinate
formation to hydroxylation 1.1 for GBB vs 1.3 for GBBNF),
1
(82 ꢁM) is similar to that obtained by H NMR (34 ꢁM) and
fluoride release (65 ꢁM) assays11. The differences likely reflect
65 the difference in assay conditions used. Thus, 19F NMR assay
employing GBBNF (9) as a substrate is useful for in vitro activity
studies. However, an advantage of the GBBNF (9) based system
is its ability to monitor specific hBBOX turnover without the
interference of any other nonꢀfluorinated components, making it
70 suitable for more biologically relevant assay conditions.
To test the turnover of fluorinated analogues in cells we used E.
coli BL21 cells producing a prokaryotic BBOX homologue from
2
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DOI: 10.1039/C3CC47581F
are also ubiquitous class of metabolites, present in all living
organisms23. In humans and animals Nꢀmethylated lysine,
45 arginine and nucleic acid compounds play crucial roles in
epigenetic regulation. We hope that the development of
appropriate fluorinated small molecules will enable work aimed
at understanding role of methylation in epigenetics.
Notes and references
50 a Department of Chemistry, University of Oxford, Chemistry Research
Laboratory, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom
b Structural Genomics Consortium, University of Oxford, Old Road
Campus Roosvelt Drive, Headington OX3 7DQ, United Kingdom
§Current address: Navarrabiomed-Fundacion Miguel Servet.
55 C/Irunlarrea 3. Complejo Hospitalario de Navarra. 31008 Pamplona.
Navarra. Spain.
Figure 3 Levels of CAR (4), GBB (3) and GBBNF (9) in HEK 293T
cells: Aꢀ control, B – treated with TML (1), C – treated with TMLNF
(11). Error bars represent standard deviation of n=3 samples.
5
Pseudomonas sp. AK1 (psBBOX). We observed that GBBNF (9)
is converted to CARNF (14) in crude cell lysates, providing 2OG
was added to the reaction mixture and that turnover level is
dependent on 2OG concentration (Fig. S7). The extent of
GBBNF (9) turnover is dependent on the amount of psBBOX
† Electronic Supplementary Information (ESI) available: synthesis
procedures,
assay
conditions,
NMR
assignments.
See
DOI: 10.1039/b000000x/
60 ‡ We thank the Wellcome Trust, Biotechnology and Biological Sciences
Research Council, European Union, Dulverton Trust (A.M.R.) and
Cancer Research UK (AT) for financial support.
10 present in the extracts (Fig. 2C, S8). Thus, 19F NMR can be used
to estimate psBBOX activities in cell lysates.
1. R. J. Wanders, P. Vreken, M. E. den Boer, F. A. Wijburg, A. H. van
In addition to the use as a label for 19F NMR studies, fluorine is a
convenient marker for small molecules in MS based studies. We
therefore investigated the metabolism of TMLNF (11) in human
15 kidney cells (HEK 293T cells) using LCꢀMS. HEK 293T cells
were grown either with or without TML (1) or TMLNF (11)
added to the growth media. Cells were harvested, lysed and
analysed for the carnitine related metabolites (Fig. 3), using
appropriate standards (Fig. S10). In none of the samples could
20 TML (1) or TMLNF (11) be detected. In the sample treated with
TML (1), elevated levels of GBB (3) were observed; indicating
TML (1) penetrates cell membranes and is converted to GBB (3).
Cells treated with TMLNF (11) contained lower amounts of GBB
(3) than controls and substantial levels of GBBNF (9), which can
25 only be formed from TMLNF (11), demonstrating TMLNF (11)
is carried through the first enzyme catalysed step of carnitine
biosynthesis. All of the samples contained similar levels of
carnitine (within error), but no CARNF (14) was observed in the
TMLNF (11) treated sample; this result may reflect the
30 differences in affinities of GBB (3) and GBBNF (11) to hBBOX,
as reflected in the in vitro by kinetic data (Table 1).
65
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