Development and application of a fluoride-detection-based fluorescence assay for γ-butyrobetaine hydroxylase.
ABSTRACT Fluoride assays for oxygenases: The 2-oxoglutarate-dependent oxygenase BBOX catalyses the final step in carnitine biosynthesis and is a medicinal chemistry target. We report that BBOX can hydroxylate fluorinated substrates analogues with subsequent release of a fluoride ion, thereby enabling an efficient fluorescence-based assay.
- SourceAvailable from: Anna Maria Rydzik[show abstract] [hide abstract]
ABSTRACT: 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 assays employing (19)F NMR and LC-MS analyses.Chemical Communications 11/2013; 50:1175-1177. · 6.38 Impact Factor
Development and Application of a Fluoride-Detection-Based Fluorescence
Assay for g-Butyrobetaine Hydroxylase
Anna M. Rydzik,[a]Ivanhoe K. H. Leung,[a]Grazyna T. Kochan,[b]Armin Thalhammer,[a]Udo Oppermann,[b]
Timothy D. W. Claridge,[a]and Christopher J. Schofield*[a]
enzyme superfamily, the members of which play a range of
important biological roles in animals, plants and bacteria. In
humans, their functions include oxygen sensing, collagen bio-
synthesis, fatty acid metabolism, and DNA/RNA repair and
modification. Several human 2OG-dependent hydroxylases and
histone demethylases are inhibition targets for therapeutic in-
tervention in diseases including myocardial infarction, anaemia,
inflammation and cancer.[1–6]
We and others are interested in the functional assignment
of 2OG-dependent oxygenases and are attempting to identify
selective small-molecule inhibitors. However, this work is limit-
ed by a lack of effective and cost-efficient assays. Reported
assays for 2OG-dependent oxygenases can be broadly divided
into those measuring cosubstrate (2OG, O2)/coproduct (succi-
nate, CO2) consumption/production and those measuring sub-
strate/product consumption/formation. The first type of assay
employ the detection of oxidised products by mass spectrome-
try,[11–13]NMR spectroscopy,[11,14]or fluorescence-/luminescence-
based methods.[15,16]However, these techniques are often not
suitable for high-throughput studies (e.g., NMR-based assays)
or require difficult to access reagents (e.g., antibodies) or ex-
pensive equipment (e.g., LC-MS, NMR).
g-Butyrobetaine hydroxylase (BBOX) catalyses the final step
in the biosynthesis of carnitine, which is essential in fatty acid
metabolism (Figure S1). BBOX is important for the develop-
ment of clinically useful 2OG oxygenase inhibitors because its
inhibition is likely to have physiological effects. Indeed 3-(2,2,2-
trimethylhydrazinium)propionate (THP), which is used clinically
to treat angina and myocardial infarction,[17–19]is proposed to
target BBOX in cells.[20,21]Hence, work on BBOX inhibition and
selectivity screens of inhibitors targeted towards other 2OG
oxygenases would both benefit from an efficient BBOX assay.
Various techniques, including NMR spectroscopy and assays
employing isotope-labelled 2OG or substrate, have been ap-
plied to study the kinetics and inhibition of BBOX;[22–28]howev-
er, these techniques are low throughput and not efficient for
The detection of fluoride ions is of interest because of their
pharmacological and environmental importance. Chemosen-
sors have attracted attention because they can enable the
fluorescence-based detection of fluoride ions.Here, we
report the synthesis and evaluation of fluorinated substrate
analogues for recombinant human BBOX.[30–34]Based on the re-
sults, we developed a fluorescence-based fluoride-release
assay that is suitable for high-throughput studies on BBOX and
other 2OG-dependent oxygenases.
Previous studies on the hypoxia-inducible factor prolyl hy-
droxylase domain containing enzyme 2 (PHD2) and a collagen
prolyl 4-hydroxylase (P4H) have reported that these oxygenas-
es can accept substrate analogues that are fluorinated on the
same carbon as that undergoing hydroxylation.[35,36]For both
P4H and PHD2, it was shown that peptides containing (4S)-4-
fluoroproline are hydroxylated to yield peptidyl-4-oxoproline
with the release of a fluoride ion (Figure S2A).We postulat-
ed that analogous fluoride-release reactions could not only
occur with peptide substrates, but could also be general for
other 2OG hydroxylases, including those employing small-mol-
ecule substrates. BBOX catalyses the hydroxylation of g-butyro-
betaine 1d (GBB or 4-(trimethylammonio)butanoic acid) to
give (R)-carnitine 1e ((3R)-3-hydroxy-4-(trimethylammonio)bu-
tanoate; Scheme 1 and Figure S1).[30–34,37,38]Therefore (3S)-3-
Scheme 1. Reactions catalysed by BBOX.
[a] A. M. Rydzik, I. K. H. Leung, A. Thalhammer, Dr. T. D. W. Claridge,
Prof. C. J. Schofield
Department of Chemistry, University of Oxford
12 Mansfield Road, Oxford, OX1 3TA (UK)
[b] Dr. G. T. Kochan, Prof. U. Oppermann
Structural Genomics Consortium, University of Oxford
Old Road Campus, Roosvelt Drive, Headington, OX3 7DQ (UK)
Supporting information for this article is available on the WWW under
ChemBioChem 2012, 13, 1559–1563? 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim
fluoro-4-(trimethylammonio)butanoate 1a ((S)-GBBF) was pre-
pared and analysed as a BBOX substrate. To evaluate the reac-
tion stereospecificity, the enantiomer of 1a ((R)-GBBF 1b) was
also prepared. The results demonstrate that the S stereoisomer
1a is the preferred substrate and can be used efficiently in
assays for BBOX activity.
The synthesis of b-fluorinated acid derivatives by direct fluo-
rodehydroxylation can be challenging because fluoride is
a strong base that induces elimination to give a,b-unsaturated
compounds.We investigated several routes based on 3-hy-
droxy-4-aminobutyric acid derivatives, but these were unsuc-
cessful (Scheme S1). We thus employed a route in which fluo-
rine was introduced prior to the carboxylic acid (Scheme 2).
(3S)-3-Fluoro-4-aminobutanoic acid (9a) and (3R)-3-fluoro-4-
aminobutanoic acid (9b) were synthesised according to
Deniau et al.,starting from (R)- and (S)-phenylalanine, re-
spectively. In this synthetic route, ring opening of the aziridini-
um intermediate by a fluoride ion to give regioisomers 5a and
5a’ was utilised. The desired potential substrates (S)-GBBF (1a)
and (R)-GBBF (1b) were obtained by N-methylation of 9a and
9b with an excess of iodomethane in the presence of potassi-
We evaluated both (S)-GBBF (1a) and (R)-GBBF (1b) as
human BBOX substrates, and (S)-GBBF was found to be a good
(trimethylammonio)butanoate (1c) with subsequent decarbox-
ylation leading to N,N,N-trimethylammonio acetone (Figures 1
fluorine-containing species formed in the reaction of (S)-GBBF
with BBOX are fluoride ions (Figure 1B). This observation is
consistent with previous studies in which BBOX was shown to
catalyse the hydroxylation of (S)-carnitine (1 f), an analogue of
(S)-GBBF, to give 3-oxo-4-(trimethylammonio)butanoate (1c)
and water (Scheme 1 and Figure S2B).However, the reaction
1H NMR studies revealed the formation of 3-oxo-4-
19F NMR analyses revealed that the only detectable
with (S)-GBBF (Figure S2C) was approximately twice as rapid as
the reaction with (S)-carnitine (Figure S4). (R)-GBBF was found
to be a poorer substrate (<5% turnover after 3 h under stan-
dard assay conditions as monitored by
troscopy—higher levels of (R)-GBBF turnover (~20%) are ob-
tained with a higher concentration of BBOX and prolonged in-
cubation; Figure S5).1H and19F NMR analyses determined that
the products of the reaction of (R)-GBBF were the same as for
the reaction of its enantiomer. Although we cannot entirely
rule out the possibility of BBOX-catalysed racemisation, the ob-
servation that (S)-GBBF (1a) is a BBOX substrate is consistent
with crystallographic studies revealing that the pro-R C?H
bond of GBB is directed towards the iron centre, which is re-
sponsible for hydroxylation to
through C?H bond oxidation,
the observation that (R)-GBBF is
a poor substrate implies that
this substrate can adopt a cata-
lytically productive arrangement
other than that observed in the
19F NMR spec-
Notably, although the reaction
products of (S)-GBBF and (R)-
GBBF as BBOX substrates are the
same as those of (S)- and (R)-car-
nitine (Figure S2C), the rate of
(S)-GBBF hydroxylation by BBOX
is significantly higher than that
of (S)-carnitine. This observation
suggested that fluoride release
from (S)-GBBF might enable the
development of efficient assays
for BBOX activity.
A fluoride-ion-selective electrode-based method has been
reported for use in P4H assays,but this method is not, at
least readily, suitable for inhibitor screening in plate format.
We therefore investigated the use of tert-butyldimethylsilyl-
protected fluorescein (2) for fluoride ion detection. When 2 is
subjected to increasing fluoride ion concentration, a propor-
tional increase in fluorescence is observed following the re-
moval of the silyl protective groups (Figure S6);this method
has been used to determine fluoride concentrations in tooth-
paste.We adapted this method to a multiwell system suita-
ble for high-throughput screening. The method was optimised
such that it can employ organic solvent–buffer mixtures and
low volumes and amounts of reagents (Figures S7 and S13).
Scheme 2. Synthesis of (3S)-3-fluoro-4-(trimethylammonio)butanoate (1a, (S)-GBBF). Reagents and conditions:
a) BnBr, K2CO3, EtOH, H2O, 62%; b) LiAlH4, THF, 76%; c) Deoxo-Fluor, CH2Cl2, 5a: 46%, 5a’: 10%; d) H2, Pd(C),
MeOH, 84%; e) Boc2O, DMAP, MeCN, 88%; f) RuCl3, NaIO4, CCl4/MeCN/H2O, 52%; g) CH2Cl2, HCl, 54%; h) CH3I,
K2CO3, MeOH, apparently quantitative (by NMR).
? 2012 Wiley-VCH Verlag GmbH&Co. KGaA, WeinheimChemBioChem 2012, 13, 1559–1563
Standard kinetic time-course and Michaelis–Menten kinetics
can be readily obtained from the fluorescence intensity by
using this method (Figure 2). The KMand Vmaxvalues obtained
for 2OG and (S)-GBBF (1a) by fluorescence are comparable to
those obtained by NMR spectroscopy under similar conditions
(Table 1, Figure 2B and C, and Figure S8). Although the Vmaxof
(S)-GBBF is reduced compared to that of GBB, the KMvalue of
(S)-GBBF is similar to that of GBB (Table 1 and Figure S8); this
suggests that the fluoride substituent does not substantially
affect the binding of (S)-GBBF in the substrate binding pocket
of BBOX. Previous kinetic studies have reported that the activi-
ty of human BBOX is stimulated by potassium ions and reduc-
ing agents.Our results reveal that human BBOX has the
same requirements when (S)-GBBF is used: potassium ions and
reducing agents such as ascorbate increase activity (Figure S9).
Thus, high-throughput and quantitative determination of
enzyme kinetics for BBOX appear feasible with fluorinated sub-
We investigated the applicability of this assay for inhibitor
screening by determining the IC50values of potential BBOX in-
hibitors. By monitoring the change in fluorescence intensity at
different inhibitor concentrations, sigmoidal curves for inhibi-
tion were observed (Figure 2D). We initially evaluated BBOX
(THP)and N-oxalylglycine (NOG), a 2OG analogue. We ob-
tained IC50values of 65 and 389 mm for THP and NOG, respec-
tively; these are consistent with the values obtained previously
by NMR spectroscopy using recombinant human BBOX
(Table 2 and Figure S11C and D).Aromatic inhibitors such as
pyridine 2,4-dicarboxylate (2,4-PDCA) and the bicyclic isoquino-
linyl inhibitor (BIQ),[1,44]which were being investigated as inhib-
itors of human prolyl hydroxylases, were then tested. Using
our assay, we found that they are also inhibitors of BBOX, with
IC50 values of 82 and 18 mm, respectively (Table 2 and Fig-
ure S11A and B).
We then investigated if the fluorescence-based fluoride de-
tection assay is applicable to other 2OG-dependent oxygenas-
es. It is reported that PHD2 accepts a fluorinated peptide ana-
logue as a substrate to yield the 4-oxoprolyl product (Fig-
ure S2).We thus carried out a reaction of PHD2 with an ap-
propriate peptide substrate bearing a cis-4-fluoroprolyl residue
at the canonical hydroxylation site. Fluoride ions were released
with PHD2 catalysis, and the amount of fluoride released could
be quantified by using the chemical probe (Figure S12). This
result demonstrates the utility of the fluorescence-based fluo-
ride detection assay for 2OG-dependent oxygenases with oli-
In conclusion we have developed a fluorescence assay
based on the detection of fluoride released from reactions of
fluorinated substrates with BBOX by the use of tert-butyldime-
thylsilyl-protected fluorescein. (S)-GBBF (1a) is a sufficiently
good substrate for BBOX, releasing fluoride when subjected to
the enzyme. Thus it can be used for kinetic and inhibition
studies. The assay was adapted to an efficient multiwall-plate
format by using fluoride detection by a chemical probe
method of desilylation and resulting in an increase in fluores-
cence. (S)-GBBF (1a) can be synthesised from the readily avail-
able (R)-phenylalanine in eight steps, and the sensitivity of the
method is such that many assays can be performed with
a small amount of material.
We used the assay as a proof of principle with THP and
broad-spectrum 2OG oxygenase inhibitors (NOG and 2,4-
PDCA) and the PHD2 inhibitor BIQ. Interestingly, we found that
BIQ is a better BBOX inhibitor than THP. Indeed, as found both
by our assay methodology and in previous work, THP is not
a particularly potent BBOX inhibitor with IC50values of 65 mm
(fluoride release assay), 34 mm (NMR-based assay), or 62 mm.
Figure 1. BBOX catalyses fluoride release from (S)-GBBF. A) The reaction of
(S)-GBBF with BBOX; B)19F NMR spectra of the reaction showing the release
of F?; C)1H NMR spectra of the corresponding reaction showing the forma-
tion of 3-oxo-4-(trimethylammonio)butanoate and N,N,N-trimethylammonio
acetone. The assay mixture included BBOX (100 nm), FeII(50 mm), 2OG
(500 mm), (S)-GBBF (200 mm), ascorbate (500 mm), KCl (80 mm) and Tris·D11
(50 mm, pH 7.5) in 90% H2O and 10% D2O.
ChemBioChem 2012, 13, 1559–1563 ? 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim
It should be noted that THP is
BBOX undergoing catalytic oxi-
dation to give multiple products,
proceeding via a Stevens-type
possible that the physiological
effects of THP are mediated by
targets other than BBOX, for ex-
ample, carnitine transporters.
If so, the metabolism of THP by
BBOX catalysis might, in fact,
Thus, the observation that (R)-
GBBF (1b) is a poor substrate for
BBOX, suggests that suitably flu-
orinated THP analogues might
NMR spectroscopy assay:
19F spectroscopy experiments were
conducted at 500 and 470 MHz,
respectively, by using a Bruker
Avance II spectrometer equipped
with a standard 5 mm z-gradient
1H/19F(13C) TXI probe. All experi-
ments were conducted at 298 K, and conventional 5 mm diameter
NMR tubes (Norell) were used.
Fluoride-release assays: Enzymatic reactions were performed with
the following final concentrations: 40 mm (S)-GBBF, 40 mm ammoni-
um iron(II) sulfate hexahydrate, 250 mm ascorbic acid disodium salt,
160 mm potassium chloride, 500 mm 2-oxoglutarate disodium salt,
100 nm BBOX and 50 mm Tris·HCl buffer (pH 7.5). FeIIsolution was
kept as a 100 mm stock solution in HCl (20 mm) and was diluted
freshly with Tris·HCl buffer before use. Reagents and BBOX were
mixed at room temperature in a 96-well plate, and the reaction
was initiated by the addition of 2OG. The reaction was quenched
by addition of 20% acetonitrile. The quenched reaction mixture
(20 mL) was then transferred to a separate solid black 96-well
round-bottom plate, and was incubated with bis(tert-butyldime-
thylsilyloxy)fluorescein (80 mL, 5 mm ) for 1 h at room temperature.
HEPES at (50 mL, 50 mm, pH 7.0) was then added, and the mixture
was incubated for a further 2 min prior to fluorescence being mea-
sured by using an EnVision Multilabel plate reader (PerkinElmer)
fitted with FITC FP 480/30 (480 nm, bandwidth 30 nm) and FITC FP
535/40 emission (535 nm, bandwidth 40 nm) filters. For each enzy-
matic reaction, a corresponding control containing all reagents but
no enzyme was also recorded. The normalised fluorescence signal
was defined as the observed fluorescence signal minus the control
Figure 2. Kinetic analyses of BBOX. A) Enzyme kinetics can be measured by using the fluoride-release assay. B) The
turnover of (S)-GBBF is dependent on the 2OG concentration. C) (S)-GBBF is a substrate for BBOX, but is an inhibi-
tor at high concentrations. D) IC50measurements can be made by using this assay; the curve obtained for BIQ in-
hibition is shown. For details on fitting and experimental conditions see Figures S10 and S11.
Table 1. Comparison of kinetic values for BBOX obtained with different assays (see also Figure S10).
(S)-GBBF and 2OG fluoride assay
(S)-GBBF and 2OG NMR assay
GBB and 2OG NMR assay
Table 2. IC50measurements (see also Figure S11).
? 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2012, 13, 1559–1563
BBOX expression and purification: BBOX was expressed and puri-
fied according to the protocol described by Leung et al.
Kinetic assays: Standard kinetic assays were conducted at 298 K in
solutions containing (S)-GBBF 40 mm), ammonium iron(II) sulfate
hexahydrate (40 mm), ascorbic acid disodium salt (250 mm), potassi-
um chloride (160 mm), 2-oxoglutarate disodium salt (500 mm),
BBOX (100 nm) and Tris·HCl buffer (50 mm, pH 7.5). Reactions were
initiated by the addition of 2OG. All measurements were repeated
Inhibition assays: Inhibition assays were conducted at 298 K in
solutions containing (S)-GBBF (40 mm), ammonium iron(II) sulfate
hexahydrate (40 mm), ascorbic acid disodium salt (250 mm), potassi-
um chloride (160 mm), 2-oxoglutarate disodium salt (500 mm),
BBOX (100 nm) and Tris·HCl buffer (50 mm, pH 7.5) and varying
concentrations of inhibitor. Reactions were initiated by 2OG addi-
tion, and quenched by the addition of 20% acetonitrile after
10 min. Response curves were fitted by using OriginPro 8.0 (Origin-
Lab, Northampton, MA, USA) and XLfit (ID Business Solution, Guild-
ford, UK). Each measurement was repeated four times.
The authors gratefully acknowledge support of this project by the
Engineering and Physical Sciences Research Council and the Bio-
technology and Biological Sciences Research Council. We thank
Naomi Rippengale for the synthesis and purification of the fluori-
nated peptide substrate for PHD2.
Keywords: carnitine · fatty acids · fluorescence · inhibition ·
metabolism · oxygenases
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Received: April 18, 2012
Published online on June 22, 2012
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