A novel fluorescent probe for NAD-consuming enzymes

Article abstract of DOI:10.1039/c1cc15499k

A novel, fluorescent NAD derivative is processed as substrate by three different NAD-consuming enzymes. The new probe has been used to monitor enzymatic activity in a continuous format by changes in fluorescence and, in one case, to directly visualize alt

Full text of DOI:10.1039/c1cc15499k

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A novel fluorescent probe for NAD-consuming enzymesw
Giulia Pergolizzi,abcd Julea N. Butt,c Richard P. Bowaterd and Gerd K. Wagner*b
Received 5th September 2011, Accepted 17th October 2011
DOI: 10.1039/c1cc15499k
A novel, fluorescent NAD derivative is processed as substrate by
three different NAD-consuming enzymes. The new probe has
been used to monitor enzymatic activity in a continuous format
by changes in fluorescence and, in one case, to directly visualize
alternative reaction pathways.
NAD-consuming enzymes have been identified as potent regulators
of key cellular functions such as calcium homeostasis and
DNA repair.3,4 Structural analogues of NAD are therefore
highly sought after as biochemical tools to study these enzymes
and processes.
To date, structural modifications of NAD have focused mostly
on the nicotinamide riboside and pyrophosphate moieties,5
while modifications on the adenine ring are relatively rare.6,7
One important example is 1,N6-etheno NAD 2 (e-NAD,
Fig. 1), a fluorescent NAD derivative that has found very
wide application in biochemical assays for NAD-consuming
enzymes.7,8 These assays are based on the fluorescence emission
of the 1,N6-etheno adenosine fragment, which in intact e-NAD
2 is internally quenched by the nicotinamide moiety.7,8
Chemical or enzymatic removal of the nicotinamide leads to
a fluorescence signal that can be used to follow this reaction.
Although introduced in the early 1970s, 2 and its analogues
have remained as the only fluorophores available for such
applications to date. However, 2 is an unsuitable substrate for
a number of important NAD-consuming enzymes,9 and its
chemical structure does not allow optimization of its enzymo-
logical or fluorescence properties.
Nicotinamide adenine dinucleotide 1 (NAD, Fig. 1) is an
essential cofactor found in all living cells. NAD-dependent
enzymes are involved in many fundamental biological processes,
including energy metabolism, cell signaling, gene expression
and epigenetic control.1 They can be grouped into redox
enzymes, which draw on the reversible reduction/oxidation
of the nicotinamide fragment,2 and NAD-consuming enzymes
such as glycohydrolases, pyrophosphatases and ADP-ribosyl
transferases, which cleave one of the high-energy bonds in
NAD (i.e. the pyrophosphate or N-glycosidic bond).3 Individual
Herein, we describe a new family of NAD fluorophores with
a fluorogenic substituent in position 8 of the adenine base and
tunable fluorescence properties (Fig. 1, 3a–e). We show that
the derivative with the most interesting fluorescence characteristics,
3e, is recognized as a substrate by three different NAD-consuming
enzymes and can be used to monitor enzyme activity in real time.
The new probe has unique practical utility as it allows, for the first
time, direct visualisation of the different reaction pathways of a
multi-functional ADP-ribosyl cyclase involved in cell signaling.
The 8-substituted NAD derivatives 3a–e were prepared from
the corresponding adenosine monophosphate (AMP) building
blocks 5a–e (Scheme 1).w The fluorogenic substituent was intro-
duced at position 8 of AMP under aqueous Suzuki–Miyaura
conditions,6c,10 followed by MnCl2-catalyzed pyrophosphate
bond formation.11 Depending on the nature of the 8-substituent,
the NAD derivatives 3a–e showed variable fluorescence character-
istics (Fig. S1 and Table S1, ESIw) with particularly pronounced
differences in the fluorescence quantum yields F of individual
analogues (Table 1). Interestingly, fluorescence in this series was
pH-dependent, with a sharp increase in emission between pH 3–6
for all dinucleotides apart from 3d (Fig. S2, ESIw). Above pH 6
fluorescence remained stable, which is important with a view
towards potential biological applications, as the pH working
range of several NAD-dependent enzymes is between pH 7–8.
Fig. 1 Chemical structures of natural NAD 1, 1,N6-ethenoadenine
dinucleotide 2, and NAD derivatives 3a–e developed in this study.
a School of Pharmacy, University of East Anglia,
Norwich Research Park, Norwich, NR4 7TJ, UK
b King’s College London, School of Biomedical Sciences, Institute of
Pharmaceutical Science, Franklin-Wilkins Building, 150 Stamford
Street, London, SE1 9NH, UK. E-mail: gerd.wagner@kcl.ac.uk;
Fax: +44 (0)20 7848 4045; Tel: +44 (0)20 7848 4747
c School of Chemistry, University of East Anglia,
Norwich Research Park, Norwich, NR4 7TJ, UK
d School of Biological Sciences, University of East Anglia,
Norwich Research Park, Norwich, NR4 7TJ, UK
w Electronic supplementary information (ESI) available: Detailed
experimental procedures, spectroscopic data, protocols for enzyme
assays, additional tables, figures and schemes. See DOI: 10.1039/
c1cc15499k
c
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Scheme 1 Reagents and conditions: (i) Na2PdCl4, TPPTS, R-B(OH)2,
K2CO3, H2O, D, 1–24 h; (ii) morpholine, dipyridyldisulfide, Ph3P, DMSO,
rt, 2 h; (iii) b-NMN, dry MgSO4, MnCl2 0.2 M in formamide, rt, 24 h.
Fig. 2 Fluorimetric nucleotide pyrophosphatase assay of 3e. Conditions:
0.007 U mLÀ1 enzyme, 3e (0.2–6.5 mM) in 10 mM MgCl2, 50 mM Tris/
HCl (pH 8), 30 1C, lex = 300 nm, lem = 410 nm, gain 15%. Enzyme
addition at 400 s. Positive control (3 mM 5e) and negative control
(buffer) included.
Table 1 Quantum yields of NAD (3) and AMP (5) derivatives
in water
Cmpd
R
F3
F5
F5/F3
2
Not applicable
3-Pyridinyl
Phenyl
0.028–0.09a 0.56–0.59a 6–20
ab
bb
cb
db
eb
0.003
0.207
0.003
0.669
0.888
0.021
0.231
1
B3
B6
7
consumption of 3e and the formation of the respective reaction
product for each enzyme by HPLC, we found that, pleasingly,
all three enzymes use 3e as a substrate (Fig. S7–S9, ESIw).
Next, we investigated if the progress of these enzyme reactions
could be monitored by fluorimetry. As expected, the consumption
of 3e by NPP led to a progressive increase in fluorescence
which could be followed in a continuous assay format (Fig. 2).
Similar results were obtained with NGH (Fig. S10, ESIw),
although the limited aqueous solubility of this membrane-bound
enzyme13 complicated the in situ monitoring of the reaction
and necessitated an additional pipetting step.
3-(Boc-aminomethyl)phenyl 0.150
0.003
0.005
2,4-DMT-pyrimidinyl
2-Pyrrolyl
B46
a
b
Values from ref. 7, 8 and 12b. 3 or 5, for scaffolds see Scheme 1.
Importantly, when we compared the fluorescence emission
of AMP/NAD pairs with the same 8-substituent, we found
that F was generally markedly higher for the AMP derivative
(Table 1). As previously described for 2,7,8 this observation
can be attributed to intramolecular fluorescence quenching in
the case of the intact NAD derivatives due to the interaction of
the nicotinamide and substituted adenine rings. To test this
hypothesis, we subjected NAD derivatives 3a–e to chemical
hydrolysis under basic conditions, and followed these reactions
by fluorimetry. As expected, fluorescence emission increased
significantly upon hydrolytic removal of the nicotinamide ring
(Fig. S3 and S4, ESIw), due to the abolition of the internal
quenching effect. The hydrolytic degradation into the corres-
ponding ADPR (adenosine diphosphate ribose) and nicotinamide
was also confirmed in HPLC control experiments with 3e
(Fig. S5, ESIw). This NAD derivative showed by far the strongest
fluorescence increase, as expected from the considerable differ-
ence in quantum yields between 3e and 5e (Table 1). Significantly,
while for most 8-substituted AMP/NAD pairs the fluorescence
emission ratio F5/F3 is in the same range as for e-AMP/e-NAD,
for 5e/3e this ratio is up to 7-fold greater than reported values for
Fe-AMP/Fe-NAD. We speculated that this could allow for a more
sensitive fluorescence-based assay of NAD-consuming enzymes
than is possible with 2. 3e would be particularly attractive for
such applications as its lex and lem do not interfere with the
intrinsic fluorescence of most proteins (Fig. S6, ESIw).
With NPP, we used the continuous assay for the enzymo-
logical characterization of 3e. After confirming the linearity of
the fluorescence response across a range of concentrations for
both 3e and its enzymatic product 5e, we found that the initial
reaction velocities were proportional to the enzyme concen-
tration (Fig. S11 and S12, ESIw). Having established a suitable
enzyme/substrate ratio, the substrate concentration was varied
to generate a saturation curve for the determination of the Km
value of 3e. The variation of the initial velocity with the
concentration of 3e was well described by the Michaelis–Menten
equation and Hanes–Woolf plots, resulting in Km of 6.22 Æ
3.08 mM and vmax of 0.0530 Æ 0.0140 mmol minÀ1 mgÀ1
(Fig. S13, ESIw). This Km value for 3e is comparable to literature
values for NAD with several pyrophosphatases.12 In contrast,
the frequently used fluorophore 2 and its derivative e-PdAD have
often higher Km values (Table S2, ESIw).12 This may be due to
interference of the etheno bridge in 2 with hydrogen bonding of
the pyrimidine ring at the cofactor binding site. Substitution in
position 8 in 3e, on the other hand, does not perturb hydrogen
bond formation. These results therefore suggest that, at least for
NPP, the new fluorophore 3e is a closer model of the natural
cofactor NAD than 2.
In order to assess the suitability of 3e as a non-natural substrate
for NAD-consuming enzymes, we chose three commercially
available enzymes—nucleotide pyrophosphatase (NPP) from
Crotalus adamanteus venom,12 NAD-glycohydrolase (NGH)
from porcine brain,13 and ADP-ribosyl cyclase (ADPRC)
from Aplysia californica14—which catalyse different covalent
modifications of NAD (Scheme S1, ESIw). Following the
Finally, we studied the behavior of 3e towards the multi-
functional enzyme ADP-ribosyl cyclase (ADPRC).14 ADPRC
catalyzes primarily the cyclization of NAD at the N1 position to
give N1-cADPR (cyclic adenosine diphosphate ribose), but also
the hydrolysis of N1-cADPR into ADPR (Scheme S2, ESIw).14a
N1-cADPR is an important 2nd messenger and mediator of
c
12656 Chem. Commun., 2011, 47, 12655–12657
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properties, and the breadth of its applicability is the subject
of ongoing studies.
We thank the UEA for a studentship, the UEA and King’s
College London for financial support, the EPSRC National
Mass Spectrometry Service Centre, Swansea, for the recording
of mass spectra, and Ms Sarah Zaehringer for expert synthetic
assistance.
Notes and references
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Chem. Commun., 2011, 47, 12655–12657 12657