Enzymatic interconversion of isomorphic fluorescent nucleosides: Adenosine deaminase transforms an adenosine analogue into an inosine analogue

Article abstract of DOI:10.1002/anie.201307064

Adenosine deaminase (ADA), a major enzyme involved in purine metabolism, converts an isomorphic fluorescent analogue of adenosine (^thA) into an isomorphic inosine analogue (^thI), which possesses distinct spectral features, allowing o

Full text of DOI:10.1002/anie.201307064

Angewandte
Chemie
DOI: 10.1002/anie.201307064
Fluorescent Nucleosides
Enzymatic Interconversion of Isomorphic Fluorescent Nucleosides:
Adenosine Deaminase Transforms an Adenosine Analogue into an
Inosine Analogue**
Renatus W. Sinkeldam, Lisa S. McCoy, Dongwon Shin, and Yitzhak Tor*
The lack of practically useful emission for the native nucleo-
sides A, C, G, and U(T)[1] prompted the development of
fluorescent nucleoside surrogates.[2] Such emissive analogues,
in conjunction with versatile and sensitive fluorescence
spectroscopy techniques,[3] have shown to be of great value
in the biophysical study of nucleic acids. Among the various
classes of fluorescent nucleosides there are the isomorphic
fluorescent nucleosides, characterized by an astute electronic
and structural resemblance to the native nucleosides.[2f,4]
Incorporation of such fluorescent probes is typically associ-
ated with minimal pairing, stacking, and higher structural
perturbation.[2f,4] While of great value within the context of
Figure 1. ADA-catalyzed interconversion of a) A to I and b) thA (1) to th
(2).
I
oligomeric structures and their biophysical applications, much
less is known about the function of such emissive analogues in
the “protein world”, where nucleosides and nucleotides
intricately interact with enzymes.[5]
In contrast to the use of fluorogenic enzyme substrates
and fluorophore precursors[6] as well as the enzymatic
unmasking or uncaging of established fluorophores for
biochemical assays or imaging applications,[7] no examples
exist, to our knowledge, where isomorphic fluorescent
nucleosides are transformed in enzymatically catalyzed
reactions to form new and distinct fluorophores. This is
likely due to the substrate specificity of many of the enzymes
responsible for metabolizing and utilizing such key nucleoside
and nucleotide cellular components.[8] Here we investigate the
utility of thA (1), which is a new emissive adenosine analogue
and a member of our fluorescent RNA alphabet,[9] for
monitoring a catabolically important deamination reaction
(Figure 1a) that is catalyzed by adenosine deaminase
(ADA).[10] The underlying hypothesis is that owing to its
similarity to its natural counterpart adenosine, thA will be
transformed to thI by ADA (Figure 1b). Since thA is emissive,
thI is likely to be fluorescent as well, yet their electronic
differences are expected to render the two chromophores
distinct. This, in principle, should allow one to monitor the
progression of the deamination reaction in real time by using
fluorescence spectroscopy, an impossible task with the natural
nucleobases. If successful, this can provide a new method for
exploring and identifying inhibitors of ADA, small molecules
of clinical utility as chemotherapeutic agents.[11] Here we
demonstrate the ability of ADA, which is a chief purine
metabolism enzyme with both biochemical and therapeutic
significance,[11] to convert thA into thI with steady-state and
kinetic analysis using absorption and emission spectroscopy.
We also demonstrate the utility of this sensitive transforma-
tion monitored by fluorescence spectroscopy for the real-time
detection of ADA inhibitors.
To be able to analytically and photophysically verify thI as
the product of the enzymatic deamination of thA, thI was
independently synthesized (Scheme 1).[9] Briefly, the synthe-
ses of thA (1) and thI (2) started from thiophene 3, which was
reacted with b-d-ribofuranose 1-acetate 2,4,5-tribenzoate in
the presence of SnCl4 to give intermediate 4 as a mixture of a-
and b-anomers. A subsequent tandem hydrolysis–annulation
reaction furnished the protected nucleoside 5. Following
thionylation and anomer resolution, the protected nucleoside
6 was obtained as the b-anomer exclusively. A final depro-
tection provided thA (1).[9] Conveniently, deprotection of
intermediate 5 followed by anomer resolution gave thI (2)
(Scheme 1). Starting from 3, thA (1) and thI (2) were
synthesized in an overall yield of 4.6% and 10.7%, respec-
tively. X-ray crystallography unequivocally shows their cor-
rect anomeric configuration. Overlaying their crystal struc-
tures with the reported structures of their natural counter-
parts A[12] and I[13] illustrates the truly isomorphic nature of
thA (1) and thI (2), respectively (Figure 2 and Figure S1.1 in
the Supporting Information).[14] Importantly, thA (1) adopts an
anti conformation having N-ribose (3’-endo) puckering, con-
formational features known to be preferred by ADA.[15]
[*] Dr. R. W. Sinkeldam, L. S. McCoy, Dr. D. Shin, Prof. Dr. Y. Tor
Chemistry and Biochemistry, University of California, San Diego
9500 Gilman Drive, La Jolla, CA 92093-0358 (USA)
E-mail: ytor@ucsd.edu
Homepage: http://torgroup.ucsd.edu
[**] We thank the National Institutes of Health for support (grant GM
069773), Dr. Su, Chemistry & Biochemistry, UCSD, for help with the
LC–MS experiments, and Drs. Rheingold and Moore at the X-ray
facility, UCSD.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201307064.
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Figure 3. a) Absorption (a) and emission (c) spectra of samples
prepared with different thI/thA ratios with a total concentration of 11 mm in
phosphate buffers of pH 7.4; b) correlation between inosine fraction and
Scheme 1. Synthesis of thA (1) and thI (2). Reagents and conditions:
a) b-d-ribofuranose 1-acetate 2,4,5-tribenzoate, SnCl4, MeNO2, 658C,
32%; b) 1.) 15% HCl (aq.) in MeOH, CHCl3, quantitative; 2.) formami-
dine·AcOH, EtOH, D, a-anomer 21%, b-anomer 39%; c) P2S5, Py,
43%; d) NH3/MeOH, 1008C, 86%; e) NH3/MeOH, 458C, 17 h (86%).
~
~
labs,max (blue ) and O.D. (orange ) at 340 nm; c) correlation between
*
*
lem,max (blue ) and PLint. at 391 nm (orange ); and d) enzymatic
deamination of thA (1) to thI (2) with ADA monitored in real-time by
absorption at 339 nm (blue) and emission at 391 nm (orange; excitation
at 318 nm). Reaction conditions: [thA]=11.7 mm, [ADA]=27 mUmLÀ1 in
phosphate buffer (50 mm, pH 7.4) kept at 258C for 1 h. The normalized
kinetic curves yield a t1/2 value of 11.5 min.
Table 1: Relevant spectroscopic properties of thA (1) and thI (2).
labs,max
[nm]
e
rel. Absint.
at 339 nm
lem,max
[nm]
rel. PLint.
at 391 nm
[mÀ1 cmÀ1
]
thA (1)
thI (2)
339
315
7.1ꢀ103
4.8ꢀ103
3.9
1.0
410
391
1.0
3.3
of the emission intensity, illustrating the higher fluorescent
quantum yield for thI (2) compared to that of thA (1).
To gain insight into the spectral changes, two correlation
plots have been constructed (Figure 3b,c). Both the shift in
absorption maximum and the drop in optical density show
a nearly linear dependence on the thA/thI ratio (Figure 3b).
Interestingly, both the emission wavelength and intensity
respond linearly to an increasing thI concentration in binary
mixtures with thA (Figure 3c). Clearly, absorption as well as
emission spectroscopy reveal significant spectral changes
suitable to study the enzymatic conversion of thA (1) to thI (2).
To investigate thA (1) as a substrate surrogate for
adenosine in ADA-mediated deamination reactions, absorp-
tion and emission were measured before and after its reaction
with ADA (Figure S2.2).[14] The spectra obtained after the
enzymatic treatment perfectly resemble the spectral charac-
teristics of thI (2; Figure 3).[14] A control experiment using
a fresh solution of thI (2) mixed with ADA, established that
the presence of the protein does not immediately affect the
spectral properties of the emissive nucleoside (Figure S2.2).[14]
Next, the enzymatic conversion of thA (1) to thI (2) was
followed in real time using absorption as well as fluorescence
spectroscopy. Absorption changes were followed at 339 nm
(labs,max of thA), and emission changes were followed at 391 nm
(lem,max of thI) upon excitation at 318 nm, which is their
Figure 2. Crystal structures of a) A, thA (1), and their nucleobase
overlay (RMS: 0.0383), and b) I, thI(2), and their nucleobase overlay
(RMS: 0.0330).[14,18]
To ensure that the photophysical characteristics of thA (1)
and thI (2) are distinguishable, binary mixtures containing
different ratios of the two nucleosides were examined by
absorption and fluorescence spectroscopy in phosphate buffer
at pH 7.4; these conditions are commonly used for enzymatic
deamination reactions (Figure 3a). The overlaid absorption
spectra of the mixtures show a distinct hypsochromic shift of
the absorption maximum from that of thA (1) at 339 nm to
that of thI (2) at 315 nm with a concomitant reduction in the
optical density at the lower energy transition > 300 nm
(Table 1). Upon excitation at 318 nm, their isosbestic point,
the recorded emission spectra also present a blue shift from
410 nm, the emission maximum of thA, to 391 nm, the
emission maximum of thI. In contrast to the lower optical
density, the increasing thI concentration results in an increase
2
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Table 2: Enzyme parameters for the deamination of thA (1) and
adenosine.
isosbestic point (Figure 3d). As expected, the absorption
spectra show a decrease (or an “OFF” signal) in the optical
density upon conversion of thA (1) to thI (2). By exploiting the
higher fluorescence intensity of thI compared to thA to follow
the enzymatic conversion, an intensification of the emission,
or “ON” signal, is obtained. Absorption and emission spectra
taken in the absence of ADA indicated that there is no
conversion without the enzyme under the experimental
conditions used.[14] LC–MS analysis unequivocally confirms
the enzymatic deamination of thA (1) and the identity of the
deamination product as thI (2; Figure S3.1).[14] ADA therefore
recognizes thA (1) as a valid substrate, thereby corroborating
the truly isomorphic nature of this fluorescent nucleoside
analogue, and quantitatively converts it into thI (2) in about
one hour.
Henri–Michaelis–Menten
Lineweaver–Burk
vmax
Km
R2
vmax
Km
R2
[mAbssÀ1
]
[mm]
[mAbssÀ1
]
[mm]
thA to th
A to I
I
5.15
417
29
0.91247
5.07
420
24
0.99530
0.99924
13.4[a]
0.99832 14.0[a]
[a] The Vmax is linearly dependent on [ADA]. To correct for the 10.58-fold
lower [ADA] used in the A-to-I experiment, the apparent Vmax (1.27 and
1.32 mAbssÀ1), obtained from Figure 4b and d, respectively, is multi-
plied by 10.58.
strate.[10,16] Nonetheless, as demonstrated below, the perfor-
mance of thA as a substrate surrogate and the enhanced and
distinct emission observed upon its ADA-mediated deami-
nation to thI, provide a robust foundation for a high-through-
put assay for inhibitor discovery.
The enzyme kinetic parameters Vmax (5.15 mAbssÀ1) and
Km (417 mm) of the deamination reactions were determined by
Henri–Michaelis–Menten analysis for both thA and A (Fig-
ure 4a,b, Table 2). The experimental results are alternatively
To illustrate the prospective for high-throughput screen-
ing and discovery of novel ADA inhibitors, which are of
particular importance for the treatment of certain leuke-
mias,[11] we developed a 96-well plate based assay, exploiting
the rapid and sensitive fluorescence monitoring of the
deamination reaction. The emission enhancement associated
with the conversion of thA (1) to thI (2) was monitored over 60
minutes with increasing concentrations of EHNA and pen-
tostatin, which are known ADA inhibitors (Figure 5a). The
inhibition of ADA is readily apparent even at low nm
concentrations (Figure 5b,c). Guanosine, used as a negative
control, had no impact on the deamination reaction up to
100 nm (Figure S5.1).[14] Despite the relatively rudimentary
nature of this high-throughput format, the data obtained can
be easily quantified. Plotting the percent inhibition at 60 min
against log[inhibitor] and applying a sigmoidal fit, yield IC50
values of (13.4 Æ 1.3) nm and (1.9 Æ 0.1) nm for EHNA and
pentostatin, respectively, thereby illustrating the established
higher potency of the latter (Figure 5d). We note that current
methods for identifying inhibitors typically rely on either
absorption spectroscopy (where other nucleoside-based
inhibitory motifs are likely to cause interference) or chroma-
tographic methods, which require relatively large quantities
and are not normally amenable for high-throughput formats.
To summarize, we have demonstrated the ability of an
isomorphic emissive adenosine analogue thA (1) to serve as
Figure 4. Henri–Michaelis–Menten plots for conversion of a) thA to th
and b) A to I. Lineweaver–Burk representations are given for conver-
sion of c) thA to thI and d) A to I. The experiments are performed in
*
I
triplicate and averaged ( ). The error bars reflect the standard error of
mean. For (a) and (b) the data points are fit to a Hill equation and for
panel (c) and (d) the data points are linearized (gray lines). Conditions
for (a,c): [thA]=22.6–942.6 mm, [ADA]=43.4 mUmLÀ1, and (b,d):
[A]=3.9–69.6 mm, [ADA]=4.1 mUmLÀ1. All experiments are performed
in phosphate buffer (50 mm, pH 7.4) at 258C.
a
viable substrate for ADA, a nucleoside-modifying
enzyme.[17] The enzymatic deamination process yields the
corresponding emissive inosine analogue thI (2), which
possesses distinct spectral features, allowing one to monitor
the enzyme-catalyzed reaction and its inhibition in real time.
To demonstrate its practical utility, we applied this process for
the fabrication of a high-throughput assay for the discovery
and biophysical evaluation of ADA inhibitors, which are key
agents for researchers and clinicians. This unique proof-of-
principle process, where the nucleobase core of a fluorescent
nucleoside analogue is enzymatically transformed into a dis-
tinctly emissive product, demonstrates a new facet for
isomorphic nucleoside analogues and expands their utility
landscape beyond their “natural” and typically explored
oligonucleotide environments.
plotted in a Lineweaver–Burk graph (Figure 4c,d, Table 2).
According to the Henri–Michaelis–Menten kinetics, Km
values of 417 and 29 mm are obtained for the thA-to-thI, and
A-to-I conversion, respectively. The lower conversion rate of
thA, compared to that of adenosine, appears to be due to the
lower affinity of the former to ADA. We speculate that the
higher Km values observed for thA are likely due to the
replacement of N7 in adenosine with a CH group in thA, as
previous structural analysis has shown contacts between side
chain residues and this heterocyclic position of the sub-
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Figure 5. a) Structures of ADA inhibitors EHNA and pentostatin;
b,c) conversion of thA (1) followed with fluorescence spectroscopy in
the presence of EHNA (b; black, blue, green, orange, red lines
represent [inhibitor]=0, 1, 10, 25, 100 nm, respectively) and pentosta-
tin (c; black, purple, blue, cyan, green lines represent [inhibitor]=0,
0.1, 1, 2.5, 10 nm, respectively. The gray lines represent the conversion
of thA (1) in the absence of ADA. The experiment is performed in
triplicate and error bars reflect the standard deviation. d) A semi-log
plot of inhibition in % at 60 min versus [inhibitor] in triplicate (data
points) and sigmoidal logistic fits performed in OriginPro (lines) for
2
*
*
EHNA ( , blue line, R :0.97155) and pentostatin ( , orange line,
R2:0.98437). The gray dashed lines visualize graphical determination of
the IC50 values. Actual values have been interpolated using the fit.
Assay conditions: [thA]=11.7 mm, [ADA]=27 mUmLÀ1, in phosphate
buffer (50 mm, pH 7.4) at 218C.
Received: August 11, 2013
Revised: October 6, 2013
Published online: && &&, &&&&
Keywords: adenosine deamination · fluorescence ·
.
high-throughput screening · kinetics · nucleosides
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Communications
Fluorescent Nucleosides
Adenosine deaminase (ADA), a major
enzyme involved in purine metabolism,
converts an isomorphic fluorescent ana-
logue of adenosine (thA) into an isomor-
phic inosine analogue (thI), which pos-
sesses distinct spectral features, allowing
one to monitor the enzyme-catalyzed
reaction and its inhibition in real time.
The utility of this sensitive fluorescence-
monitored transformation for the high-
throughput detection and analysis of ADA
inhibitors is demonstrated.
R. W. Sinkeldam, L. S. McCoy, D. Shin,
Y. Tor*
&&&& — &&&&
Enzymatic Interconversion of Isomorphic
Fluorescent Nucleosides: Adenosine
Deaminase Transforms an Adenosine
Analogue into an Inosine Analogue
6
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