Synthesis of a hydrolytically stable, fluorescent-labeled ATP analog as a tool for probing adenylyl cyclases

Article abstract of DOI:10.1016/j.bmcl.2009.10.125

(2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxy-4-[2-(methylamino)benz amido]tetrahydrofuran-2-yl-methoxy[(hydroxy)phosphoryloxy][(hydroxy)phos phoryl]dichloromethylphosphonic acid was synthesized as a chemically and metabolically stable analog of ATP substituted with a fluorescent methylanthranoyl (MANT) residue. The compound is intended for studying the binding site and function of adenylyl cyclases (ACs), which was exemplified by studying its interaction with Bacillus anthracis edema factor (EF) AC exotoxin.

Full text of DOI:10.1016/j.bmcl.2009.10.125

Bioorganic & Medicinal Chemistry Letters 20 (2010) 232–235
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of a hydrolytically stable, fluorescent-labeled ATP analog as a tool
for probing adenylyl cyclases
Thomas Emmrich a, Ali El-Tayeb b, , Hesham Taha c, Roland Seifert c, Christa E. Müller b, Andreas Link a,
*
a Institute of Pharmacy, Ernst-Moritz-Arndt-University, Friedrich-Ludwig-Jahn-Straße 17, D-17487 Greifswald, Germany
b PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
c Department of Pharmacology and Toxicology, University of Regensburg, Universitätsstraße 31, 93040 Regensburg/Institute of Pharmacology,
Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, 30625 Hannover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
(2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxy-4-[2-(methylamino)benzamido]tetrahydrofuran-2-yl
-methoxy[(hydroxy)phosphoryloxy][(hydroxy)phosphoryl]dichloromethylphosphonic acid was synthe-
sized as a chemically and metabolically stable analog of ATP substituted with a fluorescent methylanthranoyl
(MANT) residue. The compound is intended for studying the binding site and function of adenylyl cyclases
(ACs), whichwasexemplifiedbystudying itsinteractionwithBacillusanthracisedemafactor(EF)ACexotoxin.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 27 October 2009
Accepted 28 October 2009
Available online 30 October 2009
Keywords:
Adenylyl cyclase
MANT-nucleotide
Fluorescent-label
Triphosphate analog
Adenylyl cyclases (ACs) play a key role in signal transduction
and various isoforms represent attractive drug targets, for exam-
ple, for male contraceptives1 or for therapeutic intervention in car-
diovascular diseases and ageing.2,3 In addition, several AC toxins
are involved in the pathogenesis of bacterial diseases. A prominent
example is the exotoxin edema factor (EF) of Bacillus anthracis, a
calmodulin-dependent AC which is part of the bacterium’s deadly
triad that leads to the clinical symptoms of anthrax.4
ACs catalyze the conversion of ATP into the second messenger
cAMP and thus, ATP analogs have been used as experimental tools
to study the binding sites of these enzymes. Specifically, N-methyl-
anthranoyl-(MANT-) substituted nucleotides5,6 have been exten-
However, the use of mixtures of two structurally distinct nucleo-
tide derivatives is not optimal for the evaluation of isoform speci-
ficity.11 Derivatives with two MANT groups attached to the 20- as
well as to the 30-OH group would circumvent this problem but
have shown a strong reduction in inhibitory potency in the case
of related inosine derivatives.10 Another approach includes the
use of mono-deoxygenated sugars (vide infra), where intramolecu-
lar acyl migration is excluded, albeit with the concomitant loss of a
potential hydrogen bond donor. Furthermore, these carboxylic acid
esters are not resistant towards hydrolysis which may complicate
the interpretation of the obtained results.12 In addition, nucleoside
triphosphates could readily be cleaved by ecto-nucleotidases,
including nucleoside triphosphate diphosphohydrolases (NTPDas-
es)13 and alkaline phosphatases.14 Consequently, we designed, syn-
thesized and characterized a MANT-ATP analog that is stabilized
with respect to hydrolysis in aqueous media and in biological
fluids.
The notorious instability of carboxylic acid esters in biological
fluids can be overcome by various strategies. An obvious choice
based on our preceding work on 20-amido-substituted adenosine
derivatives as antiparasitic drugs15 was the exchange of the 20-
OH group by an NH2-group enabling the formation of comparably
stable carboxylic acid amides. The amides thus obtained are no
longer prone to intramolecular acyl migration or fast hydrolysis
by water or esterases present in many in vitro or in vivo assays.
Moreover, it can be excluded that the fluorescent MANT-moiety
will be transferred to nucleophilic groups of surrounding biomole-
cules, in analogy to an intermolecular acyl migration. Furthermore,
sively studied. The N-methylanthranoyl group inserts into
a
hydrophobic pocket in the catalytic centers of structurally diverse
ACs, thereby preventing transition of AC from the catalytically
inactive closed to the catalytically active open conformation.7,8
Moreover, MANT-nucleotides can be used as fluorescent tools to
analyze conformational changes in ACs with high sensitivity and
temporal resolution.7–10 However, known MANT-nucleotides pre-
pared via straightforward acylation of ATP with an activated
N-methylanthranilic acid derivative are associated with problems
arising from the ester bond formed. The resulting 20- and 30-acyl-
ated ATP derivatives are obtained as mixtures of two isomers 1a
and 1b that cannot be separated due to acyl migration (Fig. 1).5
* Corresponding author.
E-mail address: link@pharmazie.uni-greifswald.de (A. Link).
A. El-Tayeb is on leave from the University of Al-Azhar, Assiut, Egypt.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.125

T. Emmrich et al. / Bioorg. Med. Chem. Lett. 20 (2010) 232–235
233
O
P
O
P
O
P
O
P
O
P
O
P
N
N
HO
O
O
O
HO
O
O
O
NH2
NH2
OH OH
OH OH
OH
OH
O
O
N
N
N
N
N
H
N
O
O
O
O
H
O
O
NH
CH3
1a
HN
H3C
1b
Figure 1. Intramolecular acyl migration of classical MANT-substituted fluorescent ATP derivatives.
the hydrogen bond donor properties of the residual NH of the car-
boxylic acid amide closely imitate the 20-OH-group of ATP. In con-
trast, carboxylic acid esters, such as 1b only possess an H-bond
acceptor at the same position (20-oxygen atom). The feasibility of
such an approach was previously demonstrated by amide 2, which
was synthesized as a stable substrate analog of aminoacyl-tRNA
synthetases and contributed to the characterization of the binding
site and the elucidation of the function of the enzymes (Fig. 2).16
For the parallel preparation of arrays of 20-amido-20-deoxyaden-
osine derivatives as potentially bioactive nucleosides we employed
a polymer-assisted acylation protocol making use of Kenner’s
safety-catch linker. In the present study, where only a single deriv-
ative had to be synthesized on a multi-mg scale, we decided to pre-
pare the desired intermediate 20-MANT-amino-20-deoxyadenosine
(4) in solution for practical reasons.
Reaction of the fluorescent-labeled nucleoside 4 with phospho-
rus oxychloride in trimethyl phosphate afforded the reactive 50-
dichlorophosphate intermediate 5 which was directly reacted with
dichloromethylenediphosphonic acid in N,N-dimethylformamide
(DMF) to form the cyclic intermediate 6. Hydrolysis of 6 with trieth-
ylammonium hydrogen carbonate afforded the corresponding tri-
phosphate analog 7 (Scheme 1).22–24 The synthesized nucleotide 7
was purified by anion exchange chromatography using FPLC (ÄKTA
FPLC, Amersham Biosciences, equipped with an XK 26 mm/20 cm
length column filled with Sephadex DEAE A-25 gel) to remove the
monophosphate 8 obtained as a side-product. In a second step, the
productwas furtherpurifiedbyreversedphaseHPLCtoremoveinor-
ganic impurities such as inorganic phosphates and buffer compo-
nents. The structure of the synthesized nucleotide was confirmed
by 1H and 31P NMR spectra, in addition to LC/ESI mass spectra ob-
tained both in positive and negative mode.22,24
For this purpose we adapted a synthetic approach published by
Sauer et al.17, in which silica-supported ion exchange reagents
To demonstrate the suitability of compound 7 as a new tool to
probe the active centers of ACs, we initially investigated the inter-
action of 7 with B. anthracis EF. The inhibition of EF3, the isolated
catalytical domain of EF, by 7 was determined with a radiometric
were employed to facilitate the work-up after hydroxybenzotria-
0
zole-catalyzed, N2 -selective acylation at the ribose moiety.
Taking advantage of a microwave-assisted heating protocol, 4
was obtained from commercially available 20-amino-20-deoxyaden-
osine 3, in a moderate yield of 42.6 % (Scheme 1).18 Care must be
taken, as the product is susceptible to degradation by daylight
irradiation. For the preparation of nucleoside triphosphates and
analogues there are numerous protocols available in the literature.19
In our hands, the method originally established by Yoshikawa et al.20
performed best and was adapted with modifications.21 To further
enhance the stability of the targeted nucleotide probe both towards
chemical and enzyme-catalyzed hydrolysis we prepared a dichloro-
methylene analogue (7) of the MANT-ATP derivative. In contrast to
the corresponding triphosphate this structure cannot be cleaved
by NTPDases or phosphatases due to the replacement of the terminal
phosphate by a phosphonate. Therefore it is more suitable for both
in vitro and especially in vivo assays. The selected bioisosteric
replacement of the oxygen bridge promised to be particularly suit-
able due to its electronic properties, which have an impact on the
assay using [
8.98 0.8 M.
a-
32P]ATP as substrate, thus yielding a Ki-value of
l
This is approximately 9–90 times weaker compared to recently
assayed congeners of this class of compounds, for example, MANT-
ATP (0.98 0.08 l
M) or MANT-CTP (0.10 0.008), respectively.9
Furthermore, we studied the direct interaction of 7 with EF3 in
FRET-experiments. At an excitation wavelength of 280 nm, the
ubiquitous aromatic amino acids tryptophan and tyrosine are ex-
cited, thus emitting light at 350 nm,7–10 which can then excite
the MANT-moiety of 7, provided a close proximity of donor and
acceptor moiety. This radiation-free transfer of energy results in
an increased fluorescence of the MANT-residue at 420–450 nm. A
detailed description of the assay was recently published by Taha
et al.9
20-MANT-AppCCl2p 7 (300 nM) was excited at kex 350 nm, and
emission was recorded from kem 380–550 nm. In the presence of
Tris buffer and in the absence of the organic solvent DMSO, com-
pound 7 showed only a small fluorescence signal (Fig. 3A) as was
the case for the reference nucleotide 20-MANT-30-d-ATP (Fig. 3B).
The addition of DMSO at increasing concentrations augmented
the fluorescence of both nucleotides in a concentration-dependent
manner. The maximum increase in fluorescence with 20-MANT-30-
d-ATP was seen with 80–100% (v/v) DMSO. 20-MANT-AppCCl2p
was more responsive to changes in the hydrophobic environment,
that is, the maximum increase in fluorescence was already ob-
served with 40–60% (v/v) DMSO. With both nucleotides, we ob-
served a shift of the emission maximum from kem 450 nm to kem
425 nm (blue-shift). Thus, in principle, both nucleotides could be
used to monitor conformational changes in EF upon addition of cal-
modulin.9 For 20-MANT-30-d-ATP we have recently observed large
calmodulin-dependent increases in direct nucleotide fluorescence
pKa value, and therefore the protonation state of the c-phosphate/
phosphonate.
O
tRNA
P
O
O
OH
N
O
NH2
N
N
N
HO
NH
O
H2N
2
Figure 2. 20-Amido-20-deoxyadenosine derivative as a stable substrate analog of
aminoacyl-tRNA synthetases.16

234
T. Emmrich et al. / Bioorg. Med. Chem. Lett. 20 (2010) 232–235
O
P
HO
Cl
O
O
B
O
Cl
B
HO
O
B
i
ii
NH
HO
HO
NH
O
HO
NH2
O
3
5
4
HN
HN
H3C
H3C
N
NH2
N
B
=
iii
N
N
iv
O
HO
P
O
P
O
O
O
P
Cl
Cl
O
Cl
P
P
O
P
O
O
HO
O
O
HO
O
OH
OH
P
OH
OH
O
O
B
B
iv
O
iv
Cl
HO
O
B
NH
NH
HO
HO
NH
HO
O
O
O
HN
H3C
HN
H3C
7
6
8
HN
H3C
cyclic intermediate
Scheme 1. Reagents and conditions: (i) Acetonitrile/N,N-dimethylacetamide (DMA), N-methylanthranilic acid, N-hydroxybenzotriazole (HOBt), polystyrene-supported N,N0-
dicyclohexylcarbodiimide (PS-DCC), 4-dimethylaminopyridine (DMAP), microwave heating for 6 min; (ii) (OMe)3PO, POCl3, proton sponge, 5 h, 0 °C; (iii) NBu3, bis(tri-n-
butylammonium) dichloromethylenediphosphonate in DMF, 5 min, 0 °C; (iv) Et3NHþHCO ꢀ, pH 7.4–7.6, 1 h, rt.
3
2'-MANT-AppCCl2p
(kem 350 nm) upon interaction with EF. Moreover, we observed cal-
A
modulin-dependent FRET from multiple tryptophan and tyrosine
60
residues in EF to 20-MANT-30-d-ATP.9 In marked contrast, with 20-
DMSO 100%
MANT-AppCCl2p, no such changes were observed (data not
shown). A likely explanation for these negative results is the fact
that the affinity of the nucleotide as determined in functional AC
activity competition results was too low. Accordingly, the interac-
tion of 20-MANT-AppCCl2p with EF is not sufficiently tight enough
to ensure the development of direct fluorescence and FRET signals.
Another hypothesis to explain the observed results is that either
the specific charge distribution in the vicinity of the fluorophore
leads to a strong attenuation of the signals, or nearby molecules
like chlorine ions quench the otherwise expected signals, or a com-
bination of both effects.
50
40
30
20
10
0
DMSO 80%
DMSO 60%
DMSO 40%
DMSO 30%
DMSO 20%
DMSO 10%
H2O
380 405 430 455 480 505 530
Wavelength (nm)
Concluding, we have synthesized for the first time an ATP ana-
log, which combines numerous advantages of previously described
derivatives in one molecule, that is, a hydrolytically stable triphos-
phate analog, a fluorescent label that does not undergo intramolec-
ular acyl migration and maintenance of both 20- and 30-hydrogen
bond donor capabilities.
2'-MANT-3'-d-ATP
B
75
50
25
0
DMSO 100%
DMSO 80%
DMSO 60%
DMSO 40%
DMSO 30%
DMSO 20%
References and notes
1. Schlicker, C.; Rauch, A.; Hess, K. C.; Kachholz, B.; Levin, L. R.; Buck, J.; Steegborn,
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Biol. Chem. 2004, 279, 40938.
DMSO 10%
H2O
3. Göttle, M.; Geduhn, J.; König, B.; Gille, A.; Höcherl, K.; Seifert, R. J. Pharmacol.
Exp. Ther. 2009, 329, 1156.
380 405 430 455 480 505 530
Wavelength (nm)
4. Hiratsuka, T. Biochim. Biophys. Acta 1983, 742, 496.
5. Jameson, D. M.; Eccleston, J. F. Methods Enzymol. 1997, 278, 363.
6. Leppla, S. H. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 3162.
7. Mou, T.-C.; Gille, A.; Fancy, D. A.; Seifert, R.; Sprang, S. R. J. Biol. Chem. 2005, 280,
7253.
Figure 3. Increasing addition of DMSO to 300 nM solutions of (A) 20-MANT-
AppCCl2p (7) resp. (B) 20-MANT-30-d-ATP leads to both a hypsochromic shift and a
concentration-dependent increase in intensity.

T. Emmrich et al. / Bioorg. Med. Chem. Lett. 20 (2010) 232–235
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8. Mou, T.-C.; Gille, A.; Suryanarayana, S.; Richter, M.; Seifert, R.; Sprang, S. R. Mol.
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2001, 10, 159.
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Sproat, B. S.; Van Den Eynde, W.; Link, A.; Van Calenbergh, S.; Grøtli, M.;
Martinis, S. A.; Cusack, S. Mol. Cell 2003, 11, 951.
mixing. After filtration and concentration under reduced pressure a slightly
yellowish oil was obtained which was subjected to column chromatography on
silica gel (CHCl3/MeOH 19:1). Compound 4 was obtained upon drying in vacuo
for 6 h as an off-white solid, yield: 128 mg (42.6%). It was stored under inert
gas (argon) and protected from light, as the compound slowly decomposed
upon irradiation by daylight, and turned brownish within several weeks.
Decomposition came along with a decrease in fluorescence and could be
monitored by fluorimetry. 1H NMR (MeOH-d4, 200 MHz, ppm): d 2.72 (s, 3H,
CH3), 3.77–3.97 (m, 2H, C(50)H), 4.26 (s, 1H, C(40)H), 4.49 (d, 1H, C(30)H,
3J = 5.2 Hz), 5.28 (dd, 1H, C(20)H, 3J1 = 8.3 Hz, 3J2 = 5.4 Hz), 6.14 (d, 1H, C(10)H,
3J = 8.4 Hz), 6.54–6.64 (m, 2H, MANT-C(3)H and -C(5)H), 7.28 (dt, 1H, MANT-
C(4)H, 3J = 7.6 Hz, 4J = 1.6 Hz), 7.48 (dd, 1H, MANT-C(6)H, 3J = 7.6 Hz,
4J = 1.4 Hz), 8.18 (s, 1H, C(8)H), 8.29 (s, 1H, C(2)H). 13C NMR (MeOH-d4,
50 MHz, ppm): d 29.87 (CH3), 57.62 (C20), 63.84 (C50), 72.67 (C30), 89.70, 89.77
(C10 and C40), 112.04 (MANT-C3), 115.87, 115.90 (MANT-C1 and -C5), 116.46
(C5), 129.25 (MANT-C6), 134.11 (MANT-C4), 141.74 (C8), 150.44 (C4), 151.51
(MANT-C2), 153.58 (C2), 157.62 (C6), 171.95 (CO). Fluorescence data
17. Sauer, D. R.; Kalvin, D.; Phelan, K. M. Org. Lett. 2003, 5, 4721.
18. Synthesis of (2R,3R,4S,5R)-N-2-(6-amino-9H-purin-9-yl)-4-hydroxy-5-(hydroxy-
(excitation wavelength is defined as kex, fluorescence maximum as kem): kex =
434 nm?kem = 354.36 nm, kex = 354 nm?kem = 435.63 nm, kex = 265 nm?
kem = 433.85 nm. LC/ESI-HRMS (Shimadzu IT-TOF): positive mode 400.171
([M+H]+), negative mode 398.158 ([MꢀH]ꢀ), calculated: 399.1655.
19. Burgess, K.; Cook, D. Chem. Rev. 2000, 100, 2047.
20. Yoshikawa, M.; Kato, T.; Takenishi, T. Tetrahedron Lett. 1967, 50, 5065.
21. El-Tayeb, A.; Qi, A.; Müller, C. E. J. Med. Chem. 2006, 49, 7076.
22. El-Tayeb, A.; Griessmeier, K. J.; Müller, C. E. Bioorg. Med. Chem. Lett. 2005, 15,
5450.
methyl)tetrahydrofuran-3-yl-[2-(methylamino)]benzamide (4). In
microwave reaction vessel were placed 200 mg of 20-amino-20-deoxya-
denosine (3, 1.0 equiv, 752 mol), 125 mg of N-methylanthranilic acid
(1.1 equiv, 830 mol), 102 mg of anhydrous HOBt (1 equiv, 752 mol),
800 mg of PS-DCC, loading level 1.9 mmol gꢀ1
2 equiv) and catalytic
a 8 ml
l
l
l
,
amounts of 4-DMAP (1 mol %, 1 mg). Washing the polymer-supported
reagent with the solvent system of the reaction is advisable. After addition of
a 1:1 solvent mixture of MeCN and DMA (4 mL), the reaction vessel was
immediately closed and the mixture was heated in a monomode microwave
oven at 100 °C for 6 min, allowing a ramp time of 2 min. Applying constant
external cooling by pressurized air, the heating power was around 150 W at
the beginning of the reaction and just below 90 W for maintaining the
temperature. After cooling to rt, the reaction was stopped by adding the same
volume of MeOH and vigorous stirring for 5 min followed by filtering off
insoluble impurities. The filtrate was shaken with SiliaBond carbonate
(SiliCycle Inc., silica-supported trimethylammonium carbonate, loading level
0.70 mmol gꢀ1) for 1 h at 25 °C; MeOH may be added to allow thorough
23. Sauer, R.; El-Tayeb, A.; Kaulich, M.; Müller, C. E. Bioorg. Med. Chem. 2009, 17,
5071.
24. Synthesis of (2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3-hydroxy-4-[2-(methyl
amino)benzamido]tetrahydrofuran-2-ylmethoxy[(hydroxy)phosphoryloxy]-
[(hydroxy)phosphoryl]dichloromethylphosphonic acid (7). Synthesis of 7 from 4
was performed in analogy to described procedures.17–19 1H NMR (D2O + MeOH-
d4, 500 MHz, ppm): d 2.67 (s, 3H), 4.36–4.54 (m, 2H), 4.6 (m, 1H), 5.05 (m, 2H),
6.26 (d, 1H, J = 8.19 Hz), 6.78 (m, 2H), 7.42 (m, 2H), 8.16 (s, 1H), 8.45 (s, 1H). 31P
NMR (D2O + MeOH-d4, 202 MHz, ppm): d 7.37, 0.60, ꢀ10.19. LC/ESI-MS: positive
mode 706 ([M+H]+), negative mode 704.0 ([MꢀH]ꢀ), calculated: 705.