Polyfunctional binding of thymidine 5′-triphosphate with a synthetic polyammonium receptor containing aromatic groups. Crystal structure of the nucleotide-receptor adduct

Article abstract of DOI:10.1021/ja7106977

The protonated forms of the new polyfunctional polyamine receptors L, containing two terpyridine units linked together by two diamine spacers, interact with the nucleotide thymidine 5′-triphosphate (TTP) to form stable adducts in aqueous solution. Both so

Full text of DOI:10.1021/ja7106977

Published on Web 02/05/2008
Polyfunctional Binding of Thymidine 5′-Triphosphate with a Synthetic
Polyammonium Receptor Containing Aromatic Groups. Crystal Structure of
the Nucleotide-Receptor Adduct
Carla Bazzicalupi, Andrea Bencini, Antonio Bianchi,* Enrico Faggi, Claudia Giorgi,
Samuele Santarelli, and Barbara Valtancoli
Department of Chemistry, UniVersity of Florence, Via della Lastruccia 3, 50019, Sesto Fiorentino, Italy
Received November 29, 2007; E-mail: antonio.bianchi@unifi.it
Since the seminal works of Kimura and Lehn,1 in the earlier
1980s, on ATP, ADP, and AMP binding by polyammonium
macrocycles, the use of synthetic receptors has become a common
strategy to mimic biological recognition of nucleotides by proteins.2
Nucleotide binding sites in proteins generally consist of both polar
and apolar groups such as cationic, hydrogen binding, aromatic,
and aliphatic residues. For instance, as shown by several crystal
structures, TTP binding by proteins commonly takes place through
the formation of hydrogen bonds and salt bridges between the
polyphosphate chain of the nucleotide and protein main chain atoms
and/or side chains of Lys or Arg3 and between carbonyl O atoms
Solution studies (see Supporting Information) showed that
of thymine and NH groups from main or side chain residues (i.e.,
interaction of TTP with L occurs in water with the formation of
Asp, Asn, Ser, Gln, and Gly).3a,4 In addition, the thymine group
nucleotide complexes deriving from the association of the different
can give rise to π-stacking interactions with aromatic side chains,
ligand and nucleotide species present in solution at the different
such as Tyr and Phe in human thymidine kinase hTK1,5 and/or to
pH values. Around pH 6.5, the main species is the neutral [(H4L)-
CH‚‚‚π and van der Waals interactions of the methyl group with
HTTP] adduct, while on lowering the solution pH, the minor species
several aromatic and aliphatic protein residues,6 while the ribose
[(H4L)H2TTP]+ is first formed followed by the predominant
3′-OH group behaves as both donor or acceptor of hydrogen
[(H5L)H2TTP]2+ one (Figure S3, Table S1 in Supporting Informa-
bonds.3a,4,6b
tion). Attempts to isolate these adducts were successful in affording
crystals of [(H4L)HTTP]‚12H2O by slow evaporation at room
temperature of an aqueous solution, at pH 6.5, containing L and
Stacking interactions are of considerable importance since they
contribute to stabilize the protein-nucleotide adduct and assist the
nucleotide in achieving the correct orientation inside the protein
TTP in equimolar amounts. The crystal structure of this compound,
binding site. Due to the polyfunctional nature of these nucleotide-
solved by means of X-ray analysis, consists of [(H4L)HTTP] adducts
binding sites of proteins, the earlier synthetic receptors based on
(Figure 1), containing H4L4+ tetraprotonated ligand molecules and
ammonium groups, acting as Lewis acids in the binding of
HTTP4- anions, and water molecules. A list of selected hydrogen
nucleotide polyphosphate chains via charge-charge and hydrogen
bonding interactions, evolved toward more sophisticated receptors
bond distances and interatomic contacts for [(H4L)HTTP] are
reported in the Supporting Information (Table S2) along with the
containing aromatic moieties displaying enhanced mimicking
crystal packing of the compound.
characteristics and/or binding selectivity.2e,7
As shown in Figure 1 (see also Figure S9), the tetraprotonated
From the origin of this research field, the nucleotide-binding
macrocycle behaves as a polyfunctional receptor giving rise to tight
ability of synthetic receptors, as well as their ability to recognize
and activate nucleotides, has been correlated with the mutual
substrate-to-receptor arrangement in the adducts and with the nature
of the binding forces.2d,7a To this purpose, information on the modes
of interaction between these partners was obtained by means of
spectroscopic methods and computational modeling, which were
very helpful in getting a general idea of the adduct architectures,1,7
but not a single crystal structure of a nucleotide bound to similar
synthetic receptors showing detailed structural characteristics
of the adduct and the different binding interactions was reported
until now.
association with HTTP4- through different binding interactions: (i)
H-bonds between the polyphosphate chain of the nucleotide and
protonated nitrogen atoms of the ligand (N(7)‚‚‚O(2) 2.705(5) Å,
N(6)‚‚‚O(2) 2.769(6) Å, N(6)‚‚‚O(8) 2.648(5) Å), (ii) one hydrogen
bond between a carbonyl oxygen of the nucleotide and one
ammonium group of the ligand (N(12)‚‚‚O(13) 2.887(7) Å), (iii)
CH‚‚‚π interactions involving, respectively, C(1) (-CH2-) and C(8)
(-CH3) nucleotide carbon atoms and ligand N(8) and N(4) pyridine
units (3.451(8) and 3.734(9) Å), (iV) one O‚‚‚π interaction between
the nucleotide carbonyl oxygen O(14) and the N(10) pyridine ring
(O‚‚‚pyridine centroid 3.566(7) Å) of the ligand.
In the present paper, we report the first crystal structure of this
type in which the nucleotide is TTP and the synthetic receptor L is
a polyfunctional macrocyclic ligand, composed of two terpyridine
moieties linked together by two diamine chains (see Supporting
Information for the synthesis), containing both amine/ammonium
groups and aromatic residues giving rise to multiple interactions
with the nucleotide.
The protonated macrocycle is S-shaped, with an angle of ca.
17° between each line, defined by the secondary nitrogen atoms
belonging to the same aliphatic chain, and the planes defined by
the heteroaromatic nitrogen atoms of each terpyridine unit. The
three aromatic groups of each terpyridine unit are in trans
conformation and strongly deviate from planarity, the dihedral
angles measured between two adjacent pyridine rings ranging from
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J. AM. CHEM. SOC. 2008, 130, 2440-2441
10.1021/ja7106977 CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
ammonium groups of L. Altogether, a tight association between
the two polyfunctional partners is accomplished, thanks to the many
associative forces, among which, interactions between the nucleo-
base and the large heteroaromatic regions of the ligand are of
primary importance.
Molecular modeling studies (see Supporting Information) were
performed to get further information regarding bonding interactions
and mutual arrangement of L and TTP in the adduct. Annealing
simulations for [(H4L)HTTP] showed three different families of
conformations differing, at most, by 5 kcal/mol. In the three adducts,
the main interactions are again hydrogen bonds and salt bridges
involving the ammonium groups of L and TTP phosphate oxygen
atoms and π-stacking between the nucleobase and ligand heteroaro-
matic groups. In particular, in the second most populated family,
conformations of TTP and L are remarkably similar to that assumed
in the crystal structure (Figure S11c, Supporting Information).
In conclusion, we report the first crystal structure of a nucleotide
bound to a synthetic receptor. The structure shows a multiple linkage
between the two partners mimicking the different binding modes
of nucleotide-binding proteins observed, for instance, in human
thymidine kinase hTK1,5 in ribonucleotide reductases of Salmonella
typhimurium3 and Saccharomyces cereVisiae,4 in vaccinia virus
thymidine kinase,6a and deoxyribonucleoside kinase mutant N64D
of Drosophila melanogaster.6b Solution studies reveal that similar
interaction modes are active also in solution and lead to thermo-
dynamically stable nucleotide-receptor adducts.
Supporting Information Available: Experimental procedures and
data, table of stability constants, species distribution diagrams, fluo-
rescence emission and H NMR spectra, list of selected interatomic
Figure 1. (a) Crystal structure of [(H4L)HTTP]. (b) Expanded representa-
tion of intermolecular contacts.
1
distances for [(H4L)HTTP]‚12H2O, ORTEP drawings of the crystal
structure, details of the crystal packing of [(H4L)HTTP]‚12H2O, lowest
energy calculated structures for [(H4L)HTTP], and X-ray crystal-
lographic data of [(H4L)HTTP]‚12H2O as CIF file. This material is
available free of charge via the Internet at http://pubs.acs.org.
4.96(3) to 27.7(2)°. The two central pyridine groups give rise to
an intramolecular π-stacking interaction, which can be described
as intermediate between face-to-face and edge-to-face, characterized
by a distance of 3.864(9) Å between the centroid of the N9 pyridine
ring and the carbon atom in para position to the nitrogen of the
N4 pyridine ring. Details of the crystal packing are reported in the
Supporting Information.
References
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Chim. Acta 1983, 66, 2454-2466.
The multifunctional character of L in nucleotide binding is also
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bonds. For instance, the stability of the [(H4L)HTTP] species in
solution (log K ) 4.57 for H4L4+ + HTTP4- ) [(H4L)HTTP], Table
S1) is significantly higher than the stability observed for similar
nucleotide complexes with polyazamacrocycles bearing the same
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positive charge and having comparable size, with respect to H4L4+
but which do not contain aromatic groups in their structures.8 1
,
H
NMR spectra (see Supporting Information) recorded on solutions
containing L and TTP in 1:1 molar ratio show that complexation
is accompanied by significant upfield shifts of all 1H signals of the
nucleobase, suggesting that an extended interaction with the
heteroaromatic moieties of the ligand, similar to that observed in
the crystal structure of [(H4L)HTTP]‚12H2O, also exists in solution.
The largest complexation-induced chemical shifts (CIS) are found
for H6 (0.84 ppm) and CH3 (0.81 ppm), the nucleobase protons
showing CH‚‚‚π interactions with aromatic rings of L in the crystal
structure. Conversely, CIS values in 31P NMR spectra are small
(maximum CIS ) 0.67 ppm for Pâ, pH 4.2), revealing a rather
weak interaction between the triphosphate chain of TTP and the
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