Investigation of structural mimetics of natural phosphate ion binding motifs

Article abstract of DOI:10.3390/molecules20023354

Phosphates are ubiquitous in biology and nearly half of all proteins interact with their partners by means of recognition of phosphate residues. Therefore, a better understanding of the phosphate ion binding by peptidic structures is highly desirable. Two

Full text of DOI:10.3390/molecules20023354

Molecules 2015, 20, 3354-3370; doi:10.3390/molecules20023354
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Investigation of Structural Mimetics of Natural Phosphate Ion
Binding Motifs
Evgeny A. Kataev †,* and Tatiana A. Shumilova †
Institute of Chemistry, Faculty of Natural Sciences, Technische Universität Chemnitz,
09107 Chemnitz, Germany; E-Mail: sh.tat.93@gmail.com
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed;
E-Mail: evgeny.kataev@chemie.tu-chemnitz.de; Tel.: +49-371-531-39841;
Fax: +49-371-531-839841.
Academic Editor: David B. Smithrud
Received: 27 December 2014 / Accepted: 12 February 2015 / Published: 16 February 2015
Abstract: Phosphates are ubiquitous in biology and nearly half of all proteins interact
with their partners by means of recognition of phosphate residues. Therefore, a better
understanding of the phosphate ion binding by peptidic structures is highly desirable. Two
new receptors have been designed and synthesized and their anion binding properties in an
acetonitrile solution have been determined. The structure of hosts mimics a part of the
kinase active site that is responsible for the recognition of the phosphate residue. New
hosts contain additional free amino groups with the aim to facilitate coordination of
protonated anions, such as dihydrogen phosphate. According to spectrophotometric
measurements, stepwise 1:1 and 1:2 binding modes have been observed for both receptors
in the presence of acetate, hydrogen sulfate and dihydrogen phosphate. Compared with the
acyclic receptor, the macrocyclic receptor has demonstrated a remarkably enhanced
selectivity for dihydrogen phosphate over other anions. Fluorometric measurements have
revealed different responses of the acyclic and macrocyclic receptors towards anions.
However, in both cases, a 5–8 nm hypsochromic shift of fluorescence maximum has been
observed upon interaction of acetate and dihydrogen phosphate with receptors.
Keywords: phosphate binding; peptide mimetics; macrocycle; glycine-rich loop; synthetic
receptor; host-guest chemistry; anion binding

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1. Introduction
Phosphates are ubiquitous in biology and nearly half of the proteins interact with their partners by
means of recognition of phosphate residues [1,2]. They also play a role of integration between different
biochemical events ranging from metabolism and biosynthesis to gene regulation, signal transaction,
muscle contraction, and antibiotic resistance. The presence or absence of the phosphate residue in a
protein is central in regulating transmembrane and intracellular signal transaction pathways using protein
kinases (PKs) [3] and protein phosphatases (PPs) [4]. Thus, detection and sensing of natural
phosphorylated species have attracts considerable attention among scientists during the last decade [5–8].
Analysis of the literature [1] on structural aspects of phosphate binding shows that the predominant
part of proteins includes a so-called “P loop”, which is essential for the affinity for the phosphate
residue and sometimes referred to as a “giant anion hole”. The amino acid sequences of “P-loops” are
glycine-rich with several amino acids—X, referring to any alternative amino acid. Interestingly, for
proteins, which bind mononucleotides, the characteristic sequence responsible for phosphate
recognition is GXXGXGK(S,T) or GXXX, while for dinucleotide binding the sequence GXGXXG is
typical. The reason why nature uses glycine is apparently the flexibility of this moiety, namely the
ability to bend the sequence in almost perpendicular direction. For example, in a mononucleotide
binding case two glycines are in n + 4 and n + 6 positions, thus they shape the binding site into a cycle [9].
A better understanding of the phosphate ion binding by peptidic structures is therefore highly
desirable [10]. To the best of our knowledge, there is no precedent in the literature with the
investigation of anion binding properties of model compounds containing several glycine residues in
the recognition motif. There have been a number of artificial receptors published, which have
structures based on cyclic or acyclic peptides [11]. Some of these receptors can bind the phosphate
anion [12–14], phosphate esters [15], glucose-1-phosphate [16], and even nucleotides [17,18].
In this work, we explore anion-binding properties of two new compounds that structurally mimic
the active site of one of the kinases [19]. The new receptors repeat the glycine-rich “P loop” containing
the sequence GGL. This sequence was functionalized from both ends with aromatic compounds with
the aim to rigidify the overall structure of the receptors and allow one to investigate binding properties
with the help of spectrophotometric methods. One of the receptors has a tweezers-like structure with
two terminal amino groups and the other is a macrocycle containing only one free amino group. The
amino groups were introduced as hydrogen-bond-acceptor groups to facilitate the coordination of
anions bearing protons, such as dihydrogen phosphate. The amino groups in the structure of hosts
mimic a guanidinium group, which is found in active sites of most natural phosphate binding proteins.
2. Results and Discussion
2.1. Design and Synthesis
The design for model receptors relies on the structure of the active center of 4-diphosphocytidyl-
2C-methyl-D-erythritol kinase [19]. The amino acid chain forms a loop around the phosphate with
GGLG sequence (Figure 1). Interestingly, for the coordination of two oxygen atoms only four
NH-amide groups of tripeptide GGL are required. To generate a structural model of this binding motif,
we functionalized the GGL sequence with 1,2-phenylenediamine units from the one side and with the

Molecules 2015, 20
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anthranilic acid from the other side, respectively. The aromatic rings were introduced with the aim to
produce amides from both sides of the sequence and rigidify the overall conformation of receptors.
There are two reasons why receptor 1 contains two terminal amino groups. Firstly, amino groups can
be protonated and generate additional electrostatic interactions between the receptor and the anion.
Secondly, receptor 1 can react with diacids bearing either hydrogen-bond-donor or hydrogen-bond-
acceptor groups. In this work, we synthesized macrocyclic receptor 2, which contains one secondary
amino group. Thus, receptors 1 and 2 differ from each other in structure and in number of amino
groups available for protonation. For anion binding studies, we used the following anions as their
−
−
−
tetrabutylammonium salts: Cl−, CH3COO−, H2PO4 , HSO4 , and NO3 . One can expect that interaction
of receptors 1 and 2 in organic solvents with hydrogen sulfate and dihydrogen phosphate might be
stronger than for other anions because free amino groups can accept the proton from these anions.
O
O
O
O
N
HN HN
HN
H
O
O
NH
O
HN
H
HN
NH
NH
N
O
H2N
O
N
H
NH2
O
1
2
Figure 1. Structure of the active center of 4-diphosphocytidyl-2C-methyl-D-erythritol
kinase coordinating AMP-PNP substrate and the structure of model receptors 1 and 2
investigated in this work. Hydrogen bonds are depicted as dotted lines (PDB code 1OJ4).
In Figure 2 the synthesis of receptors 1 and 2 is shown. Target compound 1 was constructed by
coupling of protected amine 8 with diamine 5. The deprotection step with trifluoroacetic acid yielded
receptor 1 in 37% overall yield based on starting compound 6. The macrocyclization step with the help
of Boc-protected iminodiacetic acid 10 towards compound 9 was carried out at high dilution
conditions giving the product in 36% yield. Deprotection of macrocycle 9 with trifluoroacetic acid
gave only traces of receptor 2 and a mixture of different acyclic side-products, according to the NMR
measurements. The best method for the deprotection step was the treatment of 9 with diethyl ether
saturated with hydrogen chloride. By using this method, receptor 2 was obtained in 15% overall yield.

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BOC
HN
BOC
H2N
HN
O
c)
a), b)
OCOCl
+
O
O
NH
HN
H2N
H2N
OH
3
4
5
O
O
O
O
O
N
O
O
O
OH
O
N
N
H
HN
HN
H
f), g)
H
H
e)
d)
N
O
NH
OH
N
BOC
O
NH
BOC
H
O
N
BOC
H2N
H2N
6
7
1
8
O
O
N
h)
BOC
OH
OH
O
10
O
O
O
N
H
HN
i)
j)
O
N
H
NH
O
HN
O
NH
2
H
O
HN
NH
N
O
H
HN
NH
Cl-
N
O
N
H2
N
O
O
2•HCl
O
O
9
Reagents and conditions: (a) diisopropylethylamine (DIPEA); (b) 1,2-phenylenediamine;
(c) CHCl3/Et2O saturated with HCl, then NaHCO3; (d) 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide (EDC), 4-(dimethylamino)-pyridine (DMAP), thriethylamine, N-hydroxysuccinimide
(NHS); (e) DIPEA, diglycine; (f) EDC, DIPEA, 1-hydroxybenzotriazole (HOBt) and diamine 5; (g)
TFA in dichloromethane; (h) EDC, HOBt, DIPEA; (i) CHCl3/Et2O saturated with HCl; (j) NaHCO3
(5% aq.), extraction with ethyl acetate. BOC—t-butoxycarbonyl.
Figure 2. Synthesis of receptors 1 and 2.
2.2. Anion Binding Studies
Binding properties of receptors towards different anions were investigated by UV-Vis, fluorescence
and NMR titrations in an acetonitrile solution. The receptors bear an anthranilic acid moiety, which
exhibits fluorescence emission at 400 nm upon excitation at 250 or 328 nm. Thus, we could use two
complementary spectrophotometric methods to determine binding constants for anions. The fitting of
binding isotherms was carried out with HypSpec Program [20]. As inferred from the dilution
experiments the host molecules do not form associates at concentrations used in studies.
Receptor 1 has an acyclic structure and it was expected from the beginning that it could bind all
anions in question. However, chloride and nitrate induced very small changes in UV-Vis spectra so
that fitting of the data was not possible. In spite of this fact, the chloride anion induced considerable
changes in fluorescence titrations and the determined affinity constant was 520 M−1. Receptor 1
showed high affinity for dihydrogen phosphate, hydrogen sulfate and acetate. For the latter two anions

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we observed a stepwise binding mode—1:1 and 1:2 (receptor:anion). The Job’s plot analysis
confirmed this stoichiometry (see Supporting information). Interestingly, receptor 1 binds two
dihydrogen phosphate anions almost simultaneously, as inferred from the fitting model. The first
binding event with a 1:1 stoichiometry is likely too weak to assess it by the curve-fitting analysis.
Additional proof of a 1:2 binding mode was obtained from the NMR titration experiment. The affinity
constant for the binding of two dihydrogen phosphate anions to receptor 1 was logβ12 = 8.98(10)
(Figure 3). This value is in agreement with the value obtained by using spectrophotometric
measurements. Interestingly, the strongest shifts were observed for amide protons H1 (the anthranilic
acid fragment) and H2 (the phenylenediamine fragment) belonging to aromatic systems. Smaller shifts
were detected for amide protons of the amino acids (e.g., H3).
2.000
1.800
1.600
1.400
1.200
1.000
0.800
0.600
0.400
0.200
0.000
H-1 at 8,153 ppm
H-1 at 8153 ppm
H-2 ati 7,490 ppm
H-2 at 7490 ppm
H-3 at 7,471 ppm
H-3 at 7471 ppm
0
0.005
0.01
[anion]/M
0.015
0.02
0.025
Figure 3. 1H-NMR chemically induced resonance shifts upon addition of tetrabutylammonium
dihydrogen phosphate to receptor 1. H1 is the amide-NH of the anthranilic acid, H2 is the
amide-NH connected to the phenylenediamine. H3 is the amide-NH of one of the amino
acid residues.
Binding constants obtained by fluorescence measurements were in a good agreement with those
obtained by UV-Vis titrations (Table 1). As a rule, all anions enhance the fluorescence of 1, except
hydrogen sulfate. The latter anion quenches the fluorescence of the receptor. The addition of anions
induced also a small hypsochromic shift (Figure 4). These interesting changes in fluorescence can be
explained in terms of strong interactions through hydrogen bonds between the anions and the
anthranilic acid moiety, which is responsible for fluorescence.

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5.70
5.20
4.70
4.20
3.70
3.20
2.70
2.20
397 nm
393 nm
dihydrogen phosphate
chloride
401 nm
receptor 1
acetate
hydrogen sulfate
380
390
400
410
420
Wavelength, nm
(A)
(B)
Figure 4. (A) Fluorescence spectra of receptor 1 at 10−4 M concentration in the presence of
4 equivalents of different anions in an acetonitrile solution. (B) Changes in fluorescence of
1 upon addition of tetrabutylammonium dihydrogen phosphate.
Table 1. Association constants logK of receptor 1 with different anions in an acetonitrile
solution measured at 25 °C. a Small changes were observed upon the addition of the anion,
b
so that it was not possible to fit the data; Calculation of the first binding event was not
possible due to the high fitting error.
Anion
UV-Vis
Fluorescence
a
Cl−
-
logβ11 = 2.72 ± 0.01 a
logK11 = 5.00 ± 0.06
logK12 = 3.93 ± 0.06
logβ12 = 7.95 ± 0.03
logK11 = 5.51 ± 0.08
logK12 = 3.99 ± 0.06
logβ12 = 7.85 ± 0.01
CH3COO−
−
H2PO4
b
b
-
-
logK11 = 6.40 ± 0.08
logK12 = 2.88 ± 0.06
logK11 = 6.00 ± 0.04
logK12 = 4.87 ± 0.05
−
HSO4

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According to the titration experiments, receptor 2 binds anions stepwise in 1:1 and 1:2 modes,
except for chloride. This behavior is similar to that observed for receptor 1. While for receptor 1 we
observed almost no changes in UV-Vis titrations after addition of chloride, in case of the macrocyclic
receptor chloride induced considerable changes in spectra so that we could determine the affinity
constant. Interestingly the affinities of 1 and 2 for chloride are the same. All these facts indicate that
the coordination of chloride induces considerable changes in the conformation of the macrocyclic
receptor, likely orienting NH-donor groups towards the chloride anion. Because the affinity constant
for chloride is much less, than that for phosphate, one can suggest that the orientation of NH-donors
fits better the geometry of phosphate.
While the acyclic receptor did not show selectivity for a particular oxyanion, the macrocyclic
receptor has a remarkable preference to bind dihydrogen phosphate selectively. This can be better seen
from the comparison of the cumulative affinity constants for anions, e.g., for acetate logβ12 is 8.46, for
dihydrogen phosphate logβ12 is 10.51 (Table 2). Thus, the macrocyclic structure allowed us to achieve
selectivity for dihydrogen phosphate in an acetonitrile solution. Interestingly, hydrogen sulfate induced
no changes in fluorescence but small changes in UV-Vis. By using the latter method, we were able to
determine the affinity constant for hydrogen sulfate, which is two orders of magnitude lower than that
for dihydrogen phosphate.
Table 2. Association constants logK of receptor 2 with different anions in an acetonitrile
solution measured at 25 °C. a Small changes were observed upon the addition of the anion,
so that it was not possible to fit the data.
Anion
UV-Vis
Fluorescence
Cl−
logK11 = 2.70 ± 0.003
logK11 = 4.63 ± 0.006
logK12 = 3.83 ± 0.09
logK11 = 6.42 ± 0.07
logK12 = 4.09 ± 0.07
logK11 = 3.94 ± 0.02
logK12 = 2.84 ± 0.05
logK11 = 2.72 ± 0.01
logK11 = 4.60 ± 0.10
logK12 = 4.18 ± 0.03
logK11 = 6.00 ± 0.05
logK12 =4.16 ± 0.05
CH3COO−
−
H2PO4
−
a
HSO4
-
According to fluorescence measurements, receptor 2 demonstrated a different fluorometric answer
towards anions, as compared to receptor 1. The addition of anions induced quenching of fluorescence.
As can be seen in Figure 5, the addition of four equivalents of chloride and hydrogen sulfate has
almost no influence on the fluorescence of the host, while acetate and dihydrogen phosphate decrease
the fluorescence intensity and shift the maximum towards lower wavelength numbers on 7 nm. This
interesting behavior of the receptor to shift the fluorescence maximum upon addition of anions can be
investigated in future studies in details for the design of ratiometric probes for anions [21].

Molecules 2015, 20
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3361
402 nm
receptor 2
11
10
9
hydrogen sulfate
395 nm
acetate
dihydrogen phosphate
chloride
8
7
6
380
390
400
410
420
430
Wavelength, nm
(A)
(B)
Figure 5. (A) Fluorescence spectra of receptor 2 at 10−4 M concentration in the presence of
4 equivalents of different anions in an acetonitrile solution. (B) Changes in fluorescence of
2 upon addition of tetrabutylammonium dihydrogen phosphate.
The property of receptors 1 and 2 to bind two anions, though the cavity does not allow this, is rather
unusual. However, this behavior can have three different reasons. First, the coordination of two
dihydrogen phosphate anions can be explained by dimerization of the phosphate anion in the presence
of a synthetic receptor through the P-O…H-O-P hydrogen bond. Such a precedent has been already
−
−
reported in the literature [22]; Second, HSO4 and H2PO4 anions have protons, which can be
transferred to free amino groups during the anion coordination enabling the binding of the second
anion to the positively charged receptor. This will lead to the formation of very stable complexes, in
which the receptor and an anion have both electrostatic and hydrogen bonding interactions; Third, the
receptor has a conformation, in which two pairs of donor NH-groups are oriented in different

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directions so that two anions could be bound from different sides. Such a situation has been observed
for the urea-based receptors, which bind two acetate anions stepwise [23] and for the phosphate
receptor reported by us earlier [24]. In order to understand the binding of anions by the synthesized
receptors, we added to a solution of the receptor one equivalent of perchloric acid in acetonitrile,
measured UV-Vis spectrum and compared this spectrum with those obtained after addition of different
oxyanions. According to UV-Vis and fluorescence measurements, the receptors do not coordinate the
perchlorate anion, therefore the addition of perchloric acid can be considered as a pure protonation
effect. As can be seen in Figure 6, the spectrum of the protonated receptor 1 has the same structure as
that obtained by the addition of hydrogen sulfate. An increase in 277 nm band and a decrease in 285 nm
band were observed. In case of receptor 2, the addition of the acid or hydrogen sulfate leads to the
disappearance of a small shoulder at 282 nm, while the addition of dihydrogen phosphate or acetate
induces a considerable hypsochromic shift.
0.80
0.70
Receptor 1
0.60
Receptor 1 + 14 equiv. of dihydrogen phosphate
Receptor 1 + 14 equiv. of acetate
0.50
Receptor 1 + 14 equiv. of hydrogen sulfate
0.40
Receptor 1 + 1 equiv. of perchloric acid
0.30
0.20
0.10
0.00
380 370 360 350 340 330 320 310 300 290 280 270 260 250
Wavelength, nm
(A)
2.00
1.50
1.00
0.50
0.00
Receptor 2
Receptor 2 + 14 equiv. of dihydrogen phosphate
Receptor 2 + 14 equiv. of hydrogen sulfate
Receptor 2 + 14 equiv. of acetate
Receptor 2 + 1 equiv. of perchloric acid
380 370 360 350 340 330 320 310 300 290 280 270 260 250
Wavelength, nm
(B)
Figure 6. Comparison of UV-Vis spectra of receptor 1 (A) and 2 (B) in the presence of one
equivalent of HClO4 and 14 equivalents of dihydrogen phosphate, hydrogen sulfate and
acetate, respectively.

Molecules 2015, 20
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−
These facts indicate that both receptors are protonated after addition of HSO4 , but not after
addition of dihydrogen phosphate, acetate or chloride. The interaction of hydrogen sulfate with the
receptors leads to the protonation so that the resulting SO42− anion can be also located outside the cavity.
Such a situation was often observed in the solid state for positively-charged cryptand receptors [25].
Analysis of NMR spectra of receptor 1 and 2 in the presence of anions in a CD3CN solution supports
this hypothesis. The binding of dihydrogen phosphate or acetate leads to sharpening of the proton
signals, while binding of hydrogen sulfate induces broadening of both NH and CH signals (see
Supporting Information).
In order to explain a 1:2 stoichiometry of oxyanion binding we proposed that both receptors possess
a specific conformation that enables binding of two anions. To prove our suggestion we protonated the
receptors with one equivalent of perchloric acid and titrated the resulting salts with dihydrogen
phosphate anion. If a receptor is able to accommodate only one anion then the corresponding
positively charged receptor should bind two dihydrogen phosphate anions because it is added in excess
and one anion will be located in the binding cavity and the other—outside the cavity stabilized by
electrostatic interactions. If a receptor is able to accommodate two anions then the corresponding
positively charged receptors should bind three dihydrogen phosphate anions. Exactly the latter situation
we observed in UV-Vis titrations of salts 1•HClO4 and 2•HClO4 with tetrabutylammonium dihydrogen
phosphate. As can be seen in Figure 7 the titration curve changes its direction almost exactly at points
1, 2 and 3 equivalents of dihydrogen phosphate, respectively. Fitting of the experimental data with
HypSpec Program resulted in cumulative association constants β13(for 1) = 12.82 ± 0.01 and
β13(for 2) = 13.69 ± 0.01 (Figure 7).
Figure 7. UV-Vis titration data and fitting curves obtained for the addition of dihydrogen
phosphate to the mixture of receptor 1 (left) and receptor 2 (right) with one equivalent of
perchloric acid.
To get an understanding how two dihydrogen phosphate anions can be coordinated by our receptors
−
we carried our DFT modeling of the complexes. The optimized structures of complexes 1•(H2PO4 )2
−
and 2•(H2PO4 )2 are depicted in Figure 8. The following conclusions can be drawn from the analysis of
the structures of the optimized host-guest complexes. In the acyclic receptor one amino acid subunit
bearing two NH-sites coordinates one oxygen atom of phosphate. This binding mode is similar to that
of phosphate coordination by the kinase (Figure 1). Interestingly, in both complexes the amide oxygen

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atoms play a role of hydrogen bond acceptor, as well as amino groups, which facilitates the
coordination of the protonated phosphate anion. Macrocyclic receptor has a rigid structure and
coordinates two dihydrogen phosphate anions from different sides. One of the anions is stabilized only
with two NH-sites. This coordination mode hints how two acetate or two sulfate anions can be bound
to the receptor. Unexpected for us was the fact that the phenylenediamine subunit forms an almost
planar arrangement of NH-sites, which bind one oxygen atom of the phosphate anions. This
arrangement explains the affinity of the receptor for the chloride anion.
(A)
(B)
−
−
Figure 8. Calculated structures of complexes 1•(H2PO4 )2 (A) and 2•(H2PO4 )2 (B). The
isopropyl substituent and most of the hydrogen atoms were omitted for clarity. The atoms
are shown in different color Red: oxygen; green: phosphorus; blue: nitrogen; gray: carbon.
3. Experimental Section
3.1. General Information
All solvents were of reagent grade quality and purchased commercially. All starting materials were
purchased from Sigma-Aldrich (Steinheim, Germany) and Fluka Chemical Co. (Steinheim, Germany)

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NMR spectra used in the characterization of products were recorded on an Avance 300 instrument
(Bruker, Chemnitz, Germany). The NMR spectra were referenced to trace solvent peak, the
spectroscopic solvents were purchased from Deutero GmbH (Kastellaun Germany). Mass spectra were
recorded with Finnigan MAT SSQ 710 A (CI) and Finnigan MAT 95 (HRMS) (Regenesburg,
Germany). Column chromatography was performed on Whatman silica gel 60 Å (230–400 mesh).
UV/Vis spectra were recorder on Cary BIO 50 UV/Vis/NIR spectrometer (Varian, Chemnitz,
Germany). N-BOC protected antranilic acid, N-hydroxysuccinimide derivative [26] and BOC-protected
iminodiacetic acid [27] were prepared according to the literature procedures.
3.2. DFT Calculations
Molecular modeling calculations were performed using the DFT program “PRIRODA” [28]. A PBE
functional which includes electron density gradient was used. TZ2p-atomic basis sets of grouped
Gaussian functions were used to solve the Kohn—Sham equations. The criterion for convergence was
a difference below 0.01 kcal/mol/Angstrom in the energy between two sequential structures. Various
stationary points on potential energy surface (PES) were determined from analytical calculations of
second energy derivatives (Hessian matrixes).
3.3. (R)-2-Amino-N-(2-aminophenyl)-4-methylpentanamide (5)
N-Boc-(L)-Leucine (200 mg, 0.86 mmol) and DIPEA (163 µL, 1 mmol) were dissolved in 30 mL of
dry THF and the solution was cooled to −20 °C using an acetone-dry ice bath. Isobutyl chloroformate
(136.2 µL, 1 mmol) was added at one portion and the solution was stirred for 40 min at this
temperature. 1,2-Diaminobenzene (186 mg, 1.72 mmol) was dissolved in THF (20 mL) and added to
the first solution at one portion. At the same time, the cooling bath was removed allowing the mixture
to warm up during next 24 h. The resulting solution was evaporated and separated on a silicagel using
dichloromethane-methanol 100:2 v/v mixture as eluent, Rf = 0.6 (second fraction). The purity of the
1
product was controlled by: H-NMR (CDCl3) δ 8.04 (s, 1H), 7.17 (m, 1H), 7.07–6.95 (m, 1H),
6.79–6.63 (m, 2H), 5.17 (d, J = 7.4 Hz, 1H), 4.17 (dd, J = 49.2, 6.2 Hz, 1H), 3.97–3.75 (bs, 2H), 1.74
13
(m, 2H), 1.66–1.50 (m, 1H), 1.42 (s, Hz, 9H), 1.03–0.87 (m, 6H). C-NMR (CDCl3) δ 171.5, 156.3,
140.7, 127.1, 125.5, 123.6, 118.9, 117.3, 28.34, 24.85, 22.95, 22.06, 19.08, 18.89. Deprotection was
accomplished by stirring the compound in ether (HCl gas saturated) solution (4 mL) at 0 °C for
24 h. The white precipitate was washed with ether and dried yielding 5·(HCl)2. The salt was dissolved
in distilled water and basic ion exchange resin was added until the pH of the solution became 8–9. The
resin was filtered off, and the solution was lyophilized giving 5 as a free amine (96 mg ,50%).
1H-NMR (CDCl3) δ 7.27 (d, J = 6.3 Hz, 1H), 7.10–6.93 (m, 1H), 6.85–6.70 (m, 2H), 6.62–6.27
(m, 1H), 3.94 (d, J = 6.7 Hz, 2H), 3.65 (bs, 2H), 2.16 (s), 2.06–1.84 (m, 2H), 0.95 (d, J = 6.7 Hz, 6H).
13C-NMR (CDCl3) δ 265.8, 154.9, 140.1, 126.4, 124.7, 119.3, 117.6, 71.6, 30.9, 27.8, 18.6.
ESI-MS(+): m/z [M+H]+ 222.1. Anal. Calcd: C, 65.13%; H, 8.65%; N, 18.99%. Found: C, 65.40%;
H, 8.66%; N, 19.05%.

Molecules 2015, 20
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3.4. ((2-[(tert-Butoxy)carbonyl]amino)phenyl)formamido-Gly-Gly-OH (8)
N-Boc antranilic acid (2.00 g, 8.42 mmol), EDC hydrochloride (1.80 g, 9.43 mmol),
N-hydroxysuccinimide (1.09 g, 9.43 mmol), DMAP (30 mg, 3 mol %) and triethylamine (1.32 mL,
9.43 mmol) were mixed in DCM (100 mL) and stirred for 2 h, followed by evaporation of solvent at
40 °C on a rotary evaporator. The solid was purified on a short silica-gel column using
dichloromethane-methanol 100:5 v/v mixture as an eluent (Rf = 0.9), yielding 1.67 g (5 mmol, 60%) of
N-hydroxy-succinimide derivative. The obtained product was dissolved in DMF (30 mL) and added to
a round bottom flask containing diglycine (0.66 g, 5 mmol) and DIPEA (1 mL, 6 mmol) dissolved in a
mixture of methanol (12 mL), ethyl acetate (6 mL) and water (6 mL). The mixture was stirred for 48 h,
evaporated at 60–70 °C. The oily residue was dissolved in DCM (100 mL) and water (100 mL) was
added. After 30 min stirring of the solution the white crystals of product precipitated, which were
subsequently filtered off yielding 730 mg (41%) of 8. If the product do not precipitate the mixture
can be purified using column chromatography by subsequent washing of the column with 100:5 and
then 80:20 v/v dichloromethane-methanol mixtures.1H-NMR (DMSO-d6) δ ppm 7.08 (t, J = 7.30 Hz),
7.48 (t, J = 7.64 Hz), 7.80 (d, J = 7.59 Hz), 8.58–8.05 (m), 9.03 (s), 10.70 (s), 3.84 (dd, J = 35.67, 5.21 Hz),
3.68–2.76 (m), 1.46 (s). 13C-NMR (300 MHz, MSO-d6) δ ppm 171.05; 168.82; 168.53; 152.00; 139.69;
132.21; 128.24; 121.20; 118.70; 118.30, 79.67; 27.84. ESI-MS(+): m/z [M+H]+ 352.2. Anal. Calcd: C,
54.69%; H, 6.02%; N, 11.96%. Found: C, 54.15%; H, 6.12%; N, 12.05%.
3.4.1. ((2-[(tert-Butoxy)carbonyl]amino)phenyl)formamido-Gly-Gly-Leu-(2-aminophenyl)amine (1)
Compound 8 (750 mg, 2.14 mmol) was dissolved in dry dichloromethane (150 mL) containing EDC
hydrochloride (490 mg, 2.56 mmol), HOBt (346 mg, 2.56 mmol), and DIPEA (845 µL, 5.12 mmol),
and the solution was stirred for 1 h at 0 °C. Compound 5 (566 mg, 2.56 mmol) was added to the
solution and the stirring was continued for 24 h at room temp. The solvent was evaporated and the
residue purified using column chromatography in 100:10 v/v dichloromethane-methanol mixture
yielding crude BOC-protected amine. Deprotection was performed by stirring the product in
trifluoroacetic acid‒dichloromethane mixture (1:3 v/v) for 3 h, followed neutralization with Na2CO3,
which resulted in precipitation of white powder. The suspension was extracted by ethyl acetate and
purified using column chromatography (eluent: ethyl acetate-methanol 100:5 v/v) yielding 900 mg
(92%) of 1. 1H-NMR (DMSO-d6) δ 9.20 (s), 8.54 (t, J = 5.7 Hz), 8.21 (dd, J = 14.0, 8.4 Hz), 8.01 (d,
J = 7.6 Hz), 7.55 (d, J = 7.9 Hz), 7.15 (t, J = 7.7 Hz), 7.10–7.01 (m), 6.92 (t, J = 7.6 Hz), 6.77–6.62
(m), 6.52 (ddd, J = 12.6, 6.9, 2.9 Hz), 4.79 (s), 4.44 (q, J = 7.4 Hz), 4.03 (q, J = 7.1 Hz), 3.92–3.65
(m), 2.09 (s), 1.99 (s), 1.62 (dt, J = 24.2, 6.7 Hz), 1.17 (t, J = 7.1 Hz), 0.89 (dd, J = 9.1, 6.3 Hz).
13C-NMR (DMSO-d6) δ 174.0, 173.4, 172.7, 172.2, 150.4, 141.7, 133.7, 129.4, 128.7, 127.9, 125.2,
120.7, 120.1, 119.0, 118.4, 117.5, 116.3, 116.2, 61.6, 53.9, 44.5, 43.8, 41.6, 25.9, 23.5, 21.8, 18.4,
14.5. ESI-MS(+): m/z [M+H]+ 455.0. Anal. Calcd: C, 60.78%; H, 6.65%; N, 18.49%. Found: C,
60.60%; H, 6.66%; N, 18.47%.

Molecules 2015, 20
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3.4.2. (17S)-17-(2-Methylpropyl)-2,5,8,15,18,21,24-heptaazatricyclo[24.4.0.09,14]triaconta-1(30),
9(14),10,12,26,28-hexaene-3,7,16,19,22,25-hexone (2)
Boc-Protected iminodiacetic 10 (62 mg, 0.264 mmol) was dissolved in dry DCM (10 mL), cooled to
0 °C, and to the solution were added HOBt (72 mg, 0.53 mmol), DIPEA (86 µL, 0.53 mmol) and EDC
(101 mg, 0.53 mmol). The mixture was stirred at this temperature for 1 h and poured to the solution of
1 (100 mg, 0.22 mmol) in dry DCM (150 mL). The reaction was stirred additional 48 h at room temp.
and the solvent was evaporated. First a bluish fluorescent spot (under UV lamp) was separated on a
silicagel column using ethyl acetate-methanol 100:5 v/v mixture as eluent yielding compound 9 in 36%
yield. This compound was further deprotected by stirring it at 0 °C for 2 h and then overnight at room
temperature in the mixture of chloroform-ether (HCl saturated), 5 and 10 mL, respectively. The
precipitate formed was filtered off and washed with ether yielding salt 2•HCl. The conversion to the
free base was accomplished by dissolving the salt in a NaHCO3 (sat.)‒ethyl acetate mixture followed
by subsequent extraction with ethyl acetate five times and evaporation. Compound 2 was obtained
after silica gel column chromatography using ethyl acetate-methanol 85:15 v/v mixture as eluent with
15% yield. 1H-NMR (CD3OD) δ 7.65–7.61 (m, 1H), 7.51 (dd, J = 8.0, 1.3 Hz, 1H), 7.41 (dd, J = 7.8,
1.5 Hz, 1H), 7.21–7.13 (m, 3H), 6.75–6.70 (m, 1H), 6.59 (t, J = 7.5 Hz, 1H), 6.08 (t, J = 1.9 Hz, 1H),
4.58 (m, 1H), 4.07–3.91 (m, 2H) 3.71 (s, 2H), 3.50 (s, 2H) 3.47 (s, 2H), 1.78–1.74 (m, 3H), 0.94 (s,
3H), 0,93 (s, 3H). ESI-MS(+): m/z [M+H]+ 552.1. Anal. Calcd: C, 58.79%; H, 6.03%; N, 17.78%.
Found: C, 58.93%; H, 5.98%; N, 17.69%.
4. Conclusions
In summary, we have designed and synthesized two model receptors with a GGL binding unit
that is found in active sites of kinases and this sequence is responsible for recognition of phosphate
residues. Additional feature of our design has been the presence of free amino groups that serve as
hydrogen-bond-acceptor groups and facilitate coordination of protonated anions, such as dihydrogen
phosphate. Anion binding properties of two new receptors have been investigated with the help of
UV-Vis, fluorescence and NMR titration methods in an acetonitrile solution. These complementary
methods have produced in most cases identical results. The acyclic receptor has demonstrated a
general selectivity for oxyanions over chloride and nitrate with high affinity constants. Stepwise 1:1
and 1:2 binding modes have been observed for acetate, hydrogen sulfate and dihydrogen phosphate. A
remarkable preference to bind dihydrogen phosphate over acetate and hydrogen sulfate has been
observed for the macrocyclic receptor. Comparison of the UV-Vis spectra of the host-guest complexes
with the spectra of receptors protonated by perchloric acid has provided an evidence that protonation
of free amino groups induces only hydrogen sulfate, while other anions are bound through hydrogen
bonds. Fluorometric measurements have revealed a difference in responses of the acyclic and
macrocyclic receptors towards anions. Addition of strongly coordinating anions to the solution of the
acylic receptor has induced an increase in intensity, while in the case of the macrocyclic receptor a
decrease of intensity has been detected. For both receptors a 5–8 nm shift of fluorescence maximum
has been observed upon interaction with acetate and dihydrogen phosphate. DFT quantum chemical
calculations have been carried out to understand the interaction of the receptors with dihydrogen

Molecules 2015, 20
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phosphate in the complexes. Studies of anions binding properties of the reported receptors in an
aqueous solution at pH values, at which the receptors are protonated, are in progress.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/02/3354/s1.
Acknowledgments
We thank Burkhard König from University of Regensburg for the fruitful suggestions and
discussions. We thank Diana Drettwan for NMR experiments. This work was financially supported by
Fonds der Chemischen Industrie, Deutsche Bundesstiftung Umwelt, Technische Universität Chemnitz
and COST action “Supramolecular Chemistry in water”.
Author Contributions
Evgeny A. Kataev and Tatiana A. Shumilova synthesized the receptors and performed analytical
experiments. Evgeny A. Kataev analyzed the data and wrote the manuscript. Both authors read and
approved the final version of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Hirsch, A.K.H.; Fischer, F.R.; Diederich, F. Phosphate recognition in structural biology.
Angew. Chem. Int. Ed. 2007, 46, 338–352.
2. Hargrove, A.E.; Nieto, S.; Zhang, T.Z.; Sessler, J.L.; Anslyn, E.V. Artificial receptors for the
recognition of phosphorylated molecules. Chem. Rev. 2011, 111, 6603–6782.
3. Adams, J.A. Kinetic and catalytic mechanisms of protein kinases. Chem. Rev. 2001, 101, 2271–2290.
4. Albrecht, M.; Fiege, M.; Osetska, O. 8-hydroxyquinolines in metallosupramolecular chemistry.
Coord. Chem. Rev. 2008, 252, 812–824.
5. Riechers, A.; Schmidt, F.; Stadlbauer, S.; Konig, B. Detection of protein phosphorylation on
sds-page using probes with a phosphate-sensitive emission response. Bioconjugate Chem. 2009,
20, 804–807.
6. Gonzalez-Vera, J.A. Probing the kinome in real time with fluorescent peptides. Chem. Soc. Rev.
2012, 41, 1652–1664.
7. Kinoshita, E.; Kinoshita-Kikuta, E.; Takiyama, K.; Koike, T. Phosphate-binding tag, a new tool to
visualize phosphorylated proteins. Mol. Cell. Proteomics 2006, 5, 749–757.
8. Warwick, C.; Guerreiro, A.; Soares, A. Sensing and analysis of soluble phosphates in
environmental samples: A review. Biosens. Bioelectron. 2013, 41, 1–11.
9. Salmeen, A.; Andersen, J.N.; Myers, M.P.; Tonks, N.K.; Barford, D. Molecular basis for the
dephosphorylation of the activation segment of the insulin receptor by protein tyrosine
phosphatase 1b. Mol. Cell 2000, 6, 1401–1412.

Molecules 2015, 20
3369
10. Katayev, E.A.; Ustynyuk, Y.A.; Sessler, J.L. Receptors for tetrahedral oxyranions. Coord. Chem. Rev.
2006, 250, 3004–3037.
11. Kubik, S. Synthetic peptide-based receptors. In Supramolecular Chemistry; John Wiley & Sons,
Ltd.: Hoboken, NJ, USA, 2012.
12. Schnopp, M.; Ernst, S.; Haberhauer, G. Anion recognition by neutral macrocyclic azole amides.
Eur. J. Org. Chem. 2009, 2009, 213–222.
13. Choi, K.H.; Hamilton, A.D. Rigid macrocyclic triamides as anion receptors: Anion-dependent
binding stoichiometries and h-1 chemical shift changes. J. Am. Chem. Soc. 2003, 125, 10241–10249.
14. Martí-Centelles, V.; Burguete, M.I.; Galindo, F.; Izquierdo, M.A.; Kumar, D.K.; White, A.J.P.;
Luis, S.V.; Vilar, R. Fluorescent acridine-based receptors for h2po4–. J. Org. Chem. 2011, 77,
490–500.
15. Ishida, H.; Suga, M.; Donowaki, K.; Ohkubo, K. Highly effective binding of phosphomonoester
with neutral cyclic peptides which include a non-natural amino acid. J. Org. Chem. 1995, 60,
5374–5375.
16. Schmuck, C.; Heller, M. Solid phase synthesis of a prototype of a new class of biomimetic
receptors for anionic carbohydrates. Org. Biomol. Chem. 2007, 5, 787–791.
17. Butterfield, S.M.; Waters, M.L. A designed β-hairpin peptide for molecular recognition of atp in
water. J. Am. Chem. Soc. 2003, 125, 9580–9581.
18. McDonough, M.J.; Reynolds, A.J.; Lee, W.Y.G.; Jolliffe, K.A. Selective recognition of
pyrophosphate in water using a backbone modified cyclic peptide receptor. Chem. Commun.
2006, 2971–2973.
19. Miallau, L.; Alphey, M.S.; Kemp, L.E.; Leonard, G.A.; McSweeney, S.M.; Hecht, S.; Bacher, A.;
Eisenreich, W.; Rohdich, F.; Hunter, W.N. Biosynthesis of isoprenoids: Crystal structure of
4-diphosphocytidyl-2c-methyl-d-erythritol kinase. Proc. Natl. Acad. Sci. USA 2003, 100, 9173–9178.
20. Gans, P.; Sabatini, A.; Vacca, A. Investigation of equilibria in solution. Determination of equilibrium
constants with the hyperquad suite of programs. Talanta 1996, 43, 1739–1753.
21. Gunnlaugsson, T.; Glynn, M.; Tocci, G.M.; Kruger, P.E.; Pfeffer, F.M. Anion recognition and
sensing in organic and aqueous media using luminescent and colorimetric sensors. Coord. Chem. Rev.
2006, 250, 3094–3117.
22. Wu, B.; Jia, C.D.; Wang, X.L.; Li, S.G.; Huang, X.J.; Yang, X.J. Chloride coordination by
oligoureas: From mononuclear crescents to dinuclear foldamers. Org. Lett. 2012, 14, 684–687.
23. Zhang, D.W.; Zhao, X.; Li, Z.T. Aromatic amide and hydrazide foldamer-based responsive
host-guest systems. Acc. Chem. Res. 2014, 47, 1961–1970.
24. Sessler, J.L.; Katayev, E.; Pantos, G.D.; Ustynyuk, Y.A. Synthesis and study of a new
diamidodipyrromethane macrocycle. An anion receptor with a high sulfate-to-nitrate binding
selectivity. Chem. Commun. 2004, 1276–1277, doi:10.1039/b403665d.
25. Farrell, D.; Gloe, K.; Gloe, K.; Goretzki, G.; McKee, V.; Nelson, J.; Nieuwenhuyzen, M.; Ibolya, P.;
Stephan, H.; Towna, R.M.; et al. Towards promising oxoanion extractants: Azacages and
open-chain counterparts. Dalton Trans. 2003, 1961–1968, doi:10.1039/B210289G.
26. Tanaka, F.; Kinoshita, K.; Tanimura, R.; Fujii, I. Relaxing substrate specificity in antibody-catalyzed
reactions: Enantioselective hydrolysis of n-cbz-amino acid esters. J. Am. Chem. Soc. 1996, 118,
2332–2339.

Molecules 2015, 20
3370
27. Mcmurry, T.J.; Brechbiel, M.; Kumar, K.; Gansow, O.A. Convenient synthesis of bifunctional
tetraaza macrocycles. Bioconjugate Chem. 1992, 3, 108–117.
28. Laikov, D.N. A new parametrizable model of molecular electronic structure. J. Chem. Phys. 2011,
135, doi:10.1063/1.3646498.
Sample Availability: Samples of the compounds 1–10 in milligram quantity are available from the authors.
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