pH-Controlled recognition of amino acids by urea derivatives of β-cyclodextrin

Article abstract of DOI:10.1039/c7ra02127e

Water soluble amphiphilic urea-substituted β-cyclodextrins were synthesized and applied as amino acid receptors. A great affinity towards nonpolar amino acids was observed, ranging from 2300 M^?1 for alanine to 54?800 M^?1 for tryptoph

Full text of DOI:10.1039/c7ra02127e

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pH-Controlled recognition of amino acids by urea
derivatives of b-cyclodextrin†
Cite this: RSC Adv., 2017, 7, 15742
b
a
Pawel Stepniak,a Bruno Lainer,ab Kazimierz Chmurski and Janusz Jurczak
*
Water soluble amphiphilic urea-substituted b-cyclodextrins were synthesized and applied as amino acid
receptors. A great affinity towards nonpolar amino acids was observed, ranging from 2300 MÀ1 for
alanine to 54 800 MÀ1 for tryptophan in a highly competitive environment (pH 8 phosphate-buffered
water solution). Significant selectivity was observed, since carboxylates without the amino group (or
having hydroxyl instead) remained not bound. Endoenthalpic effects were recorded for amino acids
during titrations, pointing to a different mode of complex formation as compared to native b-
cyclodextrin. We found a dramatic change in binding strength within the physiological pH range: with pH
change from 8 to 6, affinity towards amino acids dropped from 54 000 to 500 MÀ1, making the
receptors potentially appropriate for biotechnological purposes.
Received 20th February 2017
Accepted 6th March 2017
DOI: 10.1039/c7ra02127e
rsc.li/rsc-advances
guests to less polar ones. In this manner, cucurbiturils,12–14 calix-
arenes,15 pilarenes,16 and cyclodextrins17–19 are exploited for amino
Introduction
acid binding. Another, widely applied approach involves the
utilization of hydrogen bond formation, whereby various nucelo-
philic acceptors are usually bound by receptors.20–22 This strategy is
limited by the competing environment, e.g. polar solvents.23–25
Therefore, efficient binding of nonderivatized amino acids in
aqueous solutions still remains a challenge.1
In our recent studies we have shown that combining these
two modes of interaction results in substantially improved
recognition of amino acids in water.26 The introduction of an
amphiphilic substituent containing urea moiety to b-cyclodex-
trin (b-CD) resulted in receptors 1a–c (Fig. 1), which appeared to
be 1000-fold more efficient than native (underivatized) b-CD.
Binding constants of up to 52 400 MÀ1 (for L-tryptophane) were
obtained, and the affinity was correlated with both the acidity of
the urea protons and the lipophilicity of the amino acid.
Unexpectedly, exclusion of isobutyl moiety from the side-chain
structure caused a dramatic decrease in binding constants.
Inspired by these regularities, we decided to investigate how
changes in the amphiphilic side-chain or pH of the environment
The ubiquity of amino acids is one of the main factors propel-
ling various research efforts to develop more sophisticated tools
to identify, bind, and control them. They are present in all living
organisms, playing key roles in many biochemical processes,
e.g. signal transmission and cellular structure building.1
Therefore, identifying objects that interact with amino acids
strongly and selectively is an ongoing objective of supramolec-
ular chemistry. Analytical quantitative detection of amino acids
is usually based on chromatographical or electrochemical
techniques; their main drawback is that analytes oen need to
be derivatized prior to analysis.2,3 The supramolecular approach
to amino acid identication and binding appears to offer
a solution, based on strong and selective interactions between
amino acids and electrically neutral molecular receptors.4–6 One
very demanding issue in this eld is that of pH-controlled
binding-release systems, allowing for specic and targeted
control of processes that involve amino acids.7 An analogous
approach is investigated in the eld of specialized drug delivery
systems and signal transduction regulation.8,9
The most common strategy towards the formation of com-
plexes of receptors with amino acids utilizes structures with
cavities available for the inclusion of guest species. Usually, a type
of hydrophobic effect is employed to situate the amino acid inside
the cavity.10,11 This allows for the usage of such receptors under
aqueous conditions, but, on the other hand, limits the range of
aInstitute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224
Warsaw, Poland. E-mail: jurczak_group@icho.edu.pl
bFaculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra02127e
Fig. 1 Amphiphilic urea-derived b-CD receptors 1a–c.
15742 | RSC Adv., 2017, 7, 15742–15746
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would affect binding of various amino acids and carboxylates. We obtained reference compound having just the ethylene group
assumed that introduction of an additional aromatic ring between azide and urea functionalities.26 We envisioned that the
(instead of isobutyl moiety) in the side-chain would enhance the presence of the additional aromatic moiety should enhance the
interaction with nonpolar species, in particular with hydrophobic guest binding process without causing any additional self-
amino acids. We additionally assumed that receptors having inclusion complex formation (if the latter had been the case,
such a structure would not undergo deprotonation, which would leucine-derived compounds 1a–c would have suffered from this as
make them useful in a wide range of pH. In this report, we well). We decided to investigate the amino acid binding proper-
present such new b-CD derivatives with side-chains based on ties of receptors of type 1 at pH 8, being in the upper part of the
phenylalanine and leucine and describe their utilization in physiological condition range. We used 50 mM phosphate buffer
amino acid binding.
with ionic strength of about 140 mM (which corresponds well to
the intracellular environment). We utilized isothermal titration
calorimetry to gain fuller insight into the thermodynamics of the
complexation process as well as to compare with previously re-
ported data. In this case, titrations of L-amino acid into 2 mM
solutions of receptors provided satisfactory sigmoidal curves
allowing for the determination of enthalpies, binding constants,
and interaction stoichiometries (Fig. 2). Similarly to our previous
ndings, receptors 1d–f also exhibited tendencies to form higher-
ordered superstructures, which was conrmed with dynamic light
scattering (with aggregates of 164, 122, and 255 nm identied for
respective receptors 1d–f). However, during titration a 2 : 1
(host : guest) complexation model was usually identied, which
conrmed the same mode of operation for receptors 1d–f as
compared to leucine-derived 1a–c. Consequently, we applied the
typical model of n-independent binding sites in the tting
procedure. First, we tested how these receptors complex nonpolar
amino acids. The apparent binding constants are listed in Table 1.
We found that phenylalanine-derived receptors 1d and 1f
bound alanine, phenylalanine, and tryptophan substantially
more strongly than their analogues 1a and 1c. In the case of
p-nitrourea derivatives 1b and 1e, this tendency was reversed.
Slightly lower binding constants for compound 1e could be
explained by p-stacking leading to the rearrangement of the
binding site. As we have already shown, the presence and proper
positions of two lipophilic substituents of the side-chain is
essential for the binding, since an analogue with no substituent
at the ethylene linker between urea and triazole groups showed
no affinity towards amino acids at all. In the case of both series
of receptors, an increase in the urea hydrogens' acidity resulted
in higher Ka's (Fig. 3). This is consistent with our assumption
that the urea moiety provides hydrogen bonding for the
carboxylate group of an amino acid.
Results and discussion
Synthesis
The structure of the target receptors was designed based on our
previous ndings concerning the behaviour of cyclodextrins
and various platforms exploiting hydrogen bonding in carbox-
ylate binding.27,28 We showed that in the case of C-6 substituted
b-CDs, intramolecular inclusion complex formation may occur,
making the CD cavity much less available for the guest.29
Therefore, we have developed a synthetic approach to efficient
preparation of mono-2-functionalized b-CD, which can be then
further modied via the azide–alkyne click chemistry method-
ology.30 In such a manner we used mono-2-propargyl-b-CD (2) to
synthesize compounds 1d–f, being phenyl analogues of the
previously reported receptors 1a–c. In this case N-Boc-L-phe-
nylalaninol was tosylated and subsequently transformed into
azide 3. Aer Boc deprotection the amino group was function-
alized with the corresponding commercially available aromatic
isocyanate, yielding desired compound 4. Next, azide–alkyne
coupling with monopropargylated CD 2 was carried out
(according to the known procedure), to yield receptors 1d–f
almost quantitatively (>90%). Consequently, we had in hand six
receptors of type 1, with varying side-chains originating from
leucine and phenylalanine, having different acidity of the
group. None of the compounds had any hydrogen atoms that
would be deprotonated under pH < 10 (Scheme 1).
Binding measurements
We were able to assess the role of the isobutyl/phenyl substituent
in the complexation process, comparing it with previously
Scheme 1 Synthesis of receptors 1d–f.
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Fig. 2 Representative thermograms of ITC experiments: L-Phe titrated to 1c (left) and L-Ala titrated to 1d (right).
Table
complexesa
1 Binding constants for L-amino acid and receptor 1a–f
corresponding pairs of leucine- and phenylalanine-derived
receptors the enthalpies were lower, probably indicating
better conformational preorganization of compounds 1d–f for
guest binding.
Ka (103 MÀ1
)
Receptor
Ala
Phe
Trp
Next, we investigated the scope of guests that could interact
with receptors of type 1. We analysed the previously thoroughly
examined model receptor 1c in binding of tyrosine, phenyl-
glycine, and serine as well as 2-phenylbutyric and mandelic
acids (Table 3). Tyrosine exhibited a binding constant two times
lower than phenylalanine. The conformationally more con-
strained phenylglycine appeared to be even less effectively
bound by receptor 1c. It could be inferred that, for tight inter-
action, both the carboxylic group and the pendant arm of the
guest have to be in a proper position, which would make these
two structural elements essential for complex formation. On the
1ab
1bb
1cb
1d
1e
2.3
2.1
5.0
4.6
2.1
5.1
7.0
20.2
22.4
16.5
5.3
9.8
34.4
52.4
19.1
23.4
54.8
1f
23.0
a
Aa (250 mL, 10–16 mM) was titrated to 1 (949 mL, 2 mM), stirred at
300 rpm. b Data from ref. 26.
All the reactions proved to be endothermic, strongly entropy- other hand, serine was bound with similar strength to phenyl-
driven, with enthalpies varying between 1.5 and 5.5 kJ molÀ1 alanine, even though it is far more polar and does not contain
(Table 2). This would conrm that the mode of interaction with aromatic moiety. Replacing the amino group with hydroxyl or
amino acids is similar for all the receptors. We observed that for aliphatic substituent led to dramatic decreases in affinity
constants, which proved that the presence of the amino group
in the guest structure is necessary for efficient binding. In fact,
Table 2 Enthalpies of L-amino acid and receptor 1a–f complexes
formation
DHa (kJ molÀ1
)
Receptor
Ala
Phe
Trp
1ab
1bb
1cb
1d
1e
2.99
4.89
3.72
2.92
4.23
3.08
4.18
4.90
5.51
4.10
3.10
1.69
4.02
5.42
4.46
5.19
4.18
1.55
1f
a
b
Apparent enthalpy measured by ITC. Data from ref. 26.
Fig. 3 Comparison of binding effectiveness of receptors 1a–f.
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Table 3 Thermodynamic data for complex formation between 1c and
various carboxylates
Phenylalanine and tryptophan were bound about 4 times more
weakly when pH dropped from 8 to 7.5. Under neutral conditions
the receptor was still 10-fold better than native b-CD, but affi-
nities were substantially lower. Under slightly acidic conditions
(comparable to the pH of fast-proliferating cells) binding
constants dropped to 500 MÀ1. These results were in agreement
with the abundance of anionic form of amino acids. At pH 6
almost all phenylalanine and tryptophan molecules are in
their zwitterionic forms, in which they apparently form weak
complexes with receptors of type 1. At pH 8 anionic forms
are generated, which are strongly bound by urea-derived b-CD
receptors. We observed approximately linear correlation between
effective concentration of anionic form of amino acid and Ka
values. This shows that this type of receptor is especially effective
in amino acid binding in the narrow physiological pH range. A
change of two pH units induces a 40- and 140-fold increase in
binding of phenylalanine and tryptophan, respectively.
Guest
Parametera
Phe
Tyr
PhGly Ser
a-Ph-But Man
Ka (MÀ1
)
22 400 13 330 4550
19 650 443
<100
nd
nd
DH (kJ molÀ1
)
5.51
0.52
3.86
0.44
3.83
0.77
3.43
0.53
À0.79
n
1
a
Measured by ITC.
Table 4 Affinity constants of S-ibuprofen complexes formationa,b
Receptor
Ka (MÀ1
)
DH (kJ molÀ1
)
n
b-CDc
1a
1c
8900
2100
1900
500
À14.0
À13.3
À17.7
À11.4
0.75
0.70
0.70
0.54
1d
a
b
c
Measured by ITC. Stoichiometry of interaction. b-CD used as
purchased (containing ca. 10% of water).
Conclusions
We have presented a spectrum of synthetic opportunities and
supramolecular applications of amphiphilic urea derivatives of
b-CD. We again demonstrated that our approach allows for
obtaining pure, well-dened mono-2-substituted b-CDs with
excellent yields. A repertoire of various receptors were tested for
selectivity towards amino acids, nding that receptors 1a–f
interact strongly only with amino acids among which nonpolar
ones are bound with Ka's up to 54 800 MÀ1 in phosphate buff-
ered water solution. Presence of the amino group is essential for
the binding occurrence and is an origin of its pH-dependency.
At pH 8 receptors present great affinities, while at pH 6 there
is almost no binding at all. These results make urea derived b-
CDs an interesting prospective tool for bioengineering and drug
delivery, since dramatic changes occur within the physiological
pH range.
the same phenomenon was observed in the case of ibuprofen
binding by b-CD and its derivatives (Table 4). Addition of the
side-chain into the b-CD structure provided weaker interaction
between ibuprofen and receptors 1a, 1c, and 1d, compared to
native b-CD. In all these cases the complexation reactions
were exothermic, showing that the mode of interaction is
different than observed for amino acid binding. These results
were consistent with complexation enthalpies of 2-phenyl-
butyrate and mandelate (Table 3). This proves that when typical
inclusion complex formation occurs, b-CD functions better than
receptors of type 1, since the latter are impaired by competing
self-inclusion complex formation and aggregation. However, for
amino acids specic interactions appear, leading to a different
mechanism of complexation (positive enthalpies) and much
stronger affinities.
Acknowledgements
We next investigated how pH affects the effectiveness of
receptors of type 1. Using model compound 1c, we measured
binding constants in the pH range of 6–8 (Fig. 4). The more basic
the conditions, the more efficient the receptor proved to be.
This research was nanced by the European Union within the
European Regional Development Fund, Project POIG.01.01.02-
14-102/09.
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