Complexation of 5-aminovaleric acid zwitterions in aqueous/methanol solution by heterotopic tri-cationic receptors

Article abstract of DOI:10.1039/c9ob02234a

Heterotopic tri-cationic receptors based on 4,10,16-triaza-18-crown-6 are capable of efficient and selective binding of the zwitterionic form of 5-aminovaleric acid (5-AVA) in aqueous/methanol solution. The cooperative participation of both cation and ani

Full text of DOI:10.1039/c9ob02234a

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DOI: 10.1039/C9OB02234A
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Complexation of 5-aminovaleric acid zwitterion in aqueous/methanol solution by
heterotopic tri-cationic receptor
Wojciech Walczak, Maciej Zakrzewski, Grzegorz Cichowicz and Piotr Piątek *
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
At the physiological pH of 7.4 most of amino acids exist
in their highly polar zwitterionic form. This form is strongly
hydrated in aqueous solutions therefore recognition of
amino acids by molecular receptors through noncovalent
interaction remains a challenge. Although relatively many
examples of receptors for the recognition of unmodified
α-amino acids containing hydrophobic side arm have
been reported, the number of receptors for other amino
acids is not so considerable.3 This is especially true for
amino acids lacking of any side arm such as glycine or
ω-amino acids.4 To effectively bind such amino acids in
aqueous media both ammonium and carboxylic end of
the zwitterion should strongly interact with molecular
receptor. Therefore, ditopic receptors comprising cation
and anion binding domain(s) are needed.5 The adequate
organization of these binding groups is also very
important. The cation and anion binding sites must be in
relevant spatial proximity with simultaneous minimising of
mutual interactions between them. In most examples of
heteroditopic receptors for ω-amino acids reported in the
literature, crown ethers or aza-crown ethers were applied
as ammonium cation binding domain, whereas metals
cations, quaternary ammonium or guanidinium groups
were used for carboxylate recognition.4 However, an
excellent example of hemicryptophane receptor that very
effectively encapsulates β and γ-amino acids by cation-π
and anion-π interactions has been also reported.6 We
have recently shown that 4,10,16-triaza-18-crown-6 can
be used for construction of tricyclic heteroditopic receptor
that recognizes ammonium nitrate salt in highly
competitive solvent DMSO.7 This result is just another
confirmation of Jean-Marie Lehn observation that
alkylated 4,10,16-triaza-18-crown-6 preferentially binds
ammonium cations.8 Therefore, we have chosen this
Heterotopic tri-cationic receptor based on 4,10,16-triaza-
18-crown-6 is capable of efficient and selective binding of
zwitterionic form of 5-aminovaleric acid (5-AVA) in
aqueous/methanol solution. The cooperative participation
of both cation and anion binding domains of this receptor
in 5-AVA complexation was established by 1H NMR
experiments and DFT calculations. Heterotopic receptor
and fluorescein were used for the preparation of Indicator
Displacement Assay and this assembly was applied to
selective optical sensing of 5-aminovaleric acid.
Introduction
Zwitterions are molecules that contain equal number of
ionizable groups of opposite charge and have no net
charge. The most important class of zwitterionic
molecules are amino acids.1 L-α-amino acids are
essential in nature as building blocks of peptides and
proteins, however, other non-proteinogenic amino acids
such as γ-aminobutyric acid (GABA) or taurine play an
important role in the transfer of neuronal information.2
Figure 1. Target zwitterionic substrates.
a. Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw.
e-mail: ppiatek@chem.uw.edu.pl
Electronic Supplementary Information (ESI) available: 1H and 13C NMR spectra,
UV-Vis titration spectra and the resulting binding isotherms, 1H NMR
titration spectra and the resulting binding isotherms. See DOI: 10.1039/x0xx00000x
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crown ether as both cation binding domain and molecular
scaffold to prepare receptor 1 (Figure 2). To reassure
strong anion binding in aqueous/methanol media three
guanidinium groups were used. Benzyl groups offer rigid
separation of cation and anion binding domains and
create hydrophobic environment suitable for ω-amino
acids alkyl chain. Herein we present the synthesis and
binding studies of heterotopic receptor 1 as well as the
application of this receptor to the construction of Indicator
Displacement Assay which is capable of ω-amino acids
sensing.
Results and Discussion
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DOI: 10.1039/C9OB02234A
The five-step synthesis of 1 shown on Scheme 1
commenced with LiAlH4 reduction of both formyl and
nitrile groups of 4-formylbenzonitryle. The amino group of
the resulting 4-(hydroxymethyl)benzylamine was treated
with N,N′-di-Boc-1H-pyrazole-1-carboxamidine, to yield
benzyl alcohol substituted with N,N′-di-Boc-guanidine 4.
Subsequent conversion of the hydroxy group into
bromide under Appel conditions afforded benzyl bromide
5. Initial attempts to alkylate 4,10,16-triaza-18-crown-6
with guanidine substituted benzyl bromide in the
presence of excess of K2CO3 in DMF at elevated
temperature were unsuccessful. After many trials it was
found that the use of an organic base (DIPEA) at room
temperature and extension of the reaction time to 6-7
days provided compound 6 in 46% of yield. Global
deprotection of 6 using trifluoroacetic acid (TFA) afforded
receptor 1 in a form of TFA salt. The receptor is soluble
in water and in water/organic solvent mixtures.
Figure 2. The schematic representation of the complex of
5-aminopentanoic acid (with proton groups labelling).
1 and
Figure 3. 1H NMR spectra (298 K, TRIS buffer pH=8.0, 30% D2O/MeOD) of host 1
upon progressive addition of 5-AVA. (Ctitrant = 5.9 x10-2 M, Creceptor = 3.1 x10-3 M).
Compound 1 was assessed as the receptor for
zwitterions in 30% D2O/CD3OD (buffered with 10 mM
TRIS at pH = 8.0) through 1H NMR spectroscopic
titrations. At this pH amino groups of 1 are mostly in free
amino form, whereas guanidine groups are protonated
and amino acids guests remain in zwitterionic form. In the
presence of deuterated solvents all NH protons of
guanidine groups of 1 undergo fast hydrogen-deuterium
exchange therefore only C-H protons are visible in the 1H
NMR spectrum (Figure 3). Initially, GABA was used to
probe the zwitterions-binding ability of ditopic receptor 1.
The successive addition of GABA to the solution of 1
induced significant upfield shifts of signals assigned to
both benzyl groups (protons Bg and Bc) as well as -CH2-
Scheme 1. Reagents and condition: (a) 1M LiAlH4, THF, reflux, 5 h, 95%; (b) N,N’-
di-Boc-1H-pyrazole-1-carboxamidine, DIPEA, MeCN, 2 h, 92%; (c) PPh3, CBr4, DCM,
24 h, 70%; (d) tri-HBr salt of 4,10,16-triaza-18-crown-6, DIPEA, DMF, 6 days, 46%;
(e) TFA, DCM, 3 h.
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protons (signals Cn and Co) of aza-crown ether (see distance is 2.83 Å). The guanidinium NHs point at
View Article Online
Figure 2 for protons labelling). For example, the initially carboxylate oxygen atoms, forming fiDvOeI:d1i0s.1t0in3c9/tCh9yOdBr0o22g3e4nA
broad benzylic (Bg) protons signal centered at 4.25 ppm bonds. Finally, dispersive interactions between aliphatic
was shifted upfield by Δδ = 0.20 ppm, whereas aza-crown protons of 5-AVA and aromatic rings of 1 are quite
ether (Cn) protons moved by Δδ = 0.25 ppm. This result probable. Indeed, hydrogen atom is 3.7 Å away from the
clearly suggests that zwitterion interacts with guanidine centre of the aromatic ring, and the angle of the
groups as well as with the aza-crown ether moiety. The H···centroid axis to the plane is 94o.
quantitative information on the binding process was
obtained from 1H NMR titrations. The association
constant (Ka) of 1 with the GABA zwitterion was
calculated to be 210 M−1 in 30% D2O/CD3OD solution.
Interestingly, no 1H NMR spectrum changes were
induced by the addition of α-amino acids. Therefore, the
influence of the distance between positive and negative
ends of zwitterions on binding affinity of the receptor 1
was studied. Thus, 1 was titrated with aliphatic 2-6 amino
acids monitoring benzylic (Bg) protons shifts. The
calculated binding constants are summarized in Table 1.9
Amino Acid
β-Alanine
Ka [M-1]
70
4-Aminobutyric acid
5-Aminovaleric acid
6-Aminocaproic acid
210
1 090
550
Table 1. 1H NMR spectroscopy Ka values for interactions of 1 with various amino
acids (298 K, TRIS buffer pH=8.0, 30% D2O/MeOD), errors of <10%.
Figure 4. Molecular modelling structure of 1•5-AVA.
As could be easily seen from the analysis of Table 1 a
sharp selectivity for 5-aminovaleric acid (5-AVA) was
observed (Table 1). β-alanine and GABA associated only
weakly with 1. On the other hand, the affinity constant
value of 6-ACA•1 was approximately two times lower
than 5-AVA•1 constant value. The observed selectivity
for 5-aminovaleric acid could be explained by
encapsulation of this guest in the cavity formed by the
three arms of the receptor with good fit between the
distance of positive and negative ends of 5-AVA and
binding domains of the receptor 1. To the best of our
knowledge there is only one example of molecular
receptor that binds 5-AVA. In 1996 O’Hanlon and co-
workers reported anthracene based receptor that
interacts with 5-AVA rather weekly with association
constant of 83 mol-1 (in MeOH:H2O 3:2) and selectivity 5-
AVA/GABA of 2.3.
The binding affinity of receptor 1 for zwitterions is large
enough to consider the construction of optical sensor
based on this receptor. However, receptor 1 does not
have
effective
chromophore/fluorophore
group.
Therefore, we applied Indicator Displacement Assay
(IDA) method for the preparation of optical sensor based
on 1. This method is based on the combination of an
indicator (I), a receptor (R), and a guest in which the
receptor is able to bind both the dye and guest, but with
different affinity.11 After screening many anionic dyes (at
pH = 8.0) we found out that fluorescein exhibits distinctive
colour and fluorescence changes upon mixing with the
receptor 1. Specifically, yellow solution of fluorescein in
30% H2O/CH3OH after addition of 10 equivalent of 1
becomes colourless and its green fluorescence was
almost totally quenched. The 3.75x10-5 M solution of
fluorescein (30% H2O/CH3OH, 10 mM TRIS, pH = 8) was
then titrated with receptor 1. Unfortunately, the resulting
binding isotherm could not be fit to any suitable binding
model, suggesting multiple equilibria present during the
titration (see ESI for stoichiometry studies).12 An Indicator
Displacement Assay was prepared by mixing 3.8x10-4 M
solution of 1 with 3.8x10-5 solution of fluorescein to obtain
I:R = 0.1 (30% H2O/CH3OH, 10 mM TRIS, pH = 8). Under
According to the calculated DFT-minimized structure of
the complex between 1 and 5-AVA in methanol, the
ammonium end of the guest resides in proximity to the
triaza-crown ether, whereas the carboxylate interacts
with all three guanidinium groups (Figure 4).10 The
ammonium NHs of 5-AVA are directed towards oxygen
atoms of the triaza-crown moiety (the average NH···O
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these conditions, the absorption of indicator is very low
at the end of titration (Figure 5).
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DOI: 10.1039/C9OB02234A
1.2
1.0
0.8
0.6
0.4
0.2
Figure 7. a) Solution of fluorescein; b) Solution of fluorescein after addition 20 eq
of 1; c) Displacement of fluorescein after addition 20 eq of GABA; d) Displacement
of fluorescein after addition 20 eq of 5-AVA; All solutions were prepared in 30%
water/ methanol (v/v), 10 mM TRIS, pH=8.0.
20 eq
0.0
390 410 430 450 470 490 510 530
Wavelenght [nm]
Figure 5. UV-Vis spectra of fluorescein (30% water in methanol (v/v), 10 mM TRIS,
pH=8.0). Absorbance decrease at λ=493 nm as 1 (1.9 mM) is added to solution of
fluorescein (37.5 µM).
Conclusions
In summary, positively charged heteroditopic receptor
comprising guanidinium groups (anion binding groups)
and 4,10,16-triaza-18-crown-6 (cation binding domain)
has been synthesized in five steps. As evidenced by the
1H NMR titrations this receptor associates effectively with
zwitterionic form of 5-aminovaleric acid. The selectivity of
this association is attributed to fine geometrical fit
between the distance of positive and negative ends of 5-
AVA and the distance of triaza-18-crown-6 and
guanidinium groups of the receptor that ensure
maximization of the intramolecular interactions. In
addition, we have shown that Indicator Displacement
Assay of 1 and fluorescein is capable of optical sensing
ω-amino acids, and the data obtained from displacement
experiments confirm selectivity of receptor 1 for
5-aminovaleric acid.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
370
420
470
520
Wavelenght [nm]
Figure 6. UV-Vis spectra of fluorescein (30% water in methanol (v/v), 10 mM TRIS,
pH=8.0). Absorbance increase at λ=493 nm as 5-AVA (6.4 mM) is added to solution
of fluorescein-1 complex.
Upon progressive addition of zwitterionic amino acids the
development of a typical band of fluorescein at 493 nm
was observed (Figure 6). This progressive increase of
absorption band occurs due to displacement of indicator
from the receptor by zwitterions, which restores the
original absorbance and therefore reveals the presence
of amino acids. The calculated apparent binding
constants for the indicator-guest exchange process
(Table 2) corroborate high selectivity of receptor 1 for 5-
AVA.13
Conflicts of interest
There are no conflicts to declare.
Experimental
All reagents including amino acids guests and
chemicals were of reagent grade quality and
purchased commercially from Sigma Aldrich.
Amino Acid
β-Alanine
Ka [M-1]
a
-
(4-(aminomethyl)phenyl)methanol (3). A solution of 2
(2 g, 15.25 mmol) in anhydrous THF (40 mL) was added
dropwise to a stirred 1M LiAlH4 solution in THF (40 mL)
at room temperature. Upon completion of addition, the
reaction mixture was heated to reflux and stirred for 5
hours. The reaction mixture was cooled in an ice bath,
quenched by successive addition of H2O (2 mL), 15 %
aqueous NaOH (4 mL) and H2O (6 mL) . After stirring for
4-Aminobutyric acid
5-Aminovaleric acid
6-Aminocaproic acid
114
690
-
a
Table 2. UV-Vis spectroscopy Ka values for interactions of 1 with various amino
acids (298 K, TRIS buffer pH=8.0, 30% H2O/MeOH). a The Absorbance changes were
too small to be calculated Ka, errors of <10%.
4 | J. Name., 2012, 00, 1-3
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30 min at room temperature, the mixture was filtered and H2O (5 mL) and brine (5 mL). The combined organic
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DOI: 10.1039/C9OB02234A
washed with EtOAc. The combined filtrate and washings layers were dried over Na2SO4, filtered and concentrated
were washed successively with H2O and brine, dried over in vacuo. The residue was purified by column
MgSO4, and concentrated in vacuo to give 3 as a white chromatography over silica gel with 1-6% MeOH/CHCl3.
solid (1.67 g, 95%), which was used for the next step Then the volatiles were evaporated and the residue was
without further purification.1H NMR (300 MHz, CDCl3) diluted with acetonitrile (10 mL) and washed with hexane
δ 7.35 – 7.28 (dd, J1=6 Hz, J2=9 Hz, 4H), 4.67 (s, 2H), (3x5 mL). The acetonitrile layer was concentrated in
3.85 (s, 2H), 1.71 (bs, 3H). 13C NMR (75 MHz, CDCl3) δ vacuo to give 6 as a white foam (247 mg, 46%). Rf (10%
127.43, 127.40, 77.58, 77.16, 76.74, 65.14, 46.32. HR- MeOH/DCM) = 0.44.1H NMR (300 MHz, CDCl3) δ 11.53
MS (ESI): m/z = 138.1895 [M+H]+ calc. for C8H12NO+, (s, 3H), 8.55 (s, 3H), 7.32 (d, J = 9.0 Hz, 6H), 7.23 (d, J
found 138.1876.
= 6.0 Hz, 6H), 4.59 (d, J = 6.0 Hz, 6H), 3.67 (s, 6H), 3.57
N,N'-diBoc-N''-(4-hydroxymethyl)-benzylguanidine
(s, 12H), 2.80 (s, 12H), 1.51 (s, 27H), 1.47 (s, 27H).
(4). To a solution of 3 (1.29 g, 9.40 mmol) in anhydrous 13C NMR (75 MHz, CDCl3,δ ppm): 163.74, 156.21,
acetonitrile (25 mL) was added 1-H-Pyrazole-1-(N,N-
bis(tert-butoxycarbonyl)carboxamidine (2.91 g, 9.40
mmol) and DIPEA (1.64 mL, 9.40 mmol). The reaction
mixture was stirred for 2 h at room temperature, under Ar
atmosphere. The mixture was concentrated in vacuo,
residue was diluted with DCM (20 mL) and extracted with
10% aqueous citric acid (10 mL), H2O (10 mL) and brine
(10 mL). The combined organic layers were dried over
Na2SO4, filtered and concentrated in vacuo. The residue
was purified by column chromatography over silica gel
with 0-2% MeOH/DCM as eluant to afford 4 as a pale
yellow oil (3.27 g, 92%). Rf (5% MeOH/DCM) = 0.45.
1H NMR (300 MHz, CDCl3) δ 11.53 (s, 1H), 8.57 (s, 1H),
7.33 (dd, J1=6 Hz, J2=6 Hz, 4H), 4.69 (s, 2H), 4.62
(d, J = 3 Hz, 2H), 1.52 (s, 9H), 1.47 (s, 9H). 13C NMR (75
MHz, CDCl3) δ 163.75, 156.24, 153.34, 140.46, 136.85,
128.24, 127.55, 83.35, 79.56, 77.58, 77.16, 76.74, 65.20,
153.31, 127.88, 83.27, 79.50, 53.94, 29.81, 28.43, 28.18.
HR-MS (ESI): m/z = 1367.7949 [M+Na]+ calc. for
C69H108N12O15Na+, found 1367.7939.
Trifluoroacetic salt of Tri-[(4-guanidinemethyl)-
benzyl]-triaza-18-crown-6 (1).To a solution of 6 (75 mg,
0.06 mmol) in DCM (1 mL) 1 mL of trifluoroacetic acid
was added. The reaction mixture was stirred for 3 h at
room temperature. The mixture was concentrated in
vacuo, after then three times coevaporated with CHCl3.
The residue was taken up in a minimum amount of DCM
and the product was precipitated carefully by slow
addition of diethylether. The solution was decanted off
the precipitate, it was washed once with diethylether,
decanted off again, and the product was dried in the
vacuo to give brown oil in nearly quantitative yield.
1H NMR (300 MHz, CD3CN) δ 7.51 (d, J = 9 Hz, 6H), 7.40
(d, J = 9 Hz, 6H), 4,40 (m, 12H), 3.80 (s, 12H), 3.32 (s,
12H).13C NMR (75 MHz, CD3CN,δ ppm): 158.70, 139.80,
44.91,
28.45,
28.20
HR-MS
(ESI):
+
m/z = 380.2180 [M+H]+ calc. for C19H29N3O5 , found
380.2172.
N,N'-diBoc-N''-(4-bromomethyl)-benzylguanidine
(5).To a solution of 4 (2.23 g, 5.89 mmol) in anhydrous
133.81, 128.48, 64.32, 52.24, 44.93. HR-MS (ESI):
DCM (60 mL) was added CBr4 (2.93 g, 8.84 mmol) and m/z = 745.4984 [M+H]+ calc. for C39H61N12O3 , found
+
745.4994
;
m/z=767.4804 [M+Na]+ (calc. for
PPH3 (2.32 g, 8.84 mmol). The reaction mixture was
stirred for 24 h at room temperature, under Ar
atmosphere. The mixture was concentrated in vacuo.
The residue was purified by column chromatography
over silica gel with 60-100% DCM/Hex, then
0-2% MeOH/DCM as eluant to afford 5 as a white solid
(2.11 g, 80%). Rf (DCM) = 0.67.1H NMR (300 MHz,
CDCl3) δ 11.53 (s, 1H), 8.59 (s, 1H), 7.38 (d, J = 9 Hz,
2H), 7.29 (d, J = 9 Hz, 2H), 4.62 (d, J = 6 Hz, 2H),
4.49 (s, 2H), 1.51 (s, 9H), 1.48 (s, 9H). 13C NMR (75 MHz,
CDCl3) δ 163.73, 156.30, 153.35, 137.83, 137.29,
129.58, 128.37, 83.41, 79.59, 77.58, 77.16, 76.74, 44.71,
33.26, 32.07, 29.84, 29.66, 28.44, 28.20. HR-MS (ESI):
C39H60N12O3Na+), found 767.4814.
1H NMR Titrations Experiments 1H NMR titration
experiments were performed on
a
300 MHz
BrukerAvance spectrometer, at 298K. All solutions were
buffered at pH 8.0 with TRIS (10 mM) in 30% water in
methanol (v/v). In each case 0.5 mL of 3.1x10-3
M
solution of receptor 1 was added to 5 mm NMR tube.
Then to the receptor solution titrant solution of zwitterion
in receptor solution (from 5.9 x10-2 to 6.3x10-2 M) was
added. After each addition of titrant, a spectrum was
registered. Titration isotherms for Bg, Ar, -CH2- crown
ethers protons were fitted to a 1:1 binding model. All
measurements were carried out in at least duplicate
using independent samples. The resulting titration data
were analyzed using BindFit (v0.5) package, available
online at http://supramolecular.org
+
m/z = 464.1155 [M+Na]+ calc. for C19H28BrN3NaO4 ,
found 464.1265.
Tri-[(4-((N,N'-diBoc-guanidine)methy)-benzyl]-triaza-
18-crown-6 (6). To a solution of tri-1,7,13-triaza-18-
crown-6 (200 mg, 0.40 mmol) in anhydrous DMF (20 mL)
was added 5 (538 mg, 1.2 mmol) and DIPEA (420 uL, 2.4
mmol). The reaction mixture was stirred for 6 days at
room temperature, under Ar atmosphere. The mixture
was concentrated in vacuo. The residue (pale orange
UV-Vis titration experiments UV-Vis Titrations of
Indicator and Receptor. The UV-Vis titration was
solid) was diluted with CHCl3 (20 mL) and washed with performed
using
Thermo
Spectronic
Unicam
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4
a) F. P. Schmidtchen, Tetrahedron Lett. 1984, 25, 4361−4364;
UV500 Spectrophotometer at 298K. All solutions were
buffered at pH 8.0 with TRIS (10 mM) in 30% water in
methanol (v/v). A solution of fluorescein (3.8x10-5 M) was
prepared in the cuvette, and into this was titrated a stock
solution of 1 (1.9x10-3 M) and fluorescein, thereby
keeping the host concentration constant. All
measurements were carried out in at least duplicate
using independent samples.
UV-Vis Titration of Receptor/Indicator Ensemble and
Guest. All solutions were buffered at pH 8.0 with TRIS
buffer (10 mM) in 30% water in methanol (v/v). A solution
of fluorescein (3.8x10-5 M) and receptor 1 (3.8x10-4 M)
was prepared in the cuvette, and into this was titrated a
stock solution of indicator, host, and guest, keeping the
indicator and host concentrations constant. The data
were taken at the appropriate wavelength to determine
the association constants. The guests concentration in
the stock solution was from 6.2x10-3 to 6.8x10-3 M.
Titration isotherms were fitted to a 1:1 binding model. All
measurements were carried out in at least duplicate
using independent samples. The resulting titration data
were analyzed using HypSpec package.
b) A. de Silva, C. McVeight, E. M. Magire andViEew. OAr'tiHclaenOlnolinne,
DOI: 10.1039/C9OB02234A
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