Selective binding and extraction of aqueous dihydrogen phosphate solutions via three-armed thiourea receptors

Article abstract of DOI:10.1039/c5ob01450f

A series of neutral anion receptors with one to three thiourea arms were synthesized and their binding to tetrabutylammonium chloride, acetate, and dihydrogen phosphate salts in aqueous DMSO mixtures was examined. The three-armed thiourea host was found to strongly and selectively bind H₂PO₄^- even in DMSO solutions containing up to 30% water. This enabled the dihydrogen phosphate salt to be extracted from water into chloroform in its dibasic form despite the high heat of the hydration of HPO₄^2-.

Full text of DOI:10.1039/c5ob01450f

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: E. V. Beletskiy and
S. R. Kass, Org. Biomol. Chem., 2015, DOI: 10.1039/C5OB01450F.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/obc

Page 1 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01450F
Selective Binding and Extraction of Aqueous Dihydrogen Phosphate Solutions
via Three-armed Thiourea Receptors
Evgeny V. Beletskiy1,2 and Steven R. Kass2*
1Current address: Department of Chemical and Biological Engineering, Northwestern University,
2145 Sheridan Rd., Evanston, IL 60208 USA
2Department of Chemistry, University of Minnesota, 207 Pleasant St. S.E., Minneapolis, MN
55455 USA.
*To whom correspondence should be addressed. E-mail: kass@umn.edu
Abstract: A series of neutral anion receptors with one to three thiourea arms were synthesized
and their binding to tetrabutylammonium chloride, acetate, and dihydrogen phosphate salts in
aqueous DMSO mixtures were examined. The three-armed thiourea host was found to strongly
–
and selectively bind H2PO4 even in DMSO solutions containing up to 30% water. This enabled
the dihydrogen phosphate salt to be extracted from water into chloroform in its dibasic form
2–
despite the high heat of hydration of HPO4 .
1

Organic & Biomolecular Chemistry
Page 2 of 18
View Article Online
DOI: 10.1039/C5OB01450F
Introduction
Phosphate-containing anions are essential in living organisms as they are present in energy and
genetic information storage molecules (i.e., ATP, and DNA and RNA, respectively) as well as
cell membrane phospholipid bilayers and bones. They also take part in cell signaling and serve as
a buffer in serum and urine1-5. In humans, excessive serum phosphate is removed by the kidneys
but many people have abnormal levels indicative of health problems that require treatment6. For
example, both chronic kidney disease and hyperphosphatemia (a condition corresponding to
elevated phosphate levels in blood) require phosphate concentrations to be lowered7. Several
drugs such as Sevelamer are currently on the market for this purpose, but they are slow acting
and typically plateau at phosphate levels above the recommended 5.5 mg/dL amount for patients
with the end stage chronic kidney disease8-11
.
–
2–
3–
In agriculture inorganic phosphate in its different forms (i.e., H2PO4 , HPO4 , and PO4 )
plays a critical role as more than 30,000,000 tons of phosphate–containing fertilizers are used
annually. Phosphate is also commonly found in detergents, and although some countries have
banned it for this purpose, the resulting overall world-wide phosphate pollution causes
eutrophication of natural water sources (i.e., the ecosystem response to excess nutrients). This
leads to green and blue algal blooms with toxin-producing cyanobacteria strains that threaten
aquatic life and drinking water safety12. In the United States at least one-third of the larger lakes
≥ 10 acres contain cyanobacteria, and the situation is worse in many other countries13. Detection
and extraction of phosphate from bio- and ecosystems, consequently is of critical importance14-19
.
Due to the high hydration energies and low basicities of phosphate anions this is a difficult task
that is compounded by the need for high selectivity over commonly found anions such as
chloride and acetate which are more lipophilic and basic, respectively20.
2

Page 3 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01450F
Different types of interactions have been used in developing anion receptors, but the most
promising are the more directional ones such as hydrogen bonding since they allow one to adjust
the binding pocket of the receptor to the size and shape of the target of interest12. Given our
previous work on hydrogen bond catalysts with a 1,3,5-triphenylbenzene core and the molecular
recognition studies of Tobe et al., Choi et al. and others21-27, we decided to explore a series of
neutral thioureas as potential phosphate anion receptors. Our results on mono-, bis- and
tris(thioureas) (Fig. 1) are reported herein28. All three side chains are found to play an important
role in the tris(thiourea), and this compound is a strong enough binder that it can be used to
extract dibasic phosphate out of water into chloroform.
Ar
Ar
Ar'
Ar'
NH
NH
Ar H
HN
HN
S
Ar
S
Ar
Ar
Ar'
S
NH
NH
1-mono
1-bis
1-tris
S
H
H
N
N
Ar =
Ar' = 4-C6H4NO2
NO2
Fig. 1. Anion receptors with 1–3 thiourea arms and a schematic representation of the syn all NH
trans conformation of 1-tris in which curved lines are used for the peripheral aromatic rings.
Results and discussion
Mono(thiourea) 1-mono was synthesized by reacting aniline with p-nitrophenylisothio-cyanate
(2). The bis(thiourea) derivative 1-bis was prepared from the previously reported diamine 3-bis
as indicated in Fig. 2, and tris(thiourea) 1-tris was generated in the same manner only starting
with 1,3,5-tribromobenzene29-31
.
3

Organic & Biomolecular Chemistry
Page 4 of 18
View Article Online
DOI: 10.1039/C5OB01450F
Br
NHCOC(CH3)3
NHCOC(CH3)3
B(OH)2
Pd(PPh3)4
NHCOC(CH3)3
+
Cs2CO3
Br
NO2
N
C
S
S
C
NH2
NH2
NH
NH
H2SO4
(2)
NO2
NH
NH
C
S
3-bis
NO2
Fig. 2. Synthetic scheme for the preparation of 1-bis
Anion binding investigations of neutral receptors are typically carried out in non-polar or
weak hydrogen bond accepting solvents such as chloroform and acetonitrile. Dimethylsulfoxide
(DMSO) provides a much less favorable environment for such studies because of its high
dielectric constant and strong hydrogen bond acceptor ability. Aqueous DMSO mixtures are
even more inhospitable, but 1-tris was found to strongly bind tetrabutylammonium dihydrogen
phosphate in a 99.5 to 0.5 (v/v) DMSO to H2O mixture. Given that the UV-visible absorbance
changes were nearly linearly dependent upon the added concentration of the dihydrogen
phosphate salt up to 1 equivalent, the association constant K is ≥ ~5 x 106 M–1 and too large to
accurately measure by this method. Under the same conditions tetrabutylammonium acetate
(TBAOAc) deprotonates 1-tris. This was revealed by UV-visible spectroscopy in that the
addition of 1 equivalent of TBAOAc or TBAOH to the tris(thiourea) led to the same spectral
changes, and they are distinct from those arising from dihydrogen diphosphate (Fig. S1). Infrared
4

Page 5 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01450F
spectroscopy supports this conclusion in that the reaction of 1-tris with TBAOAc leads to the
appearance of the carbonyl stretch of acetic acid at 1714 cm–1 (Fig. S2). These results are also
consistent with the known pKa value of 12.3 for acetic acid in DMSO and estimates of ~4 for
phosphoric acid32 and ≤ ~8 for 1-mono, 1-bis, and 1-tris (Fig. S3).
Additional aqueous DMSO mixtures were investigated with increasing amounts of water
given the strong binding observed in 99.5% : 0.5% (v/v) DMSO/H2O. As expected, the
association constants of 1-tris for tetrabutylammonium dihydrogen phosphate decrease with
increasing water content (Table 1). In each mixture that was examined K ≥ 103 M–1 and
interestingly over the 5 – 30% water range, the logarithm of K is linearly correlated with the
water percentage. In the higher water content mixtures (i.e., ≥ 25% H2O), acetate and hydroxide
ions led to different UV-vis spectral changes (Fig. S4) and the resulting data could be fit to 1:1
binding isotherms. This suggests that the relative acidities of 1-tris and HOAc change with the
water content and that the latter compound is less acidic when there is little water present, the
two species are similar in acidity when there is 12.5 – 20% water, and acetic acid is more acidic
than 1-tris in ≥ 25% aqueous DMSO mixtures.
Table 1. Association constants for 1:1 binding of 1-tris to tetrabutylammonium salts of
dihydrogen phosphate and acetate in DMSO–H2O mixtures.
%H2O
K (M–1)a
H2PO4
≥ 5 x 106 P.T. 20
2.3 x 105 P.T. 25
7.5 x 104 P.T. 30
%H2O
K (M–1)
H2PO4
1.5 x 104
4.0 x 103 35
1.4 x 103 13
–
–
OAc–
OAc–
0.5
5
12.5
aP.T. = proton transfer.
5

Organic & Biomolecular Chemistry
Page 6 of 18
View Article Online
DOI: 10.1039/C5OB01450F
In wet DMSO with 25% H2O 1 : 1 binding constants of 1-tris with the tetrabutylammonium
salts of dihydrogen phosphate, acetate, and chloride were measured. Association constants of 4.0
x 103, 35 and 1 M–1, respectively were obtained. This makes 1-tris the strongest and most
selective neutral receptor for dihydrogen phosphate reported to date. The importance of all three
thiourea arms in binding the acetate and dihydrogen phosphate salts were also determined by
exploring the corresponding mono and bis(thioureas); chloride was not examined in this way
since the tris(thiourea) derivative does not effectively coordinate with it. Acetate anion binds
three times more strongly with 1-tris than 1-mono (i.e., K = 12 vs 35 M–1, respectively) which
corresponds to the statistical ratio for association when only one thiourea arm is used for binding.
For dihydrogen phosphate, K1-bis/K1-mono = 28/12 or ~2 and this indicates that one arm is used for
–
these two receptors as well. In contrast, the tris derivative has a H2PO4 association constant that
is more than 100-fold larger than the statistical value of 36 M-1 for binding with one arm. All
three arms, consequently, are used in a cooperative fashion in the 1:1 complex of 1-tris with
H2PO4‾. In accord with this deduction, a B3LYP/6-31+G(d,p) optimized structure for this species
was located (Fig. 3) in which each thiourea arm participates in two NH ••• O hydrogen bonds.
–
Fig. 3. An optimized B3LYP/6-31+G(d,p) structure for the 1:1 complex between 1-tris and H2PO4 with
six NH ••• O hydrogen bonds ranging in length from 1.835 – 2.290 Å; CH hydrogens omitted for clarity.
6

Page 7 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01450F
Given the strength and selectivity of the binding for dihydrogen phosphate displayed by 1-
–
tris, we decided to examine if this compound can be used to extract H2PO4 out of water and into
an immiscible organic layer. Chloroform was chosen for this purpose, but since 1-tris is not very
soluble in this medium an analog in which the p-nitrophenyl ring was replaced by a 3,5-
bis(trifluoromethyl)phenyl substituent was employed (Fig. 4). This tris(thiourea) (4-tris) has a
CF3
CF3
S
NH
C
NH
NH
S
C
NH
NH
C
NH
S
CF3
CF3
CF3
CF3
4-tris
Fig. 4. Anion receptor 4-tris.
similar affinity as 1-tris for tetrabutylammonium dihydrogen phosphate in 0.5% and 25%
aqueous DMSO solutions where its measured binding constants are 7.7 x 105 and 1.0 x 104 M–1,
respectively. Consequently, a chloroform-d solution of 4-tris was treated with aqueous
tetrabutylammonium dihydrogen phosphate and a proton nuclear magnetic resonance (1H NMR)
spectrum of the organic layer revealed the presence of the tetrabutylammonium group. It was not
observed when 4-tris was absent indicating that extraction of a phosphorous-containing anion
salt is taking place. To identify its structure, 31P NMR spectra were examined. A broad singlet at
4.0 δ was observed for the extract in the chloroform–d solution whereas control experiments
revealed that a mixture of 4-tris and tetrabutylammonium dihydrogen phosphate gives rise to an
7

Organic & Biomolecular Chemistry
Page 8 of 18
View Article Online
DOI: 10.1039/C5OB01450F
upfield signal at 0.5 δ and the salt itself has a signal at 2.2 δ. These findings indicate that
dihydrogen phosphate is not the phosphorous-containing counterion being extracted into the
chloroform–d layer. A mixture of 4-tris, tetrabutylammonium dihydrogen phosphate, and
tetrabutylammonium hydroxide, however, has a resonance at 4.5 δ which is close to what was
observed in the extraction experiment. This suggests that tetrabutylammonium hydrogen
2
phosphate (i.e., dibasic phosphate (Bu4N+)2HPO4 ‾) is the salt being removed from the water
layer.
1
To test this conclusion, the H and 31P NMR spectra were integrated in the presence of
internal standards and the tetrabutylammonium to phosphorous ratio was found to be 2 : 1 as
required for the dibasic phosphate salt. The stoichiometry for this extraction process requires an
equivalent of phosphoric acid to be produced in the water layer, and consistent with this
requirement the pH of the aqueous layer was found to decrease. These observations taken
2
together indicate that 4-tris is able to transfer (Bu4N+)2HPO4 ‾ from water into chloroform. As a
result, it is not surprising that the extraction process is more efficient for hydrogen phosphate
than dihydrogen phosphate (Table 2). Concentrations as low as 100 µM of dibasic phosphate
were found to bind 10% of the receptor in the organic layer (entry 4). This concentration is 2.5
fold less than the typical lower limit for phosphate concentrations in blood (2.4 mg/dL)33 and 30
times lower than intestinal phosphate levels in patients with renal failure34. Compounds of this
sort, consequently, are promising leads for developing analytical methods to measure serum
phosphate levels and drug candidates for treating hyperphosphatemia35
8

Page 9 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01450F
Table 2. Extraction of aqueous tetrabutylammonium salts of mono and dibasic phosphate into
chloroform-d with 2 equivalents of a 2 mM solution of 4-tris.a
Entry [phosphate salt], mM
% Bu4N+ extracted
(Bu4N+)H2PO4‾ (Bu4N+)2HPO4 ‾
2
1
2
3
4
10
55%
30%
nd
87%
51%
38%
20%
1.0
0.33
0.10
nd
a nd = not determined.
Experimental
1,3,5-tris(2-Pivaloylaminophenyl)benzene (5). A round-bottomed flask was charged with 2-
pivaloylaminophenylboronic acid (2.66 g, 12.0 mmol)29, 1,3,5-tribromobenzene (945 mg, 3.0
mmol), cesium carbonate (9.78 g, 30 mmol), 1,2-dimethoxyethane (15 mL) and water (15 mL).
Nitrogen was bubbled through the two layered solution with stirring for 10 min to remove
oxygen from the system. Tetrakis(triphenylphosphine)palladium(0) (347 mg, 0.30 mmol) was
then added and the reaction mixture was refluxed with vigorous stirring for 13 h under nitrogen.
Upon cooling to room temperature ethyl acetate (50 mL) and water (20 mL) were added and the
two layers were separated. The aqueous solution was extracted with EtOAc (20 ml) and the
combined organic material was washed with brine (10 mL), dried over Na2SO4 and concentrated
under reduced pressure. Purification of the residue via medium pressure liquid chromatography
(MPLC) on silica gel with ethyl acetate : hexanes (15 : 85 to 67 : 33) afforded 1.47 g (81%) of 5
(1,3,5-(1,2-(CH3)3CCONHC6H4)3C6H3) as a white solid (Rf = 0.19 (EtOAc : hexanes, 33 : 67)).
9

Organic & Biomolecular Chemistry
Page 10 of 18
View Article Online
DOI: 10.1039/C5OB01450F
1H NMR (500 MHz, acetone-d6) δ 8.01 (br s, 3H), 7.96 (d, J = 8.0 Hz, 3H), 7.49 (s, 3H), 7.41
(dd, J = 1.5 and 7.5 Hz, 3H), 7.38 (dt, J = 1.5 and 8.0 Hz, 3H), 7.26 (dt, J = 1.0 and 7.5 Hz, 3H),
1.04 (s, 9H). 13C NMR (75 MHz, acetone-d6) δ 177.1, 141.2, 137.0, 135.6, 131.1, 130.5, 129.6,
126.2, 125.4, 40.5, 28.2. HRMS-ESI: calcd for C39H45N3O3Na (M + Na)+ 626.3359, found
626.3365.
1,3,5-tris(2-Aminophenyl)benzene (3-tris). A round-bottomed flask equipped with a
condenser was charged with tris-amide 5 (1.44 g, 2.39 mmol) and 65% aqueous sulfuric acid
(prepared by mixing 10 mL of concentrated H2SO4 and 10 mL of water). The resulting mixture
was heated at 125 °C under nitrogen for 17 h. It was then allowed to cool to room temperature,
poured into water (20 mL), and the aqueous solution was made basic (i.e., pH = 9) with ca. 200
mL of 30% aqueous ammonia. The resulting suspension was extracted with EtOAc (2 x 50 mL)
and the combined organic layers were dried over Na2SO4 and concentrated under reduced
pressure to afford 827 mg (99%) of 3-tris as a pale yellow powder (Rf = 0.26 (EtOAc : hexanes,
67 : 33)). Its spectral data were consistent with those that have been previously reported30.
1,3,5-tris(2-(3-(4-Nitrophenyl)thioureido)phenyl)benzene (1-tris). To a mixture of 3-tris
(70 mg, 0.20 mmol) and 4-nitrophenylisothiocyanate (2, 130 mg, 0.72 mmol) was added dry
THF (0.5 mL) under nitrogen. The solution was stirred for 16 h and concentrated under reduced
pressure. Purification of the residue via MPLC on silica gel with ethyl acetate : hexanes (12 : 78
to 100 : 0) afforded 137 mg (77%) of 1-tris as a yellow powder. 1H NMR (500 MHz, acetone-d6)
δ 9.38 (br s, 6H), 7.98 (d, J = 8.5 Hz, 6H), 7.59 (s, 3H), 7.58 (d, J = 8.5 Hz, 6H), 7.50 – 7.43
(m, 9H), 7.34 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, acetone-d6) δ 181.7, 146.9, 144.9, 140.6,
139.9 136.4, 132.0, 130.3, 130.1, 129.7, 129.2, 125.0, 124.5. HRMS-ESI: calcd for
C45H33N9O6S3Na (M + Na)+ 914.1608, found 914.1618.
10

Page 11 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01450F
1,3-bis(2-(3-(4-Nitrophenyl)thioureido)phenyl)benzene (1-bis). To a mixture of 1,3-bis(2-
aminophenyl)benzene (3-bis, 52 mg, 0.20 mmol)29 and 4-nitrophenylisothiocyanate (79 mg, 0.44
mmol) was added dry THF (0.5 mL) under nitrogen. The solution was stirred for 22 h and
concentrated under reduced pressure. Medium pressure liquid chromatography on silica gel of
the residue with ethyl acetate : hexanes (12 : 78 to 100 : 0) afforded 124 mg (100%) of 1-bis as a
yellow powder (Rf = 0.24 (EtOAc : hexanes, 50 : 50)). 1H NMR (500 MHz, acetone-d6) δ 9.36
(br s, 2H), 9.25 (br s, 2H), 8.04 (d, J = 9.5 Hz, 4H), 7.61 (d, J = 8.5 Hz, 4H), 7.59 (s, 1H), 7.52
(d, J = 7.5 Hz, 2H), 7.49 (app s, 3H), 7.44 (d, J = 9.0 Hz, 2H), 7.43 (t, J = 9.0 Hz, 2H), 7.33 (t, J
= 7.0 Hz, 2H). 13C NMR (75 MHz, acetone-d6) δ 181.8, 147.1, 144.8, 140.4, 140.0, 136.5,
132.1, 130.6, 130.2, 130.0, 129.7, 129.3, 129.0, 125.0, 124.2. HRMS-ESI: calcd for
C32H24N6O4S2Na (M + Na)+ 643.1193, found 643.1195.
1-(4-Nitrophenyl)-3-phenylthiourea (1-mono). To a mixture of aniline (91 µL, 1.0 mmol)
and 4-nitrophenylisothiocyanate (180 mg, 1.0 mmol) was added dry THF (1.0 mL) under
nitrogen. The solution was stirred for 5 h, concentrated under reduced pressure, and the residue
was recrystallized from toluene to afford 189 mg (69%) of 1-mono as a yellow powder. Its
spectral data were consistent with those that have been previously reported31.
1,3,5-tris(2-(3-(3,5-Trifluoromethylphenyl)thioureido)phenyl)benzene (4-tris). To
a
solution of tris-aniline 3-tris (176 mg, 0.50 mmol) in dry THF (1.0 mL) was added 3,5-
trifluoromethylphenylisothiocyanate (0.33 mL, 1.8 mmol) under nitrogen. The resulting solution
was stirred for 20 h and then concentrated under reduced pressure. Medium pressure liquid
chromatography on silica gel of the residue with ethyl acetate : dichloromethane (0 : 100 to 5 :
1
95) afforded 170 mg (29%) of 4-tris as a white powder (Rf = 0.47 (EtOAc : DCM, 5 : 95)). H
NMR (500 MHz, CDCl3) δ 9.18 (br s, 3H), 7.71 (br s, 3H), 7.66 (s, 6H), 7.56 (s, 3H), 7.51 –
11

Organic & Biomolecular Chemistry
Page 12 of 18
View Article Online
DOI: 10.1039/C5OB01450F
13
7.42 (m, 12H), 7.36 (d, J = 7.5 Hz, 3H). C NMR (75 MHz, CDCl3) δ 180.1, 139.3, 138.7,
137.5, 133.0, 131.8 (q, JC‒F = 34 Hz), 131.1, 130.0, 129.5, 128.7, 128.3, 124.5, 122.7 (q, JC‒F
=
19
271 Hz), 119.4. F NMR (282 MHz, CDCl3) δ -63.5. HRMS-ESI: calcd for C51H30F18N6S3Na
(M + Na)+ 1187.1299, found 1187.1263.
Binding constant determinations
Dilute non-aggregating solutions of a receptor S were titrated with a given anion X‾ via a solution
of its tetrabutylammonium salt and were monitored by UV-vis spectroscopy. Unless otherwise
stated, UV absorption spectra were recorded three times for each solution from 250-600 nm and
average values at each wavelength were used after correcting for the absorption of the solvent.
The wavelength with the maximum change in the concentration corrected absorption (Acor
=
A·C0/C, where C and C0 are the current and the initial receptor concentrations) was determined
and then used for further analysis. Absorptions were plotted versus anion concentrations and the
binding constants were determined by an iterative non-linear least squares curve fitting routine
implemented in Excel using the equations below36 where K = the binding constant for 1:1
complex formation, [S] = free receptor concentration, [S]0 = total receptor concentration, [SX] =
bound receptor concentration, [X]0 = total anion concentration, A = absorption of the solution, A0
= absorption of the initial solution without any added anion, Amax = absorption of the solution if
all the receptor is bound (infinite anion excess at the initial receptor concentration).
K[S]0 + K[X ]0 +1− (K[S]0 + K[X ]0 +1)2 − 4K2[S]0[X ]0
[SX ] =
2K
[S]
0 [S]0
[SX ]
[S]0
A = A
+ Amax
12

Page 13 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01450F
Observed and calculated absorptions are given for each titration in the subsequent tables and
figures. In cases where there is a large excess of the anion, [X] ≈ [X]0, and a plot of ΔAcor/[X]
versus ΔAcor gives a straight line where the slope of the linear least squares fit of the data is the
binding constant (K) and the intercept is K·ΔAmax (eq. 1), where ΔAcor = (A ‒ A0)·C0/C, C and C0
are the current and the initial receptor concentrations and ΔAmax = Amax ‒ A0.
ΔA
cor
= −ΔA ⋅ K + K ⋅ ΔAmax
(1)
cor
[X ]
Phosphate extraction from water into chloroform with 4-tris
A 10 mM solution of 4-tris in chloroform (1.06 mL) and a 100 mM solution of
tetrabutylammonium dihydrogen phosphate in water (0.30 mL) were placed in a 1 dram vial. The
mixture was stirred vigorously for 30 minutes after which the organic layer was pipetted out and
passed through a small piece of cotton. Diphenylmethane and triphenylphosphine were added as
internal standards and integration of the 1H NMR peaks at 3.99 ppm (the CH2 of
diphenylmethane) and 2.85 ppm (the CH2's adjacent to nitrogen in the tetrabutylammonium ion)
31
and the P signals at -6.0 ppm (PPh3) and 4.0 ppm (a broad singlet for the extracted species)
gave the tetrabutylammonium cation and phosphorous concentrations in the organic phase (i.e.,
11.8 mM and 5.1 mM, respectively). Similarly, a 2 mM solution of 4-tris in chloroform (1.0 mL)
was utilized to extract 0.5 equivalents of various volumes of aqueous solutions of
tetrabutylammonium dihydrogen phosphate and tetrabutylammonium hydrogen phosphate.
Extraction efficiencies were determined based on the amount of the tetrabutylammonium cation
found in the organic phase as described above and are summarized in Table 2. The effects of
31
complexation and deprotonation on the P NMR chemical shift of dihydrogen phosphate was
investigated by adding 4-tris and/or tetrabutylammonium hydroxide to Bu4N+H2PO4‾ in CDCl3.
13

Organic & Biomolecular Chemistry
Page 14 of 18
View Article Online
DOI: 10.1039/C5OB01450F
Triphenylphosphine at -6.0 ppm was employed as a reference, and the resulting data are given in
Table S20.
Computations
B3LYP37,38 geometry optimizations were carried out with the 6-31+G(d,p) basis set using
Gaussian 0939 on work stations at the Minnesota Supercomputer Institute for Advanced
Computational Research.
Conclusions
The first neutral receptor that binds dihydrogen phosphate strongly and selectively in the
competitive environment of aqueous DMSO is reported. If chloroform is used as a solvent,
extraction of phosphate in its dibasic form can be achieved from aqueous micromolar solutions.
This indicates that this host and its derivatives are promising compounds for applications in
therapeutics and medical and environmental analysis.
Acknowledgments
Generous support from the National Science Foundation, and the Minnesota Supercomputer
Institute for Advanced Computational Research are gratefully acknowledged.
Notes and references
1. B. P. Pedersen, H. Kumar, A. B. Waight, A. J. Risenmay, Z. R. Zurz, B. H. Chau, A.
Schlessinger, M. Bonomi, W. Harries A. Sali, A. K. Johri and R. M. Stroud, Nature, 2013, 496,
533-536.
2. S. S. Kamat, H. J. Williams and F. M. Raushel, Nature, 2011, 480, 570-573.
14

Page 15 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01450F
3. J. A. Ubersax and J. E. Ferrell, Jr., Nature Rev. Mol. Cell Biol. 2007, 8, 530-541.
4. M. J. Berridge and R. F. Irvine, Nature, 1989, 341, 197-205.
5. F. H. Westheimer, Science, 1987, 235, 1173-1178.
6. V. K. Bansal, in H. K. Walker, W. D. Hall and J. W. Hurst, Eds., Clinical Methods: The
History, Physical, and Laboratory Examinations. 3rd edition; Butterworths: Boston, 1990; chpt.
198, 895-899.
7. http://kidney.niddk.nih.gov/kudiseases/pubs/kustats/; NIH Publication No. 12–3895, last
updated 11/15/12.
8. F. Malberti, Drugs, 2013, 73, 673-688.
9. R. Petkewich, Chem. Eng. News, 2008, 86(24), 14.
10. W. Y. Qunibi and C. R. Nolan, Kidney Int., 2004, 66, S33-S38.
11. S. Spaia, Hippokratia, 2011, 15, 22-26.
12. A. E. Hargrove, S. Nieto, T. Zhang, J. L. Sessler and E. V. Anslyn, Chem. Rev., 2011, 111,
6603-6782.
13. http://water.epa.gov/type/rsl/monitoring/vms56.cfm/; last updated 3/6/12.
14. C. Warwick, A. Guerreiro and A. Soares, Biosensors and Bioelectronics, 2013, 41, 1-11.
15. G. He, L. Zhao, K. Chen, Y. Liu and H. Zhu, Talanta, 2013, 106, 73-78.
16. A. Ojida, H. Nonaka, Y. Miyahara, S. Tamaru, K. Sada and I. Hamachi, Angew. Chem. Int.
Ed., 2006, 45, 5518-5521.
17. M. S. Han and D. H. Kim, Angew. Chem. Int. Ed., 2002, 41, 3809-3811.
18. S. Ertul, M. Bayrakci and M. Yilmaz, Separation Science and Technology, 2011, 46, 625-
630.
15

Organic & Biomolecular Chemistry
Page 16 of 18
View Article Online
DOI: 10.1039/C5OB01450F
19. A. F. Danil de Namor, M. Shehab, R. Khalife and I. Abbas, J. Phys. Chem. B, 2007, 111,
12177-12184.
20. P. Bühlmann, E. Pretsch and E. Bakker, Chem. Rev., 1998, 98, 1593-1687.
21. This manuscript was taken in part from, E. Beletskiy, Ph.D. Thesis, University of Minnesota,
2014.
22. E. V. Beletskiy, J. Schmidt, X. B. Wang and S. R. Kass, J. Am. Chem. Soc., 2012, 134,
18534-18537.
23. I. Hisaki, S.-I. Sasaki, K. Hirose and Y. Tobe, Eur. J. Org. Chem., 2007, 4, 607-615.
24. Y. S. Park, S. -H. Bang and H. -J. Choi, Tetrahedron Lett., 2013, 54, 6708-6711.
25. P. Buhlmann, S. Amemiya, S. Nishizawa, K. P. Xiao and Y. Umezawa, J. Incl. Phenom.
Mol. Rec. 1998, 32, 151-163.
26. S. Sasaki, D. Citterio, S. Ozawa and K. Suzuki, J. Chem. Soc. Perkin Trans. 2, 2001, 2309-
2313.
27. S. Sasaki, S. Ozawa, D. Citterio, N. Iwasawa and K. Suzuki, Anal. Sci., 2001, 17, i1659-
i1661.
28. For recent work on charged species, see: W. Gong, D. Na, L. Fang, H. Mehdi and G. Ning,
Org. Biomol. Chem., 2015, 13, 1979-1982.
29. S. W. Youn, J. H. Bihn and B. S. Kim, Org. Lett., 2011, 13, 3738-3741.
30. P. Piatek and N. Slomiany, Synlett, 2006, 13, 2027-2030.
31. M. Roice, S. F. Christensen and M. Meldal, Chem. Eur. J., 2004, 10, 4407-4415.
32. This crude estimate is based upon the measured pKa's of several phosphoric acids derived
from binol. For the latter results, see: P. Christ, A. G. Lindsay, S. S. Vormittag, J.-M. Neudörfl,
A. Berkessel and A. C. O'Donoghue, Chem. Eur. J., 2011, 17, 8524-8528.
16

Page 17 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB01450F
33. Textbook of Clincial Chemistry, N. W. Tietz, Ed; W.B. Saunders, Philadelphia, 1986.
34. O. M. Wrong, Lancet, 1973, 3, 493.
35. E. Beletskiy, S. R. Kass, U. S. provisional patent serial no. 61/912,469, filed 12/5/13; patent
pending.
36. K. A. Connors, Binding Constants, John Wiley & Sons: New York, 1987.
37. A. D. Becke, J. Chem. Phys. 1993, 98, 5648-5652.
38. C. Lee, W. Yang and R. G. Parr, Phys. Rev. B 1988, 37, 785-789.
39. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P.
Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T.
Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K.
N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell,
J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J.
B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J.
Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski,
G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B.
Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian, Inc., Wallingford CT, 2013.
17

Organic & Biomolecular Chemistry
Page 18 of 18
View Article Online
DOI: 10.1039/C5OB01450F
Table of Content Graphic
–
A three-armed thiourea host that strongly and selectively binds H2PO4 and extracts HPO42– from
water into chloroform.
18