Thermodynamic and kinetic stabilization of divanadate in the monovanadate/divanadate equilibrium using a Zn-cyclene derivative: Towards a simple ATP synthase model

Article abstract of DOI:10.3762/bjoc.8.8

For the condensation of anions such as phosphate and ADP to form ATP and water, nature employs sophisticated supramolecular systems to overcome coulomb repulsion and activation barriers. For an attempt to create a simple, analogous chemical system, the di

Full text of DOI:10.3762/bjoc.8.8

Thermodynamic and kinetic stabilization of
divanadate in the monovanadate/divanadate
equilibrium using a Zn-cyclene derivative:
Towards a simple ATP synthase model
Hanno Sell, Anika Gehl, Frank D. Sönnichsen and Rainer Herges*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 81–89.
Otto-Diels Institut für Organische Chemie,
Christian-Albrechts-Universität zu Kiel, Otto-Hahn-Platz 4, 24418 Kiel,
Germany
doi:10.3762/bjoc.8.8
Received: 30 September 2011
Accepted: 21 December 2011
Published: 12 January 2012
Email:
Rainer Herges* - rherges@oc.uni-kiel.de
This article is part of the Thematic Series "Supramolecular chemistry II".
Guest Editor: C. A. Schalley
* Corresponding author
Keywords:
EXSY NMR; NMR titration; supramolecular chemistry; vanadate
condensation; 51V NMR; Zn-cyclene
© 2012 Sell et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
For the condensation of anions such as phosphate and ADP to form ATP and water, nature employs sophisticated supramolecular
systems to overcome coulomb repulsion and activation barriers. For an attempt to create a simple, analogous chemical system, the
dimerization of vanadate is probably the simplest model. We have investigated Zn-benzylcyclene which favors the dimerization
thermodynamically as shown by NMR titration. Moreover, EXSY NMR experiments reveal that the vanadate dimer is also kineti-
cally stabilized with respect to hydrolysis by complexation with Zn-cyclene.
Introduction
Driving endergonic reactions with external energy sources is energy is put into the system mainly in step c), thus, avoiding
one of the challenging and so far unsolved problems in supra- product inhibition [2].
molecular chemistry. The best known example in nature is
probably the condensation of phosphate and ADP to ATP, In a first attempt towards the very ambitious goal of creating an
driven by a proton gradient across a membrane [1]. There are a artificial ATP synthase model, the a) → b) → c) energy profile
number of preconditions that have to be met for such an energy has to be carefully designed to operate such a system. Here, we
driven condensation of inorganic anions. At least three steps focused on step b). To further simplify the system, we chose the
(four stages) are required (Figure 1): a) Moving both complex- dimerization of vanadate as a model for the phosphate conden-
ated anions together, b) bond formation (condensation), and c) sation for the following reasons: The condensation of phos-
product release/reactant binding. In the ATP synthase, the phate has a very high activation barrier (disodium phosphate
81

Beilstein J. Org. Chem. 2012, 8, 81–89.
Figure 1: Simplified free energy scheme for driving an endergonic condensation (elimination/addition of water is omitted for simplicity). The non-
driven reaction is depicted by a dashed line. a) Moving both complexated anions together, b) bond formation (condensation), and c) product release/
reactant binding.
condensates to pyrophosphate at temperatures >250 °C) which requires the presence of metal cations such as Zn2+, Mg2+ and
is likely difficult to be realized in a prototype artificial system Mn2+ in the active site [9]. It is generally assumed that the coor-
[3]. In contrast to phosphates, mono-, di- and higher vanadates dination of the metal cations to the phosphate oxygen atoms
are in equilibrium at room temperature depending on the pH. reduces the Coulomb repulsion in the transition states or in the
Moreover, 51V is one of the most sensitive NMR nuclei in the intermediates, thus, lowering the activation barrier [10]. In tyro-
periodic table [4]. The concentration of the different vanadate sine phosphatase, this role is taken by hydrogen bonding of the
species can be easily determined even in very diluted solutions phosphate ion to the positively charged arginine side chain [11].
by integration of the corresponding NMR peaks [5]. Although In the key step of the hydrolysis of pyrophosphate by the yeast
vanadates are slightly larger than their corresponding phos- phosphatase, two Mg2+ ions and arginine H-bonds assist the
phates (V–O bond approximately 0.170 nm, P–O bond approxi- P–O bond cleavage [12]. A number of artificial enzymes that
mately 0.152 nm) they exhibit a striking similarity in their catalyze phosphate ester hydrolysis have been synthesized and
chemistry [6]. Vanadates are accepted by a number of phos- investigated [13]. Kimura et al. demonstrated the acceleration of
phatase enzymes [7]. Thus, the plethora of information on both phosphate ester hydrolysis by Zn2+ complexes of triazacyclodo-
natural and artificial phosphate binding systems can be used to decane and tetraazacyclododecane (cyclene) [14]. The effi-
design vanadate coordinating ligands. Vice versa, the vanadate ciency was increased by tethering two or three Zn-cyclene units
model systems will provide insight into the phosphate conden- in an appropriate distance to allow multiple coordination to the
sation, e.g., the formation of ATP [8].
phosphate oxygen atoms [15]. Brown et al. were able to
catalyze the cyclization (hydrolysis/intramolecular conden-
A large number of thoroughly investigated enzymes hydrolyze sation) of 2-hydroxypropyl aryl phosphate using a tethered
phosphates and provide insight into conceivable mechanisms of dimeric Zn-triazacyclododecane [16]. Lehn et al. catalyzed the
phosphate or vanadate condensations. In phosphatases, kinases condensation of acetyl phosphate and phosphate to pyrophos-
and ATP synthase, the catalyzed transfer of phosphate usually phate using a protonated macrocyclic polyamine [17-19].
82

Beilstein J. Org. Chem. 2012, 8, 81–89.
Results and Discussion
Investigation of the thermodynamic effect
The 51V NMR titration at pH 9.5 is shown in Figure 3 (for the
corresponding spectra recorded at pH 7.6 and 8.5 see
To investigate the fundamental thermodynamic and kinetic Supporting Information File 1). The addition of Zn-benzyl-
effects of metal coordination in inorganic condensation reac- cyclene 1 leads to an upfield shift of the monovanadate and the
tions, we focused on the influence of benzyl Zn-tetraazacyclo- divanadate signal at all pH values that were investigated. This
dodecane (Zn-benzylcyclene) 1 on the aggregation of vanadate shift indicates that Zn-benzylcyclene 1 is capable of complexing
in water.
both mono- and divanadate. The monovanadate signal remains
the main signal throughout the titrations, however, the signal of
According to DFT calculations (B3LYP/6-31G*) of a number the divanadate increases upon addition of Zn-benzylcyclene. By
of different geometries, the monovanadate anion is bound to the raising the concentration of benzylcyclene 1t from 0 to 7.5 mM
Zn-cyclene unit by forming one coordination bond to the Zn2+ in a system with a total concentration of vanadium [V]t of
ion and two hydrogen bonds to the NH groups of the cyclene 1.5 mM at a pH value of 9.5, the ratio of vanadium bound in
ring (Figure 2). The formation of a bidentate complex (two divanadate species rises from 5% to 23%, while the ratio of
vanadate oxygens coordinating with Zn2+) is considerably less vanadium bound in monovanadate decreases from 95% to 77%.
favorable. This is also in agreement with X-ray structures of Hence, Zn-benzylcyclene 1 favors the condensation of mono-
Zn-cyclene phosphate complexes [15].
vanadate to divanadate.
For the analysis of the multicomponent system, 1-D 51V NMR To determine the stoichiometry and stability of the most impor-
titrations of Na3VO4 with ligand 1 were performed at three tant species in this system, we included all vanadate oxo-anions
different pH values at 25 °C (pH ~9.5 CHES buffer (N-cyclo- that are known to exist in aqueous solution [20,21] and the
hexyl-2-aminoethanesulfonic acid); pH ~8.5 EPPS buffer (3-[4- corresponding complexes with ligand 1 in different stoichiome-
(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid); pH ~7.6 tries as basis of a complex formation model. This model was
HEPES buffer (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesul- refined with respect to the experimental data.
fonic acid)) (Figure 3, see also Supporting Information File 1).
These alkaline pH values were chosen because monovanadate is In the approach mentioned above, the equilibria of the multi
the main species in this range. Like the monovanadate, the di- component system were described mathematically by mass
and tetravanadate exist primarily in the neutral region.
balance equations. When the equilibrium species are written
Figure 2: Binding motive of a vanadate zinc benzylcyclene complex (left) as suggested by the results of DFT calculations (B3LYP/6-31G*) (middle).
Bond lengths of coordinative bonds and H bridges are given in Å. Metal coordination as well as H–bond formation are operative in binding the oxo-
anion to the zinc cyclene unit. The schematic representation (right) is used to improve clarity.
83

Beilstein J. Org. Chem. 2012, 8, 81–89.
The substitution of the complex species concentration in the
mass balances of the components leads to the mass balance
equation system (3). The concentrations of the free components
are the variables; the stability constants are the parameters to be
optimized.
(3)
In the 51V NMR titrations for the analysis of the vanadate/Zn-
benzylcyclene system the total vanadium concentration [V]t at
each pH value was 1.5 mM, and the total concentration of 1
([1]t) was varied from 0 to 7.5 mM. The NMR data were
obtained by careful deconvolution and integration of the signals
using routines included in the MestreNova program [22]. The
mathematical analysis of the NMR data was performed with the
program system LAKE [23] for chemical equilibrium analysis
and with MS-Excel [24]. Data of all titrations (78 titration
points in total) were included in the calculation. The formation
constants for the vanadium oxo-anions of the VO43−/H+-
subsystem [21], the pKa value of Zn-benzylcyclene 1 [14] as
well as the pKa values of the buffers [25] were adopted from
literature. The influence of the ionic medium was taken into
account in the calculation by means of the Davies equation [26].
The most important ternary species in the equilibrium model
that fit the recorded data are presented in Table 1.
Figure 3: 51V NMR titration at pH 9.5 ([V]t = 1.5 mM, [1]t = 0 to 7.5 mM
(0 to 5 equiv), 100 mM CHES). [V]t and [1]t are the total molar concen-
trations of vanadium and Zn-benzylcyclene 1. The predominant
species in equilibrium are represented schematically (Figure 2).
Table 1: Stoichiometry and formation constants of the complex
species in the proton/vanadium/Zn benzylcyclene system.
p, q, ra
formulab
lg βpqrc (3σ)
with H+, VO43−, and Zn-benzylcyclene 1 as components, they
are formed according to the general expression (Equation 1)
[21]:
1, 1, 1
2, 2, 1
2, 2, 2
3, 2, 1
3, 2, 2
8, 4, 2
8, 4, 4
HVO41
16.7 (0.3)
32.2 (0.1)
35.7 (0.8)
40.6 (0.3)
43.6 (0.4)
99.9 (0.7)
105.6 (1.8)
V2O712−
V2O712
HV2O71−
HV2O7122+
V4O1212
V4O12144+
(1)
The formation constants of the species βpqr are defined using
the concentrations of 1, VO43− and of the protons according to
Equation 2.
aStoichiometric coefficients according to the formation from the compo-
nents H+, VO43− and 1 (Equation 1); bformula of the oxo-anion com-
plex. The water formed according to the formation reaction from the
components is not given; cionic strength, I = 150 mM, T = 298 K.
(2)
According to these data, the association constant for the proto-
nated monovanadate HVO42− with 1 is about 103.8 M−1, while
84

Beilstein J. Org. Chem. 2012, 8, 81–89.
the association constant for V2O74− is Ka = 106.2 M−1. The pKa
value for the coordinated divanadate is 8.4 and therewith,
1.9 pKa units lower than for the uncoordinated V2O74− ion. The
binding of a second Zn-benzylcyclene unit proceeds with an
association constant of 103.5 M−1. Consequently, the affinity of
the divanadate for both the proton and the second Zn-benzyl-
cyclene is lowered by the coordination of the first Zn-benzyl-
cyclene unit. A speciation diagram for the titration at pH 9.5
based on these data is given in Figure 4. Fits of calculated and
experimental 51V NMR integral data at pH 7.6, 8.5, and 9.5 are
given in the Supporting Information File 1.
(6)
(7)
The formation constants given in Table 1 can be used to calcu-
late the free enthalpy (ΔG10, ΔG20, ΔG30) of the condensation
reactions (Equations 4–9):
(8)
(9)
(4)
(5)
The analysis reveals that the free reaction enthalpy for the con-
densation of hydrogen monovanadate is lowered by the com-
plexation of the vanadium oxo-anions with Zn- benzylcyclene.
In the absence of the ligand, the dimerization of monovanadate
Figure 4: Speciation of vanadium in a solution containing 1.5 mM Na3VO4, 100 mM CHES (pH = 9.5) and a Zn-benzylcyclene 1 amount varying from
0 to 5 equiv.
85

Beilstein J. Org. Chem. 2012, 8, 81–89.
is almost thermoneutral (ΔG10 = 0.9 kJ mol−1). However, the
condensation of a free monovanadate with a monovanadate
bound to Zn-benzylcyclene is exothermic (ΔG20 = −14.8 kJ
mol−1) and the reaction of two complexed monovanadates is
less exothermic (ΔG30 = −13.1 kJ mol−1). Hence, the conden-
sation is favored by the complexation with Zn-benzylcyclene
and one equiv of the ligand is more effective than two.
(12)
(13)
Further insight into the stabilization of divanadate by
Zn-benzylcyclene is obtained by analyzing the ligand exchange
reactions (Equation 10 and Equation 12). While the transfer of a
ligand from hydrogenmonovanadate to divanadate is exergonic Accordingly, a complexed divanadate is destabilized by the
(ΔG100 = −13.7 kJ mol−1), the transfer of a second ligand from coordination of a second ligand.
another hydrogen monovanadate is endergonic (ΔG120 = 1.1 kJ
mol−1).
Investigation of the kinetic effect
The influence of the complexation on the rates of condensation
and hydrolysis of the involved vanadium oxo-anions was exam-
ined by 51V EXSY NMR experiments [27,28]. 51V EXSY
NMR data were collected for solutions buffered with EPPS and
HEPES and 1.5 mM total vanadium concentration at 25 °C. For
each buffer solution, the amount of Zn-benzylcyclene 1 was
varied. The mixing time was 1 ms (Figure 5).
(10)
(11)
The data show a decrease for the pseudo first order rate constant
of the condensation (k12 in Equation 14) as well as for the
hydrolysis (k21 in Equation 15), while the influence on the rate
constant of the hydrolysis is stronger.
Figure 5: 51V EXSY NMR spectrum (tmix = 1 ms) of a solution containing 1.5 mM Na3VO4, 3 mM Zn-benzylcyclene and 100 mM EPPS (pH = 7.9).
86

Beilstein J. Org. Chem. 2012, 8, 81–89.
mol−1) compared to the reaction of a complexed vanadate with
a free monovanadate (ΔG20 = −14.8 kJ mol−1). For the design
of an artificial system driving endergonic condensations
(Figure 1), we draw the conclusion that only one of the binding
sites should provide metal coordination to the vanadate (or
phosphate) and the other binding site should associate the
nucleophilic anion by neutral hydrogen bonds.
(14)
(15)
In the HEPES buffered solution (pH 7.6), the rate constant for
the condensation decreased from 73 s−1 to 68 s−1 upon the addi-
tion of Zn-benzylcyclene (3 mM), while the rate constant for
the hydrolysis decreased from 206 s−1 to 89 s−1. Compared to
that, in the EPPS buffered solution (pH 8.0), the rate constant
for condensation decreased from 23 s−1 to 14 s−1 upon the addi-
tion of Zn-benzylcyclene 1 (3 mM), while the rate constant for
the hydrolysis decreased from 164 s−1 to 70 s−1.
Similar effects obviously hold for natural systems – particu-
larly ATP synthase, phosphatases and kinases catalyzing the
condensation, hydrolysis and phosphate transfer in phosphate
esters and phosphate oligomers – which are unsymmetric with
respect to metal complexation of the substrates. In the P–O
bond forming/cleaving step of yeast pyrophosphatase, the phos-
phate group acting as the electrophile is activated by coordina-
The fact that the condensation of monovanadate is slower in the tion to Mg2+ as well as by hydrogen bonding to a guanidinium
presence of the ligand is probably due to steric reasons. The group [12]. The electrophilicity of ADP in ATP synthase is
nucleophilic attack on the complexed monovanadate is only even further increased by electron transfer to Mg2+ forming a
possible from the non-shielded side. Moreover, the role of the radical ion pair [29]. Similar to the ATP synthase, a simplified
monovanadate acting as the electrophile is defined by com- artificial system should prevent product inhibition by actively
plexation which reduces the probability of effective collisions releasing the product. This could be realized by a (photo)-
and, thus, decreases the entropy of activation. Both effects, switchable ligand, see step a) and c) in Figure 1.
obviously, overcompensate the increased reactivity of the vana-
Experimental
date acting as the electrophile which is induced by complexa-
tion (decreased Coulomb repulsion of the two negatively Synthesis
charged reactants). The same arguments are true for the hydrol- 1-Benzyl-1,4,7,10-tetraazacyclododecane (benzylcyclene):
ysis of divanadate (attack of water). However, the thermody- Benzylchloride (1.00 mL, 8.69 mmol) and cyclene (6.00 g,
namic stabilization of divanadate further increases the barrier of 34.8 mmol) were dissolved in dry chloroform (240 mL). Under
hydrolysis (provided that the transition state is less effected). nitrogen atmosphere, triethylamine (1.45 mL, 10.4 mmol) was
Therefore, the rate constant of the divanadate hydrolysis is added and refluxed for 20 h. After cooling, chloroform
lowered more strongly by complexation than the rate constant (200 mL) was added, and the organic phase was washed three
for the condensation of monovanadate.
times with 1 N sodium hydroxide solution (200 mL) and three
times with distilled water (200 mL). The organic phase was
dried over potassium carbonate and the solvent was removed
Conclusion
In conclusion, the condensation of monovanadate to divanadate under reduced pressure [30].
is considerably favored in the presence of the ligand 1 (Zn-
benzylcyclene). In the absence of the ligand at pH 9.5 the con- For efficient purification, the crude product (2.27 g) was
densation is almost thermoneutral (ΔG10 = 0.9 kJ mol−1) protected with di-tert-butyl dicarbonate (3 equiv) in dry di-
whereas upon addition of the ligand the dimerization reaction is chloromethane (100 mL). After removal of the solvent the prod-
exergonic (ΔG20 = −14.8 kJ mol−1). This is mainly due to the uct was purified by column chromatography (cyclo-
fact that the free binding enthalpy of ligand 1 to divanadate is hexane:ethylacetate = 1:1, Rf = 0.58). Yield: 4.24 g (87%);
13.7 kJ mol−1 larger than the complex formation with mono- 1H NMR (500 MHz, CD2Cl2) δ 7.3–7.2 (m, 5H, CHaryl), 3.65
vanadate.
(s, 2H, Ar-CH2), 3.52 (br s, 4H, CH2), 3.4–3.1 (m, 8H, CH2),
2.60 (br s, 4H, CH2), 1.41, 1.36 (2 s, 27H, CH3) ppm; 13C NMR
Complexation with 1 also favors the formation of divanadate (125 MHz, CD2Cl2) δ 155.7 (C=O), 137.5 (Caryl,q), 128.5,
kinetically by decreasing its rate of hydrolysis from 164 s−1 to 127.6, 123.6, (Caryl), 80.0, 79.5, 79.4 (C(CH3)3), 57.5 (Ar-
70 s−1 upon addition of ligand 1, as determined by 51V EXSY CH2), 50.7, 50.1, 48.2 (CH2), 28.8, 28.6 (CH3) ppm.
NMR experiments.
To release the free amine, the boc-protected benzylcyclene
Dimerization, however, is less favorable if both reacting mono- (3.76 g, 6.69 mmol) was dissolved in dry dichloromethane
vanadate units are complexed with ligand 1 (ΔG30 = −13.1 kJ (30 mL). Trifluoroacetic acid (5 mL) was added under a
87

Beilstein J. Org. Chem. 2012, 8, 81–89.
nitrogen atmosphere and the mixture stirred at room tempera- priate amount of the vanadate stock solution and the receptor 1
ture for 12 h. Under ice cooling sodium hydroxide (3 g) and stock solution in an NMR tube. Finally, the volume of each
distilled water (30 mL) were added. The organic layer was sep- sample was brought to a volume of 600 µL by addition of the
arated, and the aqueous phase extracted five times with 50 mL appropriate amount of the respective buffer stock solution. All
dichloromethane. The combined organic phases were dried over NMR spectra were recorded on a Bruker FT-NMR-spectrom-
potassium carbonate and the solvent was removed under eter DRX 500. Vanadium chemical shifts are reported relative
reduced pressure to yield a colorless solid (1.52 g, 86%); to the external reference VOCl3 (0 ppm).
1H NMR (500 MHz, CD2Cl2) δ 7.3–7.2 (m, 5H, CHaryl), 3.64
(s, 2H, Ar-CH2), 2.8–2.4 (m, 16H, CH2) ppm; 13C NMR (125 EXSY measurements
MHz, CD2Cl2) δ 139.9 (Caryl,q), 129.5, 128.6, 127.4 (Caryl), The EXSY measurements were performed at two different pH
60.1 (Ar-CH2), 51.8, 48.1, 46.6, 46.1 (CH2) ppm.
values (pH 7.6 and pH 8.0) and at a total vanadate concentra-
tion of 1.5 mM. The concentration of 1 was 3.0 mM. For all
In order to regain the excess of cyclene used in the first step, the stock solutions deionized water was used as solvent. The
aqueous phase was concentrated under reduced pressure and samples were prepared in the following way: The buffer stock
neutralized with 1 N HCl until a white precipitate formed. The solutions had a total buffer concentration of 500 mM (HEPES
precipitate was filtered to yield pure cyclene (2.8 g). 1H NMR for pH 7.6, EPPS for pH 8.0) and 250 mM of sodium
(200 MHz, DCl3) δ 2.65 (s, 16H, CH2) ppm.
hydroxide. The vanadate stock solution contained of 50 mM
sodium orthovanadate, and the concentration of the Zn-benzyl-
Zinc-1-benzyl-1,4,7,10-tetraazacyclododecane nitrate (1): cyclene 1 stock solution was 10 mM. For each experiment
1-Benzyl-1,4,7,10-tetraazacyclododecane (1.00 g, 3.81 mmol) 60 μL of the vanadate stock solution, 400 μL of the respective
was dissolved in methanol (20 mL). A zinc nitrate solution in buffer stock solution, and 600 μL of the Zn-benzylcyclene 1
water (49.7 mM, 76.6 mL) was added and the solution was stock solution were mixed with 100 μL deuterium oxide and
stirred for 1 h at room temperature. The solvent was removed 840 μL deionized water. The NMR tubes were then filled with
under reduced pressure and the colorless solid was dried. Yield: 600 μL of the mixture. In all experiments a mixing time of 1 ms
100 %; 1H NMR (500 MHz, D2O) δ 7.4–7.3 (m, 5H, CHaryl), was applied.
3.94 (s, 2H, CH2), 3.2–3.0 (m, 4H, CH2), 2.9–3.0 (m, 12H,
CH2) ppm; 13C NMR (125 MHz, D2O) δ 138.5 (Caryl,q), 131.3,
Supporting Information
129.1, 128.7 (Caryl), 56.7 (Ar-CH2), 49.1, 44.5, 43.6, 42.2
(CH2) ppm; ESI m/z (%): 363.1 (100), 364.1, 365.1, 366.1,
Supporting Information File 1
367.1, 368.1 [C15H26N4Zn(OH2)2], 326.1 (78), 327.1, 328.1,
329.1, 330.1, 331.1 [C15H25N4Zn].
51V NMR spectra and data fitting results.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-8-S1.pdf]
NMR experiments
NMR titrations
For the NMR titrations three different buffer stock solutions
were used: pH 7.6: HEPES (4-(2-hydroxyethyl)piperazine-1-
ethanesulfonic acid), pH 8.5: EPPS (4-(2-hydroxyethyl)-1-
piperazinepropanesulfonic acid) and pH 9.5: CHES (2-(cyclo-
hexylamino)ethanesulfonic acid). The stock solutions had a
total buffer concentration of 100 mM and 50 mM of sodium
hydroxide. They were prepared by dissolving the appropriate
amount of buffer and sodium hydroxide in 90% deionized water
and 10% deuterium oxide. To obtain the vanadate stock solu-
tion (50 mM) sodium orthovanadate (92.0 mg) was dissolved in
the respective buffer solution (10 mL). For the receptor stock
solution (10 mM) the receptor 1 (45.2 mg) was dissolved in
10 mL of the respective buffer solution.
Acknowledgements
We would like to thank the Deutsche Forschungsgemeinschaft
(DFG) for funding via SFB 677 (Function by Switching).
References
1. Boyer, P. D. Annu. Rev. Biochem. 1997, 66, 717–749.
doi:10.1146/annurev.biochem.66.1.717
2. O’Neal, C. C.; Boyer, P. D. J. Biol. Chem. 1984, 259, 5761–5767.
3. Osterheld, R. K. Top. Phosphorus Chem. 1972, 7, 103–254.
4. Pregosin, P. S., Ed. Transition Metal Nuclear Magnetic Resonance;
Elsevier, 1991.
5. Crans, D. C.; Shin, P. K.; Armstrong, K. B. Application of NMR
Spectroscopy to Studies of Aqueous Coordination Chemistry of
Vanadium(V) Complexes. In Mechanistic Bioinorganic Chemistry;
Thorp, H. H.; Pecoraro, V. L., Eds.; Advances in Chemistry, Vol. 246;
American Chemical Society: Washington, DC, 1995; pp 303–328.
doi:10.1021/ba-1995-0246.ch011
All measurements were carried out at a 1.5 mM total vanadate
concentration and a 100 mM buffer concentration. The samples
of the 51V NMR titrations were prepared by mixing the appro-
6. Chasteen, N. D. Struct. Bond. 1983, 53, 105–138.
doi:10.1007/BFb0111304
88

Beilstein J. Org. Chem. 2012, 8, 81–89.
7. Crans, D. C.; Keramidas, A. D.; Drouza, C.
Phosphorus, Sulfur Silicon Relat. Elem. 1996, 109, 245–248.
doi:10.1080/10426509608545136
License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
8. Ko, Y. H.; Hong, S.; Pedersen, P. L. J. Biol. Chem. 1999, 274,
28853–28856. doi:10.1074/jbc.274.41.28853
9. Lipscomb, W. N.; Sträter, N. Chem. Rev. 1996, 96, 2375–2434.
doi:10.1021/cr950042j
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
10.Lahiri, S. D.; Zhang, G.; Dunaway-Mariano, D.; Allen, K. N. Science
2003, 299, 2067–2071. doi:10.1126/science.1082710
11.Asthagari, D.; Dillet, V.; Liu, T.; Noodleman, L.; Van Etten, R. L.;
Bashford, D. J. Am. Chem. Soc. 2002, 124, 10225–10235.
doi:10.1021/ja020046n
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
12.Oksanen, E.; Ahonen, A.-K.; Tuominen, H.; Tuominen, V.; Lahti, R.;
Goldmann, A.; Heikinheimo, P. Biochemistry 2007, 46, 1228–1239.
doi:10.1021/bi0619977
(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
13.Kirby, A. J. Angew. Chem., Int. Ed. Engl. 1996, 35, 706–724.
doi:10.1002/anie.199607061
14.Koike, T.; Kimura, E. J. Am. Chem. Soc. 1991, 113, 8935–8941.
doi:10.1021/ja00023a048
doi:10.3762/bjoc.8.8
15.Kimura, E.; Aoki, S.; Koike, T.; Shiro, M. J. Am. Chem. Soc. 1997, 119,
3068–3076. doi:10.1021/ja9640408
16.Bunn, S. E.; Liu, C. T.; Lu, Z.-L.; Neverov, A. A.; Brown, R. S.
J. Am. Chem. Soc. 2007, 129, 16238–16248. doi:10.1021/ja076847d
17.Hosseini, M. W.; Lehn, J.-M. Chem. Commun. 1985, 1155–1157.
doi:10.1039/C39850001155
18.Hosseini, M. W.; Lehn, J.-M. J. Am. Chem. Soc. 1987, 109,
7047–7058. doi:10.1021/ja00257a024
19.Hosseini, M. W.; Lehn, J.-M. J. Chem. Soc., Chem. Commun. 1988,
397–399. doi:10.1039/C39880000397
20.Schmidt, H.; Andersson, I.; Rehder, D.; Pettersson, L. Chem.–Eur. J.
2001, 7, 251–257.
doi:10.1002/1521-3765(20010105)7:1<251::AID-CHEM251>3.0.CO;2-
9
21.Cruywagen, J. J.; Heyns, J. B. B. Polyhedron 1991, 10, 249–253.
doi:10.1016/S0277-5387(00)81596-X
22.Willcott, M. R. J. Am. Chem. Soc. 2009, 131, 13180.
doi:10.1021/ja906709t
23.Ingri, N.; Andersson, I.; Pettersson, L.; Yagasaki, A.; Andersson, L.;
Holmström, K. Acta Chem. Scand. 1996, 50, 717–734.
doi:10.3891/acta.chem.scand.50-0717
24.Del Piero, S.; Melchior, A.; Polese, P.; Portanova, R.; Tolazzi, M.
Ann. Chim. 2006, 96, 29–49. doi:10.1002/adic.200690005
25.Technical Librarby. Buffer pKa/pH Tables and Formulas.
http://www.sigmaaldrich.com/life-science/core-bioreagents/learning-cen
ter/technical-library.html#pH (accessed Dec 9, 2011).
26.Davies, C. Ion Association; Butterworth: Washington, D.C., 1962;
pp 37–53.
27.Crans, D. C.; Rithner, C. D.; Theisen, L. A. J. Am. Chem. Soc. 1990,
112, 2901–2908. doi:10.1021/ja00164a009
28.Perrin, C. L.; Gipe, R. K. J. Am. Chem. Soc. 1984, 106, 4036–4038.
doi:10.1021/ja00326a027
29.Buchachenko, A. L.; Shchegoleva, L. N.; Breslavskaya, N. N.
Chem. Phys. Lett. 2009, 483, 77–80. doi:10.1016/j.cplett.2009.10.044
(Example for a recent theoretical paper).
30.Massue, J.; Plush, S. E.; Bonnet, C. S.; Moore, D. A.;
Gunnlaugsson, T. Tetrahedron Lett. 2007, 48, 8052–8055.
doi:10.1016/j.tetlet.2007.09.022
89