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The pH Behavior of a 2-Aminoethyl Dihydrogen

Phosphate Zwitterion Studied with NMR-
Titrations

Article in Journal of Molecular Structure · February 2013

DOI: 10.1016/j.molstruc.2012.08.033

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Originally published: Journal of Molecular Structure, Volume 1033, Pages 171–175, 6 February 2013.
Final Draft  Antti Myller

The pH Behavior of a 2-Aminoethyl Dihydrogen

Phosphate Zwitterion Studied with NMR-
Titrations
A. T. Myller, J. J. Karhe, M. Haukka, T. T. Pakkanen

Department of Chemistry, University of Eastern Finland, P.O. Box 111, 80101

Joensuu, Finland.

Abstract
In this study a bifunctional 2-aminoethyl dihydrogen phosphate (AEPH2) was 1H and
31
P NMR characterized in a pH range of 1 to 12 in order to determine the zwitterion
properties in different pH regions in H2O and D2O solutions. NMR was also used to
determine the pH range where AEPH2 exists as a zwitterion. The phosphate group has
two deprotonation points, around pH 1 and 6, while the amino group deprotonates at
pH 11. The zwitterion form of AEPH2 (NH3+-CH2-CH2-OPO3H-) exists as the main
ion between pH 1 and 6 in water solutions and also in the solid state.

1. Introduction
Most molecules have specific signals in the NMR spectrum, depending on the
chemical structure. However, the chemical shift of the signal is affected by ionic
concentration and the pH of the solution they are measured in. Changes in chemical
shift are usually induced by changes in the electronic structure of the molecule or
interaction with a solvent e.g. accepting an electron or proton. These interactions are
usually dependent upon the solution pH and only happen in a specific and narrow pH
range. By monitoring certain signals in NMR as a function of a solution pH it is
possible to pinpoint solvent interactions to a specific pH area, but also to a specific
functional group on a multifunctional compound.

Some multifunctional molecules have functional groups that can either accept or
donate protons, but there are also amphoteric groups that can do both. When a
molecule has functional groups that are capable of donating and accepting protons
there is a chance for the formation of a zwitterion. A zwitterion is a molecule that has
both positively and negatively charged functional groups, but maintains an overall
neutral charge. Zwitterionic structures exist extensively in biological systems such as
amino acids and peptides [1]. One example of such a molecule is the common amino
acid, glycine (NH2-CH2-COOH), which exists as zwitterions in a solid state, water
solution [2], and gas phase. [3] Amino acids have both a proton donating carboxylic
acid group (-COOH) and an electron accepting primary amino group (-NH2). In
neutral aqueous solutions, the amino acid forms a carboxylic acid anion (-COO-) and

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Originally published: Journal of Molecular Structure, Volume 1033, Pages 171–175, 6 February 2013.
Final Draft  Antti Myller

an ammonium cation (-NH3+), effectively producing zwitterions, which are stabilized

by the water molecules. [4]

The formation of zwitterions via proton transfer can happen in many different ways:
through intramolecular proton transfer, proton donation/acceptance via a protic
solvent [5] and intermolecular proton transfer between molecules of a dimeric
complex [6]. With NMR titration, it is possible to track the pH range of actual
zwitterions and to determine the ionic forms in other pH regions. Because protonation
and proton transfer play an important role in biological processes between amino
acids and proteins, the use of proton NMR for the determination of a sample pH has
been studied extensively in biological systems. In biological samples, the pH is
usually tracked with amino acids or simple proteins, e.g. glycine [2, 4], histidine [7],
and carnosine [8], which have pH-dependent reactions and are commonly found in
tissue samples.

Zwitterions containing amino and phosphate groups are much less studied, even
though the interaction between the zwitterionic form of amino acids and phosphate
groups of the DNA strand has been found [9]. Aminophosphate zwitterions are known
to exist in a solid state [10], the phosphate group is known to interact with free
cationic NH3+ groups, [11] and the protons of the phosphate group dissociate easily in
water solutions [12], so it is possible for such zwitterions to exist in water solutions.

2-Aminoethyl dihydrogen phosphate (AEPH2) has managed to remain a fairly

unknown compound despite its potential as a surface modification agent. Made by
esterification of 2-aminoethanol and orthophosphoric acid [13], AEPH2 is a
bifunctional short-chained organic molecule that has both a phosphate group and an
amino group. The phosphate group is, in general, useful for binding with various
metal oxides like TiO2 [14, 15, 16] and iron oxides [17, 18] and the amino group is
useful for bonding with several metal ions [19], complexes [20], organometallic
compounds [21], and biomolecules via hydrogen bonds [22]. The short alkyl chain
length between the phosphate and the amino group promotes the solubility in protic
solvents [23]. AEPH2 has also been successfully bonded to an anatase form of TiO2
powder in two different pHs [16]. A study of the zwitterionic nature of AEPH2 can
offer valuable information about its bonding behavior in very acidic and very basic
environments. AEPH2 could also be used for pH measurements in biological systems,
as it has been found in brain samples as a metabolic side-product [24, 25]. There
might be a small difference between NMR results achieved in biological samples and
in a pure solvent used in this study due to the various solute-solute interactions
present in a typical metabolite or sucrose solution [26].

The goal of our study was to examine whether the AEPH2 molecule exists in a
zwitterionic form in water solutions and in a solid state. 1H and 31P NMR titration
were carried-out in order to characterize the acid base reactions of this multifunctional

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Originally published: Journal of Molecular Structure, Volume 1033, Pages 171–175, 6 February 2013.
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aminophosphate, and X-ray diffraction measurement was used to determine the solid
state structure. Special emphasis was placed on the pH range of the three depronation
steps of AEPH2, namely the two deprotonations of phosphate, and the deprotonation
of the NH3+ group.

2. Experimental
2.1. Materials
Ethanol 99.5% (Altia Etax Aa), D2O 99.9% (Aldrich), DCl 99% (Aldrich), NaOD
99.5% (Aldrich), and CDCl3 99.8% (Euriso-Top) were used as received from the
supplier. AEPH2 powder 98% (Aldrich) was recrystallized and filtered from a 1:3
water/ethanol mixture and then crystallized from a supersaturated water solution.
NaOH and H2SO4 solutions were made by dilution from 1.00 M and 5.00 M storage
solutions, respectively. All non-deuterated water used in the research was 18 MΩcm-1
Milli-Q water.

2.2. Instruments and Methods

The pH values were measured with an electronic Consort P901 pH-meter using a
combination electrode, temperature correction, and a 3-point calibration in buffer
solutions at pH 4.005, pH 7.000, and pH 10.012.

All NMR measurements were made with a Bruker Avance 400 NMR spectrometer
with 64 scans per sample. Delays between pulses were 2 seconds for 1H samples and
10 seconds for 31P samples. The solvent was D2O for the titration samples and CDCl3
for the aminoethanol verification. An airtight capillary tube with 0.03% of TMS
dissolved in CDCl3 was the external standard for the 1H measurements (0.00 ppm)
and 85% orthophosphoric acid was the external standard for 31P measurements
(0.00 ppm).

Single crystal X-ray diffraction was conducted with a Bruker Smart ApexII
diffractometer. A single crystal of AEPH2 was immersed in cryo-oil, mounted in a
nylon loop and measured at 100 K using an X-ray beam wavelength of 0.71073 Å.

2.3. Titration of NMR Samples

First, a 10 ml batch of 0.1M AEPH2 solution was prepared in D2O, which was then
divided into two separate solutions with an initial pH of 3.5. The first D2O solution of
AEPH2 was titrated using 5M, 1M and 0.1M H2SO4 solutions in order to make a
solution pH 1 and a new NMR sample was taken every 0.5 pH mark in the pH range
of 1 to 3.5. The second D2O solution was titrated using 1M and 0.1M NaOH

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solutions, in order to make a solution pH 12 and a new NMR sample was taken every
0.5 pH mark between the pH range of 3.5 to 12.

Second, another 10 ml batch of titrations was done with fully deuterated chemicals in
a 0.1M D2O solution of AEPH2. The solution was the divided into two separate
solutions with an initial pH of 3.5. This time the titrations were conducted using a
35 w-% DCl in D2O for a pH range of 1 to 3.5 and a 40 w-% NaOD in D2O for a pH
range of 3.5 to 12 and a new NMR sample was taken every 1.0 pH mark.

2.4. Extraction of Aminoethanol

A sample of the AEPH2 solution was extracted for 2-Aminoethanol in order to see if it
de-esterificates at a low pH. At pH 1 the 0.1M AEPH2 solution was extracted with 3
times 50 ml of diethylether. Diethylether was removed by evaporation at room
temperature and the presence of 2-aminoethanol was evaluated with 1H NMR
measurements in a CDCl3 medium. The expected signals of 2-aminoethanol (triplet δ
3.59 ppm and triplet δ 2.90 ppm) were not observed in the extracted sample.

3. Results and Discussion

The AEPH2 was expected to be in zwitterionic form in a solid state, based on the high
melting point (246.6 oC) and its good solubility in water [16]. A single crystal X-ray
diffraction analysis confirmed this hypothesis. X-ray diffraction result of AEPH2
agrees well with the crystallographic data previously measured with X-ray
photographic method [27] and neutron diffraction [28]. AEPH2 has a monoclinic
P21/c space group: a = 9.0065(2) Å, b = 7.7389(2) Å, c = 8.7756(2) Å,
β = 102.4840(10)°, Z = 4, and a zwitterionic NH3+-CH2-CH2-OPO3H- configuration
(Figure 1.), resulting in a three dimensional hydrogen bonded network of cross-linked
sheets, similar to α-aminopropanephosphonic acid [29]. Phosphorus – oxygen bond
lengths are 1.56 Å for a single P-O bond and 1.45 Å for P=O double bond [30]. The
measured P-O bond distances of the phosphate group (P1-O3 and P1-O4) are very
similar (1.493 Å and 1.508 Å, respectively) and they are between the reference bond
lengths of single and double bonds, so they both have a bond order of 1.5 with a
delocalized negative charge capable of hydrogen bonding, not double bonds as
previously assumed [27].

The form of AEPH2 in aqueous solution was studied by means of NMR titration. A
potentiometric titration study done earlier [16] on AEPH2 shows two deprotonation
steps, but the compound has three labile protons in the pH range of
1-12. One of the deprotonations occurs at a pH below the measurable range of water-
based potentiometric titrations. A NMR titration method was implemented in order to

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Originally published: Journal of Molecular Structure, Volume 1033, Pages 171–175, 6 February 2013.
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detect all of the deprotonation steps and to identify the specific functional group
connected with these steps.

The 1H NMR signals of both the CH2 groups on the hydrocarbon chain (Scheme 1.)
were plotted as a function of pH. There was no noticeable difference in chemical
shifts or coupling constants between titrations done with non-deuterated (D2O, NaOH
and H2SO4) and fully deuterated (D2O, NaOD, DCl) titrants. Signal A for the CH2
next to phosphate group is a pseudoquartet resulting from 3J(H-H) coupling and 3J(H-
P) coupling from the phosphate group. Signal B for the CH2 next to the amino group

Figure 1. Structure and hydrogen bonding network of a crystalline AEPH2 zwitterion.

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-
B O
A O B
A
H2N P
pH 12.0 O
-
O

A B OH B
O
+ A
H3N P
pH 3.5 O
-
O

A B OH
O B
+ A
H3N P
pH 1.5 -
O
O

PPM 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6
1
Scheme 1. H NMR spectra of AEPH2 measured at 400.13 MHz in D2O at 12.0 pH,
3.5 pH and 1.5 pH.

is a triplet due to 3J(H-H) coupling from the adjacent CH2 group and is transformed
into a broad singlet in acidic conditions due to the increased proton exchange rate
[31]. The 31P NMR signal was also plotted as a function of pH (Figure 2.) to obtain
more direct knowledge of the deprotonations of the phosphate group besides the 1H
NMR signal of CH2 A, which is three bonds away from the phosphorus atom. The
amino protons were not observed with 1H NMR, because the amphoteric nature of the
amino group tends to form hydrogen bonds and it exchanges its protons with the
deuterium of D2O making the 1H signal very broad or completely indistinguishable
[32].

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4,0

3,0
ppm

2,0

1,0

0,0
1 3 5 7 9 11
pH

Figure 2. The 31P chemical shifts of the phosphate group, measured at 161.97 MHz in
D2O, as a function of pH.

The plotted figures of the 1H and 31P NMR resonances as a function of pH (Figures 3.
and 2.) greatly resemble a regular titration curve of triprotic and diprotic acids,
denoting that there are three different points of deprotonation in the pH range of 1 to
12.
4,2

3,9

3,6
ppm

3,3

3,0
Signal A
Signal B
2,7
1 3 5 7 9 11
pH
Figure 3. The 1H chemical shifts of signals A and B, measured at 400.13 MHz in
D2O, as a function of pH.

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The first deprotonation point can be found around pH 1, where the phosphate group
-OPO3H2 releases a proton and becomes an ionic -OPO3H- group (AEPH2 pKa1=1.0).
The 31P NMR chemical shift of the phosphate group as a function of pH (Figure 2.)
shows a small equivalent point between pH 1 and pH 2. At pH 1, the 31P chemical
shift of the AEP-species matches closely to the chemical shift at 0.00 ppm of
orthophosphoric acid (H3PO4), which has a similar chemical environment to that of a
fully protonated phosphate ester group (-OPO3H2) in AEPH2. The first deprotonation
of APEH2 agrees well with the pKa1 value of monosubstituted phosphates which is
generally about 1.0 [33]. The amino group is in a -NH3+ state at pH 1, making the
molecule a zwitterion.

Although the pH at this point could be low enough to cause de-esterification of the
alcohol group [34, 35], resulting in the formation of orthophosphoric acid (H3PO4)
and 2-aminoethanol, it is not expected to happen at room temperature in, which these
titration experiments were conducted. Synthesis of phosphate esters usually requires a
high temperature [36] and in the case of AEPH2 a temperature of 110oC is required
[13]. Moreover, de-esterification of phosphates usually requires temperatures above
100oC [33, 37] and 2-aminoethanol was not observed in the samples extracted with
diethylether.

The second deprotonation point is found at pH 5.9 where the ionic phosphate group -
OPO3H- releases its last proton and becomes a -OPO32- group (AEPH2 pKa2=5.9)
while the amino group remains as -NH3+. The deprotonation step can be observed as
the resonance shift of signal A (Figure 3.), but the step is much more noticeable in the
31
P chemical shift of the phosphate group (Figure 2.). A very large change in the
chemical shift of phosphorus can be seen at pH 5.9, which denotes the second
deprotonation of the phosphate group and an equivalent point between -OPO3H- and -
OPO32- forms. Such a large increase in the 31P chemical shift can be explained by the
increased polarization of the P-O bonds [38] that derives from the increased electronic
density of the oxygen atoms after the deprotonation. Due to an increased electron
density surrounding the phosphorus atom, the 31P resonance shifts downfield.

The deprotonation step can also be detected as a change in the 3J(H-P) coupling
constant, which increases from 6.4 Hz at pH 3.5 to 6.9 Hz at pH 8.0 due to a change
in the geometry of the ester bond. With orthophosphoric acid, the pKa3 value is very
high [12] because of the highly covalent nature of the last O-H bond, but in the case
of the AEPH2 the last releasing proton should be compared against the pKa2 value of
the orthophosphoric acid (7.20 [12]) and the pKa2 values of the phosphate esters in
general (6.5 [33]), due to remaining covalent ester bond. These values set the
equivalent point at the neutral zone where the corresponding -OPO3H- to -OPO32-
transition of AEPH2 can be found. Due to the overall negative charge, the molecule
can no longer be considered a zwitterion at this point.

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The third deprotonation point can be found at pH 11.0, where the amino group
deprotonates from a -NH3+ ion to a neutral -NH2 group (AEPH2 pKa3=11.0). Below
pH 11.0 the -NH3+ is the main species, whereas above pH 11.0 the -NH2 is
predominant. The chemical shift of signal B as a function of pH (Figure 3.) has three
visible equivalent points at pH 1.0, 5.9 and 11.0, exactly the same points as in the case
of the proton signal A. The change in chemical shift at pH 11.0 is considerably larger
than at pH 1.0 and 5.9, indicating the deprotonation of the amino group.

7,0

6,0
Hz

5,0

4,0

3,0
1 3 5 7 9 11
pH
Figure 4. The 1H NMR coupling constant 3J(H-H) as a function of pH.

The chemical shift changes at pH 1.0 and 5.9 in other functional groups are reflected
in signal B as changes in the 3J(H-H) coupling constant (Figure 4.). 3J(H-H) steadily
increases from 4.4 Hz to 5.4 Hz, confirming the alteration of either length of the C-C
bond between the CH2 groups or torsion angle [39] at equivalent points where
deprotonation happens. The changes in the 3J(H-H) coupling constant are rather small
due to a lack of steric hindrance for the rotation around the C-C bond of the
hydrocarbon chain.

Primary amines usually have very high pKa values [40], meaning that -NH2 to -NH-
dissociation will not show in this pH range. This means that a fourth equilibrium point
can be outside the basic end of the pH measuring range and is not visible in plotted
figures of the water solutions.

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100

80
Rel. Concentration %

NH2
60
NH3+
OPO3 2-
OPO3H-
40 OPO3H2

20

0
0 2 4 6 8 10 12 14
pH

Figure 5. Predominance diagram of functional groups of AEP ions in water solution.

Relative concentrations estimated using pKa values (pKa1=1.0, pKa2=5.9, pKa3=11.0)

and Henderson-Hasselbalch equation as a function of pH (Figure 5.) show that
between pH 1 and 5 the AEPH2 is mainly in a neutral zwitterionic form with a NH3+-
CH2-CH2-OPO3H- configuration and with the maximum relative concentration around
pH 2. At a more acidic pH, the phosphate group becomes neutral and -NH3+ uses the
negative sulphate ions in the titration solutions as its counter ion. Between pH 5 and
11 the main species is anionic with a NH3+-CH2-CH2-OPO32- configuration. Similar
anionic ions with both negative and positive charges, but an overall negative charge,
have been observed earlier for e.g. pyridine-2-phosphono-4-carboxylic acid [38],
making the anionic configuration completely viable. At a more basic pH, the
amphoteric amino group undergoes deprotonation and becomes a neutral -NH2 group,
while the -OPO32- group uses the positive sodium ions in the titration solution as
counter ions.

When compared to the results achieved by potentiometric titration of AEPH2 [16] the
pKa values are somewhat different. With potentiometric titration the pKa values set at
5.61 and 10.39 while NMR titration gives out values 1.0, 5.9, and 11.0. In our
previous article [16] the first deprotonation step is missing completely and the last
deprotonation step was incorrectly interpreted as deprotonation of phosphate group
instead of deprotonation of amino group. The notable difference in the pKa values can
be explained by self-buffering properties of AEPH2 solutions. Due to the zwitterionic
nature the AEPH2 is a both weak acid and a conjugate base, simultaneously able to
donate and accept protons in multiple functional groups, this can affect results of

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potentiometric titration where concentrations are calculated using the consumed titrant
base. In NMR titration the net effect of simultaneous proton donation/acceptance does
not interfere with the observation of equivalent points because they are determined
from the changes in a single functional group.

4. Conclusions
In summary, NMR titration is a very powerful technique for evaluating the acid base
interactions of the functional groups of zwitterions and multifunctional compounds in
general, in a manner that potentiometric titration is unable to provide. The
experimental results indicate that the AEPH2 molecule is indeed in a neutral
zwitterionic form in the solid state and in water solutions at a pH range of between 1
and 5. The maximum zwitterion concentration can be found at pH 2. This study also
reveals the pH ranges in which the phosphate and amino groups deprotonate. This is a
very useful insight for those who wish to conduct specific functionalizations with a
zwitterion compound on solid metal oxide surfaces in a system with limited control
over the pH or who wish to maximize the surface coverage of zwitterions on the
support surfaces.

Supplementary Material
Crystallographic data (excluding structure factors) for structure of AEPH2 has been
deposited with the Cambridge Crystallographic Data Centre, CCDC no. 880620. A
copy of this can be obtained free of charge from The Director, CCDC, 12 Union
Road, Cambridge, CB2 1EZ, UK (fax: +44-1233-336-033; e-mail:
deposit@ccdc.cam.ac.uk; or www: http://www.ccdc.cam.ac.uk).

Acknowledgement
Funding from the Academy of Finland (project 118168) is gratefully acknowledged.

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