Does phosphoryl protonation occurs in aqueous phosphoesters solutions

Article abstract of DOI:10.1016/j.saa.2004.12.038

Ionisation of trimethylphosphate (TMP), dimethylphosphate (DMP) and diethylphosphate (DEP) is investigated by acidic titration in water by Raman (R), Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies. The vibrational frequencies of the PO₂^- ionic form and the neutral form were found in accord with the literature. While increasing further H⁺ concentration, the PO band disappears in the benefit of new ones. These results, together with deuteration experiments indicate the presence of a new ionic form positively charged with general formula R₁R₂R₃P(OH)⁺ or R ₁R₂P(OH)⁺₂. The pK of this phosphonium entities is lying in the range -2, -4. These results were confirmed by ³¹P NMR titration. The occurrence of such a phosphonium ion in aqueous solutions might be of crucial importance for biochemical reactions and interactions, owing to the large spread of phosphoryl group in biomolecules and keeping in mind that intracellular compartments are more likely concentrated media with little free water than real aqueous solutions. Furthermore, pK's can be shifted by physical-chemical parameters like dielectric constant and electric field. This may involve at least fractional positive charge apparition that might be important in biochemical regulation by charge-charge and charge-dipole interactions. This finding will gain to be further explored on more complex molecules like phospholipids, nucleic acids and proteins.

Full text of DOI:10.1016/j.saa.2004.12.038

Spectrochimica Acta Part A 62 (2005) 287–292
Does phosphoryl protonation occurs in aqueous phosphoesters solutions
a
a
a
b
a,∗
´
L. Azema , S. Ladame , C. Lapeyre , A. Zwick , F. Lakhdar-Ghazal
a
Laboratoire de Synthe`se et Physico-Chimie de Mole´cules d’Inte´reˆt Biologique, UMR-CNRS 5068, Universite´ Paul Sabatier,
118 Route de Narbonne 31062 Toulouse Cedex 4, France
b
Laboratoire de Physique des Solides, UMR-CNRS 5477, Universite´ Paul Sabatier,
118 Route de Narbonne 31062 Toulouse Cedex 4, France
Received 14 September 2004; accepted 20 December 2004
Abstract
Ionisation of trimethylphosphate (TMP), dimethylphosphate (DMP) and diethylphosphate (DEP) is investigated by acidic titration in water
by Raman (R), Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies. The vibrational frequencies of
−
the PO2 ionic form and the neutral form were found in accord with the literature. While increasing further H+ concentration, the P O
band disappears in the benefit of new ones. These results, together with deuteration experiments indicate the presence of a new ionic form
positively charged with general formula R1R2R3P(OH)+ or R1R2P(OH)+2. The pK of this phosphonium entities is lying in the range −2, −4.
These results were confirmed by 31P NMR titration. The occurrence of such a phosphonium ion in aqueous solutions might be of crucial
importance for biochemical reactions and interactions, owing to the large spread of phosphoryl group in biomolecules and keeping in mind
that intracellular compartments are more likely concentrated media with little free water than real aqueous solutions. Furthermore, pK’s can
be shifted by physical–chemical parameters like dielectric constant and electric field. This may involve at least fractional positive charge
apparition that might be important in biochemical regulation by charge–charge and charge–dipole interactions. This finding will gain to be
further explored on more complex molecules like phospholipids, nucleic acids and proteins.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Phosphoryl protonation; Perprotonation; Phosphoesters; Raman; FTIR; NMR; Spectroscopy
1. Introduction
connection with physical state of phospholipid monolayers
[4–6], bilayers [7] and with respect to mixtures of phospho-
lipids and glycolipids [8].
The phosphate group is largely spread in phospholipids,
nucleic acids and proteins. Its participation is essential in the
bioenergetic fluxes as a part of molecules stocking energy like
ATP, as intermediate in metabolic reactions, or as effectors in
signal transduction. Molecular modifications of proteins by
phosphorylation are involved in cellular regulation processes
like viral activity and protein post-translational modifications
[1,2].
At the air–water interface, phosphate group of phospho-
lipids participate in the building up of the ionic microenvi-
ronment nearby biological and model membranes [3]. The
effect of ionisation state of phospholipids was investigated in
For biophysical studies, phosphoric diesters such as
dimethylphosphate and diethylphosphate were used as model
compounds to analyse structural and conformational features
of the phosphodiester part in phospholipids [9] and nucleic
acids [7,10]. Vibrational spectroscopy and normal mode anal-
ysiswereusedtocorrelatethemodificationsofthevibrational
spectra versus physical chemical parameters and to extend the
results to biological macromolecules.
The ionisation state of the phosphate group was also stud-
ied in aprotic solutions and the results lead to identify a pos-
itively charged phosphonium ion [11,12]. Despite the fact
that detection of phosphonium ion in aqueous solutions failed
in earlier studies [13,14], we were able to characterise this
ionic form in acidic aqueous solution of phosphoric acid
∗
Corresponding author. Tel.: +33 561556485; fax: +33 561556011.
E-mail address: lakhdar@chimie.ups-tlse.fr (F. Lakhdar-Ghazal).
1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2004.12.038

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L. Aze´ma et al. / Spectrochimica Acta Part A 62 (2005) 287–292
by hydrochloric acid titration, using Raman spectroscopy
[15].
NMR tubes were prepared as follow: for a final volume
of 500 ␮L, each tube contained 10 ␮L of trimethyl phos-
phate (24 mM final concentration), 10 ␮L of D2O and vari-
able amount of HCl 12N in water(from 0to 450 ␮L). Samples
were completed to 500 ␮L with water.
Owing to the great importance of phosphoesters in many
biochemical reactions, we used trimethylphosphate (TMP),
dimethylphosphate (DMP) and diethylphosphate (DEP) to
monitor the ionisation properties of the phosphate group in
aqueous solutions by means of Raman, Fourier transform
infrared and nuclear magnetic resonance spectroscopies, to
ascertain the occurrence of the phosphonium ion in acidic
aqueous solution.
3. Results and discussion
3.1. Trimethylphosphate
Raman spectra of 1 M TMP at various concentrations of
HCl are displayed in Fig. 1 (intermediary spectra are not
reported). The P O group exhibits a band at 1273 cm−1 in
neat TMP (curve a) which is lowered by hydrogen bonding
2. Material and methods
2.1. Chemicals
Dimethylphosphate and diethylphosphate were synthe-
sised by demethylation of trimethylphosphate and di-
ethylmethylphosphate, respectively, in the presence of t-
butylamine [16]. Alternatively, diethylphosphate was pre-
pared by hydrolysis of diethylphosphate chloride. The di-
esters were purified by ether precipitation. The purity of the
products was checked by thin layer chromatography and the
structure were assessed by RAMAN, FTIR and NMR spec-
troscopies.
Diester solutions were prepared with ultra-pure water
(milli-Q reagent system of Millipore) at constant concen-
tration of 0.1 and 1 mol dm−3, and the pH was adjusted on
separate solutions with hydrochloric acid (Prolabo).
2.2. Spectroscopy
Raman spectra were recorded with a Coderg T800 spec-
trometer equipped with an Ar+-ion gas laser (Spectra-physics
model 173) and counting electronics. The excitation wave-
length was 457.8 nm and the laser power was adjusted to
have 150 mW within the sample. The spectral resolution
was 3 cm−1. The Raman data were recorded with a speed
of 1 cm−1 s−1. For spectroscopic viewing, the sample was
placed in a sealed glass tube and thermostated at 19 ◦C.
FTIR spectra were recorded on a Perkin-Elmer 1760x,
both in an ATR circle equipped with a ZnSe cylindrical crys-
tal and a variable cell with ZnSe windows. In the latter case,
the path length was 24 ␮m. The total number of scans was 200
with a narrow band MCT detector cooled with liquid nitro-
gen. TheOPDvelocityoftheinterferometerwas2 cm s−1 and
the nominal resolution was 4 cm−1. The solvent substraction
was performed by recording the spectrum against the solvent
taken as background. Self-deconvolution [17] and derivation
were performed with the help of the available Perkin-Elmer
software. Care was taken to avoid over deconvolution by fix-
ing the width factor at 13 cm−1 and the F factor at 2.
31
¨
P NMR spectrum were recorded on a Brucker AC250
spectrometer, operating at 81 MHz. Chemical shifts are re-
ported in parts per million (ppm) relative to orthoposphoric
acid.
Fig. 1. Raman spectra of 1 M trimethylphosphate: (a) neat; (b) 4N HCl; (c)
12N HCl.

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289
Fig. 2. 31P chemical shift of trimethylphosphate in various HCl aqueous
solutions. The Hammett acidity scale was used for the abscissa.
to 1236 cm−1 in pure water as well as in 4N HCl (curve b).
This band vanishes in 12N HCl (curve c). The P O vibration
is characterised by a doublet at 740 and 752 cm−1 in neat
TMP (curve a), which is shifted to 748 cm−1 with a shoul-
der at 759 cm−1 (curve b). In 12N HCl, the predominant fre-
quency is at 752 cm−1, while the shoulder at 768 cm−1 is less
pronounced. Furthermore, a new band appears at 706 cm−1
(curve c).
The titration curve of TMP with HCl concentration was
monitored by 31P NMR spectroscopy (Fig. 2). A sigmoid
shape is observed between 2 and −5 on the Hammett acidity
scale, with an inflection point at −3.6. From these results, it
can be concluded that high concentration of HCl leads to the
protonation of the P O phosphoryl group of TMP giving the
new cation (CH3O)3P+OH, following the reaction:
Fig. 3. Raman spectra of dimethylphosphate in HCl aqueous solutions: (a)
pH 1; (b) HCl 1N; (c) HCl 2N; (d) HCl 4N; (e) HCl 8N; (f) HCl 8N.
frequency, which disappears while a band at 874 cm−1 arises
when the pH is lowered below the pK of the first phosphate
acidity (curves a–d). Increasing further the HCl concentration
leads to the disappearance of the bands at 1240 and 874 cm−1
3.2. Dimethylphosphate
Raman spectra of 1 M DMP were recorded between pH 12
and −6 on Hammett acidity scale (Fig. 3). At pH 12, DMP
ion is characterised by the symmetric mode at 1078 cm−1
.
This band vanishes when the pH is lowered below the pK of
the first phosphate group acidity (curves a–c).
While increasing further the concentration of HCl, a new
band appears at 860 cm−1 (curves d–f) that disappears on
deuteration (data not shown). At the same time, the breathing
band at 754 cm−1 is shifted up to 760 cm−1
.
When this frequency is reported versus HCl concentra-
tion, a clear sigmoidal titration shape is obtained (Fig. 4)
with two inflection points. A sigmoidal fit of the data gave a
value of 0.42 which corresponds to the pKa of the first clas-
sical phosphate acidity, and a second value of −2.57 that we
attribute to the pKa of the phosphoryl group titration FTIR
spectra relative to d−ifferent HCl concentrations are displayed
in Fig. 5. The PO2 ion is characterised by the 1085 cm−1
Fig. 4. Raman frequency shift of the P–O breathing band of dimethylphos-
phate vs. HCl concentration. The Hammett acidity scale was used for the
abscissa.

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L. Aze´ma et al. / Spectrochimica Acta Part A 62 (2005) 287–292
one corresponds to the standard first acidity of the phosphate,
with a calculated value of pKa = −0.6. The second inflection
point indicates a new titrable function corresponding to the
phosphoryl group with a calculated pKa = −4.
3.3. Diethylphosphate
Raman spectra of 1 M DEP in HCl are displayed in Fig. 7.
−
The symmetric stretch of the PO2 at 1098 cm−1 decreases
with protonation up to 2N (curves a–c), together with the ap-
parition of the P O stretching mode at 1240 cm−1. Increasing
further the HCl concentration leads to the disappearance of
this band and to the rise of the bands at 810 and 1030 cm−1
that can be associated with the (CH3CH2O)2P+(OH)2 ion.
This is accompanied by the shift of the breathing band at
744 cm−1
.
The frequency variation of this band versus HCl concen-
tration displays a sigmoidal shape (Fig. 8). The fit of the data
gives two inflection points: the value of 0.76 corresponds to
the first pKa of the phosphate and the value of −4.4 that we
attribute to the pKa of the phosphoryl group.
In the FTIR spectra of 1 M DEP (Fig. 9), the band at
952 cm−1 might not be related to a C–C stretching mode as
previously claimed [18] since it vanishes after the phosphate
Fig. 5. FTIR spectra of 1 M dimethylphosphate in HCl aqueous solutions:
(a) pH 12; (b) pH 1; (c) 1N; (d) 4N; (e) 6N; (f) 8N; (g) 10N.
(curves f–g) and to the appearance of the band at 860 cm−1
(Fig. 5, curves c–g).
These results clearly indicate the presence of a new form
of the phosphate group obtained by the protonation of the
phosphoryl group of DMP.
The same trends were observed by 31P chemical shift ver-
sus HCl concentration (Fig. 6), with a sigmoidal shape of
the titration curve displaying two inflection points. The first
Fig. 6. 31P chemical shift of dimethylphosphate in various HCl aqueous
solutions. The Hammett acidity scale was used for the abscissa.
Fig. 7. Raman spectra of 1 M diethylphosphate in HCl aqueous solutions:
(a) pH 2.1; (b) 0.1N; (c) 2N; (d) 4N; (e) 8N; (f) 10N.

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291
Fig. 8. RamanfrequencyshiftoftheP–Obreathingbandofdiethylphosphate
vs. HCl concentration. The Hammett acidity scale was used for abscissa.
Fig. 10. 31P chemical shift of diethylphosphate in various HCl aqueous
solutions. The Hammett acidity scale was used for the abscissa.
protonation, below pH = 1 (curves a–c). This mode should
instead be ascribed to the band at 982 cm−1. The band at
1055 cm−1 is more likely related to the PO2− ionic form.
The bands at 1078 and 1194 cm−1 that disappear after low-
ering the pH, are associated with the symmetric and antisym-
metric stretching of the PO2−, respectively. The appearance
of the broad band near 1200 cm−1 corresponds to the P O
stretching mode.
This is confirmed by 31P chemical shift versus HCl con-
centration (Fig. 10) where a sigmoidal shape is observed
with again two inflection points corresponding to the known
first standard acidity of the phosphate with a calculated
pKa = −0.68, and a new titrable function corresponding to
the phosphoryl group with a calculated pKa = −2.58.
As with DMP, increasing the HCl concentration leads to
the appearance of new FTIR bands at 821 and 872 cm−1
.
4. Discussion
The aim of this work was to ascertain the occurrence of
phosphonium ions by monitoring ionisation states of phos-
phoesters, trimethylphosphate, dimethylphosphate and di-
ethylphosphate in aqueous solutions, by means of Raman,
FTIR and NMR spectroscopies. The results clearly show the
appearance of a phosphonium ion at high HCl concentrations
in aqueous solution of the three phosphoric esters. The only
titrable group in trimethylphosphate is the P O phosphoryl
group with an estimated value from 31P NMR spectroscopy
of pKa = −3.6. The protonation of the phosphoryl moiety oc-
curs also on dimethylphosphate and diethylphosphate and the
pKa were estimated around −4. This value differs slightly be-
tween vibrational and NMR spectroscopies, probably due to
the time scale difference of the two methods. The accuracy
might be enhanced if more data points are available for the
fit of the curves.
The same trends were observed with sulfuric and perchlo-
ric acids (data not shown). The same result was previously
obtained on phosphoric acid in aqueous solutions [15]. Phos-
phonium ions were already evidenced but only in aprotic
solutions of strong organic acids by means of NMR spec-
troscopy [11,12].
What is the relevance of this protonated state in biologi-
cal systems? Indeed the HCl concentration required to obtain
such a perprotonation is high, however the pK is known to be
may be influenced by the environment around the phosphate
group such as dielectric constant or the electric field. The
Fig. 9. FTIR spectra of 1 M diethylphosphate in HCl aqueous solutions: (a)
pH 12; (b) pH 2; (c) pH 1; (d) 1N; (e) 2N; (f) 4N; (g) 8N; (h) 10N.

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L. Aze´ma et al. / Spectrochimica Acta Part A 62 (2005) 287–292
transient variation in H+ concentration near membranes for
example, may be relevant to the transient appearance of the
phosphonium ion. In this context, H+ concentration as high
as 6N was calculated when membrane ATPases are extrud-
ing protons [19]. On the other hand, the dielectric constant
in living cells may vary considerably by water triggering.
This may allow the ionisation states to vary and the phospho-
nium ions to play some roles in the biological functions of
biomolecules like enzymatic catalysis. This indeed seems to
be the case of the rabbit enolase where a protonation of the
phosphoryl group was suggested to stabilise a carbanion in
the active site [20]. Another example of the possible protona-
tion of the phosphoryl group concerned the pyruvate kinase
where the ketonisation step involved an inner proton transfer
from a lysine in the active site [21]. From these results, it
is possible to consider some role for such a protonated state
of the phosphoryl group in biochemical reactions like enzy-
matic catalysis, or biological interactions like conformational
changed in biomolecules or signal transduction.
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´ ´
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