Complexation of phosphates by 1,3-bis(3-(2-pyridylureido)propyl)-1,1,3,3-tetramethyldisiloxane

Article abstract of DOI:10.1002/rcm.7386

Rationale Compounds containing a urea or thiourea moiety form complexes with anions thanks to the ability to form quite strong hydrogen bonds. We have synthesized 1,3-bis(3-(2-pyridylureido)propyl)-1,1,3,3-tetramethyldisiloxane (1). Compound 1 contains two urea moieties connected by a long flexible linker; thus, it should be able to adopt a structure suitable for formation of quite stable complexes with anions. Methods The ability to form complexes of compound 1 with phosphates was tested by electrospray ionization mass spectrometry (ESI-MS). Full scan ESI mass spectra and collision-induced dissociation tandem mass (CID-MS/MS) spectra of the ions of interest were obtained on a quadrupole time-of-flight (QTOF) mass spectrometer. Results It has been found that compound 1 is not only much more prone to form complexes with the phosphate anion than with other inorganic anions, but it is also able to form complexes with organic phosphates, namely nucleotides and phospholipids. However, compound 1 is not able to form complexes with organic compounds not containing a phosphate group (e.g. nucleosides, sugars, glycerolipids). Conclusions Compound 1 can be regarded as selective towards phosphate-containing organic compounds. Formation of such complexes may have some interesting applications for identification of organic phosphates in crude extracts from biological materials.

Full text of DOI:10.1002/rcm.7386

Research Article
Received: 8 April 2015
Revised: 3 September 2015
Accepted: 6 September 2015
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2015, 29, 2272–2278
(wileyonlinelibrary.com) DOI: 10.1002/rcm.7386
Complexation of phosphates by 1,3-bis(3-(2-pyridylureido)propyl)-
1,1,3,3-tetramethyldisiloxane
*
Rafał Frański , Błażej Gierczyk, Grzegorz Markiewicz and Tomasz Kozik
Adam Mickiewicz University, Faculty of Chemistry, Umultowska 89B, 61-614 Poznań, Poland
RATIONALE: Compounds containing a urea or thiourea moiety form complexes with anions thanks to the ability to form
quite strong hydrogen bonds. We have synthesized 1,3-bis(3-(2-pyridylureido)propyl)-1,1,3,3-tetramethyldisiloxane (1).
Compound 1 contains two urea moieties connected by a long flexible linker; thus, it should be able to adopt a structure
suitable for formation of quite stable complexes with anions.
METHODS: The ability to form complexes of compound 1 with phosphates was tested by electrospray ionization
mass spectrometry (ESI-MS). Full scan ESI mass spectra and collision-induced dissociation tandem mass (CID-
MS/MS) spectra of the ions of interest were obtained on a quadrupole time-of-flight (QTOF) mass spectrometer.
RESULTS: It has been found that compound 1 is not only much more prone to form complexes with the phosphate anion
than with other inorganic anions, but it is also able to form complexes with organic phosphates, namely nucleotides and
phospholipids. However, compound 1 is not able to form complexes with organic compounds not containing a
phosphate group (e.g. nucleosides, sugars, glycerolipids).
CONCLUSIONS: Compound 1 can be regarded as selective towards phosphate-containing organic compounds.
Formation of such complexes may have some interesting applications for identification of organic phosphates in crude
extracts from biological materials. Copyright © 2015 John Wiley & Sons, Ltd.
Compounds containing a urea or thiourea moiety form
complexes with anions thanks to the ability to form quite strong
hydrogen bonds.[1–10] Some urea (or thiourea) conjugates were
prepared in order to form stable complexes with the phosphate
anion (usually with dihydrogen phosphate H2PO4À).[11–16]
From the biological point of view, complexation of organic
phosphates is more important than complexation of phosphate
anions, Therefore, the literature gives many examples of
synthesized organic compounds capable of formation of
complexes with nucleotides.[17–22] Formation of such
complexes may be useful for nucleotide analysis. Besides
nucleotides, another important group of organic phosphates
are phospholipids. There are a vast number of papers devoted
to the analysis of phospholipid, e.g. by mass spectrometric
techniques.[23–26] However, to the best of our knowledge,
formation of complexes between phospholipids and any
organic compounds has not been demonstrated yet.
In this paper, it is demonstrated, by using electrospray
ionization mass spectrometry (ESI-MS), that 1,3-bis(3-(2-
pyridylureido)propyl)-1,1,3,3-tetramethyldisiloxane (compound 1,
Scheme 1) is able to form complexes not only with the
phosphate anion, but also with organic phosphates, namely
nucleotides and phospholipids. Compound 1 contains two
urea moieties connected by a long flexible linker, thus it
should be able to adopt a structure suitable to form quite
a stable complex with the anion. We also have prepared
an analogous compound containing a thiourea moiety (2,
Scheme 1); however, compound 2 was found to be less
prone to form complexes with the phosphate anion (see
Supporting Information).
As demonstrated further, formation of a complex between
1 and an organic phosphate may be useful for identification
of organic phosphates in crude extracts from biological
materials.
EXPERIMENTAL
Besides 1 and 2, other compounds used (e.g. Na2HPO4,
nucleotides, phospholipids) were obtained from commercial
sources (mainly Sigma-Aldrich, Poznań, Poland) and used
without purification.
Synthesis of 1 and 2
1,3-Bis(3-(2-pyridylureido)propyl)-1,1,3,3-tetramethyldisiloxane (1)
Portions of picolinic acid (1 g; 8 mmol) and 2.34 g of
diphenylphosphoryl azide (8.5 mmol) were dissolved in dry
toluene (40 mL). Then 0.85 g of triethylamine (8.5 mol) was
added and the mixture obtained was stirred at room
temperature for 1 h. After that the solution was heated to
80°C over 2 h. Then the solvent was evaporated and the
semisolid residue was dissolved in dry chloroform. To a
solution of crude 2-pyridyl isocyanate, 1.06 g of 1,3-bis(3-
* Correspondence to: R. Frański, Adam Mickiewicz University,
Faculty of Chemistry, Umultowska 89B, 61–614 Poznań,
Poland.
E-mail: franski@amu.edu.pl
Rapid Commun. Mass Spectrom. 2015, 29, 2272–2278
Copyright © 2015 John Wiley & Sons, Ltd.

Complexation of phosphates by urea conjugate
Scheme 1. Synthesis of compounds 1 and 2.
aminopropyl)-1,1,3,3-tetramethyldisiloxane (4.05 mmol) was
added and the resulting mixture was heated under reflux
over 48 h. After solvent evaporation, the formed urea
was purified by chromatography on SiO2 (diethyl
ether/methanol; gradient from 10:0 to 9:1 v/v). The
product was obtained as a white, crystalline solid (980
mg; 50%). 1H NMR (400 MHz, CDCl3) δ 9.86 (bs, 2H),
8.20 (ddd, J = 5.1, 1.9, 0.9 Hz, 2H), 8.17 (bs, 2H), 7.61
(m, 2H), 7.35 (bs, 2H), 6.88 (dd, J = 6.7, 5.6 Hz, 2H), 3.34
Isolation of egg lecithin
Egg yolk lipids were extracted with ethanol/diethyl ether (1:2
v/v; 75 ml). The precipitate was filtered off and the solvent
was evaporated. The oily residue was dissolved in diethyl
ether (50 mL) and the solution was dried with anhydrous
Na2SO4. The crude lipid fraction, obtained after solvent
evaporation, was used for mass spectrometric experiments
(concentration of the sample solution was 5 μg/mL).
(m, 4H), 1.64 (m, 4H), 0.59 (m, 4H), 0.10 (s, 12H).13
C
Isolation of hen liver lipids
NMR (100 MHz, CDCl3): δ 156.3, 153.8, 146.0, 138.4,
116.8, 112.3, 42.7, 24.1, 15.6, 0.3.
A portion of 5 g of fresh chicken liver was frozen in liquid
nitrogen and carefully ground. The material obtained was
extracted with 25 mL of chloroform/methanol (2:1 v/v).
The semisolid mass was filtered off and then the solvents
were evaporated in vacuum. The mixture of lipids, obtained
as an oily residue, was used without further purification for
mass spectrometric experiments (concentration of the sample
solution was 5 μg/mL).
1,3-Bis(3-isothiocyanatopropyl)-1,1,3,3-tetramethyldisiloxane
To a solution of 1 g of 1,3-bis(3-aminopropyl)-1,1,3,3-
tetramethyldisiloxane (4 mmol) in dichloromethane
(30 mL), a 5% aqueous solution of NaHCO3 was added
(30 mL). The mixture was stirred vigorously and a solution
of 1.15
g of thiophosgene (10 mmol) in 20 mL of
Electrospray ionization mass spectrometry
dichloromethane was added dropwise over 0.5 h at room
temperature. Then the solution was refluxed for 2 h, the
organic phase was separated, washed with water and dried
with Na2SO4. After solvent evaporation, the isothiocyanate
was obtained as a brownish liquid (1.4 g; 95%). 1H NMR
(400 MHz, CDCl3) δ 3.49 (t, J = 6.7 Hz, 4H), 1.70 (m, 4H),
0.60 (m, 4H), 0.08 (s, 12H). 13C NMR (100 MHz, CDCl3): δ
129.4, 112.3, 47.8, 24.5, 15.4, 0.2.
Full scan ESI mass spectra and ESI-CID-MS/MS spectra were
recorded on a Q-tof Premier mass spectrometer (Waters/
Micromass, Manchester, UK) (software MassLynx version
4.1). The sample solutions, containing 5 × 10À6 mol/dm3
(unless indicated otherwise) of the compound analyzed in
methanol, were infused into the ESI source by a syringe
pump at a flow rate of 5 μL/min. The electrospray voltage
was 2.7 kV and the cone voltage À30 V. The source
temperature was 80°C and the desolvation temperature was
250°C. Nitrogen was used as the cone gas and desolvating
gas at flow rates of 50 and 400 L/h, respectively. Argon was
used as a collision gas at a flow rate of 0.5 mL/min in the
T-wave collision cell. The applied collision energy (CE,
laboratory frame), the most important parameter for CID-
MS/MS experiments, is indicated in each CID-MS/MS
spectrum shown.
1,3-Bis(3-(2-pyridylthioureido)propyl)-1,1,3,3-tetramethyldisiloxane (2)
A
portion of 1 g of 1,3-bis(3-isothiocyanatopropyl)-
1,1,3,3-tetramethyldisiloxane (2.8 mmol) was dissolved
in dry toluene (100 mL) and 0.55 g of 2-aminepyridine
(5.9 mmol) was added. The solution obtained was
refluxed for 14 days. After that the solvent was
evaporated and the product was purified by
chromatography on silica (diethyl ether/hexane 9:1
v/v). The thiourea was obtained as a yellowish red,
viscous oil (660 mg; 45%). 1H NMR (400 MHz, CDCl3):
δ 11.69 (bt, 2H), 8.93 (bs, 2H), 8.11 (dd, J = 5.2, 2.0 Hz,
2H), 7.57 (m, 2H), 6.90 (m, 2H), 6.79 (d, J = 8.3 Hz, 2H),
3.68 (m, 4H), 1.70 (m, 4H), 0.60 (m, 4H), 0.05 (s, 12H).
13C NMR (100 MHz, CDCl3): δ 179.3, 153.4, 145.7, 138.6,
117.8, 112.1, 48.5, 22.8, 15.7, 0.3.
RESULTS AND DISCUSSION
At first it had to be demonstrated that 1 forms more stable
complexes with the phosphate anion than with other
inorganic anions. Figure 1(a) shows the ESI mass spectrum
(for clarity in a narrow m/z range of interest) obtained for
Rapid Commun. Mass Spectrom. 2015, 29, 2272–2278
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R. Frański et al.
also at a concentration of 5 × 10À5 mol/dm3. Although
there was a high excess of chloride, the [1+H2PO4]À ion is still
quite abundant.
Attachment of the chloride anion to organic compounds is
a well-known phenomenon in negative ESI-MS[27–29] and it is
not surprising that chloride attachment is also observed for 1.
However, as clearly Àseen in Fig. 1, 1 is more prone to form an
adduct with H2PO4 than with ClÀ. Obviously, it would be
better if the binding of ClÀ by 1 could be avoided. However,
it seems to be rather difficult. If we add a compound which
binds chlorides, it would also at least slightly bind phosphates.
Obviously, we also compared the ability of 1 to form
À
complexes with H2PO4 with that to form complexes with
other inorganic ions. The respective mass spectra are shown
in the Supporting Information. Adducts with other inorganic
ions were less abundant than the [1+H2PO4]À ion,
At the next step of the research we tested the ability of 1 to
form complexes with organic phosphates, at first with simple
nucleotides. Figure 2 shows the ESI mass spectra obtained for
a solution containing 1 and uridine monophosphate – UMP.
As follows from the spectra, the ion [1+UMP–H]À is not very
abundant (not more abundant than [1+Cl]À), but gives a clearly
seen signal; the CID MS/MS spectrum of the [1+UMP–H]À ion
confirmed its composition, as the fragment ion [UMP–H]À was
formed (Fig. 2). It has to be stressed that the complex
between 1 and uridine was not detected, as demonstrated
in the Supporting Information. Thus, a conclusion can be
drawn that the complex between 1 and UMP is formed
thanks to the presence of the phosphate group.
Next, we extended the studies to more complex nucleotides,
namely nicotinamide adenine dinucleotide
– NAD. As
presented in Fig. 3, the adduct between 1 and NAD was
formed; the CID MS/MS spectrum of the ion [1+NAD–H]À
Figure 1. ESI mass spectrum (for clarity in the narrow m/z
range of interest) obtained for a solution containing 1 and
phosphate (Na2HPO4) at the same concentrations of 5 × 10À6
mol/dm3 (a); 1 and phosphate at the same concentrations of
5 × 10À6 mol/dm3 and chloride (NaCl) added also at a
concentration of 5 × 10À6 mol/dm3 (b); 1 and phosphate at
the same concentrations of 5 × 10À6 mol/dm3 and chloride
added at a concentration 5 × 10À5 mol/dm3 (c).
solution containing
1
and phosphate at the same
concentrations of 5 × 10À6 mol/dm3. The most abundant
ion is [1+H2PO4]À (complex between 1 and dihydrogen
phosphate anion), while the [1–H]À ion occurs at a low
abundance. Although chlorides were not added to
the solution analyzed, there is also a quite abundant ion
[1+Cl]À. Chlorides are common contaminants and, at the level
10À5 to 10À6 mol/dm3, it is difficult to control their
concentration (or to remove them). Figure 1(b) shows the ESI
mass spectrum obtained for a solution containing 1 and
phosphate at the same concentrations of 5 × 10À6 mol/dm3
and added chloride also at a concentration of 5 × 10À6
mol/dm3 (obviously total concentration of ClÀ is higher). As
shown in Fig. 1(b), the ion [1+H2PO4]À is twice as abundant
as the [1+Cl]À ion. Figure 1(c) shows the ESI mass spectrum
obtained for a solution containing 1 and phosphate at the
same concentrations of 5 × 10À6 mol/dm3 and chloride added
Figure 2. ESI mass spectra obtained for solution containing 1
and uridine monophosphate (UMP) (top: full scan mass
spectrum, bottom: CID MS/MS spectrum).
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Complexation of phosphates by urea conjugate
Figure 3. ESI mass spectra obtained for a solution containing
1 and nicotinamide adenine dinucleotide (NAD) (top: full
scan mass spectrum, bottom: CID MS/MS spectrum).
Figure 4. ESI mass spectra obtained for a solution containing
1 and phosphatidylinositol (PI) (top: full scan mass spectrum,
bottom: CID MS/MS spectrum).
confirmed its composition as the fragment ion [NAD–H]À was
formed (Fig. 3). It has to be stressed that the respective adducts
between 1 and adenosine, or between 1 and nicotinamide, were
not detected (Supporting Information). Thus, analogous with
the UMP adduct, ion [1+NAD–H] was formed thanks to the
presence of the phosphate group (in this compound it was a
diphosphate group).
It is worth adding that, theoretically, it is possible to form a
number of hydrogen bonds between 1 and uridine, between 1
and adenosine, as well as between 1 and nicotinamide.
However, the respective adducts were not detected
(Supporting Information). Thus 1 is prone to form complexes
with organic phosphates but not with the related organic
compounds.
a choline moiety were detected in both negative and positive
ion mode. In the positive ion mode the respective sodium-
containing adducts were observed and these signals
were more abundant than the signals observed in the negative
ion mode. Figure 5(a) shows the full scan ESI mass spectrum
obtained
for
a
solution
containing
1
and
lysophosphatidylcholine (LPC). Similarly as for phosphatidyl-
inositol, there are a number of signals which are difficult
to rationalize. However, the complex in question was
detected and its CID MS/MS spectrum (Fig. 5(b)) confirmed
that we really deal with the complex between 1 and
lysophosphatidylcholine.
One can argue that the complex between
1 and
lysophosphatidylcholine is formed thanks to the presence of
a quaternary nitrogen atom (electrostatic attraction). However,
as shown in the Supporting Information, the complex between
C14H29-N+≡(CH3)3 and 1 was not detected.
At the next stage of our study, we isolated phospha
tidylcholines from the yolk of hen egg (these compounds
are sometimes called egg lecithins). It is known that
phosphatidylcholine (PC) of mass 759 is the most common
in yolk, the protonated and sodiated adducts were observed at
m/z 760 and 782, respectively.[30–32] As shown in Fig. 6 the
complexofthisphosphatidylcholinewasdetectedinthefullscan
mass spectrum and the CID MS/MS spectrum confirmed the
presence of egg lecithin.
Upon analysis of the yolk we knew which phospholipid was
dominant in it, thus we knew what we were looking for (the
published data). Hen liver was analyzed in terms of fatty acids
present in phospholipids;[33–36] however, to the best of our
knowledge, there is no data concerning the specific
phospholipid present in hen liver. Therefore, at the final stage
ofourstudy,wedecidedtoanalyzeacrudeextractfromhenliver.
At the next stage we studied phospholipids. It is clear that
for these compounds access of 1 to the phosphate group is
hampered, thus it is expected that respective adducts will
not be abundant.
Figure 4 shows the full scan ESI mass spectrum obtained for a
solution containing 1 and phosphatidylinositol (PI). Besides the
peaks originated from 1 and/or phosphatidylinositol there are
other intense peaks which are difficult to rationalize. However,
the complex in question was detected (ion [1+PI–H]+) and
its CID MS/MS spectrum (Fig. 4) confirmed that we really
deal with the complex between 1 and phosphatidylinositol.
It should be stressed that the complex between glycerol 1-
monooleate and 1, as well as between sugar molecules and
1, was not detected, as shown in the Supporting Information.
Thus, the complex between 1 and PI is formed thanks to the
presence of the phosphate group.
A
very common group of phospholipids are those
containing the choline moiety, i.e. the -OCH2CH2-N+≡(CH3)3
moiety. Complexes between 1 and phospholipids containing
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R. Frański et al.
Figure 5. ESI mass spectra obtained for a solution containing
1 and lysophosphatidylcholine (LPC) (top: full scan mass
spectrum, bottom: CID MS/MS spectrum).
Figure 7. ESI mass spectra obtained for a solution containing
1 and crude extract from hen liver (top: full scan mass
spectrum, bottom: CID MS/MS spectrum of ion at m/z 1230).
m/z 1230. The CID MS/MS spectrum of the ion at m/z 1230
(Fig. 7) revealed a fragment ion at m/z 742 and a small fragment
ion [1–H]À (m/z 487). The ion at m/z 742 may correspond to the
deprotonated molecule of phosphatidylethanolamine (PE)
containing two oleic acid residues (Fig. 7). By using the ion
[1–H]À as a lock mass (m/z 487.2309), we obtained the
exact mass of the ion at m/z 742, namely 742.5388, which is in
good agreement with the deprotonated molecule of
phosphatidylethanolamine (PE) containing two oleic acid
residues (composition C41H77NPO8).
CONCLUSIONS
It has been demonstrated by using ESI-MS that compound 1
(1,3-bis(3-(2-pyridylureido)propyl)-1,1,3,3-tetramethyldisiloxane) is
very prone to form complexes with the phosphate anion.
Compound 1 forms complexes with other inorganic anions;
however, its complexation ability toward other inorganic
anion is definitely lower than toward the phosphate anion.
The high ability of 1 to form complexes with the phosphate
anion resulted in the ability of 1 to form complexes with
organic phosphates. In this work we tested nucleotides
and phospholipids. It has to be stressed that 1 does not
form complexes with organic compounds containing no
phosphate group (e.g. 1 forms a complex with uridine
monophosphate but not with uridine). Thus, it can be
concluded that 1 is selective towards phosphate-containing
organic compounds.
Figure 6. ESI mass spectra obtained for a solution containing
1 and phosphatidylcholine (PC) isolated from egg yolk (top:
full scan mass spectrum, bottom: CID MS/MS spectrum).
Figure 7 shows the full scan ESI mass spectrum (negative ion
mode)obtainedforasolutioncontaining1andthecrudeextract.
In the m/z range where the signals of the complex between 1
and phospholipids were expected, we detected a small signal at
We are aware that the methodology described here does not
allow performance of comprehensive, qualitative and
quantitative, phospholipid analysis as for example the method
described by Liu et al.[37] However, sometimes, relatively time-
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Complexation of phosphates by urea conjugate
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consuming comprehensive phospholipid analysis may be not
necessary. Our method allows in less than half an hour
(obviously provided that compound 1 has been earlier
synthesized) to identify the main phospholipids present in
biological samples, which sometimes may be enough.
Formation of a complex between 1 and organic phosphates
may be applied in initial fast qualitative analysis, by using
direct-inlet ESI/MS, of organic phosphates in crude extracts
from biological materials (Figs. 6 and 7). When we deal with
crude extracts, there are often a vast number of signals in
the ESI mass spectra. Therefore, it may be difficult to identify
those derived from phosphates. As demonstrated in Fig. 7,
formation of a complex between 1 and organic phosphates
(even with low abundance) may be useful for identification
of signals derived from organic phosphates, as a consequence
allowing the identification of organic phosphates in biological
samples (obviously MS/MS analysis of uncomplexed
phosphate may be also desirable).
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Godino-Salido,
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R. Lopez-Garzón, B. Valtancoli. Binding and recognition of
AMP, ADP, ATP and related inorganic phosphate anions
by
a
tren-based ligand containing
a
pyrimidine
Acknowledgement
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This work was supported by the Polish National Science
Centre (NCN; Grant No. N N204 155840).
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