First bisphosphonate hydrogelators: Potential composers of biocompatible gels

Article abstract of DOI:10.1039/c3tb20957a

Recently, investigation of hydrogels has gained ever increasing attention mostly because of their biomedical and pharmaceutical properties, and novel hydrogelators are constantly studied to find functional applications. Bisphosphonates (BPs) are well-known compounds applicable in different fields but are mostly used in clinics as drugs for bone-related diseases. In this study, a novel class of BP-hydrogelators together with a BP-organogelator was found, and the gelating abilities of the compounds were studied. Several techniques to analyze the structure and the properties of the formed gels were used, including solid state ¹³C and ³¹P CPMAS and solution state NMR spectroscopy, IR spectroscopy, PXRD, thermoanalysis, as well as SEM. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3tb20957a

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DOI: 10.1039/C3TB20957A
Journal of Materials Chemistry B
Page 1 ofJ11ournal Name
►
ARTICLE TYPE
First bisphosphonate hydrogelators: potential composers of
biocompatible gels
Aino-Liisa Alanne,*a Manu Lahtinen,b Miika Löfman,b Petri Turhanen,a Erkki Kolehmainen,b Jouko
Vepsäläinen,a and Elina Sievänenb
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Recently, investigation of hydrogels has gained ever increasing attention mostly because of their biomedical and pharmaceutical
properties, and novel hydrogelators are constantly studied to find functional applications. Bisphosphonates (BPs) are well-known
compounds applicable in different fields but are mostly used in clinic as drugs for bone-related diseases. In this study, a novel class of
BP-hydrogelators together with a BP-organogelator was found, and the gelating abilities of the compounds studied. Several techniques to
analyze the structure and the properties of the formed gels were used, including solid state 13C and 31P CPMAS and solution state NMR
spectroscopy, IR spectroscopy, PXRD, thermoanalysis, as well as SEM.
Based on our recent search from Chemicals Abstracts there are no
previous reports about BPs acting as gelating agents, while
Introduction
approximately 33 600 BP studies have been published over the
Hydrogels, in other words gels forming in aqueous solutions,
years. Nevertheless, gels containing BPs have been investigated
have become an area of increasing interest during the last decade
for a wide variety of purposes. Some BP containing hydrogels
or two, in particular due to their applicability for biomedical and
have been related to applications of treatment of bone resorption,
pharmaceutical purposes.1-3 The traditional hydrogels consist of
wound healing, and removal of radionuclides from wounds.12-14
synthetic and natural polymers, but recently low-molecular-
Others include odontological applications15-17 and scaffolds for
weight gelators (LMWG) have intrigued because of their special
growing cells18. Organo- and xerogels from BP-metal complexes
properties related to self-assembly.4 The formation of
have been investigated, too.19-22
supramolecular gels by LMWGs is based on non-covalent
The actions of BPs in the body are carefully investigated
interactions, such as hydrogen bonding, van der Waals
because of their use in clinic. BPs are generally known to be non-
interactions, π-π stacking, and solvophobic effects, being a big
advantage compared to more prevalent polymer gels. Thus many
toxic and well-tolerated compounds. In consequence, the novel
BP-gels could be used, for example, as matrices that bind drugs
supramolecular gels are biodegradable and better suitable for
to administrate, deliver, and release them. Hydrogels have been
biological applications.
Bisphosphonates (BPs), analogues of naturally occurring
used extensively in the development of drug delivery systems,
and synthesis of new hydrogelators with better biocompatibility is
inorganic pyrophosphate, are enzymatically stable molecules
essential for successful applications.23
characterized by a P-C-P bond.5 BPs have been used for many
In this article, we present four novel BP-based gelator
purposes during the past 50 years. In the beginning BPs acted as
water softeners by inhibiting the crystallization of calcium salts,
but the basis for their nowadays main use is their high affinity for
bone mineral hydroxyapatite. Clinically BPs are used for the
treatment of bone-related diseases, like osteoporosis and Paget’s
disease, because of their ability to inhibit osteoclast-mediated
molecules, three of which have a long alkyl chain (with the length
of 11 to 13 carbons) attached to the central carbon (1-3) and one
being an acetyl derivative of etidronate (4) (Fig. 1). Compounds
1-3 formed hydrogels and compound 4 a gel in a mixture of two
organic solvents. The gelating properties of the compounds were
studied, and several techniques used to analyze the structure of
bone resorption. Also, the anticancer effects of BPs have been
the formed gels. These included solid state 13C and 31P CPMAS
widely studied and reviewed6-7
, as well as their act as
and solution state NMR spectroscopy, IR spectroscopy, PXRD,
thermoanalysis, as well as SEM.
antiparasitic agents8-9 and anti-inflammatory drugs10-11
.
aSchool of Pharmacy, Biocenter Kuopio, University of Eastern Finland,
P.O. Box 1627, FIN-70211 Kuopio, Finland
E-mail: aino.alanne@uef.fi
bDepartment of Chemistry, P.O. Box 35, FIN-40014 University of
Jyväskylä, Finland
†Electronic Supplementary Information (ESI) available
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

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DOI: 10.1039/C3TB20957A
Journal of Materials Chemistry B
Page 2 of 11
1496, 2850, 2916, 2954.
(1-Acetyloxyethylidene)-1,1-bisphosphonic acid P,P’-dimethyl
ester disodium salt 4. Dimethylphosphite (26.6 g, 0.24 mol) and
dibutylamine (3.1 g, 0.024 mol) in ether (350 ml) were cooled to
0°C. Keeping this temperature, dimethyl acetylphosphonate (36.7
g, 0.24 mol) was slowly introduced with stirring into the mixture
after which the reaction mixture was allowed to warm to room
temperature. Stirring was continued for 3 hours, the product was
filtered and recrystallized from dry toluene, to give (1-
hydroxyethylidene)-1,1-bisphosphonic acid tetramethyl ester as
colourless crystals (56.2 g, 89%). The obtained product (7.0 g,
0.027 mol) was allowed to react with NaI (8.1 g, 0.054 mol) in
acetone (30 ml) for 30 h to remove two of the methyl esters to
yield (1-hydroxyethylidene)-1,1-bisphosphonic acid P,P’-
dimethyl ester disodium salt as a white powder (7.0 g, 94%). This
intermediate (800 mg, 2.88 mmol), AcOH (3 ml) and Ac2O (3
ml) were stirred at 55 ˚C for 19 h and evaporated to dryness. The
residue was dissolved in MeOH (5 ml), isopropanol was added (5
ml) and mixture was heated to reflux. After the addition of water
(600 μl), the refluxing was stopped, 1 ml of isopropanol added
and the mixture kept in the freezer overnight. Formed precipitate
was filtered, washed with isopropanol and dried in vacuum to
give 4 (763 mg, 83%) as a slightly yellow powder.
Fig. 1 Structures of bisphosphonate gelators 1–4.
Experimental section
Materials
1-Bromoundecane (98%), 1-bromododecane (97%), 1-
bromotridecane (98%), and NaH (60% in oil) were purchased
from Sigma-Aldrich. D2O (99.9% D) and methanol-D4 (99.8%
D) used in the NMR measurements were purchased from Euriso-
Top. Other solvents and reagents were purchased from J.T.
Baker, Merck, Riedel-de Haën, and Sigma-Aldrich and used
without further purification.
Synthesis and characterization
Compounds 1-3 were synthesized according to the method
described earlier24, with purities >98%. The structural data for
compound 2 has been published before by us.25 Compound 4 was
prepared as published previously and the spectral data was in
accordance with the published data.26 Elemental analyses (C, H)
were accomplished with a ThermoQuest CE Instruments EA
1110-CHNS-O elemental analyzer (CE Instruments, Milan,
Italy).
Gelation procedure
Compounds 1-3 were weighed (20-25 mg) in test tubes and the
solvent (0.5 ml) added. The sample was sonicated for a few
minutes and heated with a heat gun until the solid was dissolved
or the boiling point of the solvent reached. The solution was left
to cool at room temperature with a cap on. Observations with
regard to the gelation were made after an hour by inverting the
test tube. If the solid mass was immobilized, the material was
defined as a gel. Compound 4 (20 mg) was dissolved in methanol
(200 µl) with sonication and heating. 2-Propanol (200 µl) was
then added to the solution dropwise until the gel formed.
Xerogels were made by placing the hot solution on an
evaporation dish, letting the gel form, and the solvent evaporate.
The resulting solid was dried under vacuum.
Dodecane-1,1-diyldiphosphonic acid 1.
A
mixture of
tetraisopropylbisphosphonate (14.64 g, 42.5 mmol) and dry THF
(20 ml) was added dropwise to a solution of NaH (2.04 g, 51.0
mmol, 60% in oil) in dry THF (80 ml) followed by stirring under
an argon atmosphere at room temperature for 1.5 h. 1-
Bromoundecane (10.0 g, 42.5 mmol) in THF (20 ml) was added
gradually and the mixture maintained at 85 °C for 24 h. Water
(200 ml) was added to the cooled mixture, the product extracted
with DCM (3 × 150 ml), dried over MgSO4, and the solvent
removed under reduced pressure. The residue was purified by
column chromatography (ethylacetate/methanol 95:5). In the last
step, the isopropyl protecting groups were removed by refluxing
in 4 M HCl overnight yielding 1 (5.17 g, 37%) as a white solid.
1H NMR (CD3OD): δ 2.16 (1H, tt, 2JHP = 23.5 Hz, 3JHH = 5.9 Hz),
1.99-1.85 (2H, m), 1.65-1.55 (2H, m), 1.38-1.21 (16H, m), 0.93-
Solution state NMR spectroscopy
1H, 13C, and 31P NMR spectra were recorded on a Bruker Avance
500 FT NMR spectrometer operating at 500.1, 125.8, and 202.5
MHz, respectively. TMS (in CD3OD) was used as an internal
1
standard for H and 13C, and 85% H3PO4 as an external standard
1
0.85 (3H, m); 13C NMR (CD3OD): δ 39.3 (t, JCP = 127.3 Hz),
n
for 31P measurements. The JHP couplings from proton spectra
2
n
33.1, 30.8, 30.8 (2C), 30.7, 30.5 (3C), 26.8 (t, JCP = 4.4 Hz),
and the JCP couplings from carbon spectra were calculated with
23.7, 14.5; 31P NMR (CD3OD): δ 21.89 (s). IR (cm-1): 422, 513,
the coupling constants given in Hertz.
716, 924, 1020, 1123, 1157, 1377, 1470, 2851, 2918, 2955.
Solid state NMR spectroscopy
The solid state 13C and 31P CPMAS NMR spectra were recorded
at 297 K with a Bruker Avance 400 spectrometer operating at
100.62 and 161.98 MHz, respectively. The spectrometer was
equipped with a dual CPMAS probehead. The original synthesis
products were packed in 4.0 mm zirconia rotors, and the xerogel
and gel samples in HRMAS rotors of 50 µl internal volume. The
gel samples 1-3 (1 4% w/v; 2 and 3 5% w/v) were prepared by
dissolving the gelator in water and adding the hot solution into
Tetradecane-1,1-diyldiphosphonic acid 3. Prepared as 1 from 1-
bromotridecane (5.0 g, 19.0 mmol) to give 1.70 g (25%) of 3 as a
1
2
white solid. H NMR (CD3OD): δ 2.16 (1H, tt, JHP = 23.7 Hz,
3JHH = 6.1 Hz), 1.99-1.85 (2H, m), 1.65-1.57 (2H, m), 1.37-1.23
1
(20H, m), 0.92-0.87 (3H, m); 13C NMR (CD3OD): δ 39.3 (t, JCP
= 127.9 Hz), 33.1, 30.8, 30.8 (2C), 30.8, 30.8, 30.7, 30.5 (2C),
2
30.5, 26.8 (t, JCP = 4.7 Hz), 23.8, 14.5; 31P NMR (CD3OD): δ
21.85 (s). IR (cm-1): 438, 490, 715, 927, 1014, 1122, 1158, 1377,

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DOI: 10.1039/C3TB20957A
Page 3 of 11
Journal of Materials Chemistry B
the rotor filled with an insert screw. In the case of gel sample 4
temperature. The standard uncertainty for the thermal degradation
onset temperature was estimated to be u(T) = 1.0 °C. The sample
weights used in the measurements were about 5 - 8 mg.
the gel (5% w/v) was made beforehand (as described in the
chapter Gelation procedure) and transferred into the rotor by a
spatula. The gels were let to stabilize for a few hours before the
measurements. The contact times for the CPMAS experiments
were 2 ms for 13C and 5 ms for 31P, respectively. The relaxation
delay was 5 s, and the spin rate 10 kHz for the solid samples and
2 kHz for the gel samples. Typically, for 13C spectra 200-600
scans were acquired for the synthesis products, 500-1500 for
xerogels, and 16 000-17 000 for the gel samples. For 31P spectra
the number of scans for synthesis products was usually 8, for
xerogels 96, and for gels 2048 scans. The 13C chemical shifts
were referenced to that of glycine carbonyl (176.03 ppm from
TMS) and the 31P NMR chemical shifts to that of
ammoniumdihydrogen phosphate (2.5 ppm).
Differential scanning calorimetry
Power compensation-type Perkin Elmer Pyris Diamond
differential scanning calorimeter (DSC) was used to measure
DSC heating-cooling scans for the compounds 1-4. Each
measurement was carried out under nitrogen atmosphere (flow
rate 50 ml min-1) using 50 μl aluminum pan sealed by perforated
30 μl aluminum pan. Temperature calibration was carried out
with two standard materials (n–decane and In) and energy
calibration by an indium standard (28.45 J g-1). Each sample was
heated at a rate of 10 °C min-1 from -40 °C to about 10 - 20 °C
above the last observed thermal transition (predetermined by
TG/DTA), cooled back to -40 °C at a rate of 5 °C min-1, and then
heated for a second time. The standard uncertainty u(T) for the
onset temperatures was equal to 1.0 °C, and the relative expanded
uncertainties for the enthalpies and heat capacities with a 0.95
confidence level were estimated to be Ur(ΔH) = 0.02 and Ur(ΔCp)
Infrared spectroscopy
Infrared spectra were recorded with Bruker Tensor 27
spectrometer equipped with Pike Technologies GladiATRTM
accessory in the range of 400-4000 cm-1 with 4 cm-1 resolution,
using 32 scans.
=
0.03, respectively. The sample weights used on the
measurements were about 3 - 6 mg.
SEM
Thermomicroscopy
Scanning electron micrographs were taken with Bruker
Quantax400 EDS microscope equipped with a digital camera.
The sample stub was covered with a carbon tape. Samples of the
xerogels were prepared on the stub by scooping a small amount
of the gel formed in a test tube with a spatula. Direct application
of 20 µl of the hot sol was used in the case of the in situ-formed
gels/solid material samples. The samples were left to dry at room
temperature for two days before coated with gold in a JEOL Fine
Coat Ion Sputter JFC-1100.
A sub-milligram powder sample of compounds 1-4 was placed on
a standard microscope slide and was covered with a 0.2 mm
cover glass. Additionally, to ease observation of potential
desolvation/dehydration processes (bubble formation) a few
samples were prepared on a slide by immersing it to a drop of
perfluoropolyether (Fomblin®) fluid. A sample was heated in
Linkam LTS420E hot stage controlled by T95-HS Linksys heat
controller and LN95-LTS liquid N2 cooling unit. Hot stage was
mounted on Olympus BX51 microscope equipped with
polarization filters and Olympus DP26 digital color camera.
Samples were heated from 25 to 200 °C with a heat rate of 10 °C
min-1 and photographs were taken either while heating or on a
hold, with or without polarization filter and with varying
magnifications (from 50x to 500x).
Powder X-ray diffraction
The X–ray powder diffraction data of compounds 1-4 was
measured with PANalytical X´Pert PRO alpha 1 diffractometer in
Bragg–Brentano geometry using Johansson monochromatized Cu
K1 radiation (1.5406 Å; 45 kV, 30 mA). A gently hand-ground
powder sample was prepared on a silicon-made zero–background
holder using petrolatum jelly as an adhesive. The diffraction
intensities were collected from a spinning sample by X´Celerator
detector using continuous scanning mode in 2 range of 2 - 80°
with a step size of 0.017° and counting times of 200 s per step.
The recorded data was processed and analyzed in X´Pert
HighScore Plus v. 2.2d software package. The Search-match
phase identification routines were made with the same program
and patterns were retrieved from ICCD-PDF4+ powder
diffraction database.27
Results and discussion
Gelation studies
Compounds 1-3 were tested for gelation with 13 different
solvents (acetic acid, acetonitrile, acetone, benzene, 1-butanol,
dimethylformamide, dioxane, methanol, 2-propanol, pyridine,
tetrahydrofurane, toluene, and water), water being the only
solvent wherein the gels were formed. Different concentrations in
0.5 ml of water were investigated, resulting in 20 mg for
compound 1 and 25 mg for compounds 2 and 3 being sufficient
amounts to form a non-flowing gel. Compounds 1 and 2 made
transparent gels, while the gel of compound 3 appeared turbid.
Compound 4 needed a mixture of two organic solvents for gel
formation. The solid was first dissolved in methanol, after which
2-propanol, acetone, or ether was added forming a yellow gel (see
Table 1).
Thermogravimetry
Simultaneous thermogravimetric and differential thermal analysis
curves for compounds 1-4 were acquired with Perkin Elmer STA
6000 TG/DTA. Each measurement was carried out in an open
platinum pan under air atmosphere (flow rate of 45 ml min-1) by
heating a sample from 25 to 600 °C with a heat rate of 5 °C min-1.
The temperature calibration of STA 6000 instrument was made
using melting points of the indium (156.60 °C) and zinc (419.5
°C) standards. The weight balance of the instrument was
calibrated by measuring the standard weight of 50 mg at room
The thermal stability (Tgel) of the gels 1-3 was studied by
heating the gels in a convector with a thermometer set in the gel
and by observing the viscosity of the gels by eye. A distinct
melting point was not distinguished, but the viscosity of the gels

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DOI: 10.1039/C3TB20957A
Journal of Materials Chemistry B
Page 4 of 11
was observed to change. After 80 °C gel 1 (6% w/v) passed into
liquid, whereas gel 2 (5% w/v) became more liquid-like after 90
°C and gel 3 (5% w/v) after 85 °C (Table 1). The two latter gels
were heated to the boiling point of water without turning totally
into liquid state. Gel 4 could not be heated, because the organic
solvents would have evaporated.
signal width at the concentration of  3% for compounds 1 and 3,
and at the concentration of  4% for compound 2, whereafter the
signals appeared broader than at the lower concentrations (Fig.
S2-S4 in ESI). This indicates an increase in viscosity of the
samples when the concentration increases, due most probably to
restrictions in the free tumbling of the gelator molecules.
Interestingly, the intensity of the water signal in the spectra of
compound 2 seemed to decrease significantly at the concentration
of  4%, whereas for compounds 1 and 3 the intensity of the
water signal remained about the same in every concentration.
This probably indicates that the exchange of water molecules
between the gel and the sol state is restricted in the case of
compound 2, thus supporting the previous assumption according
to which the water molecules are more tightly bound within the
gel network when compared with gels formed by 1 and 3.
Table 1 Properties of compounds 1-4.
1
2
3
4
Gelating
solvent(s)
water
water
water methanol + 2-
propanol/
acetone/ ether
Cgel
Appearance
Tgel
4% (w/v)
transparent transparent turbid
80 °C 90 °C
5% (w/v) 5% (w/v) 5% (w/v)
yellow
85 °C not defined
Solution state NMR spectroscopy
1
Variable-temperature (VT) H NMR spectra of the hydrogels 1,
2, and 3 (5% w/v) were recorded in 5 °C steps between
temperatures 30 - 95 °C (Fig. 2 - 4). For compound 1 in the
temperature scale of 30 - 50 °C (when heated in 5 °C steps) only
the signals with the highest intensity were visible (Fig. 2).
Moreover, they appeared very broad. From the temperature of 50
°C onwards the signals started to become sharper step by step and
the coupling pattern gradually more resolved so that the shape of
the triplet of triplets -pattern arising from the proton attached to
the central carbon could be distinguished at the final temperature.
This indicates the change of the sample from a viscoelastic gel to
more and more liquid form, and coincides with the results
obtained by the thermal stability tests described above, which
suggest that the gels do not have explicit melting temperatures
but rather change phases gradually. Gel 3 behaved quite similarly
with gel 1, except that the shapes of the signals became more
resolved not until at 80 °C (Fig. 4). The change observed for
compound 2 was more explicit, since the broad signals appeared
as dramatically narrower ones at 75 °C, indicating that the gel had
transformed to the sol between 70 and 75 °C (Fig. 3).
Nevertheless, at 95 °C the shape of the signals was not as clear
for 2 and 3 as for compound 1. The phenomenon of narrowing of
the signals at the temperature  75 C was also observed for the
water signal in the spectra of gel 2 suggesting that the water
molecules are an essential part of the gel assembly. This was not
seen in the spectra of compounds 1 and 3, in which the shapes of
the water signals did not change remarkably.
Fig. 2 VT 1H NMR spectra of 5% (w/v) D2O gel of compound 1. For
comparison 1H NMR of a dilute solution in MeOD is presented at the
bottom.
The relative chemical shift values for compounds 1-3 do not
markedly change as the temperature is increased. The absolute
values of the chemicals shifts, however, seem to move downfield,
since an increase in the temperature results in an upfield change
of the chemical shift value of the signal of the residual water in
the D2O (see ESI, Fig. S1). Furthermore, since the ppm scale is
calibrated on the signal of the residual water, the other signals
appear to move downfield instead.
Fig. 3 VT 1H NMR spectra of 5% (w/v) D2O gel of compound 2. For
comparison 1H NMR of a dilute solution in MeOD is presented at the
bottom.
In order to investigate the gel forming process in more detail,
1H NMR spectra for compounds 1-3 were measured in five
different concentrations (1 - 5% w/v). The concentration-
1
dependent H NMR experiment showed a clear difference in the

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Page 5 of 11
Journal of Materials Chemistry B
synthesis products and xerogels of 1-4. The xerogels of 1-3
behaved similarly to each other, according to 31P CPMAS spectra
(Fig. 9). Compared to the spectra of synthesis products, two new
signals for xerogels were observed. Nevertheless, in the original
spectrum of compound 3 there were already hints of the signals
arisen in the spectrum of the xerogel (Fig. 9: 3 a, b). In the case of
xerogels 1 and 3 the new signals were located slightly further off
from the original peak (22.30 - 21.38 ppm), while for xerogel 2
the signals were closer to the original ones at 23.80 and 23.36
ppm, respectively. The intensities of the new signals varied. The
31P CPMAS spectra of the hydrogels 1 and 3 were very similar
having one signal at around 24 ppm, but the spectrum of hydrogel
2 was differing again, as it was in the 13C CPMAS spectrum, and
the peak was wider and deformed. Based on the above results
compounds 1-4 seemed to pack slightly differently in the
crystalline state of the synthesis products compared to the
corresponding xerogels. In addition, the hydrogel of compound 2
was different from the hydrogels of 1 and 3 since water
molecules appeared to be more tightly bound within the gel
network.
Fig. 4 VT 1H NMR spectra of 5% (w/v) D2O gel of compound 3. For
comparison 1H NMR of a dilute solution in MeOD is presented at the
bottom.
Solid state NMR spectroscopy
13C and 31P solid state NMR spectra were recorded for the bulk
synthesis products, xerogels, and hydrogels. The 13C CPMAS
spectra of compounds 1-3 were quite similar to each other (Fig. 5
- 7) showing only one signal for each carbon. The triplet at about
38 ppm in the 13C CPMAS spectra of the synthesis products and
xerogels of 1-3 is caused by one-bond spin-spin couplings
between carbon and phosphorus nuclei. In particular, the spectra
of the hydrogels resembled the ones of the synthesis products.
The spectra of the xerogels slightly differed from the other ones,
new signals appearing in the area of alkyl chain signals (around
20 to 35 ppm) indicating some changes in the alkyl chain
assembly or conformations. In the 13C CPMAS spectrum of the
hydrogel 2 clearly wider and more unclear signals compared to
hydrogels of 1 and 3 were found. This observation supports the
suggestion of water molecules playing essential role of the gel
assembly since the wide signals appear for many structures
closely resembling each other.
Fig. 5 13C CPMAS NMR spectra of compound 1: (a) synthesis product,
For compound 4 the 13C CPMAS spectra of all three different
forms resembled closely each other (Fig. 8). The only exception
was observed in the spectrum of the organogel, where the two
methyl signals in 24.4 - 13.6 ppm (one attached to the central
carbon and the other to the acetyl-group) were more clearly
separated. In the 31P CPMAS spectra of compound 4 the
differences were even more evident: the phosphorous signal of
the synthesis product showed only one signal, the xerogel was
shaped as there were two signals overlapping, and for the
methanol/2-propanol gel two signals could be clearly
distinguished (Fig. 9: 4).
(b) xerogel, (c) hydrogel (4% w/v).
In the 31P CPMAS spectra of the synthesis products 1-3 the
signals had the same chemical shift at around 24.8 ppm (Fig. 9: 1
- 3). The signal, however, had clearly divided in two for
compound 1, whereas for 3 its shape was as two signals were
merged together. In the spectrum of compound 2, on the other
hand, the phosphorus gave rise to a single signal. Because 13C
CPMAS spectra possessed a singlet peak for each carbon, the
origin of doubled structure of 31P CPMAS spectra of 1 and 3 was
probably due to the nonequivalence of two phosphorus nuclei
whereas in 2 and 4 the chemical shift difference was that small
that it was not resolved. Based on 13C CPMAS spectra there was
only one molecule in an asymmetric unit in the crystals of
Fig. 6 13C CPMAS NMR spectra of compound 2: (a) synthesis product,
(b) xerogel, (c) hydrogel (5% w/v).

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Journal of Materials Chemistry B
Page 6 of 11
resolved than those of the xerogel of 1 (Fig. 10: a, b). Some of the
bands were clearly shifted as well, such as the ones in the range
of ~900-980 cm-1. The possible differences in the level of
protonation, which can have large effects on the IR spectra of
bisphosphonates28-29, may contribute to some of the observed
differences in the IR spectra along with the solid state packing.
The synthesis product 1 was assumed to be fully protonated, but
direct conclusions for the state of the xerogel of 1 could not be
done. Indicative bands related to P-OH- and P-O- groups28-29
could be useful for interpretation but the long alkyl chain of
compound 1 caused the fingerprint region to be crowded. In
general, trying to assign likely candidates for any stretching and
bending modes of P=O, P-O(H), or P-O- groups was not therefore
feasible as the modes related to the phosphonate groups all lay in
the fingerprint region below ~1320 cm-1.28-31
Fig.7 13C CPMAS NMR spectra of compound 3: (a) synthesis product, (b)
On the other hand, the two bands at 1462 cm-1 and 1476 cm-1
in the spectrum of xerogel of 1 and the single band at 1470 cm-1
in the spectrum of the synthetic product 1 were clearly
distinguishable as aliphatic modes corresponding to CH2-
deformation, and asymmetric out-of-phase CH3-deformation.31-32
The exact identity of the two xerogel bands was difficult to affirm
definitely but the band at 1462 cm-1 was more likely to contain
intensity from the asymmetric out-of-phase CH3-deformation.31
The very weak band at 1379 cm-1 (xerogel of 1), and at 1377 cm-1
(synthesis product 1) evidently corresponded to the in-phase
deformation mode of CH3.31-32 The two forms of 1 did not show
any practical differences above 1500 cm-1, and the aliphatic
stretching region was identical between compound 1 and its
xerogel. Although very broad and weak, there were two areas
visible at 2000-2700 cm-1 (ignoring the CO2-artefact at ~2350 cm-
1), which could correspond to the P-OH hydroxyl stretching
bands.30-31 The asymmetric stretching modes for the CH2-groups
and the CH3-group could be found at 2919 cm-1 and 2955 cm-1,
respectively. The symmetric stretching mode of the CH2-groups
is found at 2851 cm-1, the very weak bulge near 2870 cm-1
xerogel, (c) hydrogel (5% w/v).
Fig. 8 13C CPMAS NMR spectra of compound 4: (a) synthesis product,
(b) xerogel, (c) gel in MeOH/2-propanol (5% w/v).
possibly coming from the symmetric stretch of the CH3-group.30-
31
Fig. 9 31P CPMAS NMR spectra of compounds 1-4: (a) synthesis product,
(b) xerogel, (c) hydrogel 4% w/v for 1 and 5% w/v for 2-3; gel in
MeOH/2-propanol (5% w/v) for 4.
Infrared spectroscopy
Infrared spectra were measured to shed light on the structure of
the solid state forms of compounds 1-4. Some changes in the
fingerprint region were observed for all of the compounds 1-4,
when comparing the synthetic products to their corresponding
xerogels. However, appreciable differences were noted for
compound 1 only. In general, the bands in the fingerprint region
of the synthesis product 1 (Fig. 10: a) were broader and less
Fig. 10 (a) Selected region of the IR spectra of the solid synthesis product
1 (black line) and xerogel of 1 (grey line). The wavenumbers of selected
bands are presented for the solid synthesis product 1. (b) IR spectrum of
the xerogel of 1.
Structural properties by X-ray Diffraction
The structural characteristics of xerogels and synthesis product

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DOI: 10.1039/C3TB20957A
Page 7 of 11
Journal of Materials Chemistry B
samples of compounds 1-4 were investigated by powder X-ray
xerogels of compounds 1-3.40
diffraction (XRD). The perceptibly similar XRD patterns of the
synthesis products and corresponding xerogels of compounds 1-3
showed narrow and well-resolved diffraction peaks up to about
40° in 2 indicating moderate crystallinity of the examined
samples (Fig. 11). In contrast, for compound 4 only a few broad
scattering humps characteristic for an amorphous form, most
notably in 2-range of 5 to 9°, were visible in the XRD pattern.
The corresponding xerogel was somewhat more crystalline, as a
few prominent diffraction peaks could be seen for instance in 2-
angles of 6.92°, 11.34°, 13.83°, and 17.48°. The XRD patterns of
1-3 and their xerogels were also subjected to unit cell indexing
but no reasonable cell settings were found. Regardless of that the
XRD patterns failed to index, and hence the exact correlation
(Miller indices) between the lattice planes and observed peak
angles were missing, the average crystallite sizes of the samples
were determined by the Scherrer method33-34, as it can be assumed
that the strongest peaks at low 2 mostly originate from the
principal crystallographic planes with the smallest Miller
integers. For compounds 1-3 and the corresponding xerogels
average crystallite sizes of about 170-250 nm were obtained,
whereas crystallite sizes of less than 10 nm and 90 nm were
determined for 4 and its xerogel, respectively.
The structural characteristics of 4 and its xerogel remained
somewhat scarce as both substances were poorly crystalline.
Therefore, the structural comparisons were inconclusive, except
of that both of the XRD patterns differed from the corresponding
patterns for compounds 1-3, obviously due to the significant
chemical dissimilarity of 4 (disodium salt of acetylated and
methyl protected etidronate). It can also be contemplated whether
weak crystallization properties of this compound originates from
its anion, which is rather irregularly shaped and has impaired
ability to form hydrogen bonds due to its acetyl, methyl, and
methyl protected phosphonate groups located around the central
carbon.
4xg
4
3xg
3
As implied above, from the structural point of view, the XRD
patterns of the synthesis products and xerogels of 1-3 were
remarkably similar. It could also be seen that by increasing the
alkyl chain length with a single methylene group (n = 10 - 12),
the greatest change took place in form of a rather systematic shift
of peak positions towards the lower 2-range but without
noticeable changes either in overall number of the peaks or in
their characteristic peak height distribution. This change is best
seen on 3° - 10° in 2, wherein the strongest diffraction peaks are
located: at 3.97° and 7.89° for the xerogel of 1, at 3.88° and 7.57°
for the xerogel of 2, and at 3.55° and 7.07° for the xerogel of 3,
respectively. Based on the literature and our own observations, it
is frequently witnessed that molecules with aliphatic chain(s) of
10 carbons or more in connection with group(s) capable of
hydrogen bonding at the far end of the molecule, show tendency
to organize the aliphatic chains in parallel fashion and
consequently, hydrophilic and -phobic sections in the crystal
lattice are formed. This phenomenon is observed in many
aliphatic carboxylic acids, carboxylates, amines, quaternized
amines, and alkylphosphonates.35-39 Moreover, when a series of
these kinds of structural motifs has been investigated with
crystallographic methods, it has been found in a number of cases
that an increase in aliphatic chain length may induce one-
dimensional change of the unit cell, since the long aliphatic chain
favors the parallel packing motif due to the repetitive van der
Waals interactions that are gradually enhanced by the increase of
the alkyl chain length. Thereby, the molecular packing system in
the lattice remains otherwise unchanged.35 In the now studied
aliphatic BPs, above packing mechanism along with the
capability to form hydrogen bonds between the phosphonic acid
groups (and/or with solvent molecules; water in this case) are
presumably the prevailing packing forces that most likely guide
the structures to form parallel-packed structure modifications,
which in part may have contributed to the relatively high
crystallinity of the synthesis products and the corresponding
2xg
2
1xg
1
4
8
12
16
20
24
28
2 (°)
Fig. 11 XRD patterns of synthesis products 1-4 with the corresponding
xerogels 1xg-4xg.
Thermal analysis
Thermal degradation and potential hydration levels of 1-4 and
their
corresponding
xerogels
were
investigated
by
thermogravimetry (TG) and differential scanning calorimetry
(DSC). Additionally, wherein the interpretation of TG and DSC
data proved to be challenging, thermomicroscopy was used as an
auxiliary method to further explore the true nature of the thermal
event in question. No significant differences were observed to
exist between the TG curves of the synthesis products and the
corresponding xerogels, as can be seen in Fig. 12. For compounds
1 and 3 as well as their xerogels, two noticeable weight loss steps
could be seen in the TG curves, prior to the thermal degradation
that commenced at about 302 (307) and 317 (318) °C,
respectively (see Table 2). The TG curves of 2 and its
corresponding xerogel were similar to those of 1 and 3, except
that only one weight loss step was observed (at about 314 °C)
before the degradation. The TG curves of 4 and its xerogel, for
one, deviated from the first three ones by showing just a single
weight loss step corresponding to the thermal degradation that
starts at 206 (227) °C. In compounds 1-3 and their xerogels the
degradation proceeded via multiple, partially overlapped steps
that eventually leveled out temperatures above 500 °C and
showed residual weights of 5 - 9%. Separate degradation products
could not be identified for these aliphatic BPs but it can be
assumed that simple chemical evaporates like, H2O, CO,

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DOI: 10.1039/C3TB20957A
Journal of Materials Chemistry B
Page 8 of 11
CH3COOH, and a variety of hydrocarbons are the main
degradation products, as similar type of components have
recently been identified in the degradation of amino trimethylene
phosphonic acid (ATMP) and 1-hydroxy ethylidene-1,1-
diphosphonic acid (HEDP, etidronate).41 Moreover, the relatively
low residual weights, in contrast to initial phosphorus content
(17.7 - 16.4%) indicated that also phosphorus was almost entirely
removed via degradation products; most likely as phosphorus
oxide. Compound 4 deviated in this respect as well. The
compound was originally composed as a disodium salt and
consequently degraded via different path terminating to a non-
combustible residue with high residual weight after the removal
of the organic part of the initial composition. The residue was
removed from the TG crucible and was identified as a mixture of
sodium metaphosphate (Na3P3O9) and graphite (minor
component) by powder diffraction (Fig. S5 in ESI).42
Furthermore, the calculated and experimental weight losses were
in agreement with this interpretation (Δmcalc. = 63.7% and Δm exp.
= 62.44% in the case of the xerogel of compound 4).
As mentioned above, additional weight loss steps prior to the
degradation were visible in the TG curves of both synthesis
products and xerogels of compounds 1-3 (Fig. 12). The first
relatively weak weight loss (Δm = 0.5 - 2.5%, not seen in 2)
occurred between 53 and 60 °C and the second, larger one (Δm =
3.6 - 5.8%) initiated at around 142 - 160 °C and ended gradually
till 280 °C. The observed weight changes were expected to be
associated with the removal of water, which occurred presumably
in two separated stages for 1 and 3, and in a single step for 2. In
order to confirm the above postulation, the experimental and
calculated weight changes were examined. It could be noted that
they were reasonably well in accordance with the molar ratio of
single water (Table 2) for 1 and its xerogel, as well as for 2,
whereas somewhat higher hydration levels corresponded with the
xerogel sample of compound 2, as well as with both the synthesis
product and the xerogel of compound 3 (1.2-1.3 moles). The DSC
scans of compounds 1-3 and the corresponding xerogels
displayed three (two in the case of 2 and its xerogel) endothermic
transitions, of which the first and third were located in the
temperature ranges consistent with the observed weight losses
(Fig. 13). The second endotherm (no weight loss) was in the
immediate vicinity of the first one, forming partially overlapping
doublet peak with the first endotherm, with the exception of the
synthesis product and xerogel of 2 wherein the “second”
endotherm appeared as a single peak at 62.3 and 79.5 °C,
respectively. Interestingly, no weight loss was related to this
second moderately strong endotherm. At this point, visual
monitoring of all the xerogels was made by the hot stage
microscope, in order to clarify the true nature of each endotherm
and their linking to the thermogravimetric changes. Readily a few
very important observations could be made. The xerogel of
compound 4 stayed as an unchanged solid until its degradation
began at 227 °C. The xerogels of compounds 1-3, for one,
remained as solids till 142 - 160 °C, wherein they melted (Fig.
14, upper four-field). By heating the samples above their melting
point, formation of small bubbles initiated (best seen in the
xerogel of compound 3), suggesting that some crystalline water
was released simultaneously with melting. Further heating to
above 280 - 300 °C turned the samples gradually brownish
implying the beginning of degradation. Based on the observations
above, it is suggested that the release of the first part of the water
(at lower temperatures) occurs in solid state. The second nearby
endotherm originates from a solid-state phase transition induced
by the release of water. Speculations can be made, whether this
more loosely bound water is originally located in channels or
cavities that may exist in these structures. In that case the larger
portion of the water would be more tightly hydrogen bonded to
the phosphonic groups and could not be evaporated prior to
melting.
In addition, the xerogels of compounds 1-3 were observed to
show birefringence with crossed polarizer when heated over the
melting range, and fractals typical for a liquid crystal could be
seen all the way to the degradation temperature. The liquid
crystalline properties of these compounds will be examined in
more detail and reported in due course.
100
80
60
40
20
0
100
80
60
40
20
0
~160 °C
~150 °C
~305 °C
~314 °C
O
O
O H
P
O H
O
O
H
P
H
P
O H
O H
H
P
O
H
H
O
O
O H
2xg
1xg
2
1
100
200
300
400
500
600
100
200
300
400
500
600
Temp. (°C)
Temp. (°C)
100
80
60
40
20
0
100
80
60
40
20
0
227 °C
~149 °C
222 °C
~317 °C
O
P
O
O
O
O
O H
P
O H
–
+
N a
–
P
O
N a +
O
P
O
O
O H
O H
H
O
3xg
3
4xg
4
100
200
300
400
500
600
100
200
300
400
500
600
Temp. (°C)
Temp. (°C)
Fig. 12 TG runs of compounds 1-4 and their corresponding xerogels (xg).
4xg
4
3xg
3
2xg
2
1xg
1
0
20
40
60
80
100
120
140
160
Temp. (°C)
Fig. 13 DSC scans of compounds 1-4 and their corresponding xerogels
(xg), measured with a heat rate of 10 °C min-1.

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DOI: 10.1039/C3TB20957A
Page 9 of 11
Journal of Materials Chemistry B
Table 2 Thermal properties of examined bisphosphonates.
DSC (1st heating)
comp.
TG (Δm(%))
degrad.
Td
307
Elem. anal. (%)
Tdh + ss (ΔH)
69.9 (36.80)
78.4 (76.00)
59.9 (38.4)
Tm+dh (ΔH)
158.5 (63.00)
calc.
exp.
calc.b
exp.
1xg
1
5.17
5.21
159.7 (58.32)
5.17
5.05
302
C 41.4
H 8.7
C 41.94
H 8.76
82.7 (76.36)
62.4 (100.25)
79.5 (82.50)
2xg
2
142.0 (35.34)
146.8 (27.62)
4.97
4.97
5.87
5.16
315
314
C 43.1
H 8.9
C 44.53 H,9.00
3xg
3
57.6 (36.44)
73.00 (79.67)
55.7 (43.34)
63.7 (76.41)
154.6 (43.06)
156.9 (45.34)
4.79
4.79
6.29
6.11
318
317
C 44.7
H 9.1
C 45.52
H 9.22
4xg
4
63.7 a
63.7 a
62.44
60.10
227
222
c
c
C 21.3
H 4.2
Dehydration with solid-state crystal form transformation (Tdh+ss), melting
with dehydration (Tm+dh), and degradation (Td) temperatures have been
obtained as an extrapolated onset values (°C). ΔH = enthalpy of a
transition (J g-1). xg = xerogel. a = weight-% of Na3P3O9 heating residue at
600 °C. b = calculated with a 1 mole of water. c = Due to the residual
solvents, exact elemental analysis could not be obtained.
Xerogel morphology
As the dynamics of supramolecular gel systems may be very
complicated43,44, microscopy techniques (such as SEM) relying
on dried samples do not necessarily give the right information
about the structures present in the corresponding wet gels.45
Sample preparation and -history may additionally influence the
outcome for the xerogels.46 However, it is still beneficial to
observe what kind of structural components may form among the
solid materials. An additional important factor to consider is the
surface/particle morphology of the system, which gives clues
about the structure and behavior of the material in a way that
crystallographic data cannot address. To probe the sensitivity of
the formation of the xerogels of 1-3, two preparation techniques
were used for the SEM samples: The first one was in situ
gelation/material formation directly on the SEM sample stub, and
the second one sampling a fresh gel prepared in a test tube.
Xerogel 4 was prepared only with the second preparation
technique because the gel was not obtained in the solution form
due to the evaporation of the solvents.
For compound 1, it is clear that the material obtained in situ
was different from the xerogel. The xerogel of 1 contained
ribbon-like fibers of 3-4 µm in diameter, which often entangled
and blend into each other (Fig. 15: A). In the corresponding in
situ-material, spherical/oblate objects commonly having
a
diameter in the 20-25 µm range were observed on cracked
platelets (Fig. 15: B). Some of the spherical objects appeared to
be burst open, thereby forming a cavity. One speculative
explanation for their existence might be the removal of the water,
contained within, during air drying and when the sample was
subjected to the vacuum while gold coated. For compound 2, both
techniques yielded similar material, where the ribbon-like fibers
dominated as the major structural component (Fig. 15: C and D).
The fibers (~2-3 µm in diameter) were formed as part of
spherulites47 (bundles of fibers emanating radially from a central
nucleation center) usually ~75-100 µm in diameter. In addition,
small crystalline-appearing grains were visible in the xerogel 2,
proving that it contains more than one component (Fig. 15: C).
The formed materials for compound 3 were likely composed of
crystalline aggregates in the nanoscale, because of the plain
appearance (Fig. S6: A and B in ESI). The xerogel of compound
4 showed signs of possibly fibrillar growth with sub-micrometer
lateral dimensions (~0.5 µm fiber diameter) (Fig. S6: C and D).
Fig. 14 Hot stage microscope images of xerogel samples of compounds 1
(a), 2 (b), 3 (c), and 4 (d) in solid state at 100 °C (upper four-field), and in
molten state at 160 °C (lower four-field), with exception of the xerogel of
4 that remains solid until degrades thermally at 227 °C.

Journal of Materials Chemistry B
View ArtiPcleaOgnleine10 of 11
DOI: 10.1039/C3TB20957A
crystal properties. The xerogels of 1-4 contained non-spherulitic
and spherulitic fibers as their structural elements in the
micrometer range, in addition to other micro- and nanoscale
aggregates. There is a possibility that all or some of the observed
xerogel structural components may be found in the wet gels as
well, but further studies are needed to prove their composition.
Taking into account the importance of BPs in medicine and
pharmacy we believe that this innovation opens many new
opportunities for the use of BP derivatives in different
applications.
Acknowledgements
Chief Laboratory Technicians Maritta Salminkoski and Helena
Vepsäläinen (School of Pharmacy, University of Eastern
Finland), and students Maciek Duda and Agata Zerka (Wroclaw
University of Technology, Poland) are thanked for their help in
the synthetic and analytical work. Laboratory Engineer Esa
Haapaniemi (Department of Chemistry, University of Jyväskylä)
is acknowledged for his help in measuring the NMR spectra and
Laboratory Technician Hannu Salo (Department of Chemistry,
University of Jyväskylä) for SEM images. This work was
supported by the Academy of Finland (project no. 132070 for
A.A., project no. 218178 for E.S., and projects no. 119616 and
255648 for M.L.), strategic funding of UEF, and PhoSciNet
COST action.
Fig. 15 SEM micrographs of (a) xerogel of 1, (b) in situ-prepared material
of 1, (c) xerogel of 2, (d) in situ-prepared material of 2. The scale bars are
(a) 20 µm, (b) 100 µm, (c) 40 µm, (d) 100 µm.
The wet gels of 1-3 differed from the xerogels according to the
13C CPMAS-studies, but whether this affects the observable
morphology of the xerogels remains an open question. Although
the dimensions of the crystallites determined using PXRD data
could in principle be compared to those of the structures observed
in SEM micrographs, the structural objects found in SEM are
often composed of larger crystallite agglomerates, instead of “X-
ray visible” single crystallites, thereby indicating particle size
instead of crystallite size.48 The appearance of the xerogel of 3
could conform to the crystallite size range, but the xerogels of 1,
2, and 4 did not readily fit into the same category based on the
micrographs, probably because of the abovementioned reasons.
At any rate, the one-to-one correspondence between the observed
structures in SEM and the data obtained from XRD- or
spectroscopic techniques cannot be verified, since different
xerogel samples were used in them.45,46 Further studies would be
needed to establish such a connection.
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, in which D = average crystal size in

direction perpendicular to the diffracted crystal planes, K = shape-
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(1.5406 Å), βs
=
the peak broadening (in radians) at half the
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