Sweet and salted: Sugars meet hydroxyapatite

Article abstract of DOI:10.1055/s-0030-1260953

Carbonated hydroxyapatite (CHA) has been successfully biodecorated with carbohydrate derivatives, via silylation of the hydroxy groups of the apatite and subsequent covalent bonding with a suitably functionalized carbohydrate moiety. The presented procedu

Full text of DOI:10.1055/s-0030-1260953

LETTER
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Sweet and Salted: Sugars Meet Hydroxyapatite
S
ugars Meet
H
ydroxya
o
patite nica Sandri,a Antonino Natalello,b Davide Bini,b Luca Gabrielli,b Laura Cipolla,*b Francesco Nicotra*b
a
b
Received 12 May 2011
Dedicated to Prof. Alfredo Ricci
carbohydrates, perbenzylated C-glycoside derivatives of
biologically relevant monosaccharides such as D-glucose,
D-galactose and L-fucose, were used.
Abstract: Carbonated hydroxyapatite (CHA) has been successfully
biodecorated with carbohydrate derivatives, via silylation of the hy-
droxy groups of the apatite and subsequent covalent bonding with a
suitably functionalized carbohydrate moiety. The presented proce-
dure opens the way toward covalent biofunctionalisation of CHA
with carbohydrates.
Key words: carbohydrates, C-glycosides, biomaterials, hydroxy-
apatite, biofunctionalisation
Recent promising trends in biotechnology and tissue engi-
neering are based on the development of advanced mate-
rials with biomimetic features created by designing and
tailoring of specific surface properties, which improves
biological responses and tissue compatibility.1 Among
various biocompatible materials, synthetic hydroxyapatite
(HAp) and carbonate hydroxyapatite (CHA) are widely
used in many biomedical applications.2 HAp is a natural
mineral ingredient of bones, teeth and calcified tissues in
vertebrates. Synthetic HAp and CHA are used for human
implant coatings possessing beneficial biocompatibility
and osteoconductivity.
The combined use of inorganic materials, such as CHA,
and organic signaling molecules is very a promising ap-
proach to tissue engineering.3,4 To date, main efforts to-
wards the covalent functionalisation of apatite has
focused on the biodecoration of this inorganic material
with whole proteins5 or short peptide epitopes;6 the most
commonly used peptide for surface modification is
RGD,6a,b which is the signaling domain derived from fi-
bronectin and laminin.7 Less exploited as cellular ligands
for material functionalisation are carbohydrate epitopes;
these are well-known to bind to cell surface receptors and
allow cell–cell interactions.8 In this context, the possibili-
ty of decorating hydroxyapatite with carbohydrates can be
relevant.9 To the best of our knowledge, no examples have
been reported on hydroxyapatite biofunctionalisation with
carbohydrates.
In this report, we present our preliminary results on the
functionalisation of CHA with monosaccharide deriva-
tives, to explore the possibility of covalently linking car-
bohydrate epitopes to hydroxyapatite, as a novel method
of ‘bioactivation’ of this promising material. As model
Scheme 1 Methodology for CHA functionalisation with carbohy-
drate C-glycosides
C-Glycosides present several advantages over their O-
glycosidic counterparts:10 their structure closely resem-
bles that of the parent sugar, thus maintaining the biolog-
ical information, but they lack the anomeric glycosidic
bond that is usually subjected to degradation in vivo. In
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Advanced online publication: 14.07.2011
DOI: 10.1055/s-0030-1260953; Art ID: S03811ST
© Georg Thieme Verlag Stuttgart · New York

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M. Sandri et al.
LETTER
addition, the benzyl protecting groups were used as
probes to analyze the surface-decorated CHA.
CHA granules with dimensions in the 400–600 mm range,
which were obtained from a nanostructured biomimetic
CHA powder with a specific surface area of 36 m2/g, were
used for biodecoration with monosaccharide deriva-
tives.11 Because CHA possesses hydroxy groups, we en-
visaged the possibility of covalently linking suitably
functionalized C-glycosides via an aminopropylsilane
linker (Scheme 1). In more detail, the strategy for C-gly-
coside immobilization (Scheme 1) involved: (i) grafting
of aminopropyltriethoxy silane (APTES) onto the surface
of CHA granules in anhydrous hexane, and (ii) reaction of
the amino groups on the CHA surface with the activated
hydroxysuccinyl esters from C-glycosides in anhydrous
THF as solvent.6a
To assess the degree of functionalization with the amino
functionality, spectrophotometric analysis was performed
after silanization of the hydroxyapatite with (3-triethoxy-
silylpropyl)carbamic acid 9H-fluorenylmethyl ester
(Fmoc-APTES),12 as shown in Scheme 2. Removal of the
Fmoc group is usually achieved by treatment with piperi-
dine in N,N-dimethylformamide (DMF). The mechanism
of the Fmoc-deprotection reaction involves the formation
of aromatic cyclopentadiene intermediates, which are rap-
idly eliminated to form dibenzofulvene, which is further
scavenged by piperidine to form the Fmoc-adduct 1-[(9H-
fluoren-9-yl)methyl]piperidine. This product strongly ab-
sorbs UV radiation at 289 nm, offering the potential to
monitor the deprotection reaction by UV/Vis spectrosco-
py and quantifying the extent of functional-group loading
on CHA. In this way, the degree of amino functionaliza-
tion of hydroxyapatite was found to be 0.15 mequiv/g
CHA.
Scheme 2 Method used for the quantification of amino groups on
the CHA surface
of the granules after functionalization could be detected
— the material remained monophasic apatite. In addition,
ICP analysis showed that the Ca/P molar ratio remained
substantially unaffected after the chemical reactions. RX
mapping of the elements by SEM-EDS analysis provided
evidence for the homogeneous presence of Si, and for Ca
and P, in the functionalized granules. TG analysis of the
unfunctionalized CHA granules in air revealed a weight
loss occurring up to 500 °C, which was attributed to dehy-
dration of the absorbed and occluded water and to the re-
moval of absorbed species.16
C-Glycosidic derivatives possessing a carboxylic group
for the coupling reaction to the aminopropylsilane-func-
tionalized CHA (CHA-APTES) were synthesized in a few
steps from the suitably protected natural monosaccharide
(for experimental details see the Supporting Information).
Briefly, the a-allyl-C-glycoside was prepared by Sakurai
reaction from the suitably protected parent monosaccha-
ride;13 the allyl C-glycosidic appendage was then func-
tionalized with a carboxyl terminus,14 which is a suitable
functional group for the condensation reaction with the
CHA-APTES, by hydroboration (9-BBN-H), followed by
oxidation to the corresponding carboxylic acid (TEM-
PO).15 The carboxylic group was finally activated as the
succinyl ester (diisopropylcarbodiimide, N-hydroxysuc-
cinimide) and then coupled to the amino group of CHA-
APTES in anhydrous THF.
At higher temperatures, carbonate ions decompose, caus-
ing a weight loss due to CO2 elimination; this allowed the
extent of initial carbonation of the CHA granules to be es-
timated to be about 5.5wt%, which is comparable to the
carbonate content found in samples of biological apatite
(2–8wt%).
The CHA functionalized with carbohydrate derivatives
was then characterized to determine (i) the chemical sta-
bility of the material upon chemical derivatization, and
(ii) the effective biodecoration with carbohydrate deriva-
tives.
The STA analysis confirmed the presence of additional
compounds besides CHA in the functionalized material,
because the TGA and the DTA profiles changed moving
from unfunctionalized CHA, to the functionalized deriva-
tive (Figure 1). In particular, DTA analysis showed, in
both curves 2 and 3, the presence of significant peaks in
To assess the stability of the material after chemical mod-
ifications, XRD analysis was performed; no phase change
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Sugars Meet Hydroxyapatite
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Figure 1 TGA analysis of the HA samples: unfunctionalized CHA (curve 1), CHA-APTES (curve 2), and biodecorated CHA (curve 3)
the 250–450 °C temperature range that were attributed to A), functionalization with 3-aminopropyltriethoxysilane
the degradation of organic phases. In particular, the peak leads to the appearance of several new bands, including
centered at 270 °C in curve 2 was correlated to the degra- the 2933 cm–1 and 2863 cm–1 peaks that are due, respec-
dation of the silane group (APTES), and the multiple tively, to asymmetric and symmetric CH2 stretching.
peaks in the 250–450 °C range of curve 3 were linked to These bands are also present in the spectra of CHA bio-
the presence of both APTES and the connected carbohy- decorated with derivatives 1–3 via 3-aminopropyltri-
drate moiety.
ethoxysilane. In addition, these last materials display
absorption peaks in the 3091–3033 cm–1 region due to CH
vibrations of the aromatic rings present in the carbohy-
drate structure.
To further characterize the biofunctionalization of CHA
and, in particular, to assess the effective biodecoration
with carbohydrates, FTIR analyses were performed. In
Figure 2A, we report the IR spectra of unfunctionalized A more detailed inspection of the spectra reveals several
CHA and those of CHA functionalized with 3-aminopro- additional features that further confirm the biodecoration
pyltriethoxysilane (CHA-APTES) and with C-glycosides of CHA. For instance, the formation of an amide bond be-
1–3 conjugated to CHA via 3-aminopropyltriethoxysilane tween the silane group and the different compounds leads
(spectra from CHA-1 CHA-3). All the spectra are domi- to a new absorption around 1650 cm–1 (Figure 2 B) due to
nated by the 1017 cm–1 absorption peak due to the phos- the C=O amide stretching vibration.18 To discard the pos-
phate (PO43–) vibration of CHA. Other absorption bands sibility of carbohydrate absorbtion onto CHA, and fully
of CHA can be observed in the spectra, such as those due confirm the bonding between the amine group of CHA-
to the structural OH (3570 cm–1), to phosphate (962 cm–1), APTES and the C-glycosides carboxyl group, we per-
and to the B-type carbonate (couple at 1415 cm–1 and at formed a second derivative analysis of the measured spec-
~1455 cm–1, and the peak at 872 cm–1).16,17 The presence tra (a resolution enhancement mathematical procedure).
of the typical peaks of the B-type carbonate confirmed Because the C=O amide band has a reduced width com-
that the carbonate ions are situated on the phosphate site pared to the CHA absorption in the same spectral region,
of hydroxyapatite.16
in this way it is possible to clearly detect the formation of
the new amide bond,18 as can be seen in Figure 2 C (only
CHA-2 is reported as an example). The presence of an ab-
sorption component around 1575 cm–1 further confirms
To characterize the biodecorated materials, the spectral
region 3100–2800 cm–1 at which CH bond absorptions are
typically found, is of valuable interest. Indeed (Figure 2
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M. Sandri et al.
LETTER
carbohydrates; CHA functionalization with biologically
active carbohydrate epitopes can upgrade this material
from being biomimetic to being a bioactive material that
is able to cross-talk with the surrounding cellular environ-
ment.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
Acknowledgment
We gratefully acknowledge FONDAZIONE CARIPLO, project
2008/3175 and MIUR, project FIRB RBPO68JL9 for financial sup-
port.
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Figure 2 FTIR analysis of CHA samples. Panels A and B: ATR/
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functionalized with 3-aminopropyltriethoxysilane (CHA-APTES)
and with compounds 1–3 (CHA-1, CHA-2, CHA-3). Panels C and D:
Second derivatives of the absorption spectra reported in Panels A and
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the formation of an amide bond. Indeed, this component,
which is absent in the CHA spectrum, can be assigned to
the NH2 bending vibration in the case of CHA-APTES
and to the NH bending (Amide II band) in that of CHA-2
(Figure 2 C). Similar results were obtained for the biodec-
oration with carbohydrates 1 and 3.
Many additional bands were also observed after biodeco-
ration that confirmed the success of our procedure. For in-
stance, the absorption in the 720–760 cm–1 region can be
assigned to the aromatic ring vibrations (Figure 2 D). We
should also report that materials CHA-APTES, CHA-1,
CHA-2, and CHA-3 display a component around 817 cm–1,
which is absent in CHA, that can be assigned to a vibra-
tional –O-Si- mode.19
With these data in hand, we can affirm that carbonated hy-
droxyapatite has been successfully biodecorated with car-
bohydrate derivatives. It should be noted that classical
organic coupling reactions have been applied for the co-
valent functionalization of inorganic materials such as
CHA. The presented procedure opens the way to the ap-
plication of organic chemistry to the covalent biofunction-
alization of CHA, which is an excellent biomimetic
material, with suitable classes of biomolecules, such as
Synlett 2011, No. 13, 1845–1848 © Thieme Stuttgart · New York