Controlled click-assembly of well-defined hetero-bifunctional cubic silsesquioxanes and their application in targeted bioimaging

Article abstract of DOI:10.1002/chem.201300339

A general procedure for the assembly of hetero-bifunctional cubic silsesquioxanes with diverse functionality and a perfectly controlled distribution of functional groups on the inorganic framework has been developed. The method is based on a two-step sequence of mono- and hepta-functionalization through the ligand-accelerated copper(I)-catalyzed azide-alkyne cycloaddition of a readily available octaazido cubic silsesquioxane. The stoichiometry of the reactants and the law of binomial distribution essentially determine the selectivity of the key monofunctionalization reaction when a copper catalyst with strong donor ligands is used. The methodology has been applied to the preparation of a set of bifunctional nano-building-blocks with orthogonal reactivity for the controlled assembly of precisely defined hybrid nanomaterials and a fluorescent multivalent probe for application in targeted cell-imaging. The inorganic cage provides an improved photostability to the covalently attached dye as well as a convenient framework for the 3D multivalent display of the pendant epitopes. Thus, fluorescent bioprobes based on well-defined cubic silsesquioxanes offer interesting advantages over more conventional fully organic analogues and ill-defined hybrid nanoparticles and promise to become powerful tools for the study of cell biology and for biomedical applications. Copyright

Full text of DOI:10.1002/chem.201300339

FULL PAPER
DOI: 10.1002/chem.201300339
Controlled Click-Assembly of Well-Defined Hetero-Bifunctional Cubic
Silsesquioxanes and Their Application in Targeted Bioimaging
M. Eugenia Pꢀrez-Ojeda,[b] Beatriz Trastoy,[a, c] ꢁlvaro Rol,[a] Marꢂa D. Chiara,[d]
Inmaculada Garcꢂa-Moreno,[b] and Jose Luis Chiara*[a]
Abstract: A general procedure for the
assembly of hetero-bifunctional cubic
silsesquioxanes with diverse functional-
ity and a perfectly controlled distribu-
tion of functional groups on the inor-
ganic framework has been developed.
The method is based on a two-step se-
quence of mono- and hepta-functional-
ization through the ligand-accelerated
copper(I)-catalyzed azide–alkyne cyclo-
addition of a readily available octa-
mine the selectivity of the key mono-
functionalization reaction when
rescent multivalent probe for applica-
tion in targeted cell-imaging. The inor-
ganic cage provides an improved pho-
tostability to the covalently attached
dye as well as a convenient framework
for the 3D multivalent display of the
pendant epitopes. Thus, fluorescent
a
copper catalyst with strong donor li-
gands is used. The methodology has
been applied to the preparation of a
set of bifunctional nano-building-
blocks with orthogonal reactivity for
the controlled assembly of precisely de-
fined hybrid nanomaterials and a fluo-
bioACTHNUTRGNEpUNG robes based on well-defined cubic
silsesquioxanes offer interesting advan-
tages over more conventional fully or-
ganic analogues and ill-defined hybrid
nanoparticles and promise to become
powerful tools for the study of cell bi-
ology and for biomedical applications.
Keywords: bioimaging
· carbohy-
ACHTUNGTRENNUNGazido cubic silsesquioxane. The stoichi-
ometry of the reactants and the law of
binomial distribution essentially deter-
drates · click chemistry · hybrid
materials · silsesquioxanes
Introduction
(POSS)[2] have emerged as an increasingly important group
of 3D nano-building-blocks for the preparation of a variety
of hybrid functional-materials.[3] POSS are cage-like mole-
cules with a core-shell composition consisting of a siloxane
inorganic scaffold decorated with organic substituents at the
vertices. Due to their rigid inorganic core, these unique
nanometer-sized hybrid molecules have superior mechanical
and thermal stabilities that are partially transmitted to their
derived materials. Applications in areas as diverse as poly-
mers, composite materials, dendrimers, optical materials,
coatings, liquid crystals, metal catalysts, drug carriers, and
tissue engineering have been described, particularly in the
patent literature.[3,4] POSS are usually synthesized by hydro-
lytic condensation of organosilicon monomers RSiX3 (R=
organic group; X=halogen or alkoxide group) and can be
readily modified both on the inorganic cage (e.g., T8, T10,
T12, and so on)[5] and the peripheral organic functionality
(mono-, multi-, homo- or heterofunctionalized).
The “bottom-up” modular construction of 1D, 2D, and 3D
nanomaterials with molecular precision at the nanometer
scale is of paramount interest for the fine-tuned control of
their macroscopic properties. For this endeavor, it is essen-
tial to have a ready and scalable access to a set of structural-
ly well-defined, homogenous nano-building-blocks with di-
verse functionality as well as a set of efficient chemical reac-
tions (ideally “click”-type)[1] for their functionalization.
Within the last two decades, polyhedral oligosilsesquioxanes
[a] Dr. B. Trastoy, ꢀ. Rol, Dr. J. L. Chiara
Instituto de Quꢁmica Orgꢂnica General, IQOG-CSIC
Juan de la Cierva 3, 28006 Madrid (Spain)
E-mail: jl.chiara@csic.es
[b] M. E. Pꢃrez-Ojeda, Prof. Dr. I. Garcꢁa-Moreno
Instituto de Quꢁmica-Fꢁsica “Rocasolano”
The most promising POSS monomers are the highly sym-
metrical and topologically ideal cube-octameric frameworks
(T8), with the general formula type (RSiO1.5)8 and a cage
size of approximately 0.5–0.7 nm.[2b] Most known cubic
POSS are homo-octafunctionalized. Among these, octa-
IQFR-CSIC, Serrano 119, 28006 Madrid (Spain)
[c] Dr. B. Trastoy
Present address: Institute of Human Virology
University of Maryland. School of Medicine
725 West Lombard Street, Baltimore, Maryland 21201 (USA)
AHCTUNGTRENNUNG
[d] Dr. M. D. Chiara
AHCTUNGTRENNUNG
Othorhinolaryngology Unit
AHCTUNGTRENNUNG
Institute of Oncology of Principado de Asturias
Hospital Central de Asturias, Celestino Villamil s/n
33006 Oviedo (Spain)
AHCTUNGTRENNUNG
silsesquioxane (4),[5c,9] and octakis(3-aminopropyl)octasilses-
quioxane (5)[10] (Scheme 1) have been the most useful pre-
cursors to a variety of different T8 monomers and derived
materials. However, the derivatization of the POSS cage
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300339.
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with mixed sets of functional groups can provide still greater
diversity, widely expanding their applications for polymeri-
zation,[3c,e] grafting,[3c] surface bonding,[3c] bioconjugation,[4c]
and other useful transformations. POSS containing an as-
sortment of two different organic groups (hetero-bifunction-
al POSS)[11] are the simplest class among these multifunc-
tional derivatives and possibly the most useful, but those al-
lowing independent derivatization of both functionalities
have scarcely been reported. For example, the functionaliza-
tion of the POSS cage with both a biorecognition element
(e.g., a pepACTHNUTRGNEUGtN idic, nucleic acid, or carbohydrate epitope) and
an “active” molecule, such as a fluorescent dye, a magnetic
resonance imaging probe, a drug or other functional mole-
cules, is of great interest for potential applications in bio-
AHCTUNGTREGiNNUN maging, vectorized drug-transport, or for the study of bio-
logical receptors.[4,12] The preparation of Janus-type POSS
particles is also a very promising application that is being ac-
tively pursued.[13,14] Five different strategies have been de-
scribed in the literature for the preparation of hetero-bifunc-
tional POSS: 1) direct co-hydrolytic condensation of mix-
tures of different organosilicon monomers;[5d,15] 2) nucleo-
phile-induced cage reshuffling of mixtures of different pre-
formed POSS;[16] 3) cross-condensation of two cyclic
tetrasiloxane tetraols (“half-cube” silsesquioxanes);[13b,17]
4) corner-capping of incompletely condensed silsesqui-
AHCTUNGTRENNUNG
oxanes;[12c,18,38] and 5) partial functionalization of homo-octa-
19]
functional POSS.[6d,9f,
However, a majority of examples
prepared by these routes contain simple alkyl or aryl groups
as one of the two functionalities and, hence, are not readily
amenable to further derivatization without compromising
the integrity of the inorganic cage and/or the accompanying
organic functionality, which considerably limits their poten-
tial applications. This is generally the case for the corner-
capping route. In addition, only with the exception of this
route and some peculiar cases of mono-[19c] and di-functiona-
lized[19e,20] POSS, statistical mixtures of multi-functionalized
cages and their various structural (and stereo-) isomers
(Figure 1) are usually formed, which are difficult to sepa-
rate.[15a,b,19h,n] Thus, the selective synthesis of bifunctional
POSS with well-defined substitution patterns is still a com-
plex and largely unsolved challenge, in spite of recent
progress.
Our group has previously described the synthesis of octa-
kis(3-azidopropyl)octasilsesquioxane (6, Scheme 1)[21] from
readily available 5 by a diazo-transfer reaction[22] and its
highly efficient octafunctionalization through copper(I)-cat-
alyzed azide–alkyne 1,3-dipolar cycloaddition[23] (CuAAC, a
paradigmatic example of “click” reaction[1]) with a variety
of terminal alkynes.[21,24–26] Following this approach, several
new octaglycosyl-POSS conjugates were readily assembled
and their multivalent interaction with a complementary pro-
tein receptor, the model plant-lectin ConA, was thoroughly
studied by using an array of biophysical techniques.[26]
Armed with this simple and efficient methodology for the
preparation of diverse homo-octafunctionalized POSS deriv-
atives, we now describe that 6 is also an optimum scaffold
for the controlled synthesis of structurally well-defined
Scheme 1. Readily available homo-octafunctionalized POSS (1–6) used
for the preparation of diverse hybrid materials and bifunctional POSS
derivatives (7–16) prepared from 6 in this work.
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Hetero-Bifunctional Cubic Silsesquioxanes
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6
containing a single 4,4-di-
fluoro-4-bora-3a,4a-diaza-s-in-
dacene (BODIPY) chromo-
phoric substituent (compound
7).[24] We now disclose in detail
the synthetic methodology and
optimization studies that led to
the hybrid dye 7 and the exten-
sion of the new methodology to
the preparation of other bifunc-
tional POSS (8–17, Scheme 1).
As will be shown below, both
the stoichiometry of the initial
CuAAC monofunctionalization
reaction as well as the nature of
the employed copper(I) catalyst
need to be carefully adjusted
Figure 1. All possible POSS products and their different structural isomers (and stereoisomers) that can be
generated in the reaction of a homo-octafunctional cubic POSS with a monofunctional reagent.
hetero-bifunctional POSS derivatives equipped with orthog-
onally reactive groups as versatile building blocks for the
controlled assembly of diverse hybrid functional-materials.
To this end, we have followed a simple two-step strategy
(Scheme 2), which involves the highly selective monofunc-
tionalization of 6 by a controlled CuAAC reaction with a
for optimum results. As a proof of concept, we have used
this strategy for the preparation of a set of well-defined
hetero-bifunctional POSS with a variety of orthogonally re-
active functional groups as well as a POSS derivative that
combines a cluster of sugar epitopes and a fluorescent dye,
which has been shown to be a useful probe for bioimaging
of cell surface receptors.
Results and Discussion
Synthetic methodology: Octaazide 6 is an ideal starting scaf-
fold for the preparation of bifunctional POSS derivatives
since the irreversible character, wide substrate-scope and
outstanding efficiency of the CuACC reaction can enable
the fine-tuned control of the extent of the derivatization
with a diversity of functional terminal alkynes. However,
with eight reactive and symmetry-equivalent vertex groups
on cubic 6, the formation of statistical mixtures of polyfunc-
tionalized products (and their isomers; see Figure 1) is un-
avoidable under usual homogeneous reaction conditions. As
for other cases of monoderivatization of a homo-multifunc-
tional substrate, polysubstitution can in principle be mini-
mized by a judicious selection of the appropriate stoichiom-
etry of the reactants, depending on the number of equiva-
lent reactive groups on the substrate.[27] Surprisingly, there is
no consensus in the literature as to what stoichiometry
should be preferably employed for the selective monoderiv-
Scheme 2. Sequential click strategy for the preparation of structurally
well-defined bifunctional POSS.
terminal functional alkyne followed by the subsequent
CuAAC of the remaining seven azide groups with a second
functional alkyne. This strategy, based on two sequential
click reactions, is a powerful method for attaching virtually
any pair of mutually compatible functionalities to the POSS
framework. However, due to the octafunctional character
and cubic symmetry of 6, the key monofunctionalization
step requires a careful optimization of the reaction condi-
tions to avoid the formation of complex statistical mixtures
of polytriazolyl-POSS derivatives (Figure 1) that would com-
plicate the purification of the required monotriazolyl-POSS
product. We have recently reported on the interesting pho-
tophysical and laser properties of a fluorescent derivative of
AHCTUNGTREGaNNNU tization of a homo-octafunctional POSS, with reported re-
actant/POSS molar-ratios varying widely from 1:1 to 1:8. If
one assumes that all functional groups on the starting octa-
functional POSS as well as on its resultant partially substi-
tuted derivatives have all the same reactivity independently
of the extent of substitution,[28] a statistical mixture of prod-
ucts will be formed that will ideally follow a binomial distri-
bution.[27] Table 1 shows the theoretical binomial distribution
of all possible products that can be obtained in the derivati-
zation reaction of an octafunctional POSS (or any other
molecule with 8 equivalent reactive groups) with a mono-
functional reagent, ranging from unreacted (unsubstituted)
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J. L. Chiara et al.
Table 1. Theoretical yields[a] of substituted POSS products formed in the
reaction of a homo-octafunctional POSS with a monofunctional reagent
(X) as a function of the reaction stoichiometry (n), predicted by the law
of binomial distribution.[b]
about 90% selectivity for monosubstitution in 20% yield
with respect to total starting POSS, with approximately
20% of derivatization reagent lost in producing polysubsti-
tuted derivatives, which could be a balanced compromise in
the case of inexpensive reagents. One can also conclude
from the analysis of Table 1 that no other partially substitut-
ed POSS derivative would be selectively accessible by
simply modifying the stoichiometry of the functionalization
reaction, since complex statistical mixtures of products will
be always obtained for 1ꢀn<8 (Table 1, entries 5–10). Of
course, this corollary does not necessarily hold true for the
case of sterically hindered or polyfunctional derivatization
reagents.
Although the CuAAC monofunctionalization of 6 could
in principle be achieved by simply performing the reaction
with a large excess of POSS over alkyne, as explained
above, it is known that attempted CuAAC monofunctionali-
zation of some polyazides can show unusual product distri-
butions that deviate significantly from those expected on a
purely statistical basis. Thus, 1,2- and conformationally con-
strained 1,3-diazides preferentially afford bis-triazoles at the
expense of the expected monotriazole, even when an excess
of diazide with respect to alkyne is employed.[31] This unusu-
al result has been tentatively ascribed to direct participation
of the initial Cu-triazolide intermediate in the subsequent
click reaction of a vicinal-azide group.[31] Whereas TBTA
(Scheme 3), a widely used accelerating copper(I)-ligand, did
Number of vertex substituents on POSS that have reacted
n
0[c]
1
2
3
4
5
6
7
8
0.1
90.43 95.65
4.24
0.11
0.65
2.46
8.55
0.00
0.03
0.20
1.53
9.61
0.00
0.00
0.01
0.17
2.56
0.00
0.00
0.00
0.01
0.43
3.11
0.00
0.00
0.00
0.00
0.04
0.53
3.14
0.00
0.00
0.00
0.00
0.00
0.04
0.39
2.33
10.01
34.36
0.25 77.57 89.25 10.08
0.5
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
59.67 78.92 18.41
34.36 59.83 29.91
10.01 29.67 34.61 23.07
2.33 11.44 24.03 28.83 21.63 10.38
0.39
0.04
0.00
0.00
0.00
3.14 10.98 21.96 27.45 21.96 10.98
0.52
0.04
0.00
0.00
3.04 10.14 21.13 28.17 23.48 11.18
0.38
0.01
0.00
2.31
0.11
0.00
8.65 20.76 31.15 26.70
1.00
0.00
5.61 19.63 39.27
0.00 0.00
0.00 100.00
[a] Calculated with respect to the limiting reagent. [b] For the statistical
calculations, we have assumed that all functional groups on the different
partially functionalized POSS derivatives have the same reactivity, inde-
pendently of the extent of substitution. [c] Yield of unreacted POSS rela-
tive to total starting POSS.
starting substrate to fully (octa-) substituted product, as a
function of the stoichiometry of the reaction. For an equi-
molar reaction mixture of POSS and derivatization reagent
(n=1, Table 1, entry 4), the maximum yield of monosubsti-
tuted POSS theoretically expected is only 39.27%, accompa-
nied by 34.36% unreacted POSS, and with the remaining
substrate (26.37%) yielding a mixture of polysubstituted
products (mainly di- and tri-substituted derivatives).[29] Thus,
under these conditions, up to 60.73% of the derivatization
reagent will be wasted in the production of undesired poly-
substituted POSS cages, which would be an untenable situa-
tion for the introduction of expensive and highly elaborated
substituents and/or for valuable POSS substrates. Recalcu-
lating the yields with respect to the total reacted POSS (i.e.,
subtracting the recovered starting material, see Table 1), the
mixture of substituted products obtained in this case will
consist mainly of 59.83% monosubstituted, 29.91% disubsti-
tuted, and 8.55% trisubstituted POSS, with negligible
amounts (<2%) of higher-substituted derivatives. Lowering
the reagent/POSS stoichiometry n (Table 1, entries 1–3) will
improve the selectivity of monosubstitution at the expense
of reducing overall POSS conversion. However, this is a
simple and very effective strategy for the selective prepara-
tion of monosubstituted POSS compounds provided that
excess unreacted POSS could be readily recovered from the
reaction mixture and recycled.[30] Thus, by using a 1:10 re-
agent/POSS molar ratio (n=0.1; Table 1, entry 1) will pro-
duce the monosubstituted compound with very high
(95.65%) selectivity with only 8.4% of derivatization re-
agent lost in producing higher substituted POSS derivatives,
although with a low 9.16% yield of monosubstituted cage
calculated with respect to total starting POSS. A 1:4 re-
agent/POSS molar ratio (n=0.25; Table 1, entry 2) gives
Scheme 3. Copper catalysts, ligands, and model (fluorescent) alkyne used
in the optimization studies of the CuAAC monofunctionalization of
octaazide 6.
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Hetero-Bifunctional Cubic Silsesquioxanes
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Table 2. Optimization of the CuAAC monofunctionalization of octaazide POSS 6 with fluorescent alkyne 7a
to afford bifunctional POSS 7.[a]
not have a significant effect on
the outcome of diazide reac-
tions, Finn and co-workers have
described that some (benz-
Entry Catalyst
Base
Solvent (v/v ratio)
T
t
Yield
[8C][b]
[h] [%][c]
1
2
3
4
5
CuSO4·5H2O, sodium ascorbate
[CuTC]
[CuCl(IPr)]
CuSO4·5H2O, sodium ascorbate, (BimC4A)3
[Cu(C186tren)]Br
–
CH2Cl2/H2O (1:1)
25
20
2
9
ACHTUNGTRENNUNG
G
iPr2NEt THF
iPr2NEt PhMe
–
80 (MW)
80 (MW) 11
25
25
57
48
82
ACHTUNGTRENNUNG
E
N
gave the monotriazole as the
major product in the expected
2:1 mono-/bis-triazole statistical
ratio in one example of confor-
mationally rigid 1,3-diazide
using a 1:1 diazide/alkyne reac-
tion stoichiometry.[31b]
THF/H2O (5:1)
15
6
N
iPr2NEt PhMe
80 (MW)
[a] A molar ratio 6/7a=10:1 was used in all cases. [b] MW indicates that the reaction was carried out under
microwave heating. [c] Yields of the isolated product (column chromatography).
For the initial optimization
study of the CuAAC mono-
functionalization of octaazide 6,
we selected the fluorescent
BODIPY dye 7a (Scheme 3) as
a model alkyne, which we have
used previously for the prepara-
tion of an octa-BODIPY-POSS
cluster.[21] The reaction could be
easily followed by TLC by
simple naked eye inspection,
and affords a POSS derivative 7
(Scheme 1) labeled with
a
single fluorescent probe and
seven additional reactive azide
groups ready for further func-
tionalization with any molecule
of interest having a terminal al-
kynyl group. In addition, as we
have already shown, compound
7 is an interesting hybrid dye
for the preparation of solid-
state laser materials with im-
proved thermal and photo-
chemical stability.[24] Based on
the previous statistical calcula-
tions, we selected a rather con-
servative tenfold molar excess
of 6 over alkyne 7a to guaran-
tee a very high statistical selec-
tivity for the monofunctional-
ized
product
7
(Table 1,
entry 1). However, under the
previously optimized condi-
tions,[21] using CuSO4/sodium
ascorbate as catalyst in a bipha-
sic CH2Cl2/H2O (1:1)[32] solvent
mixture, a very low yield (9%)
of monotriazolyl-POSS 7 was
obtained together with unreacted 6 and a complex mixture
of uncharacterized polytriazolyl-POSS products (Table 2,
entry 1). Thus, in the absence of added copper ligands, oc-
taazide 6 showed the same strong preference for the forma-
tion of polytriazolyl products previously observed by Finn
Figure 2. a) 1H (400 MHz, CDCl3) and b) 29Si NMR (79.5 MHz, CDCl3) spectra of compound 7 showing partial
peak assignments. The numbers under the spectral line in (a) show relative integral regions for the correspond-
ing peaks.
and co-workers[31] for apparently more conformationally
constrained polyazides. By using the same 6/7a=10:1 molar
ratio as above, we screened different copper catalysts
(Scheme 3) and reaction conditions (e.g., aqueous or anhy-
drous solvents, with or without added base, at room temper-
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J. L. Chiara et al.
ature or under microwave heat-
ing) with the objective of mini-
mizing the formation of poly-
triazolyl-POSS products to
obtain 7 in the expected statisti-
cal yield. Copper(I) thiophene-
2-carboxylate
(ACTHNGUTER[NNUG CuTC],
Scheme 3), which has been re-
cently used in CuAAC reac-
tions of sulfonyl azides,[33] gave
a
slightly better yield of 7
(25%) after heating for two
hours under microwave irradia-
tion in THF (Table 2, entry 2),
but this outcome was still far
from the expected statistical
value. Better results were ob-
tained with a copper(I) catalyst
bearing an N-heterocyclic car-
bene[34] ligand
(ACHTUNGTRENNUNG[(IPr)CuCl],
Scheme 3) or with Finnꢅs tetra-
dentate tripodal amino ligand
[35]
G
(Scheme 3;
Table 2, entries 3 and 4, respec-
Scheme 4. Simplified mechanistic rationale for the observed ligand effect on the selectivity of the CuAAC
monofuntionalization of octaazide 6.
tively). The tetramine [Cu-
A
catalyst[36]
base in toluene as solvent and under microwave heating
gave the highest yield of 7 (82%; Table 2, entry 5), close to
the expected statistical value (95.65%). In all cases, the rest
of alkyne 7a was transformed into a complex mixture of
1
polytriazolyl POSS products, as shown by H NMR analysis
of the remaining mixed fractions from column chromatogra-
phy. The structure of 7 was unambiguously confirmed by
high-resolution mass spectrometry and multinuclear (1H,
13C, 29Si) NMR spectroscopy (see Figure 2 and the Support-
ing Information). The degree of substitution can be readily
recognized by the 7ACHTUNGTRENNUNG(4+3):1 and 4:3:1 pattern distribution of
1H (Figure 2a) and 29Si NMR (Figure 2b) signals, respective-
ly, as expected from molecular symmetry considerations.[15a]
The 29Si chemical shifts are within the expected region for
an alkyl-substituted cubic POSS (ca. d=À65 to
À70 ppm).[2b]
An informed speculation to explain the observed ligand
effect on the selectivity of the CuAAC reaction is the fol-
lowing. According to previous mechanistic studies by Finn
and co-workers,[31] the rate-limiting step of the CuAAC reac-
tion apparently changes from the protonolysis of the Cu-tri-
azolide intermediate to the alkyne deprotonation–cycloaddi-
tion sequence upon incorporation of chelating ligands on
the copper catalyst. Therefore, under Sharpless conditions
(no ligand) a build-up of Cu-triazolide A (Scheme 4) is ex-
pected to occur that kinetically favors its coordination to
azide or alkyne (B) and the subsequent intramolecular for-
mation of polytriazolyl products (through D). In contrast,
chelating ligands hamper the further coordination of the
metal in A and accelerate the protonolysis of A to C, divert-
Scheme 5. Synthesis of mono-glycosyl-POSS 14 and 15. A 2:1 (BimC4A)3/
copper molar-ratio was employed.
ing the copper catalyst towards another cycle of alkyne de-
protonation and intermolecular cycloaddition to afford a
statistically controlled mixture of triazole products with a
final composition essentially determined by the initial stoi-
chiometry of the reactants and the law of binomial distribu-
tion (Table 1). The apparent correspondence observed be-
tween the selectivity of the monofunctionalization reaction
and the donor strength of the copper ligand, which approxi-
mately correlates with its proton affinity, is in agreement
with this mechanistic view.[37]
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Table 3. Synthesis of hetero-bifunctional POSS 8–13 with orthogonal re-
activity by selective CuAAC monofunctionalization of octaazide 6.
Entry
R, POSS product
t
Isolated product
yield [%][a]
[h]
1
2
8
9
3
2
51 (87)
74 (99)
Scheme 6. Possible applications of complementary reactive mono-/hepta-
functionalized POSS for nanoconstruction of dumbbell-shaped POSS
dyads and complex 3D POSS assemblies.
3
10
2
87 (89)
4
5
11
12
2
2
64 (96)
83 (94)
A similar catalyst-effect on selectivity was observed for
the CuAAC reaction of 6 with other alkynes (Scheme 5).
Thus, the acetal-protected a-d-mannose glycoside 14a gave
also a low yield (10%) of monosubstituted POSS 14 under
Sharpless conditions by using a biphasic aqueous solvent
mixture and a tenfold molar excess of 6, but the yield im-
6
13
3
68 (85)
proved to 79% by using the [CuACHTNUGTRENUNG(C186tren)]Br catalyst
under the optimized reaction conditions with the same stoi-
chiometry. However, these optimized conditions are not ap-
propriate in the case of water-soluble alkynes such as 15a,
which is not readily soluble in apolar organic solvents. In
[a] Recovery yield of excess 6 is shown in parenthesis.
ester (12) or amide bond formation (11, 12), amine depro-
tection followed by diazo-transfer and subsequent CuAAC
reaction (11), or Diels–Alder cycloaddition (13) could pro-
vide access to a large variety of new hetero-bifunctional
POSS. In addition, the obtained bifunctional POSS nano-
building-blocks could be readily homo- or hetero-dimerized
to afford, respectively, symmetric or asymmetric dumbbell-
shaped POSS dyads,[13c,38] or they could be assembled with
this case, addition of the water-soluble ligand ACTHNUGTRNEUNG(BimC4A)3 (in
a 2:1 ligand/copper molar ratio) to a biphasic CH2Cl2/H2O
solvent reaction mixture containing a catalytic amount of
CuSO4·5H2O and an excess of sodium ascorbate afforded
the monosubstituted POSS 15 in a short reaction time and
in a reasonably good yield.
To study the scope and versatility of the method, we syn-
thesized a set of hetero-bifunctional POSS 8–13 with orthog-
onal reactivity by using a variety of alkynes functionalized
with alkene (8a and 9a), halobenzene (10a), protected
amine (11a), protected carboxylic acid (12a), or anthracene
(13a) end-groups (Table 3). The monotriazolyl-POSS prod-
ucts were obtained in good yield and with excellent selectiv-
ity by using the optimized conditions. The structures of the
new POSS products were unambiguously confirmed by
high-resolution mass spectrometry and multinuclear (1H,
13C, 29Si) NMR spectroscopy (see the Supporting Informa-
tion). Recovery of excess 6 from the crude reaction mixture
was close to quantitative in all cases (see Table 3). This set
of hetero-bifunctional POSS allows for the selective, step-
wise modification of the cage functionality at either the
azido groups or the other orthogonal functionality for the
preparation of new bifunctional hybrid materials with well-
defined structures. For example, the combination (in the ap-
propriate order) of CuAAC heptafunctionalization with
either alkene homo/cross-metathesis (8, 9), transition-metal-
catalyzed cross-coupling (8, 9, 10), deprotection followed by
Scheme 7. Synthesis of fluorescently labeled POSS glycoclusters 16 and
17.
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J. L. Chiara et al.
complementarily
reactive
homo-octafunctional-POSS
monomers to produce even
more complex 3D constructs
(Scheme 6).
Confocal microscopy study: As
a proof of concept to demon-
strate the effectiveness of this
synthetic strategy for the prepa-
ration of complex, well-defined
bifunctional materials, we have
prepared a multivalent fluores-
cent probe for application in
targeted cell-imaging. To this
end, we proceeded to react the
seven azide groups of mono-
BODIPY-POSS 7 with a termi-
nal alkyne functionalized with
a
carbohydrate
epitope
(Scheme 7). The acetal-protect-
ed a-d-mannose derivative 16a,
equipped with a propargylated
tetraethyleneglycol connector,
was selected for this task. The
hepta-click functionalization of
7
proceeded efficiently to
afford the isolated 16 in 75%
yield (=96% yield per azido
group) by using the same opti-
mized reaction conditions pre-
viously described by our group
for the octafunctionalization of
6.[21,26] Removal of the protect-
ing acetal groups under mildly
acidic conditions[21,26] produced
the fluorescently labeled glyco-
POSS cluster 17 in quantitative
yield. The structures of 16 and
17 were unambiguously con-
firmed by MALDI-TOF and
multi-nuclear (1H, 13C, 29Si)
Figure 3. a) 1H (400 MHz, CDCl3) and b) 29Si NMR (79.5 MHz, CDCl3) spectra of compound 16 showing parti-
al peak assignments. The numbers under the spectral line in the left inset of (a) show relative integral regions
for the triazole protons.
NMR
spectroscopy
(see
Figure 3 and the Supporting Information). As in the case of
compound 7 (Figure 2), the degree of substitution could be
readily recognized by the 4:3:1 pattern distribution of 1H
(Figure 3a) and 29Si NMR (Figure 3b) signals, as expected
from molecular symmetry considerations.[15a] The 29Si chemi-
cal shifts are, again, within the expected region for an alkyl-
substituted cubic POSS.[2b] As expected, the dispersion of
29Si chemical shifts in these octatriazolyl-POSS derivatives
ACHTUNGTRENNUNG(16, 17) are narrower than those of the monotriazolyl-
heptaazido-POSS derivatives 7–15.
Water-soluble glyco-POSS 17 was assayed as a fluorescent
probe for the imaging of C-type lectin receptors (CLRs)[39]
present on the surface of antigen-presenting cells by using
fluorescence microscopy. An important group of CLRs rec-
ognize oligosaccharides containing mannose and/or fucose,
including the mannose receptor (MR)[40] and DC-SIGN
(dendritic cell-specific intercellular adhesion molecule-3-
grabbing nonintegrin, also called CD209)[41] on dendritic
cells (DC). DC-SIGN in particular has attracted much inter-
est since its discovery in 2000,[42] because it binds to a large
range of clinically relevant pathogens, including HIV, Ebola
virus, Candida albicans and Leishmania, among others, facil-
itating their uptake for subsequent antigen presentation.[41]
Both, MR and DC-SIGN bind to glycan patterns present on
the surface of pathogens through multivalent carbohydrate–
protein interactions. With its seven mannose epitopes, glyco-
POSS 17 is properly equipped to sustain a multivalent bind-
ing to MR and DC-SIGN. In addition, the covalent attach-
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Hetero-Bifunctional Cubic Silsesquioxanes
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Figure 4. Live confocal microscopy analysis of the interaction of glyco-POSS 16 (panel A) and 17 (panels B and C) with stable K562 transfectant cells ex-
pressing DC-SIGN (K562-CD209). The cells were incubated with the glyco-POSS in phosphate-buffered saline for 10 min at 48C. The cell nuclei were
subsequently stained with 4’,6-diamidino-2-phenylindole. Insets show large images for the indicated cell. Results are representatives of multiple cells in
three independent experiments. Scale bar=10 mm.
ment of the BODIPY chromophore to the inorganic cage of
POSS confers an improved photostability to the multivalent
construct as we have described recently,[24,43] which is partic-
ularly important for the new imaging techniques developed
in optical microscopy that require high-laser-intensity irradi-
ation.
We have used confocal laser-scanning microscopy to visu-
alize the binding interaction of fluorescent glyco-POSS clus-
ter 17 with cell surface receptors of human K562 cells stably
transfected with DC-SIGN (K562-CD209),[44] which express
a high level of endogenous DC-SIGN (Figure 4). Analogue
16, which has all its key sugar hydroxyls blocked with acetal
protecting groups and should therefore be unable to interact
specifically with the sugar receptors, was used as control.
The cells were incubated with the probe for a short time
(10 min) and at low temperature (48C) to minimize the re-
The key initial monofunctionalization of 6 required a careful
optimization of the reaction conditions to minimize forma-
tion of complex mixtures of polysubstituted POSS products.
The selectivity of this reaction is not only dependent on the
stoichiometry of the reactants, but also on the donor ability
of the ligands on the copper catalyst. A simple statistical cal-
culation proved to be essential for an informed decision on
the appropriate reaction stoichiometry required for the se-
lective monofunctionalization of homo-octafunctional POSS
molecules. A screening of copper catalysts showed that [Cu-
AHCTUNGRTEG(NNUN C186tren)]Br provided the reaction outcome closest to the
expected statistically. This catalyst effect on selectivity can
be ascribed to the ligand-accelerated protonolysis of the
copper-triazolide intermediate of the CuAAC reaction in
combination with the hampering of further coordination of
the metal in this intermediate to alkyne or azide. The pres-
ence of a tethered carboxylic acid on the ligand does not
seem to be really required for this accelerated hydrolysis.[31b]
The wide substrate scope and high efficiency of the click
functionalization allow for the easy preparation of a large
variety of unprecedented hetero-bifunctional POSS with a
perfectly controlled distribution of functional groups on the
cubic framework. To demonstrate the versatility of this
methodology, we have prepared a set of bifunctional POSS
with orthogonal reactivity, which can be used as nano-build-
ing-blocks for the controlled assembly of hybrid nanomateri-
als, and a multivalent fluorescent probe for application in
targeted cell-imaging. The inorganic cage of POSS provides
an improved photostability to the covalently attached
dye,[24,43] as well as a convenient framework for the 3D mul-
tivalent display[26] of the pendant epitopes. In addition, the
recently observed gradual disassembly of similar water-solu-
ble octasilsesquioxanes under physiological conditions[26]
and the expected low toxicity of the resulting monomeric or-
ganosilanes suggest that these hybrid multivalent constructs
would be very attractive systems for in vivo applications.
Accordingly, fluorescent bioprobes based on well-defined
ceptor-mediated endoACTHNUGRTENUNGcytosis of the glyco-POSS. The slow
off-rate of 17 from the receptors, as expected for a multiva-
lent binding interaction, allowed imaging (at room tempera-
ture) to take place following washout of the unbound fluo-
rescent probe. Live confocal images revealed the strong
fluoACHTUNGTRENNUNGrescence of 17 located mainly at the cell membrane (Fig-
ure 4b and c), although a minor internalization of the probe
into the cytoplasm was unavoidable in spite of the precau-
tions taken to avoid the receptor-mediated endocytosis. In
comparison, only a very faint and diffuse (nonspecific) stain-
ing was observed for the control compound 16 (Figure 4a).
Conclusion
We have shown that readily available octakis(3-azido-
ACHTUNGTRENNUNGpropyl)octasilsesquioxane (6) is an ideal starting material
for the controlled assembly of well-defined hetero-bifunc-
tional POSS. Successive grafting of two different groups on
the POSS core has been efficiently achieved by using two
sequential ligand-accelerated CuAAC functionalizations.
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J. L. Chiara et al.
Sulaiman, A. Bhaskar, J. Zhang, R. Guda, T. Goodson, III, R. M.
Laine, Chem. Mater. 2008, 20, 5563–5573; i) P. Zak, B. Marciniec,
M. Majchrzak, C. Pietraszuk, J. Organomet. Chem. 2011, 696, 887–
891.
POSS materials offer interesting advantages over more con-
ventional, fully organic analogues and ill-defined hybrid
nanoparticles and promise to become powerful tools for dis-
secting cellular interactions at the molecular level and for
biomedical applications. We foresee that the described syn-
thetic methodology can be easily transferred to similar
“click”-type functionalization reactions, such as the highly
efficient radical-mediated thiol-ene/yne reaction,[45,46] widely
expanding the potential applications of this strategy in
POSS chemistry.
[8] a) K. Olsson, Ark. Kemi 1958, 13, 367–378; b) A. R. Bassindale, Z.
Liu, I. A. MacKinnon, P. G. Taylor, Y. Yang, M. E. Light, P. N.
Horton, M. B. Hursthouse, Dalton Trans. 2003, 2945–2949; c) M.
Kozelj, B. Orel, Dalton Trans. 2008, 5072–5075.
[9] a) U. Dittmar, B. J. Hendan, U. Floerke, H. C. Marsmann, J. Orga-
nomet. Chem. 1995, 489, 185–194; b) S. Lucke, K. Stoppek-Langner,
B. Krebs, M. Lage, Z. Anorg. Allg. Chem. 1997, 623, 1243–1246;
c) A. Gultek, T. Seckin, H. I. Adiguzel, Turk. J. Chem. 2005, 29,
391–399; d) Y. Liu, X. Yang, W. Zhang, S. Zheng, Polymer 2006, 47,
6814–6825; e) B. Marciniec, M. Dutkiewicz, H. Maciejewski, M. Ku-
bicki, Organometallics 2008, 27, 793–794; f) S. Fabritz, D. Heyl, V.
Bagutski, M. Empting, E. Rikowski, H. Frauendorf, I. Balog, W.-D.
Fessner, J. J. Schneider, O. Avrutina, H. Kolmar, Org. Biomol.
Chem. 2010, 8, 2212–2218; g) D. Heyl, E. Rikowski, R. C. Hoff-
mann, J. J. Schneider, W.-D. Fessner, Chem. Eur. J. 2010, 16, 5509.
[10] a) M.-C. Gravel, R. M. Laine, Polym. Prepr. Am. Chem. Soc. Div.
Polym. Chem. 1997, 38, 155–156; b) F. J. Feher, K. D. Wyndham,
Chem. Commun. 1998, 323–324; c) F. J. Feher, K. D. Wyndham,
M. A. Scialdone, Chem. Commun. 1998, 1469–1470; d) F. J. Feher,
K. D. Wyndham, D. Soulivong, F. Nguyen, J. Chem. Soc. Dalton
Trans. 1999, 1491–1498.
Acknowledgements
This work was supported by the Spanish Ministerio de Ciencia e Innova-
ciꢇn (projects MAT2010-20646-C04-01 and MAT2010-20646-C04-03), the
European Social Fund and Comunidad de Madrid (project S2009/PPQ-
1634 “AVANCAT”), Fondo de Investigaciꢇn Sanitaria (FIS PI11/929),
and Obra Social Cajastur-IUOPA. The Ministerio de Ciencia e Innova-
ciꢇn is gratefully thanked for a FPU predoctoral contract to B.T. and
CSIC for a JAEPRE contract to M.E.P.O. We also thank Prof. Angel
Costela for a thoughtful critical review of the manuscript.
[11] C. Marcolli, G. Calzaferri, Appl. Organomet. Chem. 1999, 13, 213–
226, 3457.
[12] a) H. Ghanbari, B. G. Cousins, A. M. Seifalian, Macromol. Rapid
Commun. 2011, 32, 1032–1046; b) K.-Y. Pu, B. Liu, Adv. Funct.
Mater. 2011, 21, 3408–3423; c) F. Olivero, F. Reno, F. Carniato, M.
Rizzi, M. Cannas, L. Marchese, Dalton Trans. 2012, 41, 7467–7473.
[13] a) R. M. Laine, M. Roll, M. Asuncion, S. Sulaiman, V. Popova, D.
Bartz, D. J. Krug, P. H. Mutin, J. Sol-Gel Sci. Technol. 2008, 46, 335–
347; b) M. Z. Asuncion, M. Ronchi, H. Abu-Seir, R. M. Laine, C. R.
Chim. 2010, 13, 270–281; c) Y. Li, W.-B. Zhang, I. F. Hsieh, G.
Zhang, Y. Cao, X. Li, C. Wesdemiotis, B. Lotz, H. Xiong, S. Z. D.
Cheng, J. Am. Chem. Soc. 2011, 133, 10712–10715.
[14] Some Janus-type cubic silsesquioxanes are now commercially availa-
ble from Mayaterials, Inc (http://www.mayaterials.com).
[15] a) B. J. Hendan, H. C. Marsmann, J. Organomet. Chem. 1994, 483,
33–38; b) S. Kraus-Ophir, I. Jerman, B. Orel, D. Mandler, Soft
Matter 2011, 7, 8862–8869; c) C. Chen, S. Huang, M. Chen, Q. Lu,
High Perform. Polym. 2012, 24, 119–124.
[1] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113,
2056–2075; Angew. Chem. Int. Ed. 2001, 40, 2004–2021.
[2] a) P. D. Lickiss, F. Rataboul, Adv. Organomet. Chem. 2008, 57, 1–
116; b) D. B. Cordes, P. D. Lickiss, F. Rataboul, Chem. Rev. 2010,
110, 2081–2173.
[3] a) R. M. Laine, C. Zhang, A. Sellinger, L. Viculis, Appl. Organomet.
Chem. 1998, 12, 715–723; b) R. M. Laine, J. Mater. Chem. 2005, 15,
3725–3744; c) S.-W. Kuo, F.-C. Chang, Prog. Polym. Sci. 2011, 36,
1649–1696; d) R. M. Laine, M. F. Roll, Macromolecules 2011, 44,
1073–1109; e) F. Wang, X. Lu, C. He, J. Mater. Chem. 2011, 21,
2775–2782.
[4] a) Applications of Polyhedral Oligomeric Silsesquioxanes, in Advan-
ces in Silicon Science, Vol. 3, (Ed.: C. Hartmann-Thompson), Spring-
er, 2011; b) K. Tanaka, Y. Chujo, J. Mater. Chem. 2012, 22, 1733–
1746; c) S. Fabritz, S. Horner, O. Avrutina, H. Kolmar, Org. Biomol.
Chem. 2013, 11, 2224–2236; d) K. Tanaka, Y. Chujo, Polym. J. 2013,
45, 247–254.
[5] a) E. Rikowski, H. C. Marsmann, Polyhedron 1997, 16, 3357–3361;
b) F. J. Feher, R. Terroba, J. W. Ziller, Chem. Commun. 1999, 2153–
2154; c) V. Ervithayasuporn, X. Wang, Y. Kawakami, Chem.
Commun. 2009, 5130–5132; d) M. Z. Asuncion, R. M. Laine, J. Am.
Chem. Soc. 2010, 132, 3723–3736; e) M. Ronchi, S. Sulaiman, N. R.
Boston, R. M. Laine, Appl. Organomet. Chem. 2009, 24, 551–557;
f) J. C. Furgal, J. H. Jung, T. Mizmuo, K. Chou, M. Schwartz, T.
Goodson, III, R. M. Laine, Polym. Prepr. Am. Chem. Soc. Div.
Polym. Chem. 2012, 53, 15–16; g) R. M. Laine, J. H. Jung, J. C.
Furgal, K. Chou, M. Schwartz, T. Goodson, III, Polym. Prepr. Am.
Chem. Soc. Div. Polym. Chem. 2012, 53, 543.
[6] a) R. Muller, C. Dathe, L. Heinrich, J. Prakt. Chem. 1959, 9, 71–74;
b) C. L. Frye, W. T. Collins, J. Am. Chem. Soc. 1970, 92, 5586–5588;
c) P. A. Agaskar, Inorg. Chem. 1991, 30, 2707–2708; d) G. Calzafer-
ri, D. Herren, R. Imhof, Helv. Chim. Acta 1991, 74, 1278–1280.
[7] a) K. A. Andrianov, N. M. Petrovnina, T. V. Vasileva, V. E. Shklover,
B. I. Dyachenko, Zh. Obshch. Khim. 1978, 48, 2692–2695; b) F. J.
Feher, D. Soulivong, A. G. Eklund, K. D. Wyndham, Chem.
Commun. 1997, 1185–1186; c) P. G. Harrison, C. Hall, Main Group
Met. Chem. 1997, 20, 515–529; d) B. W. Manson, J. J. Morrison, P. I.
Coupar, P.-A. Jaffres, R. E. Morris, J. Chem. Soc. Dalton Trans.
2001, 1123–1127; e) Y. Itami, B. Marciniec, M. Kubicki, Chem. Eur.
J. 2004, 10, 1239–1248; f) E. O. Dare, L.-K. Liu, J. Peng, Dalton
Trans. 2006, 3668–3671; g) G. Cheng, N. R. Vautravers, R. E. Morris,
D. J. Cole-Hamilton, Org. Biomol. Chem. 2008, 6, 4662–4667; h) S.
[16] a) Z. Li, Y. Kawakami, Chem. Lett. 2008, 37, 804–805; b) J. H. Jung,
R. M. Laine, Macromolecules 2011, 44, 7263–7272.
[17] S. Tateyama, Y. Kakihana, Y. Kawakami, J. Organomet. Chem. 2010,
695, 898–902.
[18] a) F. J. Feher, D. A. Newman, J. F. Walzer, J. Am. Chem. Soc. 1989,
111, 1741–1748; b) S. T. Iacono, A. Vij, W. Grabow, D. W. Smith, Jr.,
˙
J. M. Mabry, Chem. Commun. 2007, 4992–4994; c) P. Zak, C. Pie-
traszuk, B. Marciniec, G. Spꢇlnik, W. Danikiewicz, Adv. Synth.
Catal. 2009, 351, 2675–2682; d) Y.-C. Lin, S.-W. Kuo, J. Polym. Sci.
Part A: Polym. Chem. 2011, 49, 2127–2137; e) H. Liu, M. Puchberg-
er, U. Schubert, Chem. Eur. J. 2011, 17, 5019–5023; f) B. M. Moore,
S. M. Ramirez, G. R. Yandek, T. S. Haddad, J. M. Mabry, J. Organo-
met. Chem. 2011, 696, 2676–2680; g) W. Wang, Q. Shen, W. Zha, G.
Zhu, J. Polym. Res. 2011, 18, 1119–1124; h) J. K. Hu, Q. C. Zhang,
S. L. Gong, Chin. Chem. Lett. 2012, 23, 181–184; i) S. C. Kettwich,
S. N. Pierson, A. J. Peloquin, J. M. Mabry, S. T. Iacono, New J.
Chem. 2012, 36, 941–946; j) Y. Zheng, L. Wang, S. Zheng, Eur.
Polym. J. 2012, 48, 945–955.
[19] a) C. Zhang, R. M. Laine, J. Organomet. Chem. 1996, 521, 199–201;
b) A. Tsuchida, C. Bolln, F. G. Sernetz, H. Frey, R. Muelhaupt, Mac-
romolecules 1997, 30, 2818–2824; c) F. J. Feher, K. D. Wyndham,
R. K. Baldwin, D. Soulivong, J. D. Lichtenhan, J. W. Ziller, Chem.
Commun. 1999, 1289–1290; d) R. Knischka, F. Dietsche, R. Hansel-
mann, H. Frey, R. Mꢆlhaupt, P. J. Lutz, Langmuir 1999, 15, 4752–
4756; e) M. A. Said, H. W. Roesky, C. Rennekamp, M. Andruh, H.-
G. Schmidt, M. Noltemeyer, Angew. Chem. 1999, 111, 702–705;
&
10
&
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ꢄ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Angew. Chem. Int. Ed. 1999, 38, 661–664; f) N. Auner, J. W. Bats,
D. E. Katsoulis, M. Suto, R. E. Tecklenburg, G. A. Zank, Chem.
Mater. 2000, 12, 3402–3418; g) A. R. Bassindale, D. J. Parker, P. G.
Taylor, A. C. Watt, Can. J. Chem. 2003, 81, 1341–1349; h) C.
McCusker, J. B. Carroll, V. M. Rotello, Chem. Commun. 2005, 996–
998; i) K. Tanaka, K. Inafuku, Y. Chujo, Bioorg. Med. Chem. 2008,
16, 10029–10033; j) S.-y. Kuwahara, K. Yamamoto, J.-i. Kadokawa,
Chem. Lett. 2010, 39, 1045–1047; k) X.-F. Yu, S. Zhong, X.-P. Li, Y.-
F. Tu, S.-G. Yang, H. R. M. Van, C.-Y. Ni, D. J. Pochan, R. P. Quirk,
C. Wesdemiotis, W.-B. Zhang, S. Z. D. Cheng, J. Am. Chem. Soc.
2010, 132, 16741–16744; l) W.-B. Zhang, Y. Li, X. Li, X. Dong, X.
Yu, C.-L. Wang, C. Wesdemiotis, R. P. Quirk, S. Z. D. Cheng, Macro-
molecules 2011, 44, 2589–2596; m) Y. Li, X.-H. Dong, K. Guo, Z.
Wang, Z. Chen, C. Wesdemiotis, R. P. Quirk, W.-B. Zhang, S. Z. D.
Cheng, ACS Macro Lett. 2012, 1, 834–839; n) H. Lin, X. Wan, X.
Jiang, Q. Wang, J. Yin, J. Mater. Chem. 2012, 22, 2616–2623; o) S.
Hçrner, S. Fabritz, H. D. Herce, O. Avrutina, C. Dietz, R. W. Stark,
M. C. Cardoso, H. Kolmar, Org. Biomol. Chem. 2013, 11, 2258–
2265.
[31] a) V. O. Rodionov, V. V. Fokin, M. G. Finn, Angew. Chem. 2005, 117,
2250–2255; Angew. Chem. Int. Ed. 2005, 44, 2210–2215; b) V. O.
Rodionov, S. I. Presolski, D. Diaz Diaz, V. V. Fokin, M. G. Finn, J.
Am. Chem. Soc. 2007, 129, 12705–12712.
[32] B.-Y. Lee, S. R. Park, H. B. Jeon, K. S. Kim, Tetrahedron Lett. 2006,
47, 5105–5109.
[33] J. Raushel, V. V. Fokin, Org. Lett. 2010, 12, 4952–4955.
[34] a) V. Jurkauskas, J. P. Sadighi, S. L. Buchwald, Org. Lett. 2003, 5,
2417–2420; b) S. Dꢁez-Gonzꢂlez, A. Correa, L. Cavallo, S. P. Nolan,
Chem. Eur. J. 2006, 12, 7558–7564.
[35] V. O. Rodionov, S. I. Presolski, S. Gardinier, Y.-H. Lim, M. G. Finn,
J. Am. Chem. Soc. 2007, 129, 12696–12704.
[36] a) G. Barrꢃ, D. Taton, D. Lastecoueres, J.-M. Vincent, J. Am. Chem.
Soc. 2004, 126, 7764–7765; b) N. Candelon, D. Lastecoueres, A. K.
Diallo, J. Ruiz Aranzaes, D. Astruc, J.-M. Vincent, Chem. Commun.
2008, 741–743.
[37] a) A linear relationship between the donor ability of the ligands (as
determined by their proton affinity) and the activation barrier of the
protonolytic cleavage of the metal–carbon bond has been found by
DFT calculations for some transition-metal complexes, with better
donors decreasing the barrier: H. M. Senn, D. V. Deubel, P. E.
Blçchl, A. Togni, G. Frenking, J. Mol. Struct. 2000, 506, 233–242.
The low donor ability of the TBTA ligand could thus explain its neg-
ligible effect on the outcome of diazide reactions. b) Likewise, the
efficiency of CuTC in catalyzing the formation of 1-sulfonyl-1,2,3-
triazoles over other competitive processes in the CuAAC reaction
of alkynes with sulfonyl azides has been ascribed (see ref. [33]) to its
ability to stabilize the Cu-triazolide intermediate and/or to promote
the protonolytic cleavage of this intermediate, which could also ex-
plain its positive effect on the selectivity of the monofunctionaliza-
tion of POSS polyazides.
[20] E. A. Rebrov, N. A. Tebeneva, A. M. Mouzafarov, Y. E. Ovchinni-
kov, Y. T. Struchkov, T. V. Strelkova, Russian Chem. Bull. 1995, 44,
1286–1292.
[21] B. Trastoy, M. E. Perez-Ojeda, R. Sastre, J. L. Chiara, Chem. Eur. J.
2010, 16, 3833–3841.
[22] J. R. Suꢂrez, B. Trastoy, M. E. Perez-Ojeda, R. Marin-Barrios, J. L.
Chiara, Adv. Synth. Catal. 2010, 352, 2515–2520.
[23] a) C. W. Tornøe, M. Meldal, “Peptidotriazoles: Copper(I)-catalyzed
1,3-dipolar cycloadditions on solid-phase”, Peptides 2001, Proc. Am.
Pept. Symp., American Peptide Society and Kluwer Academic Pub-
lishers, San Diego, 2001, pp. 263- 264; b) C. W. Tornøe, C. Christen-
sen, M. Meldal, J. Org. Chem. 2002, 67, 3057–3064; c) V. V. Rostovt-
sev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. 2002,
114, 2708–2711; Angew. Chem. Int. Ed. 2002, 41, 2596–2599. For a
comprehensive review, see: d) M. Meldal, C. W. Tornøe, Chem. Rev.
2008, 108, 2952–3015.
[38] H. Araki, K. Naka, Macromolecules 2011, 44, 6039–6045.
[39] A. Cambi, M. Koopman, C. G. Figdor, Cell. Microbiol. 2005, 7, 481–
488.
[40] L. East, C. M. Isacke, Biochim. Biophys. Acta Gen. Subj. 2002, 1572,
364–386.
[24] M. E. Pꢃrez-Ojeda, B. Trastoy, I. Lopez-Arbeloa, J. Banuelos, A.
Costela, I. Garcia-Moreno, J. L. Chiara, Chem. Eur. J. 2011, 17,
13258–13268.
[41] U. Svajger, M. Anderluh, M. Jeras, N. Obermajer, Cell Signal 2010,
22, 1397–1405.
[25] For alternative syntheses of 6, see ref. [5c, 9f and 9g]. For other ex-
amples of CuAAC functionalization of this substrate, see ref. [9f,g].
[26] B. Trastoy, D. A. Bonsor, M. E. Pꢃrez-Ojeda, M. L. Jimeno, A.
Mꢃndez-Ardoy, J. M. Garcꢁa-Fernꢂndez, E. J. Sundberg, J. L. Chiara,
Adv. Funct. Mater. 2012, 22, 3191–3201.
[42] T. B. H. Geijtenbeek, R. Torensma, S. J. van Vliet, G. C. F. van
Duijnhoven, G. J. Adema, Y. van Kooyk, C. G. Figdor, Cell 2000,
100, 575–585.
[43] See the Supporting Information for a laser and photostability study
of compound 16.
[27] In an excellent “teaching editorial”, Donald M. Simons nicely ex-
plained the statistical relationship between chosen stoichiometry
and reaction outcome for the derivatization of a homobifunctional
spacer with a monofunctional reagent. Interestingly, this communi-
cation has been almost completely overlooked by the synthetic
chemistry community (cited only once, according to Web of Knowl-
edge): D. M. Simons, Bioconjugate Chem. 1999, 10, 3–8.
[28] This ideal case rarely occurs in real molecules, particularly for the
most substituted derivatives due to increasing steric hindrance with
increasing substitution; thus, ratios of multisubstituted products are
somehow overestimated by this assumption.
[29] Not surprisingly, the examples of synthesis of monofunctional POSS
by selective monofunctionalization of a homo-octafunctional POSS
that has been described using a 1:1 stoichiometry rarely exceed a
30% isolated yield.
[30] The rather lipophilic octaazide 6 is an ideal substrate in this context
too, since each azide to 1,2,3-triazole functional group transforma-
tion will produce a significant increase of the polarity of the mole-
cule thus allowing for a facile chromatographic separation of un-
reacted POSS from the more polar mono-triazolyl-POSS product
and also of the latter from all possible polytriazolyl-POSS deriva-
tives that may have formed in the click reaction, which will all have
an even higher polarity and, thus, a lower mobility in normal phase
silica column chromatography.
[44] E. Caparros, P. Munoz, E. Sierra-Filardi, D. Serrano-Gomez, A.
Puig-Kroger, J. L. Rodriguez-Fernandez, M. Mellado, J. Sancho, M.
Zubiaur, A. L. Corbi, Blood 2006, 107, 3950–3958.
[45] For recent reviews, see: a) C. E. Hoyle, C. N. Bowman, Angew.
Chem. 2010, 122, 1584–1617; Angew. Chem. Int. Ed. 2010, 49, 1540–
1573; b) A. Dondoni, A. Marra, Chem. Soc. Rev. 2012, 41, 573–586;
c) A. Massi, D. Nanni, Org. Biomol. Chem. 2012, 10, 3791–3807.
[46] However, the thiol-ene/yne reaction has narrower functional group
compatibility than the CuAAC reaction. For some selected applica-
tions of the thiol-ene/yne reaction in POSS chemistry, see: a) Y.
Gao, A. Eguchi, K. Kakehi, Y. C. Lee, Org. Lett. 2004, 6, 3457–
3460; b) J. Xu, X. Li, C. M. Cho, C. L. Toh, L. Shen, K. Y. Mya, X.
Lu, C. He, J. Mater. Chem. 2009, 19, 4740–4745; c) I. Nischang, O.
Brueggemann, I. Teasdale, Angew. Chem. 2011, 123, 4688–4692;
Angew. Chem. Int. Ed. 2011, 50, 4592–4596; d) M. Lo Conte, S. Sta-
derini, A. Chambery, N. Berthet, P. Dumy, O. Renaudet, A. Marra,
A. Dondoni, Org. Biomol. Chem. 2012, 10, 3269–3277; e) Z. Wang,
Y. Li, X.-H. Dong, X. Yu, K. Guo, H. Su, K. Yue, C. Wesdemiotis,
S. Z. D. Cheng, W.-B. Zhang, Chem. Sci. 2013, 4, 1345–1352; f) A.
Marra, S. Staderini, N. Berthet, P. Dumy, O. Renaudet, A. Dondoni,
Eur. J. Org. Chem. 2013, 1144–1149. See also references [13c, 19l,
and 19m].
Received: January 29, 2013
Published online: && &&, 2013
Chem. Eur. J. 2013, 00, 0 – 0
ꢄ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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&
ÞÞ
These are not the final page numbers!

J. L. Chiara et al.
Play the click dice game: The selectiv-
ity of the functionalization reaction of
a symmetric polyfunctional substrate is
statistically related to the stoichiome-
try of the reactants. For the copper(I)-
catalyzed azide–alkyne cycloaddition
monofunctionalization of the cubic
octaazide shown, this statistical rela-
tionship holds only if a copper catalyst
with strong donor ligands is employed
for the click reaction, allowing the
preparation of well-defined hetero-
bifunctional hybrid nanoclusters.
Click Chemistry
M. E. Pꢀrez-Ojeda, B. Trastoy, ꢁ. Rol,
M. D. Chiara, I. Garcꢂa-Moreno,
J. L. Chiara* .................. &&&& — &&&&
Controlled Click-Assembly of Well-
Defined Hetero-Bifunctional Cubic
Silsesquioxanes and Their Application
in Targeted Bioimaging
&
12
&
www.chemeurj.org
ꢄ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!