Micelles for the self-assembly of "off-on-off" fluorescent sensors for pH windows

Article abstract of DOI:10.1002/chem.200500613

A micellar approach is proposed to build a series of systems featuring an "off-on-off" fluorescent window response with changes in pH. The solubilizing properties of micelles are used to self-assemble, in water, plain pyrene with lipophilized pyridine and tertiary amine moieties. Since these components are contained in the small volume of the same micelle, pyrene fluorescence is influenced by the basic moieties: protonated pyridines and free tertiary amines behave as quenchers. Accordingly, fluorescence transitions from the "off" to the "on" state, and viceversa, take place when the pH crosses the pK_a values of the amine and pyridine fragments. To obtain an "off-on-off" fluorescent response in this investigation we use either a set of dibasic lipophilic molecules (containing covalently linked pyridine and tertiary amine groups) or combinations of separate, lipophilic pyridines and tertiary amines. The use of combinations of dibasic and mono-basic lipophilic molecules also gives a window-shaped fluorescence response with changes in pH: it is the highest pyridine pK_a and the lowest tertiary amine pK_a that determine the window limits. The pK_a values of all the examined lipophilic molecules were determined in micelles, and compared with the values found for the same molecules in solvent mixtures in which they are molecularly dispersed. The effect of micellization is to significantly lower the observed protonation constants of the lipophilized species. Moreover, the more lipophilic a molecule is, the lower the observed logK value is. Accordingly, changing the substituents on the basic moieties or modifying their structure, tuning the lipophilicity of the mono- or dibases, and choosing among a large set of possible combination of lipophilized mono- and dibases have allowed us to tune, almost at will, both the width and the position along the pH axis of the obtained fluorescent window.

Full text of DOI:10.1002/chem.200500613

FULL PAPER
DOI: 10.1002/chem.200500613
Micelles for the Self-Assembly of “Off–On–Off” Fluorescent Sensors for pH
Windows
Yuri Diaz-Fernandez,[b] Francesco Foti,[a] Carlo Mangano,[a] Piersandro Pallavicini,*[a]
Stefano Patroni,[a] Aurora Perez-Gramatges,[b] and Simon Rodriguez-Calvo[b]
Abstract: A micellar approach is pro-
posed to build a series of systems fea-
turing an “off–on–off” fluorescent
window response with changes in pH.
The solubilizing properties of micelles
are used to self-assemble,in water,
plain pyrene with lipophilized pyridine
and tertiary amine moieties. Since
these components are contained in the
small volume of the same micelle,
pyrene fluorescence is influenced by
the basic moieties: protonated pyri-
dines and free tertiary amines behave
as quenchers. Accordingly,fluorescence
transitions from the “off” to the “on”
state,and viceversa,take place when
the pH crosses the pKa values of the
amine and pyridine fragments. To
obtain an “off–on–off” fluorescent re-
sponse in this investigation we use
either a set of dibasic lipophilic mole-
cules (containing covalently linked pyr-
idine and tertiary amine groups) or
combinations of separate,lipophilic
pyridines and tertiary amines. The use
of combinations of dibasic and mono-
basic lipophilic molecules also gives a
window-shaped fluorescence response
with changes in pH: it is the highest
pyridine pKa and the lowest tertiary
amine pKa that determine the window
limits. The pKa values of all the exam-
ined lipophilic molecules were deter-
mined in micelles,and compared with
the values found for the same mole-
cules in solvent mixtures in which they
are molecularly dispersed. The effect
of micellization is to significantly lower
the observed protonation constants of
the lipophilized species. Moreover,the
more lipophilic a molecule is,the lower
the observed logK value is. According-
ly,changing the substituents on the
basic moieties or modifying their struc-
ture,tuning the lipophilicity of the
mono- or dibases,and choosing among
a large set of possible combination of
lipophilized mono- and dibases have al-
lowed us to tune,almost at will,both
the width and the position along the
pH axis of the obtained fluorescent
window.
Keywords: fluorescence · micelles ·
molecular devices · sensors
Introduction
small volume of the same micelle has been exploited in sur-
factant science since the early 1970s. The observation of
either steady-state or dynamic fluorescence quenching due
to intramicellar interactions between for example,pyrene
and a series of quenchers (typically N,N’-dibutyl aniline[1]
and dodecylpyridinium[2]) is a well-established method to
calculate the aggregation number (AN),that is,the average
number of surfactant units per single micelle. Kinetics pa-
rameters,such as the rate constants of the processes involv-
ing the fluorophore/quencher systems inside a micelle,have
The possibility offered by working in water and confining
separate hydrophobic fluorophores and quenchers in the
[a] Dr. F. Foti,Dr. C. Mangano,Dr. P. Pallavicini,Dr. S. Patroni
Dipartimento di Chimica Generale
Università di Pavia,via Taramelli
12-27100 Pavia (Italy)
Fax : (+39)038-252-8544
also been elucidated by means of fluorescence quenching[3]
Sophisticated structural parameters,such as the position of a
.
E-mail: psp@unipv.it
[b] Dr. Y. Diaz-Fernandez,Prof. A. Perez-Gramatges,
Prof. S. Rodriguez-Calvo
hydrophobic molecule inside a micelle with respect to the
water/micelle interface[4] or the shape of a micelle,[5] have
also been investigated through intramicellar quenching proc-
esses between separated components. Another branch of
chemistry,fluorescent sensing of cations,anions and neutral
species,has developed dramatically [6] after the first examples
Instituto Superior de Tecnologias y Ciencias Aplicada
Department of Radiochemistry,Ave Salvador Allende y Luaces
Plaza de la Revoluciòn,Ciudad de la Habana
Apartado Postal 16040,Habana 11600 (Cuba)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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published as early as 1988.[7] These examples were based on
a traditional molecular approach,in which a fluorophore
and a receptor component,also capable of acting as quench-
er in the absence (“off–on” sensor) or in the presence (“on–
off” sensor) of the target species,were covalently linked by
a spacer. Stressing that this approach often requires tedious
synthetic efforts,it is surprising that only the new millenni-
um brought the idea of using micelles as containers for pre-
paring supramolecular sensors in which simple,separated
fluorophores and receptors/quenchers are kept together and
communicate inside the micelle without the need of building
a covalent structure. Tecilla,Tonellato et al. have shown
how Cu2+ can be signalled for by fluorescence quenching
when it is coordinated by a lipophilized dipeptide inside
CTAB (cetyltrimethylammonium bromide) micelles. These
micelles also contained the lipophilic 8-anilino-1-naphthale-
nesulfonic acid fluorophore,[8] or,in a similar system,the
Rhodamine 6G fluorophore.[9] Anslyn and Niikura demon-
strated that the neutral surfactant Triton X-100 allows the
detection of the IP (inositol triphosphate) anion in water,
thanks to enhanced ion-pair-driven molecular recognition
between a hydrophobic anion receptor,IP,and the compet-
ing anionic lipophilic fluorophore 5-carboxyfluorescein
inside the micelles.[10] Recently,we published a contribution
describing a sensing system selective for Ni2+ and Cu2+
based on a lipophilized diamino–diamido ligand and pyrene,
which interacted inside Triton X-100 micelles.[11]
Scheme 1. Working scheme for the covalent approach to window-shaped
fluorescent sensors for pH. Component F is a fluorophore (typically an-
thracene,see refs. [13a–d],the “spacer” may be as simple as a CH unit.
2
Transition from A to B takes place when pH crosses the pyridinium pKa
value; transition from B to C takes place when pH crosses the ammoni-
um pKa value.
cence goes “on”; iii) as the pH is increased further above
the amine pKa,this group also becomes nonprotonated,the
molecule is neutral (form C) and the fluorescence turns
“off” again due to the quenching properties (by PET) of the
electron-donating tertiary amino groups.
Since we are also interested in the study of fluorescent
sensors for pH,and,in particular,of those capable of giving
a window-shaped If versus pH response (If =fluorescence in-
tensity),[12] we decided to also apply the supramolecular mi-
cellar approach to these kinds of systems. Window-shaped
sensors for pH may be important for life sciences,as biologi-
cal processes are known to be effective only in restricted
concentration ranges of the involved species and,in particu-
lar,in restricted pH ranges. However,designing and build-
ing systems of this kind is a complicated matter and only
few papers have been published reporting an “off–on–off”
or “on–off–on” If versus pH window. Moreover,beside a
very recent example based on an EuIII-luminescent complex
of a tetraquinoline-substituted cyclen ligand,[13] and another
example published by our group based on multicomponent
coordination complexes,[12b,c] all the described systems are
organic molecules that have to be built by step-by-step or-
ganic synthesis.[14] These molecular systems are based on the
original approach of de Silva and co-workers.[14a] Here an-
thracene was the light-emitting component and was cova-
lently linked to one or more pyridine and tertiary amino
groups,that behaved as pH-dependent quenching fragments,
through spacer units. Three states are available in these kind
of molecules as a function of pH (described in Scheme 1):
i) at low pH both pyridine and amine are protonated (form
The approach to “off–on–off” fluorescent sensors for pH
windows that we describe in this paper merges the ideas
presented by de Silva and co-workers with the flexibility of
the micelle-as-container approach: lipophilized pyridines
and amines are used as pH-dependent quenchers and
pyrene is the fluorophore. However,since these components
are not (or only partially) covalently linked,but rather self-
assembled inside a micelle,synthetic efforts are reduced to
the minimum.
Results and Discussion
Protonation constants in the water/micelle medium: We
have investigated a set of lipophilized molecules,each one
containing either one pyridine or one trialkylamine group,
or both. N,N’-Dimethyl-N’’-dodecylamine (DAm) is com-
mercially available,while the remaining six molecules were
obtained through simple syntheses (see Experimental Sec-
tion). They are all insoluble in water,at least in their neutral
form. They readily dissolve in water containing the neutral
surfactant TritonX-100 above its critical micelle concentra-
tion (cmc),thanks to the insertion of their lipophilic tails in
the micellar core. Triton X-100 is a classical surfactant that
is widely employed and whose properties are well-establish-
ed.[15] Moreover,since it is neutral,no electrostatic effects
are to be expected for protonation/deprotonation properties
of any species included inside its micelles. Unless otherwise
stated,in the experiments run for this investigation we have
operated with a surfactant concentration of 6.47 gLꢀ1. Triton
+
A) and the fluorescence is “off”,with the pyridineH frag-
ment acting as a quencher by photoelectron transfer (PET)
thanks to its electron-acceptor properties; ii) as the pH rises
above the pyridine pKa,this fragment is no longer protonat-
ed (while the amine still is: form B) and thus the fluores-
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the organic tail inserted in the hydrophobic micellar core
and the more polar head presumably lying in the hydrophil-
ic,external layer of the micelle. Penetration of the solvent
and any dissolved species into the hydrophilic layer of the
micelle is partially allowed,but the local concentration of
water and H+ could be significantly lower with respect to
the bulk solution.[4] Hence,taking into account both lower
local [H+] and a less efficient solvation of protonated spe-
cies,we expected lower observed protonation constant
values with respect to the appropriate references,that is,
nonlipophilic bases triethylamine and 2-methylpyridine in
water. Moreover,it has already been shown how the lower-
ing of the measured protonation constants is proportional to
the lipophilicity of the considered base,[4] that is,to the
depth of its inclusion in the micelle,as this is inversely pro-
portional to the efficiency of solvation of the protonated
species. The found values are reported in Tables 1 and 2,
Table 1. Logarithmic protonation constants for the monobasic molecules.
Uncertainties are reported in parentheses. The K values refer to the for-
mation equilibria,base + H+ =[baseH]+.
logK
Dpy
DAm
DPi
3.73(0.02)
7.84(0.01)
7.38(0.02)
Table 2. Step logarithmic protonation constants for the dibasic molecules.
Uncertainties are reported in parentheses. The K values refer to protona-
tion equilibria. In particular, K1 refers to Equilibrium (1),see text,and it
is relative to the protonation of the amino moiety. K2 refers to Equilibri-
um (2),see text,and it is relative to the protonation of the pyridine
moiety.
X-100 has a cmc of ~2”10ꢀ4 m and an aggregation number
of 111.[15b] Since the average molecular weight of the com-
mercial Triton X-100 that we used was 647,we were operat-
ing with an average micelle concentration of ~10ꢀ4 m. We
used concentrations of lipophilized bases equal or lower
than 10ꢀ3 m,so that a maximum average of 10 base mole-
cules per micelle is to be expected. This was done in order
to minimize base–base interactions and to avoid significant
changes in the micelle parameters with respect to pure sur-
factant (we have already shown that,for example,the AN
does not change significantly for inclusion of up to 10 mole-
cules equipped with a C12 tail and possessing analogous acid/
base properties to those presented in this paper[5]).
logK1
logK2
o-DMAPy
p-DMAPy
DDAPy
6.27(0.01)
5.41(0.01)
4.58(0.01)
6.31(0.02)
2.13(0.03)
2.23(0.03)
1.8 (0.1)
DMAMPy
2.06(0.04)
and confirm the expected trend. The logK values for proto-
nation of the reference compounds triethylamine and 2-
methylpyridine in water are 10.68[17] and 6.06,respectively. [18]
Moreover,an even better molecule for comparison with the
cyclic monobase N-dodecylpiperidine, DPi,is N-methylpi-
peridine,with a log K value of 10.13.[17] The values found for
the monobases 2-dodecylpyridine (DPy), DAm and DPi
show that lipophilization and inclusion in micelles signifi-
cantly lower the logK value by ~2–3 log units with respect
to the water-soluble reference bases. This micellization
effect is also confirmed by the determination of the protona-
tion constants for the monobases DPy, DAm and DPi in the
organic-enriched solvent mixture dioxane/water (8:2 v/v). In
this mixture the three molecules cannot form micelles and
are molecularly dispersed. We found logK=4.14(ꢁ0.02) for
DPy,log K=8.55(ꢁ0.01) for DAm and logK=8.48 (ꢁ0.04)
for DPi. These values are still lower then those found in
water for 2-methylpyridine,triethylamine and N-methylpi-
As a first step,we examined the acid/base properties in
the water–surfactant medium for each lipophilized base,
using a standard potentiometric titration apparatus and
well-established experimental procedures (see Experimental
Section),that is,measuring a space-averaged value of
E
with a macroscopic glass electrode in an excess acid solution
to which standard base was added in 10–20 mL portions.
From the titration data we calculated the protonation con-
stants of the examined bases,corresponding to the p Ka of
their protonated species.[16] These values are of course not
“intrinsic” but “observed”,that is,influenced by the fact
that the lipophilic bases are included inside micelles,with
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P. Pallavicini et al.
peridine; the difference is due to the intrinsic lower solva-
tion ability of any organic-enriched aqueous solvent with re-
spect to water. However,we also measured the protonation
constants for 2-methylpyridine,triethylamine,and N-methyl-
piperidine in dioxane/water (8:2 v/v). We found the logK
values very similar to those of DPy, DAm and DPi in the
same medium,that is,4.41( ꢁ0.01),9.20( ꢁ0.01) and 8.66
(ꢁ0.03),respectively. Consequently,the huge differences
found between the logK values for water-dispersed and
water-micellized analogous base moieties can be assigned to
their inclusion in micelles.
When the protonation constants of the dibasic molecules,
which can be schematized as R2N-CH2-Py,are considered
(Table 2),the proximity of the pyridine and amine moieties,
which are separated only by a -CH2- unit,is the origin of
even lower values. In Table 2 the logK1 values for the dibas-
es pertain to the more basic amino group,that is,refer to
Equilibrium (1).
Finally,from the log K values listed in Tables 1 and 2,the
distribution diagrams,that is,% of species (relative to total
[19]
lipophilic base) versus pH,can be drawn.
These diagrams
will be used in the following sections to relate the found If
versus pH profiles to the species in equilibrium in the exam-
ined pH ranges.
“On–Off” fluorescent pH sensors from the monobasic mol-
ecules: Prior to any experiment involving lipophilic bases,
the fluorescence of pyrene was checked at a concentration
of 9”10ꢀ6 m (the value was kept constant for all the experi-
ments described),in water containing Triton X-100 at the
usual concentration. Under these conditions,an average of
~0.1 pyrene molecules per micelle can be calculated. The
value is kept so low both because of the intense pyrene fluo-
rescence and to avoid any possible formation of pyrene–
pyrene excimers (which derives from the inclusion in the
same micelle of two or more pyrene units). Coupled pH–
spectrofluorimetric titrations in the 0–12 pH range con-
firmed that the emission of pyrene is constant.[20] We then
also examined pyrene fluorescence in the same pH range in
the presence of either triethylamine or 2-methylpyridine
(10ꢀ3 m in each case). Both these species are water soluble
and are not expected to interact significantly with the mi-
celles. Accordingly,also in this case,no variations in the
pyrene fluorescence was observed in the 0–12 pH range.
Coupled pH–spectrofluorimetric titrations were then per-
formed to determine the variation of the fluorescence of
pyrene with pH within the same range for solutions contain-
ing one of the monobases (DPy, DMa,or DPi) at a concen-
tration of 10ꢀ3 m. An average of 10 molecules of base is ex-
pected for each micelle at this concentration,so that any mi-
cellized pyrene should experience this average number of
pH-sensitive quenchers in its micelle. As expected,in all
cases a sigmoidal fluorescence intensity trend was found
with a change in pH; that,on going from low to high pH,is
of the “off–on” type for the 2-dodecylpyridine derivative
and of the “on–off” type for the two dodecylated tertiary
amines. In particular,fluorescence goes “on” or “off” when
the pH is increased above the basesꢁ pKa values. Figure 1a
reports the series of spectra obtained at various pH values
in the representative case of DPy (analogous emission spec-
tra have been obtained for all the other fluorescence versus
pH measurements in this work). A better visualization is ob-
tained by plotting If at 392 nm,that is,at the wavelength of
pyrene maximum emission,as a function of pH. Figure 1b–d
displays If,392 versus pH profiles for the three monobases,su-
perimposed to the relative distribution diagrams.
þ
R2N-CH2-Py þ Hþ Ð ½R2NH-CH2-PyŠ
ð1Þ
and vary between 4.58 (DDAPy) and 6.31 (DMAMpy). The
further lowering of the logK values of the tertiary amine
moieties with respect to DAm and DPi is due to the in-
creased steric hindrance and lipophilicity introduced by the
pyridine group (which is nonprotonated at the pH values at
which Equilibrium (1) takes place). It is noteworthy that the
didodecylated molecule DDAPy has a logK1 value as low as
4.58. This low value is due to its increased hydrophobicity
because of the location of the amino group closer to the mi-
cellar core and far away from the water–micelle interface.[4]
On the other hand,all the log K2 values,corresponding to
the protonation of the pyridine group [Equilibrium (2)].
þ
2þ
½R2NH-CH2-PyŠ þ Hþ Ð ½R2NH-CH2-PyHŠ
ð2Þ
are dramatically low in the micelle/water medium. This is
due to the electrostatic repulsion exerted by the proximate
ammonium group (the tertiary amines are already protonat-
ed at the pH values where the pyridines become protonat-
ed),that adds to the micellization effect. Noticeably,lower-
ing of logK2 is also observed for the para-derivative
p-DMAPy,suggesting that not only an electrostatic
through-space effect,but also a through-bond inductive
effect is playing a significant role. The roles of both micelli-
zation and the ammonium proximity were confirmed by de-
termining the protonation constants of the representative
o-DMAPy molecule in dioxane/water (8:2 v/v). In this case
we found logK1 =7.08 (ꢁ0.05) and logK2 =2.59 (ꢁ0.05). In-
terestingly,it can be noted that the DlogK values,that is,the
difference between the logK found in dioxane/water and in
water/micelle media are 0.41 for DPy and 0.71 for Dam.
These values match well with the DlogK of 0.46 and 0.81
found in o-DMAPy for the protonation of its pyridine and
amine moieties,suggesting an analogous contribution of the
micellization effect in both cases.
A close superposition of the If versus pH profiles is ob-
served with the distribution diagrams,as shown in Fig-
ure 1b–d. In particular,Figure 1b shows that the fluores-
cence goes “on” (black triangles) when the [DPyH]+ de-
protonates to form free DPy,while Figure 1c and d show
that the fluorescence goes “off” when the tertiary amino
group of [DMaH]+ and [DPiH]+ deprotonates to form free
DMa and DPi. Although we do not have any experimental
information on the nature of the quenching process,the ob-
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Fluorescent pH Sensors
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Figure 1. a) Series of pyrene emission spectra obtained for the system containing the DPy monobase at various pH. Superimposable,low-intensity spec tra
are obtained at low pH values (<2),intensity starts to increase at pH >2.5,and superimposable full-emission spectra are obtained at pH > 6.5. b–
d) Black triangles represent the relative emission intensity (If/I0) at 392 nm with varying pH (the maximum found emission in each series of spectra is
considered as I0). Solid lines represent the % (with respect to total base) of each species present at the equilibrium with varying pH. The species pertain-
ing to each profile are indicated on the plots.
vious hypothesis that intramicellar photo-electron transfer
mechanisms are still valid can be proposed; protonated pyri-
dines are good electron acceptors and free tertiary amines
good electron donors and both are well-known PET quench-
ers from/to the excited state of polyaromatic molecules.[14]
their protonated forms. As it can be seen in Figure 2a–d,a
fair superposition is observed between If,392 versus pH pro-
files (black triangles) and species distribution (solid lines).
In particular,fluorescence is “off” when the base molecules
are in the diprotonated [R2NH-CH2-PyH]2+ form,with mi-
celles containing a pyridinium fragment; fluorescence goes
“on” when the pH is raised above the pKa value of pyridini-
um,so that the pyridine fragment deprotonates and the mo-
noprotonated species [R2NH-CH2-Py]+ is obtained. Finally,
fluorescence is “off” again when the pH is sufficiently high
to be above the ammonium pKa value,so that the tertiary
amine group is deprotonated to give the neutral R2N-CH2-
Py species.
Quite interestingly,the data shown in Figure 2 merged
with those in Table 1 illustrate the versatility of this ap-
proach. The window limits are regulated by the logK values
of the micellized molecules and thus can be tuned by play-
ing with substituents or with the lipophilicity of the whole
molecule. Figure 3 shows the amplitude and the limits of the
fluorescent pH windows described by the systems presented
in this paper. The horizontal segments connect,for each
system,the pH values corresponding to the p Ka of the spe-
cies responsible for the quenching process. However,switch-
ing of If from the “off” to the “on” state,or viceversa,is
not,of course,a vertical transition with respect to the pH
An “off–on–off” fluorescent pH sensor from the dibasic
molecules: In the case of the dibasic molecules o-DMAPy,
p-DMAPy, DDAPy and DMAMPy,the If versus pH behav-
iour has also been examined by means of pH–fluorimetric
titrations for each molecule in the 0–12 pH range. The solu-
tions contained 10ꢀ3 m bases,that is,with an average of 10
base molecules per micelle. In this situation,any micelle
containing pyrene is a supramolecular system resembling
the “classical” covalently linked molecular systems for fluo-
rescent signalling of pH windows[14] described in Scheme 1:
although the molecules are not directly connected,pyrene
experiences the presence of pyridine and tertiary amino
groups in the small micellar volume. Accordingly,an “off–
on–off” If versus pH profile is to be expected on going from
low to high pH values or viceversa,as it is pictorially shown
in Scheme 2.
This behaviour is indeed what is found for the four sys-
tems,with the window limits determined by the log K values
of the pyridine and amine moieties,that is,by the p Ka of
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P. Pallavicini et al.
Scheme 2. Working scheme for the micelle-contained systems based on lipophilic dibases (F = pyrene). The “spacer” component is always CH2,connect-
ed to the pyridine ring in an ortho- or para-position. Transitions from one form to the other take place with the pH increased above the pKa of the in-
volved base.
Figure 2. Black triangles represent the relative emission intensity (If/I0) at 392 nm with varying pH (the maximum found emission in each series of spectra
is considered as I0). Solid lines represent the % (with respect to total base) of each species present at the equilibrium with varying pH. The species per-
taining to each profile are indicated on the plots.
axis. This transition follows a smooth curve along the distri-
bution diagram of the pertinent species,so that the resulting
If versus pH profile follows a curve resembling a bell-shaped
one. Accordingly,the horizontal segments of Figure 3 more
appropriately represents the half-height width of the bell-
shaped If versus pH curves.
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Fluorescent pH Sensors
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acid–base properties can be excluded: we again performed a
potentiometric titration on solutions containing Triton X-
100 and the two bases at 5”10ꢀ4 m concentration and ob-
tained the same logK values found with the titrations car-
ried out with just one micellized species at a concentration
of 10ꢀ3 m. By means of pH–fluorimetric titrations,a very sat-
isfactory “off–on–off” If versus pH profile was found (see
Figure 4a,black triangles),which clearly comes from the
merging of the If versus pH profiles displayed in Figure 1a
and b. In Figure 4a the fluorescence intensity profile is su-
perimposed on a distribution diagram calculated for the
system containing both DPy and DAm in 5”10ꢀ4 m concen-
tration. It can be seen that the fluorescence is switched
“off” at low pH values where the quencher [DPyH]+ coex-
ists with [DAmH]+,is switched “on” where DPy/[DAmH]+
coexist,and is switched “off” again where the quencher
DAm coexists with DPy. When the same kind of experiment
is repeated with the DPy/DPi system,we obtained the same
type of profile (Figure 4b,black triangles),but with the
upper pH limit of the window shifted by ~0.5 units to the
left due to the analogous difference in the logK values be-
tween DAm and DPi (see Table 1).
Noticeably,two different amines or two different pyri-
dines can coexist in the same micelle,with the “off” limits
being dictated by the higher pyridine pKa and lower amine
pKa. This suggests the possibility to also vary the combina-
tion of monobases and dibases to shift the window position
along the pH axis,or to obtain larger or narrower windows.
Accordingly,we have combined a monobase, DPy,with a
dibase, o-DMAPy (both at 5”10ꢀ4 m concentration). In this
case,the tertiary amine of o-DMAPy sets the upper window
limit with its logK value of 6.27. It is therefore the more
basic of the two coexisting pyridines (i.e.,that belonging to
DPy) that regulates the lower window limit with its logK
value of 3.73,thus allowing to obtain another fluorescent
Figure 3. Horizontal segments represent the ideal half-height width of the
If versus pH curves of each system. Their limiting values on the pH scale
correspond to the pKa values of base moieties responsible of the quench-
ing processes.
“Off–On–Off” fluorescent pH sensor from the combination
of two basic molecules: The flexibility of the micellar ap-
proach can be further exploited by using combinations of
monobases,or even combinations of monobases and dibases
with pyrene to build,in the same micelle,a system similar
to the covalent ones described in Scheme 1. In the simplest
case,all that is needed is to introduce one lipophilized pyri-
dine and one lipophilized tertiary amine in the same micelle
in the presence of pyrene. As it is pictorially described by
Scheme 3,what is obtained is an “off–on–off” signalling
system delimited by the pKa values of the pyridine and terti-
ary amine moieties.
A combination of the two monobases DAm and Dpy was
obtained by operating in a pyrene/Triton X-100 solution
containing both bases in 5”10ꢀ4 m concentration,that is,
with an average of five molecules of each base per micelle.
Any reciprocal influence of the two molecules on their
Scheme 3. Working scheme for the system based on the combination of one pyridine and one tertiary amine monobases (F = pyrene,the pyridine-con-
taining component is DPy,the tertiary-amine-containing component may be DAm or DPi). Low pH passage from the “off” to the “on” form takes place
at pH values crossing DPy pKa. High pH passage from the “on” to the “off” form takes place at pH values crossing DAm or DPi pKa.
Chem. Eur. J. 2006, 12,921 – 930
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P. Pallavicini et al.
Figure 4. Black triangles represent the relative emission intensity (If/I0) at 392 nm with varying pH (the maximum found emission in each series of spectra
is considered as I0). Solid and dashed lines represent % (with respect to total base) of each species present at the equilibrium with varying pH. The possi-
ble forms of each molecule (free,protonated and,where possible,diprotonated) are described by the same type of line (i.e.,either solid or dashed).
species pertaining to each profile are indicated on the plots.
The
pH window with different limits (see Figure 3). Figure 4c re-
ports the corresponding If versus pH profile that shows that
the If is switched “off” in correspondence to the formation
of [DPyH]+ and of free o-DMAPy,while it is “on” only in
the pH range in which free DPy and [o-DMAPyH]+ (proto-
nation is on the amino group) coexist.
On the other hand,it should be emphazized that not all
the combinations are allowed if an efficient signalling
system is needed. The transition from the “off” to the “on”
state,or viceversa,is superimposable to the distribution dia-
grams of the involved bases. Combinations of bases where
the pyridine/amine pKa values are similar (e.g. DPy/
DDAPy) would result in an almost “always off” profile: at
the same pH at which the pyridine group of one species is
being deprotonated,the amine of the other species starts to
be deprotonated as well. A significant quantity of a quench-
ing group is therefore always present inside the micelles,al-
lowing for only partial or even negligible fluorescence reviv-
al.
Secondary amines are less efficient quenchers than terti-
ary ones for polyaromatic hydrocarbon fluorophores,due to
their intrinsically reduced electron donating properties [see
for example,ref. [21]]. We studied the acid/base properties
and the If versus pH behaviour in micelles of (N-dodecyl)-2-
aminomethylpyridine (o-DAPy) and (N-dodecyl)-4-amino-
methylpyridine (p-DAPy) under the usual experimental
conditions. For o-DAPy we found logK1 =5.08 (ꢁ0.01) and
logK2 =2.62(ꢁ0.02),while for p-DAPy logK1 =6.65(ꢁ0.02)
and logK2 =3.20 (ꢁ0.02). The If versus pH profile,deter-
mined by means of pH–fluorimetric titrations of these mi-
cellized molecules (10ꢀ3 m),does not feature a fluorescent
window but it is just of the “on–off” type. Superposition
with the relative distribution diagram (see Supporting Infor-
mation) shows that only the protonated pyridine moiety is
Finally,in order to further extend the flexibility of this ap-
proach,the secondary amine precursors of the R N-CH2-Py
2
molecules can be advantageously used. Preparation of
o-DMAPy, p-DMAPy, DDAPy and DMAMPy (as described
in the Experimental Section) passes through the tertiariza-
tion of the corresponding secondary amines, o-DAPy and
p-DAPy,that can be schematically indicated as RNH-CH -Py.
2
928
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Chem. Eur. J. 2006, 12,921 – 930

Fluorescent pH Sensors
FULL PAPER
ed HCl (37%). The acid was evaporated under reduced pressure. The
residue was dissolved in NaOH (2m,27.6 mL) and the obtained solution
was extracted with chloroform (3”50 mL). The combined organic ex-
tracts were dried over Na2SO4,filtered and evaporated (0.81 g,77%). 1H
NMR (CDCl3): d = 8.62,7.43 (2d,2”2H; pyr),3.64 (s,2H; py-C H2-N),
2.36 (t,2H; N-C H2-(CH2)n-CH3),2.26 (s,3H; N-C H3),1.5–1.2 (m,20H;
CH2 dodecyl chain),0.90 (t,3H; -(CH 2)n-CH3); ESI MS: m/z: calcd for
C19H34N2; found: 291 [p-DMAPy+H+].
an efficient quencher,while the free secondary amine
moiety does not quench pyrene fluorescence (lowering in in-
tensity of less than 5% is observed for RNH-CH2-Py with
respect to [RNH2-CH2-Py]+ for both systems). Accordingly,
o-DAPy and p-DAPy are dibasic molecules with just one
pH-sensitive quenching fragment. In order to obtain a
system signalling a pH window with different limiting
values,we choose to combine p-DAPy and DPi,which are
dictated by p-DAPy logK2 (3.20) and by DPi logK (7.38).
The corresponding If versus pH profile,obtained from a 5”
10ꢀ4 m solution of both bases in the usual pyrene/Triton X-
100 medium,is displayed in Figure 3d,which also shows a
satisfactory superposition of the profile to the expected
curves of the distribution diagram.
(N-Methyl,N’-dodecyl)-2-methyl-6-aminomethylpyridine
(DMAMPy):
DAPyM (290.5 mg,1.0 mmol) was treated with the same procedure de-
scribed for p-DMAPy. Yield: 247 mg,81%. 1H NMR (CDCl3): d = 7.81
(t,1H; pyr),7.43 (d,1H; pyr),7.20 (d,1H; pyr),3.80 (s,2H; py-C
H2-N),
2.60 (s,3H; C H3-py),2.37 (t,2H; N-C H2-(CH2)n-CH3),2.25 (s,3H; N-
CH3),1.5–1.2 (m,20H; CH
dodecyl chain),0.91 (t,3H; -(CH 2)n-CH3);
2
ESI MS: m/z: calcd for C20H36N2; found 305 [DMAMPy+H+].
(N,N’-Didodecyl)-2-aminomethylpyridine (DDAPy): o-DAPy (1.0 g,
3.62 mmol) was dissolved in CH3CN (70 mL) in a 100 mL flask contain-
ing K2CO3 (400 mg,2.86 mmol). Dodecylbromide (812 mL,3.62 mmol)
was added under stirring and the solution was refluxed for 24 h. The re-
action was stopped when the reagents were completely consumed (con-
trolled by TLC: alumina; hexane/acetate 4:1). The carbonate was filtered
off and the solvent was evaporated under reduced pressure to obtain a
pale yellow oil (1.22 g,76%). 1H NMR (CDCl3): d = 8.66 (d,1H),7.59
Experimental Section
Synthesis and materials: Pyrene (97%,Fluka) was used as received.
Triton X-100,that is, tert-octylphenoxy polyoxyethylene glycol with and
average of 9–10 oxyethylene units,was purchased from Caledon (average
molecular weight=647). N,N’-Dimethyl-N’’-dodecylamine (DAm) was
purchased from Aldrich and used as received. 2-Dodecyl-pyridine (DPy)
was prepared according to a described procedure.[22] The secondary
amines (N-dodecyl)-2-aminomethylpyridine (o-DAPy),( N-dodecyl)-4-
aminomethylpyridine (p-DAPy) and (N-dodecyl)-2-methyl-6-aminome-
thylpyridine (DAPyM) were prepared by formation of the Schiff bases
(i.e.,by mixing commercially available 2-pyridinealdehyde,4-pyridineal-
dehyde or 6-methyl-2-pyridinecarboxaldehyde with dodecylamine in
(t,1H; pyr),7.42 (d,1H; pyr),7.20 (t,1H; pyr),3.70 (s,2H; py-C
H2-N),
2.38 (t,4H; N-C H2-(CH2)n-CH3),1.5–1.2 (m,40H; CH dodecyl chains),
2
0.90 (t,6H; -(CH 2)n-CH3); ESI MS: m/z: calcd for: C30H56N2; found 445
[DDAPy+H+].
Titrations: Protonation equilibria of monodispersed species were studied
in 0.05m tetrabutylammonium nitrate water/dioxane mixtures (2:8 v/v),
at 258C,by titrating a solution containing the chosen molecule and
excess nitric acid with standard base (KOH). Electrode calibration and
potentiometric measurements were carried out as already described.[24]
Protonation equilibria of micellized species were studied in water con-
taining 6.47 gLꢀ1 of Triton X100,by addition of standard base (KOH) to
a 10ꢀ3 m solution of the chosen molecule containing excess standard nitric
acid. Solutions were prepared to contain 0.05m NaNO3 as the supporting
methanol),followed by reduction with excess NaBH and hydrolysis,ac-
4
cording to a well established procedure. The obtained products were
characterized by IR and ESI mass spectroscopy and used for the prepara-
tion of the tertiary amine derivatives (o-DAPy is already described in lit-
erature; see reference [23]).
electrolyte, T=258C. Potentiometric measurements were carried out au-
[24]
tomatically,as already described.
The titration curves were fitted and
the equilibrium constants were calculated by using the nonlinear fitting
N-dodecylpiperidine (DPi): Dodecylbromide (1.0 g,4.01 mmol) and pi-
peridine (0.342 g,4.02 mmol) were dissolved in CH 3CN (50 mL) in a
100 mL flask containing K2CO3 (0.4 g). The mixture was refluxed over-
night and,after cooling at room temperature,the solvent was evaporated
under reduced pressure. The product was treated with water (50 mL) and
the obtained solution was extracted with CH2Cl2 (3”50 mL). The com-
bined organic extracts were dried over Na2SO4,filtered and evaporated
(0.93 g,92%). 1H NMR (CDCl3): d = 2.32 (t,2H; N-C H2-(CH2)n-CH3),
program HYPERQUAD.[16]
Coupled pH–spectrofluorimetric titrations were carried out on water sol-
utions containing 6.47 gLꢀ1 of Triton X-100,9”10 ꢀ6 m pyrene (dissolved
by adding aliquots of concentrated pyrene solutions in t-butanol,final t-
butanol concentration
<
0.5% v/v),10 ꢀ3 m of the chosen mono- or
dibase (or 5”10ꢀ4 m each when two different molecules were combined)
and 0.05m NaNO3,at 25 8C. Bulk solutions of 50–70 mL were used (kept
under a constant flow of nitrogen),treated with excess nitric acid and ti-
trated by manual micropipette additions of 10–50 mL aliquots of standard
KOH. A glass electrode for pH measurement was dipped in the bulk so-
lution. At each base addition the pH was recorded and a portion of 2–
3 mL of the bulk solution was transferred into a cuvette. The emission
spectra were recorded in the spectrofluorimeter (lexc =340 nm) and the
portion of solution was transferred to the bulk,prior to the next KOH
addition. Total KOH addition,at the end of the titrations,did not exceed
0.3 mL. Further pH–spectra measurements were added in some cases at
the end of the titration to better explore the more acidic zone (pH 0–2)
by addition of 10m HNO3 in 10–50 mL aliquots.
2.25 (t,4H; N-C H2- piperidine ring),1.6–1.2 (m,26H; CH
dodecyl
2
chain and piperidine ring),0.88 (t,3H; -(CH 2)n-CH3); ESI MS: m/z:
calcd for C17H35N; found: 254 [DPi+H+].
(N-Methyl,N’-dodecyl)-2-aminomethylpyridine (o-DMAPy): (N-dode-
cyl)-2-methylaminopyridine (o-DAPy,276.5 mg,1.0 mmol) was dissolved
in formic acid (1.1 mL,27.4 mmol) and formaldehyde (1.1 mL,11 mmol).
After 16 h of reflux and stirring the reacted mixture was cooled at room
temperature and treated with 20 drops of 37% HCl. Evaporation under
reduced pressure yielded a solid which was dissolved in NaOH (2m,
28 mL). The obtained solution was extracted with chloroform (3”
50 mL). The combined organic extracts were dried over Na2SO4,filtered
and evaporated to give DMP as a white wax (250 mg,86%). 1H NMR
(CDCl3): d = 8.65 (d,1H; pyr),7.60 (t,1H; pyr),7.41 (d,1H; pyr),7.15
(t,1H; pyr),3.67 (s,2H; py-C H2-N),2.40 (t,2H; N-C H2-(CH2)n-CH3),
Instrumentation: Mass spectra were recorded on a Finnigan MAT TSQ
700 instrument; NMR spectra on a Bruker AMX 400. Spectrofluorimet-
ric measurements were performed with a Perkin Elmer LS 50B instru-
ment. The pH titrations were made with a Radiometer TitraLab 90 titra-
tion system.
2.25 (s,3H; N-C H3),1.6–1.2 (m,20H; CH
dodecyl chain),0.88
2
(t,3H; -(CH 2)n-CH3); ESI MS: m/z: calcd for C19H34N2; found: 291
[o-DMAPy+H+].
(N-Methyl,N’-dodecyl)-4-aminomethylpyridine (p-DMAPy): (N-dode-
cyl)-4-aminomethylpyridine (p-DAPy,1.0 g,3.62 mmol) was dissolved in
formic acid (3.97 mL,99.2 mmol) and formaldehyde (3.99 mL,
39.8 mmol). After 16 h under reflux and stirring,the reacting mixture
was cooled at room temperature and treated with 23 drops of concentrat-
Chem. Eur. J. 2006, 12,921 – 930
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929

P. Pallavicini et al.
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served even in the absence of water-soluble bases (i.e.,with Tri-
tonX-100 and pyrene only),and may be attributed to changes in the
polarity of the micellar microenviroment that can cause a decrease
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New J. Chem.
Received: May 31,2005
Published online: September 30,2005
930
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Chem. Eur. J. 2006, 12,921 – 930