Solid-supported Zn(ii) porphyrin tweezers as optical sensors for diamines

Article abstract of DOI:10.1039/c000954g

A melamine-bridged bis-Zn(ii) porphyrin dyad covalently attached onto amino-functionalized TentaGel polymer beads or controlled pore glass (CPG) undergoes a reversible and marked colour change from purple to green upon exposure to a biogenic diamine such as cadaverine both in organic and aqueous media.

Full text of DOI:10.1039/c000954g

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Solid-supported Zn(II) porphyrin tweezers as optical sensors
for diamineswz
Lubian Elisa,a Francesco Baldini,b Ambra Giannetti,b Cosimo Tronob and
Tommaso Carofiglio*a
Received 18th January 2010, Accepted 24th March 2010
First published as an Advance Article on the web 15th April 2010
DOI: 10.1039/c000954g
A
melamine-bridged bis-Zn(II) porphyrin dyad covalently
In previous work,9 we described an efficient and modular
strategy for the synthesis of melamine-bridged bis-porphyrin
dyads.y This method takes advantage of the temperature-
dependent stepwise nucleophilic substitution of two of the
three chloride atoms of cyanuric chloride by the amino group
attached onto amino-functionalized TentaGel polymer beads or
controlled pore glass (CPG) undergoes a reversible and marked
colour change from purple to green upon exposure to a biogenic
diamine such as cadaverine both in organic and aqueous media.
of the 5-(4-aminophenyl)-10,15,20-triphenyl porphyrin
(Scheme 1, Path A).
1
There is an increasing social awareness of the need to improve
welfare and well-being through healthy eating. In this light,
and with respect to the priorities for the future of the food
chain, the European Technological Platform (ETP) called
‘‘Food for Life’’ has pointed out several key challenges with
a special emphasis on food safety and quality control. In
particular, monitoring the degree of spoilage of foodstuffs
susceptible to microbial degradation during aging and storage
is a priority issue. It has been established that the production
of specific polyamines by bacteria may be directly linked to the
freshness and quality of foods such as fish, meat, cheese, and
wine.1–2 For example, putrescine (1,4-diaminobutane) and
cadaverine (1,5-diaminopentane) possess a distinctive smell
and are commonly used by trained inspectors and individual
consumers for a qualitative evaluation of food freshness. On
the other hand, the quantitative analysis of polyamines via
chromatographic techniques is complicated by the lack of
natural chromophoric or electrophoric groups and by the high
polarity of these compounds. Hence, extensive chemical
derivatisation is indispensable to assist the chromatographic
separation and to enhance the detection sensitivity of biogenic
amines.3 Such a derivatisation protocol is usually performed
off-line, prior to the chromatographic analysis, thus increasing
the risk of analyte loss and contamination, and prolonging the
overall analysis duration. Accordingly, there is a need for
a sensing device which accurately and rapidly detects the
presence of biogenic amines in their pristine form and in
different matrices.4–8 From an applicative point of view, such
a device would prompt analysis automation thus enhancing
the efficiency of food processing and giving to the manu-
facturers real-time and online information on their products.
The third chloride atom reacts with an excess of piperidine
at 60 1C in the presence of N,N-diisopropylethylamine
(DIPEA) to form compound 3. Metalation of the porphyrin
a
`
Universita di Padova, Dipartimento di Scienze Chimiche, V. Marzolo 1,
35131 Padova, Italy. E-mail: tommaso.carofiglio@unipd.it;
Fax: ++39(0)49 827 5239; Tel: ++39(0)49 827 5670
b Dept. Optoelectronics & Photonics, IFAC-CNR, V. Madonna del Piano
10, 50019 Sesto Fiorentino (Firenze), Italy
w Dedicated to Professor Gianfranco Scorrano on the occasion of
his 70th birthday.
z Electronic supplementary information (ESI) available: Experimental
details for supporting the dyads onto TentaGel and CPG and for
carrying out the flow-analysis. See DOI: 10.1039/c000954g
Scheme 1 Synthesis of melamine-bridged bis-porphyrin dyads (Path A)
and their supporting onto aminated materials such as TG-NH2 and
CPG-NH2 (Path B).
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3678 | Chem. Commun., 2010, 46, 3678–3680

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macrocycles with zinc acetate in a CHCl3–MeOH mixture
affords the corresponding Zn(II) porphyrin–Zn(II) porphyrin
dyad. The generality of this approach has been demonstrated
by the construction of a small library of structurally related
molecular tweezers by the systematic variation of the porphyrin
substituents, and homo- or hetero-combination of the two
appended porphyrin moieties.10 The Zn(II) derivatives feature
a ditopic binding interaction with a,o-aliphatic diamines,
resulting in supramolecular complexes with more than 3 orders
of magnitude higher stability constants as compared to a
monodentate reference amine.
Immobilization of porphyrin-based sensors into an
appropriate solid support has been described as an efficient
strategy for the construction of recyclable optical sensors and
for their miniaturization and arraying for the development of
multianalyte detection systems.11–13
In the present work we describe a straightforward approach
for supporting the porphyrin–porphyrin dyads which is
achieved by replacing the piperidine terminus in Scheme 1
(Path A) with surface amino-functions anchored on a solid
material (Scheme 1, Path B).
Fig. 1 Single-bead Uv–vis spectra (Q-band region), monitoring the
evolution of the TG-NH2 resin into material 4 (spectrum A), 5
(spectrum B), and the spectral variation of 5 in the presence of an
excess of cadaverine. All spectra are recorded in DCM at 25 1C.
As aminated solid material, we have initially turned our
attention to the commercially available amino-functionalized
TentaGel beads (TG-NH2), generally used for solid-phase
synthesis applications, including the preparation of solid-
supported reagents for continuous-flow synthesis. The
TentaGel resin consists of a cross-linked polystyrene network
with grafted polyethylene glycol units and offers several
advantages such as (i) uniform swelling in solvents ranging
from toluene to water; (ii) optical transparency; (iii) a variety
of available functional groups; (iv) sizes of beads ranging from
5 to 400 mm with a narrow size dispersion.
Path B in Scheme 1 depicts the covalent conjugation of the
porphyrin dimer to the TG-NH2. In more detail, the mono-
chloro dimer 2, formed upon reaction of the amino-porphyrin
1 with half an equivalent of cyanuric chloride in the presence
of DIPEA, is readily scavenged by an appropriate amount of
the TG-NH2 resin added in situ. Heating of the mixture is
prolonged until complete decoloration of the supernatant
solution with formation of purple-coloured beads. The resin
is collected by filtration and washed extensively with THF in
order to remove unreacted and adsorbed products. Metalation
with Zn(Ac)2 takes place smoothly in CHCl3–MeOH mixture
affording the bis-Zn(II) porphyrin adduct, 4. According to the
TG-NH2/dimer stoichiometry adopted, the supported tweezer
corresponds to 1% of the overall amino-functionalities of
the beads.
recording of the Soret band around 420 nm is impracticable.
On the contrary, the Q-band region, in the 500–650 nm
wavelength range, gives a direct proof of the tweezer attachment
to the substrate, of its Zn(II) metalation, and of the coordination
status of the Zn(II) centers.
It is noteworthy that the Q-bands pertaining to 4 are typical
of a Zn(II) porphyrin,10 thus confirming the attachment of the
metalated tweezer, but turn out to be more red-shifted than
expected, with maxima at 564 and 606 nm, as a result of some
adventitious binding to the Zn(II) centers. This phenomenon is
also evident from a visual analysis of the resulting bead color
that appears to be blue–green rather than purple, as generally
obtained for uncomplexed Zn(II) porphyrins (Fig. 1A).
Since the spectrum is recorded in a non-coordinating
solvent, DCM, the observed coordination can be uniquely
ascribed to the residual amino groups of the beads which
account for 99% of the total resin functionalization. Since
such an intra-bead coordination will prejudice the ability of
material 4 to signal the coordination with an exogenous amine,
the capped material 5 is the sensor of choice. The success of
this approach is evident from the single bead Uv–vis spectrum
of 5 (Fig. 1B) showing the Q-bands with maxima at 557 and
598 nm. As a further remark, the capped beads show the
expected purple colour, indicating the free-Zn(II) state within
the tweezer receptor. Finally, the ability of 5 to sense biogenic
diamines is readily demonstrated upon its exposure to a
solution of 1,5-diaminopentane (cadaverine) in DCM. The
colour of the beads turns to blue–green, as a consequence of
the spectral red-shift, which is observed for the Q-bands with
maxima at 565 and 606 nm (Fig. 1C). These results confirm
that the sensing properties of the melamine-bridged bis-Zn(II)
porphyrin tweezer are maintained upon immobilization on a
solid support, as compared to the behaviour in homogeneous
solution.10
In order to avoid any competitive intra-bead Zn-coordination,
the residual free-amino groups of the TG-NH2 resin have been
capped by reaction with acetic anhydride, to yield the corres-
ponding acetamidate derivative 5.
Evolution of the TentaGel resin according to the diverse
steps of Path B in Scheme 1 is conveniently monitored through
single bead Uv–vis spectroscopy by a home-made apparatus
consisting of a confocal microscope coupled to a diode-array
spectrophotometer through an optical-fiber. Fig. 1 reports the
single bead Uv–vis spectra of the porphyrin-attached resin, of
the Zn(II) derivative 4 (spectrum 1A) and 5 (spectrum 1B) in
dichloromethane (DCM). Due to the intense bead colour,
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however, it is effective for a much higher concentration
(range of sensitivity 10ꢁ2–10ꢁ1 M) if compared to what is
observed in a non-aqueous environment (in toluene the range
of sensitivity is 10ꢁ4–10ꢁ3 M). This is not surprising due to the
strong competition of bulk water with diamine for the
coordination to the Zn(II) centers. In addition, at the pH of
analysis, one can assume protonation of the amine, and
consequently, a lower interaction of the ammonium functions
with the Zn(II) center. Nevertheless, the final goal is to access
the gas-phase sensing of the biogenic amines by taking
advantage of the analyte volatility in the head-space of the
sample, where the interference of water is supposed to be less
severe than in aqueous solution. In this light, the tweezer-
based sensing material offers an unprecedented opportunity in
terms of the variety of component assembly, the consequent
modulation of the molecular recognition properties, the
resulting performance and vis-a-vis micro-fluidic applications.
In summary, we have provided a convenient methodology
to synthesize and covalently support melamine-bridged
bis-porphyrin tweezers onto aminated materials such as
TG-NH2 beads and CPG-NH2. These materials are suitable
for diamines sensing, as exemplified in the case of cadaverine,
a biologically active diamine involved in food freshness
monitoring. Efforts to apply these systems to real-life
problems and for the construction of smart-packaging sensors
are currently ongoing in our laboratory.
Fig. 2 Single bead Uv–vis analysis of cadaverine (0.1 mM) by 5 under
flow-conditions (1 mL minꢁ1) in toluene.
Another feature that needs to be tested for a supported
sensor is its reversibility, especially in terms of time required to
recover to the initial state after the sensor has been exposed to
the analyte. This issue has been addressed within a microflow
cell by placing a sequence of two beads within a 400 mm square
glass capillary, one bead of 5 and a second one of the pristine
TentaGel resin (for zeroing purposes). The flowcell is
completed by inserting two fused silica capillaries that act
both as inlet and outlet for the microfluidic circuit and as
stoppers to keep the two beads in place (see Electronic
Supplementary Information, ESIz). Continuous monitoring
of the sensing bead is performed at 565 nm while a solution of
cadaverine (0.1 mM in toluene) and toluene are alternatively
infused at 1 mL minꢁ1 flow-rate (Fig. 2).
Financial support for this project came from the University
of Padua (Progetti di Ricerca di Ateneo-CPDA088228/08 and
Progetti Strategici 2008-HELIOS).
The time course of the flow analysis shows that the sensing
process by 5 is reproducible, reversible and effective for a
sub-millimolar concentration of the analyte. In addition, the
sensor response is rapid (less than a minute) and complete
recovery to the initial state upon solvent fluxing takes place in
a few minutes.
Notes and references
y For a related use of the melamine scaffold see also refs. 15 and 16.
1 M. H. S. Santos, Int. J. Food Microbiol., 1996, 29, 213–231.
2 S. Moret, D. Smela, T. Populin and L. S. Conte, Food Chem., 2005,
89, 355–361.
As a further upgrade, diamine sensing with the tweezer-
based sensor has been applied also in water, in order to
evaluate its potential towards aqueous matrices typically
found in food analysis. Unfortunately, the TentaGel beads
become translucent when swollen in phosphate buffer solution
at pH = 7.4, thus hampering the Uv–vis detection.
3 See for example: L. Mure-san, R. R. Valera, I. Frebort, I. C. Popescu,
´
E. Csoregi and M. Nistor, Microchim. Acta, 2008, 163, 219–225.
¨
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L. A. Villaescusa, K. Rurack, J. M. Barat, I. Escriche and
Pedro Amoros, Chem. Commun., 2006, 2239–2241.
´
a-Acosta, M. Comes, J. L. Bricks, M. A. Kudinova,
´
´
´
´
As an alternative support, controlled pore glass (CPG) has
been considered. This material, already tested with success for
pH sensing14 in aqueous conditions, is commercially available
in different pore sizes and can be easily functionalized with
amino-groups by reacting the CPG with 3-aminopropyl-
trimethoxysilane in dry toluene.14 In turn, the CPG-NH2 can
be functionalized with the porphyrin dimer as accomplished
for the TG-NH2 resin (Scheme 1, Path B). Also in this case, the
residual amino-groups of CPG-NH2 have been capped by
reaction with acetic anhydride.
6 G. Lu, J. E. Grossman and J. B. Lambert, J. Org. Chem., 2006, 71,
1769–1776.
7 C. Di Natale, G. Olafsdottir, S. Einarsson, E. Martinelli, R. Paolesse
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analyzed under flow conditions with a properly designed
apparatus (see ESIz) based on an optical fiber, so as to exploit
the scattered light signal provided by the sensing element for
analytical purposes. In particular, a microwell containing
the CPG sensor has been fluxed with buffered solutions
(phosphate buffered at pH 7.4) at different concentrations of
cadaverine. The sensor responds reversibly to the diamine,
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14 M. Bacci, F. Baldini and S. Bracci, Appl. Spectrosc., 1991, 45,
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