Cascading of molecular logic gates for advanced functions: A self-reporting, activatable photosensitizer

Article abstract of DOI:10.1002/anie.201306177

Logical progress: Independent molecular logic gates have been designed and characterized. Then, the individual molecular logic gates were coerced to work together within a micelle. Information relay between the two logic gates was achieved through the intermediacy of singlet oxygen. Working together, these concatenated logic gates result in a self-reporting and activatable photosensitizer. GSH=glutathione. Copyright

Full text of DOI:10.1002/anie.201306177

Angewandte
Chemie
DOI: 10.1002/anie.201306177
Chemical Logic Gates
Cascading of Molecular Logic Gates for Advanced Functions: A Self-
Reporting, Activatable Photosensitizer**
Sundus Erbas-Cakmak and Engin U. Akkaya*
The complexity of biochemical reaction networks usually
arises from the cascade of reactions that control or are
controlled by other reactions in the network. This enables
signal relay between functional biological modules and
generates intricate emerging processes. The same analogy is
also applicable to silicon-based logic operations. Indeed,
complex digital computational processes show similar levels
of dependency on series of simple logic operations, from
which sophisticated algorithms can be effectively produced.
Chemical logic gates, on the other hand, are already able
to perform quite complex tasks,[1] as exemplified by multi-
plexer/demultiplexer,[2] flip-flop logics,[3] subtracter/adder,[4]
or keypad-lock[5] systems. Smart oligonucleotide-based con-
structs playing simple games such as Tic-Tac-Toe have been
reported.[6] Physical cascading (concatenation and integra-
Scheme 1. Working principles for the individual AND and INHIBIT
logic gates.
tion) of a series of simple logic gates with chemical or
electronic communication in between has also been de-
scribed.[7] Nevertheless, the de novo assembly of a practical
and functional logic construct that relies on the conveyance of
signals between each independent module, or between logic
gates as a whole, has not been demonstrated thus far. Herein,
we propose a cascade of two chemical logic gates that
concatenates an acid-activatable photosensitizer and an
activity-reporting moiety through a singlet-oxygen-mediated
information relay.
To enable such an intentional and physical cascading with
modular logic gates, photodynamic action and chemical probe
moieties were considered. As the output of photodynamic
action is singlet oxygen (1O2), we aimed at developing
a reaction that senses this output and thus reports the activity
of the first action. The working principles for the individual
logic operations are shown in Scheme 1. The first logic gate
includes an AND logic operation; 660 nm light and acid are
applied as inputs, and the singlet oxygen produced on
photosensitization is the output. Considering a likely practical
potential, the slightly acidic microenvironment of a tumor
tissue[8] was targeted with the use of an acid-responsive
photosensitizer (PS), as the pKa of the micelle-embedded
photosensitizer has to be optimal for its use in such an
application. The excitation of the PS at the irradiation
wavelength (660 nm) is more pronounced for its protonated
form as a result of the blue shift in the absorption spectrum.
This is our pH-activatable photosensitizer (Scheme 1).
The second logic gate can be considered either as an AND
gate that uses 1O2 (the output of the first logic operation) and
520 nm light as an input, or as an INHIBIT gate with 1O2 and
glutathione (GSH) as the inputs. Glutathione concentrations
have been reported to be much higher in cancer cells than in
normal cells. The design of the second logic gate is such that
two energy-transfer modules are attached to one another
1
using a O2-reactive linker. The linker is (Z)-1,2-bis(alkyl-
thio)ethane, which was reported to have very high reaction
rates with 1O2, and which is easily synthesized.[9] The reaction
1
of this linker with O2 is shown in Scheme 2. Following the
initial addition of 1O2 to the double bond, an unstable
dioxethane is formed, and spontaneous decomposition of this
intermediate produces S-alkyl methanethioate. In aqueous
solutions and in the presence of amines, this compound has
been reported to be easily hydrolyzed to the corresponding
thiols.[9]
[*] Dr. S. Erbas-Cakmak, Prof. Dr. E. U. Akkaya
UNAM-Institute of Materials Science and Nanotechnology
Bilkent University, Ankara, 06800 (Turkey)
E-mail: eua@fen.bilkent.edu.tr
Prof. Dr. E. U. Akkaya
Department of Chemistry, Bilkent University
Ankara, 06800 (Turkey)
[**] S.E.-C. thanks TUBITAK for a scholarship, and the authors thank Mr.
Yusuf Cakmak for fruitful discussions.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201306177.
1
Scheme 2. The reaction of O2 with electron-rich olefins.[9]
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Following the cleavage of the linker, excitation of the
donor results in bright emission, because the acceptor is not in
the vicinity to act as an energy sink. Two energy-transfer
modules and two molecular logic gate constructs are shown in
Scheme 3.
Figure 1. Normalized electronic absorption spectra of Gate 2 (c), A
(b), D (g), Gate 1 (d), and Gate 1 in the presence of
piperidine (l) in THF.
Table 1: Photophysical characterization of Gates 1 and 2 and modules D
and A.
Compound labs [nm] e [mÀ1 cmÀ1
]
lF [nm] fF[a] [lexc(nm)] tF[b] [ns]
Gate 1
Gate 2
645[a]
720
525
654
525
654
40000
30000
85000
93000
70000
76000
667
–
537
670
535
670
0.13
0.00
0.14
0.56
0.96
0.50
1.2
–
3.5
4.4
4.9
4.5
Scheme 3. Independent logic gates and energy donor (D) and acceptor
(A) modules. Tg=methoxytriethyleneglycol.
D
A
The acidic nature of tumor tissue has been known for
some time, but the pH difference between healthy and
malignant regions is not very large.[10] The pH is usually above
six and even close to neutrality for most tumors. Thus, the pH-
activatable PS was selected accordingly, keeping possible
biological applications in mind. With rational modifications,
the pKa of the photosensitizer was chemically adjusted to
slightly acidic values, so that the micellar (Cremophor-EL)
photosensitizer (Gate 1, Scheme 3; see also the Supporting
Information, Figure S1) has a pKa of 6.92 in aqueous
solutions. Electronic absorption spectra of the gates and the
energy-transfer modules in tetrahydrofuran (THF) are given
in Figure 1. The absorption maximum of the deprotonated
form of the micellar photosensitizer (Gate 1) moves from
649 nm to 720 nm in aqueous solution (Figure S2). Further-
more, the compound is essentially non-emissive in this form
(Table 1; see also Figure S3).
[a] Values were obtained in THF. [b] Values were obtained in THF with
piperidine as an additive. [c] The following reference compounds were
used for quantum-yield calculations: For emission-band peaking at
537 nm, Rhodamine 6G (water, fF =0.95, n=1.333) was used, and for
emission-band peaking at 670 nm, Cresyl Violet (MeOH, fF =0.66,
n=1.329) was used.
The spectral shift in absorption and other possible
deactivation processes that are active at alkaline and neutral
pH facilitate the selective sensitization of the PS in acidic
media. Figure 2 shows the singlet-oxygen-generation activity
of the micelle-embedded Gate 1 in slightly acidic (pH ca. 6.0)
and slightly basic (pH ca. 7.5) aqueous solutions, which was
detected by the selective bleaching of 2,2’-anthracene-9,10-
Figure 2. Relative 1O2-generation efficiency of micellar Gate 1 (7.5 mm)
1
diylbis(methylene)dimalonic acid by the produced O2. As
!
~
in aqueous solutions in the presence ( ) or absence ( ) of acid,
detected by the absorption decrease of the trap at 378 nm. The
expected, the results indicate that singlet oxygen is efficiently
generated only under irradiation with 660 nm light in acidic
media. Using the change in trap absorption in the presence of
Gate 1, an AND logic is constructed (Figure 3). The percent-
age decrease in trap absorption at 378 nm was determined as
&
*
changes in the trap absorption in slightly basic ( ) and acidic ( )
solutions (pH=ca. 7.5 and 6.0, respectively) are also shown. During
the first 15 min, the samples were kept in the dark, and for the
following 50 min, the samples were irradiated with 660 nm light using
an LED array.
1
a measure of O2-generation ability, and the threshold rate
2
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Figure 3. AND logic construct of the photosensitizer (Gate 1). The
output (1O2) is measured as a percentage decrease in trap absorption
within 15 min (in the dark or with 660 nm irradiation). The inputs are
660 nm light and acid. The threshold was set at 1.
was set at 1. Taking the values obtained by incubation for
15 min in the dark, or under irradiation with 660 nm light, in
either slightly acidic or basic aqueous solutions, the AND
logic shown in Figure 3 was obtained. The output levels
clearly demonstrate that the photosensitizer is activated in
acidic media with essentially no dark reaction.
The second part of the logic-gate cascade was developed
using the 1O2 produced (the output of the first AND gate) and
either 520 nm light or GSH as inputs. To analyze the cascade,
two compounds (corresponding to Gates 1 and 2) were
embedded together within a micelle in a mole ratio of
approximately 1:1. The concentrations were estimated by
comparison of the absorption values with the THF absorptiv-
ities. Micellar constructs of Gates 1 and 2 were prepared for
the analysis of the second logic gate and D2O was used to
enhance the lifetime of 1O2.
Figure 4. Comparison of the emission spectra of a) equally absorbing
Gate 2 (c) and the EET donor (D, b) excited at 520 nm, and b)
of Gate 2 (g) and the EET acceptor (A, d) excited at the same
wavelength in THF.
A comparison of the emission spectra of Gate 2 with that
of the energy-transfer module shows that the emission of the
excitation energy transfer (EET) of the donor part D at
537 nm is considerably quenched in Gate 2. This was further
supported by a decrease in the quantum yield of the donor
emission from 0.96 to 0.14 (Figure 4, Table 1). On the other
hand, the EET acceptor module A cannot be excited at
520 nm to produce an emission at 670 nm, whereas Gate 2
shows intense emission at this wavelength upon excitation at
520 nm (Figure 4b). Both of these spectroscopic results
indicate an efficient EET process between the modules with
a calculated efficiency of 85.0%. An efficiency of less than
one unit in this process is probably due to poor spectral
overlap between the donor and acceptor moieties. Excitation
spectra also confirm that the acceptor emission is a result of
the excitation of both donor and acceptor moieties (Figure 5).
In contrast, excitation spectra of the acceptor A do not
display any peaks around 524 nm, when emission at 670 nm
followed.
Figure 5. Comparison of the excitation spectra of the equally absorbing
compounds A (b) and Gate 2 (c) for emission at 670 nm in
THF.
As the PS produces singlet oxygen only in the presence of
acid, acidic conditions are interpreted as the presence of O2
in the dark do not display any change in the emission of the
EET donor module of Gate 2 at 537 nm (Figure S4). Like-
wise, under slightly basic conditions, no significant change in
the emission is observed because 1O2 is not generated
1
at the second logic gate. Liberation of the EET donor upon
the reaction of Gate 2 with 1O2 is expected. The samples kept
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efficiently under these conditions (Figures S5 and S6). How-
ever, when acid is present to activate the PS, a more than
twofold increase in the emission intensity of the donor at
537 nm indicates that it has been detached from the energy
acceptor module; the increase in the emission seems to reach
almost a constant value (Figure 7). Nevertheless, the final
process is still effective to some extent, owing to Gate 2
molecules that had remained uncleaved thus far. This result
may be due to a decrease in the amount of oxygen dissolved in
the solution after a certain time, which leads to a decrease in
the reaction rate. In actual biological media, the oxygen flow
within blood and tissue is usually under homeostasis, and thus
essentially constant.
The overall results for the fluorescence enhancement in
the presence of acid or 660 nm light are given in Figure 7. The
singlet-oxygen-mediated fluorescence enhancement at
537 nm is apparently more pronounced in the presence of
1O2, which is efficiently produced in the presence of 660 nm
light in acidic aqueous media.
To make sure that the resultant increase is not solely due
to irradiation, or to test the stability of Gate 2 under
irradiation with the same experimental conditions, a micelle
containing only this compound (Gate 2) was tested in
aqueous solutions. The results show that the increase in the
emission of the donor in the absence of the photosensitizer is
essentially zero (Figure S8), which indicates that the main
1
contributor to the enhancement at 537 nm is O2.
Considering all of the above results, the second AND
logic was constructed. As an excitation at 520 nm is required
for the second logic gate, the absence of light at this
wavelength corresponds to a zero output (no excitation).
The results are depicted in Figure 8, with an assigned
threshold value of 0.3.
Figure 6. Changes in the emission spectra of Gate 2 (0.75 mm) in the
presence of Gate 1 (0.75 mm) in D2O upon irradiation with 660 nm
light for 50 min. The spectra of the samples were recorded under
excitation at 520 nm at 10 min intervals.
quantum yield is smaller than the quantum yield of the free
EET donor D, and the excitation spectra do not display
a significant decrease for the donor part (Figure S7). By
comparing the quantum yields of the free EET donor D and
Gate 2 after light irradiation (660 nm) for 50 min, a cleavage
of 18% can be estimated. This is already enough to obtain an
intense fluorescence output (Figure 6). The quantum yield of
the donor emission within the micellar construct increases
from 0.14 to 0.29, and the increase in the emission almost
seems to level out (Figure 7). This could indicate that the EET
Figure 8. Construction of the second AND logic gate with light
(520 nm) and acid as the inputs; the output is reported as a fractional
change in the emission intensity at 537 nm. The threshold was set at
0.3.
Although light has been applied as an input in a number of
logic-gate designs in the literature, this input may seem trivial,
as the excitation is required anyway for any fluorescence
process. Therefore, an alternative input was chosen for the
second gate, namely glutathione. High levels of this molecule
lead to resistance against photodynamic therapy (PDT)
because of its properties as a singlet oxygen scavenger.[11] To
suppress the PDT-deactivating property of GSH at high
concentrations, researchers use chemicals to decrease the
biological production of this molecule.[12] Herein, GSH was
used as the input because of its role as a singlet oxygen
Figure 7. Changes in the fluorescence at 537 nm of the micellar
Gates 1 and 2 in D2O for different combinations of conditions: dark
~
&
*
and basic ( ), light and basic ( ), dark and acidic ( ), and light and
!
acidic ( ). Light was supplied by a 660 nm LED source. All four
samples were excited at 520 nm.
4
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likely to find their first convincing application in a health-
related niche. Further work along these lines is expected to
produce such information-processing and smart therapeutic
agents.
Received: July 16, 2013
Published online: && &&, &&&&
Keywords: energy transfer · fluorescence ·
.
photodynamic therapy · photosensitizers · singlet oxygen
[1] a) A. Credi, Angew. Chem. 2007, 119, 5568 – 5572; Angew. Chem.
Int. Ed. 2007, 46, 5472 – 5475; b) J. Andrꢀasson, U. Pischel,
Chem. Soc. Rev. 2010, 39, 174 – 188; c) K. Szacilowski, Chem.
Soc. Rev. 2008, 108, 3481 – 3548; d) S. Kou, H. N. Lee, D.
van Noort, K. M. K. Swamy, S. H. Kim, J. H. Soh, K.-M. Lee, S.-
W. Nam, J. Yoon, S. Park, Angew. Chem. 2008, 120, 886 – 890;
Angew. Chem. Int. Ed. 2008, 47, 872 – 876.
[2] a) J. Andrꢀasson, S. D. Straight, S. Bandyopadhyay, R. H.
Mitchell, T. A. Moore, D. Gust, Angew. Chem. 2007, 119, 976 –
979; Angew. Chem. Int. Ed. 2007, 46, 958 – 961; b) M. Amelia, M.
Baroncini, A. Credi, Angew. Chem. 2008, 120, 6336 – 6339;
Angew. Chem. Int. Ed. 2008, 47, 6240 – 6243; c) E. Perez-
Inestrosa, J. M. Montenegro, D. Collado, R. Suau, Chem.
Commun. 2008, 1085 – 1087; d) S. Erbas-Cakmak, O. A. Bozde-
mir, Y. Cakmak, E. U. Akkaya, Chem. Sci. 2013, 4, 858 – 862.
[3] a) G. de Ruiter, L. Motiei, J. Choudhury, N. Oded, M. E.
van der Boom, Angew. Chem. 2010, 122, 4890 – 4893; Angew.
Chem. Int. Ed. 2010, 49, 4780 – 4783; b) P. Remꢁn, M. Balter, S.
Li, J. Andreasson, U. Pischel, J. Am. Chem. Soc. 2011, 133,
20742 – 20745.
Figure 9. INHIBIT logic gate with the inputs 1O2 and GSH; the output
is emission at 537 nm.
scavenger. For efficient impact on the 1O2 produced, the GSH
level should be low. The results of a fluorescence enhance-
ment at 537 nm under different conditions are shown in
Figure S9.
The results in Figures S9 and 9 show that the presence of
GSH interferes with fluorescence enhancement. This data
1
may be interpreted as follows: GSH acts as a O2 scavenger
1
and decreases the O2 level; thus, the rate of fluorescence
[4] a) D. H. Qu, Q. C. Wang, Q. H. Tian, Angew. Chem. 2005, 117,
5430 – 5433; Angew. Chem. Int. Ed. 2005, 44, 5296 – 5299; b) M.
Semeraro, A. Credi, J. Phys. Chem. C 2010, 114, 3209 – 3214;
c) D. Margulies, G. Melman, A. Shanzer, J. Am. Chem. Soc. 2006,
128, 4865 – 4871.
enhancement is diminished. The lack of a quantitative
decrease in the fluorescence enhancement may be due to
limited diffusion of GSH to the interior of the micelle within
the duration of experiment (50 min). However, an appropri-
ate choice of threshold still allows the construction of an
INHIBIT logic gate.
[5] D. Margulies, C. E. Felder, G. Melman, A. Shanzer, J. Am.
Chem. Soc. 2007, 129, 347 – 354.
[6] M. N. Stojanovic, D. Stefanovic, Nat. Biotechnol. 2003, 21, 1069 –
1074.
In conclusion, we were able to develop a modular cascade
of independently functioning logic gates (Figure 10), and
a logical construct with a higher function was thus obtained.
An AND logic gate was used to control the activity of a PS
with acid and light (Gate 1). The output of this gate, which is
1O2, was used by another logic gate, either another AND gate
or an INHIBIT gate, depending on the choice of input. By
means of this concatenation, the two gates were designed to
communicate with each other, such that the activity of a pH-
activatable PS was monitored by the increase in the emission
of a fluorescent reporter within the same micellar structure.
This result supports the idea that molecular logic gates are
[7] a) G. de Ruiter, E. Tartakovski, N. Oded, M. E. van der Boom,
Angew. Chem. 2010, 122, 177 – 180; Angew. Chem. Int. Ed. 2010,
49, 173 – 176; b) V. Balzani, A. Credi, M. Venturi, ChemPhys-
Chem 2003, 4, 49 – 59; c) R. Guliyev, S. Ozturk, Z. Kostereli,
E. U. Akkaya, Angew. Chem. 2011, 123, 10000 – 10005; Angew.
Chem. Int. Ed. 2011, 50, 9826 – 9831; d) T. Gupta, M. E.
van der Boom, Angew. Chem. 2008, 120, 2292 – 2294; Angew.
Chem. Int. Ed. 2008, 47, 2260 – 2262; e) T. Gupta, M. E.
van der Boom, Angew. Chem. 2008, 120, 5402 – 5406; Angew.
Chem. Int. Ed. 2008, 47, 5322 – 5326; f) S. Silvi, E. C. Constable,
C. E. Housecroft, J. E. Beves, E. L. Dunphy, M. Tomasulo, F. M.
Raymo, A. Credi, Chem. Eur. J. 2009, 15, 178 – 185; g) S. Silvi,
E. C. Constable, C. E. Housecroft, J. E. Beves, E. L. Dunphy, M.
Tomasulo, F. M. Raymo, A. Credi, Chem. Commun. 2009, 1484 –
1486.
[8] M. G. Vander Heiden, L. C. Cantley, C. B. Thompson, Science
2009, 324, 1029 – 1033.
[9] A. Rotaru, A. Mokhir, Angew. Chem. 2007, 119, 6293 – 6296;
Angew. Chem. Int. Ed. 2007, 46, 6180 – 6183.
[10] R. J. Gillies, N. Raghunand, G. Karczmar, Z. Bhujwalla, J. Magn.
Reson. Imaging 2002, 16, 430 – 450.
[11] T. P. Devasagayam, A. R. Sundquist, P. Mascio, S. Kaiser, H.
Sies, J. Photochem. Photobiol. B 1991, 9, 105 – 116.
[12] F. Jiang, L. Lilge, M. Belcuig, G. Singh, J. Grenier, Y. Li, M.
Chopp, Lasers Surg. Med. 1998, 23, 161 – 166.
Figure 10. Two different logic-gate cascades. Two functional logic-gate
modules are coerced to work together, yielding useful information
about the rate of singlet oxygen production.
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Communications
Chemical Logic Gates
S. Erbas-Cakmak,
E. U. Akkaya*
&&&& — &&&&
Cascading of Molecular Logic Gates for
Advanced Functions: A Self-Reporting,
Activatable Photosensitizer
Logical progress: Independent molecular
logic gates have been designed and
characterized. Then, the individual
molecular logic gates were coerced to
work together within a micelle. Informa-
tion relay between the two logic gates was
achieved through the intermediacy of
singlet oxygen. Working together, these
concatenated logic gates result in a self-
reporting and activatable photosensitizer.
GSH=glutathione.
6
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Angew. Chem. Int. Ed. 2013, 52, 1 – 6
These are not the final page numbers!