The utilization of pH sensitive spirocyclic rhodamine dyes for monitoring D-fructose consumption during a fermentation process

Article abstract of DOI:10.1039/c3nj00613a

The colorimetric and fluorometric detection of d-fructose was achieved by employing a two component sensing system composed of an arylboronic acid as the host molecule and a pH sensitive spirocyclic rhodamine dye as the indicator molecule.

Full text of DOI:10.1039/c3nj00613a

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The utilization of pH sensitive spirocyclic rhodamine
dyes for monitoring D-fructose consumption during a
fermentation process†
Cite this: NewJ.Chem., 2013,
3
7, 2632
Received (in Montpellier, France)
8
th June 2013,
¨
˘
Erman Karaku-s, Muhammed Uç u¨ nc u¨ , Ritchie C. Eanes and Mustafa Emrullahoglu*
Accepted 9th July 2013
DOI: 10.1039/c3nj00613a
www.rsc.org/njc
The colorimetric and fluorometric detection of D-fructose was achieved
by employing a two component sensing system composed of an
arylboronic acid as the host molecule and a pH sensitive spirocyclic
rhodamine dye as the indicator molecule.
Rhodamine dyes are well-known fluorophores with remarkable Scheme 2 Diol adduct of arylboronic acid.
photophysical properties, such as long absorption and emission
wavelengths, high fluorescence quantum yields and large
2
,3
(
i.e. in solution, atmosphere and living cells). Relying on their
absorption coefficients. The unique ring opening equilibrium
of their spirocyclic derivatives has been widely utilized in the
promising chemical and photophysical properties we envisioned
that pH sensitive rhodamine dyes could serve as pH indicators in
a saccharide sensing system, based on a boronic–saccharide
affinity pair. It is well known that boronic acids show high
affinities for diols. A boronic acid reversibly forms a cyclic
boronate ester with diols (Scheme 2), and in aqueous media
the formation of the boronate ester is accompanied by an
1
design of turn-on type fluorescent sensors. The rhodamine
spirolactam can exist in two isomeric forms, depending on the
medium pH (Scheme 1). The rhodamine spirolactam in its
closed form is non-fluorescent at neutral pH; however, the
structure undergoes a change from the spirolactam to an open
ring amide at lower pH (less than 5.0), resulting in a visibly
4
4
2
increase in hydronium ion concentration (i.e. lowered pH).
colorful and highly fluorescent rhodamine amide.
It was our aim to develop a general sensing strategy that
allows one to monitor the binding of a target saccharide to
simple unmodified boronic acids both colorimetrically and
fluorometrically. Considering the high sensitivity of spirocyclic
rhodamine dyes to pH changes we designed a two component
sensing system that employs an aryl boronic acid molecule as
the host and a rhodamine B spirolactam dye as the signal
reporter.
The ability of rhodamine based dyes to change their color and
fluorescence in response to pH changes has been extensively
exploited in monitoring the pH changes in various environments
Practical examples of sensors that are based on this sensing
5
,6
principle have been reported recently. The majority of the
reported studies, however, have been focused on the colorimetric
5
a,b
Scheme 1 Two isomeric forms of rhodamine B spirolactam.
responses of the indicator dyes.
Examples of detection
methods that employ pH sensitive fluorophore indicators are
6
quite rare. In general, fluorometric detection methods are
:
Department of Chemistry, Faculty of Science, Izmir Institute of Technology
more sensitive than colorimetric detection methods in terms
of both the detection limit and signal magnitude. In this
regard, a method based on fluorometric detection might be
well suited to meet the need for the highly efficient analysis of
saccharide-containing samples.
(
IZTECH), Urla 35430, Izmir, Turkey. E-mail: mustafaemrullahoglu@iyte.edu.tr;
Fax: +90 232 750 7509; Tel: +90 232 7507610
Electronic supplementary information (ESI) available: Synthesis and character-
ization of probe 1, and all data for UV-vis and fluorescence titrations. See DOI:
0.1039/c3nj00613a
†
1
2
632 New J. Chem., 2013, 37, 2632--2635
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Scheme 3 Spirocyclic rhodamine indicators (1a–1c) and arylboronic acid
derivatives.
To the best of our knowledge, a strategy for specifically
exploiting the use of rhodamine spirolactam dyes as pH
indicators for a saccharide sensing system has not been presented
in the scientific literature. Here we report a simple yet highly
efficient method for a naked-eye as well as fluorometric detection
of D-fructose employing a sensing system that utilizes pH sensitive
rhodamine dyes as indicators. In this strategy the rhodamine
spirolactam dye acts as a signal switcher, which is envisioned to
turn-on when the target sugar is bound to arylboronic acid.
The pH indicator probe (1a) was prepared from rhodamine-B
by using a ‘‘one-pot’’ synthetic procedure (POCl , reflux; then
3
amine, NEt , CH CN, see ESI†). The resultant compound (1a)
3
3
was colorless in solution, showed no absorption in the visible
region, and was nonfluorescent, suggesting that the spirocyclic
form of the rhodamine dye is the predominant form (Scheme 3).
In order to check the feasibility of our strategy, we first
examined the binding between phenylboronic acid (PBA) and
D-fructose in the presence of rhodamine (1a) as the signal
reporter. D-Fructose was selected as the substrate to be detected
Fig. 1 (a) Absorption and (b) emission spectra of sensing system SS-1 in water–
methanol (3 : 7, v/v) with increasing concentrations of D-fructose [0, 1.0, 5.0, 10,
2
5, 50, 100, 250, and 500 mM], lex: 525 nm.
in the optimization study as it is known to bind to PBA with attempt to improve the effectiveness of the overall sensing
7
3
high affinity. The sensing system (SS-1) was prepared by process. The results showed p-CF -phenylboronic acid ( p-CF3-
mixing dye 1a (25 mM) and PBA (50 mM) in an aqueous- PBA) to be the most efficient host among those tested ( p-CH3-
methanolic solution. The water content in the semi-aqueous PBA, p-Br-PBA, p-CH3O-PBA and PBA) and that it caused
media was carefully inspected and a water–methanol (3 : 7, v/v) the most dramatic change in acidity after complexation with
8
mixture was chosen as the reaction medium (Fig. S1, ESI†).
D-fructose (Fig. S6, ESI†). Moreover, in the absence of a boronic
Upon the direct addition of D-fructose (100 mM, 2 equiva- acid derivative, the addition of saccharides did not change the
lents with respect to arylboronic acid) to SS-1, the colorless and acidity of the media such that a spectroscopic response might
nonfluorescent solution instantly became pink in color and be observed. Eventually, the sensing system employing p-CF3-
started to fluoresce under UV light. Fig. 1a shows the absorption PBA as the host molecule and 1a as the indicator dye (SS-2) was
spectrum of SS-1 upon addition of increasing concentrations of discovered to be the most efficient.
D-fructose. It can be seen that the solution of dye (1a) and PBA
In this sensing system, saccharides are indirectly detected by
exhibited essentially no absorption in the visible region. How- the creation of hydronium ions (Scheme 2) and the subsequent
ever, upon addition of D-fructose, a new absorption band protonation of rhodamine spirolactam (Scheme 1), which gives
appeared at 558 nm. The effect of the added D-fructose on the an optical response. Accordingly, detection of saccharides
fluorescence properties was then investigated. Fig. 1b shows how should be dependent upon the absolute hydronium ion
the fluorescence emission of the dye (1a) in SS-1 changes at concentration as is true for any indicator and there would be
578 nm as a function of D-fructose. It can be seen that the an associated useful pH range for rhodamine spirolactam.
solution of SS-1 displays no obvious spectral characteristics in Optimization studies revealed that both the initial and the
the emission spectrum. When D-fructose was added to the final acidity of the media played an important role in the
8
solution (SS-1), a significant enhancement of fluorescence with overall efficiency.
an emission maximum at 578 nm was observed.
We evaluated the effect of the solution acidity on the total
Encouraged by these results we synthesized two alternative efficiency of the sensing process. The fluorescence response of
rhodamine derivatives (1b and 1c) and evaluated their optical SS-2 to the addition of D-fructose under various pH conditions
performance and found that the fluorescence responses of dyes was recorded. We observed that the fluorescence response of
1
b and 1c were relatively weaker than dye 1a (when used in SS-1) SS-2 towards the addition of saccharides increases as the solution
8
(Fig. S3, ESI†).
becomes more acidic, i.e. at pH 5.2. Below pH 5.0, SS-2 is not
With these results in mind, we further examined several useful since even the dye (1a) itself responds to this acidity
8
commercially available arylboronic acid derivatives in an even before the addition of any sugar derivative (Fig. S2, ESI†).
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Fig. 2 Emission intensities of SS-2 (1a/p-CF
3
-PBA (25 mM/5 mM)), towards the
Fig. 3 Emission intensities of SS-2 (1a/p-CF
3
-PBA (25 mM/50 mM) in water–
addition of D-fructose, D-glucose, D-galactose, and D-sucrose [10 mM] in water–
methanol (3 : 7, v/v, pH 5.2).
methanol (3 : 7, v/v, pH 5.2)) throughout the consumption of D-fructose
[100 mM]. Inset photograph: colorimetric monitoring of D-fructose consumption
(
0–120 hours).
On the other hand, at pH > 7.0, the ability of any boronic acid to
decrease the pH after complexation with sugar was not enough
in order to initiate a spectroscopic response (Fig. S2, ESI†).
via fermentation, the pH of each sample solution was adjusted
to an optimum sensing condition (pH 5.2) by adding the
8
D-Fructose can be reliably seen to exhibit a fluorescence required amount of NaOH solution. Then, each sample was
subsequently subjected to the probe solution.
response at concentrations approaching 1 mM for solutions
adjusted to pH 7.0 and at concentrations approaching 0.1 mM
for solutions of pH 5.2. The fluorescence and absorption
profiles of SS-2 upon the addition of increasing concentrations
As displayed in Fig. 3, the fluorescence intensity decreased
dramatically with time which is an indication that D-fructose
was consumed throughout the fermentation process and after
8
120 hours its concentration dropped from about 100 mM to
of D-fructose at pH 7.0 and 5.2 are depicted in Fig. S4 (ESI†).
8
about 0.6 mM based on a standard calibration curve. More-
In order to validate the selectivity of the sensing system
(
SS-2), the spectroscopic response of SS-2 to the most common over, the decrease in the concentration of D-fructose could be
saccharides including D-glucose, D-galactose and D-sucrose was easily monitored by the naked eye (Fig. 3).
In summary, we have shown for the first time that spirocyclic
also investigated. Fig. 2 illustrates the fluorescence profile of
SS-2 after the addition of D-fructose, D-glucose, D-galactose,
and D-sucrose (10 mM of each). Under optimized conditions
rhodamine derivatives can be successfully utilized as pH indicators
for monitoring pH lowering resulting from the complexation
of arylboronic acid with diol containing compounds such as
saccharides. We developed a simple yet highly efficient sensing
strategy for the selective detection of D-fructose employing a two
3
(1a/p-CF -PBA (25 mM/5 mM), water–methanol (3 : 7, v/v,
pH 5.2)), only the addition of D-fructose (10 mM) resulted in a
change in absorbance and fluorescence, whereas almost no
spectroscopic response was observed for any other saccharide component sensing system which relies on the spectroscopic
derivative indicating the exceptional selectivity of this sensing response of the spirocyclic rhodamine dyes to changes in acidity
within the media. Among the tested saccharide derivatives,
D-fructose can be selectively detected both colorimetrically and
fluorometrically in an efficient manner. As a practical applica-
tion we demonstrated the utility of this strategy for monitoring
the consumption of D-fructose throughout a fermentation process.
It is hoped that this sensing system may be further modified such
system to D-fructose.
Selective detection and monitoring of D-fructose throughout
a fermentation process are of great importance for wine
makers. In a wine fermentation process, yeasts convert most
of the sugars such as D-fructose and D-glucose into alcohol and
2
CO . D-Fructose is primarily responsible for the sweetness and
characteristics of the wine, thus rapid and reliable methods for that other target saccharides may be selectively sensed by adjusting
the routine analysis of D-fructose are highly demanded to the type of arylboronic acid and the rhodamine dye.
achieve the desired taste.
Motivated by the obvious fluorescence response and the
unique selectivity of the sensing system towards D-fructose we
Experimental part
performed an experiment to show the utility of this sensing All reagents for synthesis were purchased from commercial
strategy for monitoring the consumption of D-fructose during suppliers (Aldrich and Merck) and used without further purifica-
fermentation. To this end, we prepared a solution of D-fructose tion. Absolute methanol and distilled water were used throughout
(
100 mM) in water to which wine yeast was added. The solution the experiment. The pH was recorded using a WTW InoLab pH
was maintained at 30 1C with continuous stirring for 120 hours 720 precision pH meter (Weilheim, Germany). UV absorption
under an inert atmosphere. spectra were obtained on a Shimadzu UV-2550 Spectrophotometer.
Samples were withdrawn periodically from the fermentation Fluorescence emission spectra were obtained using a Varian Cary
broth, and centrifuged to remove particulate matter. To prevent Eclipse Fluorescence spectrophotometer. The slit width was
any discrepancy arising from the acidic by-products produced 5.0 nm for both excitation and emission. The emission spectra
2
634 New J. Chem., 2013, 37, 2632--2635
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were integrated over the range 525 nm to 700 nm in 2.00 mm
path length quartz cells (0.5 mL volume). All measurements
were conducted at least in triplicate.
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