Spirolactonized Si-rhodamine: A novel NIR fluorophore utilized as a platform to construct Si-rhodamine-based probes

Article abstract of DOI:10.1039/c2cc34159j

Spirolactonized Si-rhodamine was prepared as a platform to construct Si-rhodamine-based probes by following the design strategy widely used in rhodamine systems. Among them, the reaction-based probe SiR-Hg was operated for NIR sensing and bioimaging of Hg

Full text of DOI:10.1039/c2cc34159j

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Spirolactonized Si-rhodamine: a novel NIR fluorophore utilized as a
platform to construct Si-rhodamine-based probesw
Ting Wang,a Qing-Jie Zhao,a Hong-Gang Hu,a Shi-Chong Yu,a Xiang Liu,b Li Liu*b and
Qiu-Ye Wu*a
Received 10th June 2012, Accepted 12th July 2012
DOI: 10.1039/c2cc34159j
Spirolactonized Si-rhodamine was prepared as a platform to con-
struct Si-rhodamine-based probes by following the design strategy
widely used in rhodamine systems. Among them, the reaction-based
probe SiR-Hg was operated for NIR sensing and bioimaging of
Hg2+ in living cells based on the similar irreversible spirolactam
ring-opening process to traditional rhodamine derivatives.
low background. Unfortunately, modifying the specific spiro-
cycle for other applications is of great difficulty. To address
this problem, we focus on the close analogue of rhodamine B,
spirolactonized Si-rhodamine (SiR). By following the design
strategy widely used in rhodamine systems, spirolactone in SiR
could be utilized as a platform to construct SiR-based probes.
If such a design strategy can be experimentally demonstrated,
it will extend the range of available NIR fluorescent turn-on
probes utilizing the unique ring-opening process of the corre-
sponding spirolactone or spirolactam (Fig. 1).
The design of fluorescent probes for sensing and bioimaging of
important species has emerged as a significant research area.1
Among the probes developed, the rhodamine spirolactam based
fluorescent probes have already attracted great interest due to
the valuable characteristics of rhodamine derivatives, such as
high molar extinction coefficient, large fluorescence quantum
yield and a unique ring-opening process with turn-on signals.2
However, the absorption and emission of most rhodamine
derivatives are located in the visible range (o600 nm), which
limits their further applications for bioimaging. In comparison
to visible fluorescent probes, near-infrared (NIR, 650–900 nm)3
fluorescent probes are expected to be superior for bioimaging
because light in this wavelength region shows less overlap with
the spectrum of background autofluorescence, lower light
scattering, deeper tissue penetration and less phototoxicity.3
Thus, it is highly desirable to develop novel NIR probes for
bioimaging.4 Very recently, a new class of NIR fluorescent dyes
and probes, silicon-substituted rhodamines, has been developed.5
Their excellent photophysical properties qualify them for NIR
sensing and bioimaging. Among the limited examples, most
Si-rhodamine-based probes are controlled by a photoinduced
electron transfer (PeT) mechanism.5c,5d Besides PeT, the
HOCl-induced ring-opening approach applied to sulphur-
involved spirocycles5b is more attractive because of the
chromogenic and fluorogenic turn-on signals with relatively
With this in mind, we reported the synthesis and photo-
physical properties of SiR and Si-rhodamine-hydrazine (SiRH).
Subsequently, SiR-based probes SiR-Cu and SiR-Hg were pre-
pared by introducing phenolic hydroxy and thiosemicarbazide
moieties6 for recognizing Cu2+ and Hg2+, respectively. The
recognition groups are selected for the following reasons: firstly,
the ring-opening mechanisms represent two common ways:
metal-coordination bonding and ion-triggered reaction.
Secondly, it is interesting to make a comparison of properties
between Si-rhodamines and rhodamine analogues. Lastly, there
have been few reports on NIR fluorescent probes for sensing and
bioimaging of these biologically relevant ions.7 In particular, the
ring-opening process accompanied by turn-on NIR signals was
achieved in SiR and SiR-Hg. It clearly demonstrates that SiR has
great potential for developing SiR-based NIR probes.
Although Qian and Nagano et al. have described the synthesis
of several Si-rhodamines,5 an efficient synthetic strategy to
challenge spirolactonized SiR has not yet been reported. As
presented in Scheme 1, a lithiated benzene moiety bearing a
protected carbonyl group was employed to react with compound
a Department of Organic Chemistry, College of Pharmacy, Second
Military Medical University, Guohe Road 325, Shanghai 200433,
P.R. China. E-mail: wuqy6439@sohu.com; Fax: +86-21-81871225
b State Key Laboratory of New drug and Pharmaceutical Process;
Center for Pharmacological Evaluation and Research, Shanghai
Institute of Pharmaceutical Industry, Zhongshanbeiyi Road 1111,
Shanghai 200437, P.R. China. E-mail: liuli1129@hotmail.com;
Fax: +86-21-6544936
w Electronic supplementary information (ESI) available: Synthetic and
experimental details, absorption and fluorescence spectra, Job’s plot
and crystallographic data. CCDC 876103. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c2cc34159j
Fig. 1 Schematic representation of our strategy to develop SiR-based
probes.
c
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Chem. Commun., 2012, 48, 8781–8783 8781

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suitable for bioimaging at physiological pH. In sharp contrast,
SiRH showed no obvious characteristic color or fluorescence
change between pH 1.6 to 12.3 (Fig. 2b), suggesting that SiRH
preferred the nonfluorescent spirocyclic form over a wide pH
range. This characteristic implied that Si-rhodamine amide
derivatives with low background fluorescence satisfied the
criteria for turn-on sensing and bioimaging applications.
Except for the similarities to rhodamine B, some different
properties are observed. It is worth noting that the pH-
dependent spectral profile of SiR changed when the content
of MeCN increased in buffer solution (ESIw, Fig. S5). We also
notice that SiR amide derivatives showed considerable
tolerance to acid (ESIw, Fig. S6 and S7). These facts indicate
that spirocyclic structure in Si-rhodamines is more stable than
that in rhodamine analogues.
Scheme 1 Synthesis of SiR, SiRH, SiR-Cu and SiR-Hg.
As we know, rhodamine-hydrazine (RH) has been proved to
be an efficient chemodosimeter for sensing Cu2+ based on a
Cu2+-induced ring-opening process.9 Thus we reason that the
analogue SiRH owns the same ability to recognize Cu2+ via
the similar mechanism. Surprisingly, the addition of Cu2+
resulted in slight fluorescence enhancement and a yellow color
change with the formation of two absorption bands, the
dominating one centered at 420 nm and the weak one
appeared at 654 nm (ESIw, Fig. S8 and S9). In order to better
understand the interaction, the ESI mass spectra of the
complex were recorded and they displayed two unique peaks
at m/z 497.5 and 1055.5, which may be assigned to the
dehydrogenation product of SiRH (calc 497.3) and the 2 : 1
(SiRH–Cu2+) chelating complex (calc 1055.5), respectively
(ESIw, Fig. S10). In addition, the 2 : 1 binding stoichiometry
was further confirmed by the Job’s plot analysis (ESIw, Fig. S11).
Based on the above findings, we infer that the binding between
SiRH and Cu2+ produces the metal-chelating complex (ESIw,
Fig. S12) with a small amount of ring-opened form, which are
responsible for the observation of short and long absorption
bands, respectively. Furthermore, other metal ions exerted a
little or slow effect on the color change of SiRH under
identical conditions (ESIw, Fig. S13), confirming the selectivity
to Cu2+. Similar results were obtained when probe SiR-Cu
interacted with Cu2+, suggesting the similar Cu2+ binding
behavior to SiRH (ESIw, Fig. S14 and S15).
SiX, finally affording SiR in medium yield after deprotection and
cyclization. Subsequent treatment of SiR with hydrazine hydrate
gave SiRH, which served as a key intermediate to create probes
SiR-Cu and SiR-Hg through convenient chemical modification.
X-Ray crystallographic investigation further confirmed the
existence of the silicon-substituted ring and unique spirolactam
structure in SiR-Hg. To the best of our knowledge, this is the first
example of the X-ray structure of the Si-rhodamine bearing the
spirocyclic moiety (ESIw, Fig. S1).
It is known that the ring-opening of spirolactone or spiro-
lactam in rhodamines gives rise to a pink color and strong
fluorescence emission. Similarly, SiR formed colorless and
nonfluorescent solution in most aprotic organic solvents, but
underwent a reversible blue color change after adding AcOH
or Et3N (ESIw, Fig. S2). This phenomenon implied that a
similar reversible ring-opening process of the corresponding
spirolactone may occur in SiR. We examined the spectral
profile of SiR at different pH values in HEPES buffer solution.
As shown in Fig. 2a and b, the solution of SiR kept a bright
blue color (labs = 650 nm, e = 50 000 MÀ1 cmÀ1) and high
NIR fluorescence emission (lem = 666 nm, f = 0.19) over a
wide pH range from pH 5 to 10, exiting in its zwitterionic
form. However, under more acidic conditions (pH o 5), the
blue color solution turned colorless and the fluorescence
quenched significantly. On the other hand, the fluorescence
intensity also decreased at higher pH values (pH > 10). The
fluorescence on–off switching of SiR is very similar to that of
the classic rhodamines.3b,8 Additionally, SiR has sufficient
photostability for potential biological applications (ESIw,
Fig S3). The cell fluorescence imaging as shown in Fig. S4
(ESIw) demonstrated that SiR with desirable features was
The distinct recognition behavior should be attributed to
the stability of spirolactam in Si-rhodamine, which favors the
spirocyclic form rather than the ring-opened form when
interacting with Cu2+ via metal-coordination bonding.
Although probes SiRH and SiR-Cu could perform as colori-
metric probes for turn-on sensing of Cu2+, the unique ring-
opening process with characteristic turn-on NIR fluorescence
emission was not achieved. Thus, we turn our attention to the
reaction-based probe SiR-Hg, the spirolactam in which could
be liberated by the breaking of covalent bonds.10
In HEPES buffer solution at pH 7.4, the free SiR-Hg
formed a colorless solution, indicating that it predominantly
existed in the spirocyclic form. As expected, the addition of
Hg2+ caused an iconic blue color change instantaneously at
room temperature (ESIw, Fig. S16 and S17). Concomitantly,
an intense absorption band appeared at 664 nm (e =
160 000 MÀ1 cmÀ1), suggesting the formation of ring-opened
structure from SiR-Hg (ESIw, Fig. S18). Further evidence was
Fig. 2 (a) The absorption and emission spectra of respective rhod-
amine B and SiR in HEPES buffer solution at pH 7.4. Inset: color
pictures of rhodamine B and SiR. (b) pH-dependence of the fluores-
cence intensity of SiR (circles) and SiRH (triangles).
c
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In conclusion, we have described the first synthesis of the
spirolactonized Si-rhodamine, which undergoes the similar
pH-dependent ring-opening process to its rhodamine analogue
accompanied by chromogenic and fluorogenic turn-on NIR
signals. Taking advantage of this feature, SiR could be utilized
as a platform to construct SiR-based probes by following the
design strategy widely used in rhodamine systems. Among
them, probes SiRH and SiR-Cu preferred the spirocyclic form
when interacting with Cu2+ via the metal-coordination bonding
due to the stability of spirolactam. In contrast, the reaction-
based probe SiR-Hg underwent the irreversible ring-opening
process via efficient desulfurization and cyclization promoted by
Hg2+, giving rise to a blue color and strong NIR fluorescence
emission. Importantly, a similar design strategy is expected to
be applicable to other SiR-based NIR probes for sensing and
bioimaging important species.
Fig. 3 (a) Emission spectra changes of SiR-Hg in the presence of
increasing concentration of Hg2+ in HEPES buffer solution at pH 7.4.
Inset: plot of fluorescence intensity depending on the number of
equivalents of Hg2+. (b) Fluorescence intensities of SiR-Hg upon the
addition of various metal ions. Black bars represent the fluorescence
response of SiR-Hg to the excess metal ion (50 mM) of interest. Gray
bars represent the subsequent addition of 50 mM Hg2+ to the solution.
obtained by comparing the ESI mass spectra (ESIw, Fig. S19).
The unique peak at m/z 600.4 (calcd 600.3) corresponding to
the desulfurized and cyclized product from SiR-Hg was clearly
observed when 1.5 equiv. of Hg2+ was added. Fluorescence
titration of Hg2+ was further conducted (ESIw, Fig. S20). As
depicted in Fig. 3a, with the gradual addition of Hg2+, a
simultaneous enhancement in fluorescence intensity at 680 nm
(f = 0.12) was noticed. The final intensity of SiR-Hg upon
addition of 10 equiv. of Hg2+ was found to enhance by 500
fold, partly due to the extremely low background fluorescence
of SiR-Hg. The selectivity of SiR-Hg observed for Hg2+ over
other ions is very similar to its rhodamine analogue. Various
metal ions did not cause any significant fluorescence intensity
changes. In addition, the enhancement in fluorescence inten-
sity resulting from the addition of Hg2+ was not influenced by
the presence of other metal ions (Fig. 3b).
This research was supported in part by the Natural Science
Foundation of Shanghai City, China (11ZR1446800) and
Shanghai Leading Academic Discipline Project NO. B906.
We also sincerely thank Weiquan Dai for her invaluable help
and Xiaoyan Cui for the useful discussion and suggestion.
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Fig. 4 Cells incubated with SiR-Hg (50 mM) and Hg2+ (50 mM). (a)
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SH-SY5Y cells treated with SiR-Hg in the absence of external
Hg2+. (c) Microscopic image of SH-SY5Y cells treated with both
SiR-Hg and Hg2+. (d) Fluorescence microscopic image of SH-SY5Y
cells treated with both SiR-Hg and Hg2+. Scale bar = 50 mm.
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c
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