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Design strategy for photoinduced electron transfer-based small-molecule fluorescent probes of biomacromolecules Wei Zhang, Zhao Ma, Lupei Du and Minyong Li* As the cardinal support of innumerable biological processes, biomacromolecules such as proteins, nucleic acids and polysaccharides are of importance to living systems. The key to understanding biological processes is to realize the role of these biomacromolecules in thte localization, distribution, conformation and interaction with other molecules. With the current development and adaptation of fluorescent technologies in biomedical and pharmaceutical fields, the fluorescence imaging (FLI) approach of using small-molecule fluorescent probes is becoming an up-to-the-minute method for the detection and monitoring of these imperative biomolecules in life sciences. However, conventional small-molecule fluorescent probes may provide undesirable results because of their intrinsic deficiencies such as low signal-to-noise ratio (SNR) and false-positive errors. Recently, small-molecule fluorescent
Received 26th December 2013 Accepted 3rd March 2014
probes with a photoinduced electron transfer (PET) “on/off” switch for biomacromolecules have been thoroughly considered. When recognized by the biomacromolecules, these probes turn on/off the PET
DOI: 10.1039/c3an02379f
switch and change the fluorescence intensity to present a high SNR result. It should be emphasized that these PET-based fluorescent probes could be advantageous for understanding the pathogenesis of
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various diseases caused by abnormal expression of biomacromolecules. The discussion of this successful
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology of Natural Products (MOE), School of Pharmacy, Shandong University, Jinan, Shandong
250012, China. E-mail: [email protected]; Fax: +86-531-8838-2076; Tel: +86-5318838-2076
Zhao Ma is currently studying for a PhD in the Department of Medicinal Chemistry, School of Pharmacy, Shandong University since 2011. His research area involves design, synthesis and bioactivity study of novel uorescent probes for a1-adrenoceptor and reactive oxygen species (ROS). Lupei Du is currently an associate professor at the School of Pharmacy, Shandong University. She obtained her PhD degree from China Pharmaceutical University in 2006, and got her postdoctoral training at the Department of Chemistry, Georgia State University (2006–2009). She joined Shandong University in 2009. Her main research interests include the rational design and synthesis of medicinal molecules and bioactive probes. Minyong Li is a professor at the School of Pharmacy, Shandong University. He received his PhD degree from China Pharmaceutical University in 2005. He began his academic career in 2005 with Dr Binghe Wang at the Department of Chemistry, Georgia State University as a postdoctoral research associate. From 2007 to 2009, he was a research assistant professor at the Department of Chemistry, Georgia State University. In 2009, he moved to his current institution as a professor. His research interests are in the general areas of medicinal chemistry and chemical biology. Wei Zhang received his bachelor's degree at the School of Pharmacy, Shandong University in 2011. He stayed at Shandong University to pursue his master's degree in the Li lab. His research interests mainly focus on the design, synthesis and bioactivity study of uorescent probes for a1-adrenoceptor. This journal is © The Royal Society of Chemistry 2014
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Critical Review strategy involved in this review will be a valuable guide for the further development of new PET-based small-molecule fluorescent probes for biomacromolecules.
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1. Introduction Biomacromolecules that include proteins, nucleic acids and polysaccharides oen play pivotal roles in living systems, which cause a wide variety of diseases in the case of abnormal expression. Although the structures of many biomacromolecules are successfully analyzed, it remains difficult to understand their complicated positions in biological processes. In view of the vivid visualization of biomolecules, uorescence imaging (FLI) technology has been a practical and ideal method for the detection of biological substances.1 With considerable attempts by scientists, an obvious development of small-molecule uorescent probes for biosystems has been achieved within the past few decades.2,3 FLI can reveal the localization and distribution of intracellular macromolecules, sometimes at the single-molecule level,4–6 which allows us to understand the roles of biomacromolecules in living systems. Fluorescent probes are indispensable tools for bioimaging, and a large number of uorescent probes have been well designed and synthesized so far. Nevertheless, conventional techniques of imaging biomacromolecules with organic dyes or uorescent probes regularly consist of a number of painstaking steps. In the concept to design small-molecule uorescent probes for bioimaging, the photoinduced electron transfer (PET) effect is one of the most widely used sensing mechanisms.7–9 Recently, numerous PET-based uorescent probes have been developed for biomacromolecule imaging. In general, this type of probe is structurally divided into two parts: a uorophore and a quencher. The uorescence of the probe is suppressed by the quencher under free conditions and can then be released upon binding by blocking the PET process when the quencher reacts with the target substrate. By using these PET-based uorescent probes, the localization, distribution and conformational change of target molecules can be conveniently unveiled. Compared with those regular probes without uorescence switches, PET-based probes can easily achieve a high signal-tonoise ratio (SNR), which is a tremendously decisive factor for in vitro and in vivo uorescence imaging. The present review article summarizes the PET-based uorescent probes for biomacromolecules and their applications in bioimaging. A brief discussion on the advantages of uorescence imaging will be presented herein, as well.
in emission intensity is called uorescence quenching. As one of the mechanisms that can result in uorescence quenching, the PET effect is a widely used method to diminish the uorescence of uorescent probes.7,8,10,11 A typical PET system consists of an aromatic uorophore, an aliphatic amine and a short methylene chain as the linker.12 In other words, a PET-based uorescent sensor for biomacromolecules oen includes three parts: a receptor that serves as an electron donor or a quencher, a uorophore that acts as the electron acceptor, and a spacer that links these two parts. In this system, the intramolecular electron transfer from the receptor to the uorophore will lead to uorescence quenching. Nevertheless, when the receptor binds upon its targets, this PET process is restricted or totally suppressed, and as a result, the sensor regains its uorescence13,14 (Fig. 1). The mechanism of PET-based uorescent probes can be explained by frontier orbital theory (Fig. 2).12,15–18 When the uorophore is excited by an appropriate light wavelength, the electron of the highest occupied molecular orbital (HOMO) is transferred to the lowest unoccupied molecular orbital (LUMO). Because the receptor is adjacent to the uorophore and the HOMO energy level of the receptor is between the LUMO and HOMO levels of the uorophore, the electron transfers from the HOMO of the receptor to the HOMO of the uorophore which prevents the electron in the LUMO of the uorophore from returning to the HOMO, and then the PET effect occurs to quench the emitted uorescence. When the receptor binds upon the target, the HOMO energy of the receptor changes to be lower than the HOMO energy level of the uorophore. Therefore, the PET process is blocked, and the uorescence is recovered.
Fig. 1
The mechanism of PET-based fluorescent probes.
Fig. 2
Frontier orbital theory for the PET effect.
2. Photoinduced electron transfer (PET) effect When irradiated by ultraviolet or visible light, certain substances can emit light with different wavelengths and intensities, and if the irradiation is stopped, the emitted light will cease instantaneously. This emitted light is known as uorescence. The intensity of the uorescence emission can be weakened by a large number of methods and such a reduction
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3. The advantage of PET-based fluorescence imaging (FLI) FLI is one of the most powerful techniques for medical imaging. Compared to other imaging methods, such as X-ray radiography, computer tomography (CT), magnetic resonance imaging (MRI) and ultrasonography (US), FLI is more convenient, specic, sensitive, faster-responding and is more easily handled. In addition, FLI has been successfully implemented in endoscopy19 and surgery.20,21 However, overall, FLI has some inadequacies, such as a low SNR and the tendency for false-positive errors. These defects can be overcome by using “switchable” uorescent probes, for example, PET-based probes. Switchable uorescent probes can be turned on only when specically bound to the targets of interest, otherwise the probe uorescence is quenched. Consequently, the imaging result with a particularly high targetto-background ratio will be presented. PET-based FLI can monitor biomolecules of interest noninvasively in living systems with high spatial and temporal resolution, as opposed to other imaging techniques. Moreover, this PET-based FLI has a high potential for no-wash live-cell imaging, thus enabling the rapid identication of biomacromolecules in living cells with a high SNR.22 The combination of these advantages would contribute to conveniently revealing the biological roles that the biomolecules of interest perform within complex organisms. As a result, PET-based FLI is effective enough to promote the development of life sciences, which will be generally considered in the future of medical imaging.
4. PET-based small-molecule fluorescent probes 4.1
The uorophores in PET-based probes
A wide range of small-molecule uorophores with several core structures, including BODIPY, uorescein, rhodamine, cyanine, naphthalimide and coumarin (Fig. 3), ranging from nearinfrared (NIR) light to blue light, can be applied in designing a PET-based uorescent probe. These commercially available uorophores are hydrophilic and contain no net charge, so as to be reasonable candidates for in vivo imaging.
Fig. 3 Small-molecule fluorophores including BODIPY, fluorescein, rhodamine, cyanine (Cy7), naphthalimide and coumarin.
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A successful PET-based uorescent probe not only depends on a high sensitivity and selectivity towards the target, but also relies upon the appropriate choice of uorophore. The appropriate uorophore is bonded to the selected recognition group through chemical reactions to form a PET-based uorescent probe. For example, Tang and co-workers designed a PET-based probe for copper(II).23 N-Hydroxyacetamide was chosen as the Cu2+ receptor and tricarbocyanine (Cy) as the NIR uorophore. This was the rst PET-based NIR uorescent probe for Cu2+ and was successfully applied to living cells and tissues to visualize Cu2+.
4.2
PET-based probes for enzymes
PET-based uorescent probes are particularly protable for the sensitive and selective monitoring of target enzyme activities. In a typical case, a PET-based probe can be recognized by a specic enzyme to cleave the PET quencher, which leads to the disappearance of the PET effect. As a result, this system recovers to release the uorescence. Based on this PET mechanism, so far various uorescent probes have been well developed for manifesting the functional role of enzymes in the physiological environment. 4.2.1 Glutathione S-transferase. As a kind of the dimeric enzyme related to detoxication reactions,24 glutathione Stransferase (GST) is involved in a large number of biological processes, such as drug metabolism, protecting our body against endogenous reactive oxygen species (ROS) and xenobiotic biotransformation.25 In other words, GST can catalyze the reaction of reduced glutathione (GSH) with the targets, including both exogenous and endogenous molecules. Recently, Nagano et al. designed and synthesized an “off/on” uorescent probe for GST based on the PET mechanism.26 On the basis of 3,4-dinitrobenzanilide (NNBA) as a specic substrate for GST, they prepared some uorogenic substrate probes (DNAFs). DNAFs have excellent kinetic parameters on releasing uorescence catalyzed by GST. However, these DNAFs were membrane-impermeable which is inappropriate for living cells. Therefore, the authors designed and synthesized another uorogenic substrate, DNAT-Me, which expressed a dramatic uorescence surge aer GST-catalyzed glutathionylation and subsequent denitration (Fig. 4). This was available for imaging GST activities in living cells (Fig. 5). These results indicated that DNAT-Me was a suitable uorogenic substrate for uorescence imaging of the intracellular distribution of GST and also for research on GST activities, including drug resistance in cancer cells and the detection of GST-overexpressing pre-neoplastic foci in diagnosis. This design strategy would serve as a direction for developing probes on the structural basis of enzyme substrates in living cells. 4.2.2 Alkaline phosphatase. Alkaline phosphatase (ALP) is a hydrolase with high sensitivity towards numerous types of target molecules by removal of the phosphate groups.27 A conventional utilization of ALP is used to conjugate with a secondary antibody to identify the target protein in Western blot immunoanalysis. The combination of ALP with a uorescent probe can achieve the discernable signal amplication.28
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Fig. 4 DNAT-Me catalyzed by GST to release fluorescence.
Fig. 5 Confocal microscopic imaging of DNAT-Me and nuclearspecific dye SYTO-loaded HuCCT1 cells. The results displayed the imaging efficacy of DNAT-Me to visualize the GST activity in HuCCT1 cells. (Reprinted with permission from ref. 26. Copyright 2008, American Chemical Society.)
However, the application of available uorescent probes to Western blot analysis is limited at the present time. Recently, Nagano et al. designed and synthesized an “off/on” uorescent probe for ALP based on the PET mechanism that is suitable for Western blot analysis.29 They obtained almost non-uorescent TG-Phos by linking the phosphate group to the phenolic group of 2-Me-4-OMe TG, which could be hydrolysed by ALP to release strong uorescence as well as conrm its high affinity for Western blots (Fig. 6). It should be underlined that this new probe TG-Phos provided suitable sensitivity to ALP for multicolor labeling. In our opinion, this approach may provide a rapid and practical identication of proteins other than ALP on Western blotting analysis. 4.2.3 b-Galactosidase. As an exoglycosidase that catalyzes the hydrolysis of b-galactosides to generate monosaccharides, b-galactosidase (b-Gal) is widely recognized as a marker enzyme,
Fig. 6 Reaction of TG-Phos with ALP to produce highly fluorescent hydrolyzed products by breaking the PET process.
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in particular as a gene expression marker.30 Though there have been many uorescent substrates for b-Gal,31 most of them have been applied with in vitro systems and only a few in vivo. Recently, Nagano, Urano and colleagues developed a unique, sensitive and membrane-permeable PET-based “off/on” uorescent probe, TG-bGal, for b-Gal.32 TG-bGal has an O-alkylated xanthene as the uorophore and a 3-methoxytoluene moiety as the PET donor. When hydrolyzed by b-Gal, the b-galactoside group was released, and the probe released the uorescence (Fig. 7). In view of its reasonable membrane-permeability, this probe enables imaging of b-Gal activities in living cells in a realtime manner. It needs to be noted that the “off/on” switch of such a probe gives results with a high SNR, which is crucial for cell imaging. Based on these results, this group worked torward examining whether the probe, TG-bGal, was applicable for targeted tumor imaging. However, the results were unsatisfactory because the uorescent hydrolysis product of TG-bGal was hydrophobic, which made the probe easily washed out from cells thus weakening the uorescence. In other words, the probe TG-bGal was not suitable for targeted tumor imaging. Based on the photochemical and photophysical experiments, they modied the previous probe by adding an ester moiety. The new probe, AM-TG-bGal, also based on the PET mechanism, manifests a great uorescence enhancement when hydrolyzed by b-Gal and the ester moiety is subsequently hydrolyzed by widespread intracellular esterases33 (Fig. 8). The nal hydrolysis product is hydrophilic and can be well retained in the cells even if being washed, thus avoiding the loss of uorescence. In tumor imaging experiments, the mouse tumor model was labeled with an avidin–b-galactosidase conjugate and followed by incubation with AM-TG-bGal. Aerwards, the intraperitoneal tumors were clearly visualized in the uorescence imaging. These results indicated the bright prospect for applications of the new probe,
Fig. 7 A highly sensitive fluorescent probe based on the PET mechanism for b-galactosidase.
Fig. 8 Reaction of AM-TG-bGal as a b-Gal and esterase-sensitive probe based on PET.
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such as in video-assisted laparoscopic tumor resection. The design strategy provided a unique method for other researchers on tumor imaging by using a two-step procedure: rstly the localization on target cancer cells of a target enzyme and subsequently its recognition with a highly sensitive uorescent probe, which results in a high tumor-to-background ratio. 4.2.4 Protease. A protease, oen related to a number of disease processes, functions as an enzyme for the removal of a specic peptide from a substrate peptide or protein. Among the proteases, microsomal leucine aminopeptidase (LAP), an exopeptidase to eliminate the N-terminal amino acids of unsubstituted oligopeptides, plays an essential role in tumor metastasis.34 Nagano et al. designed and synthesized some lanthanide-based functional probes for LAP on the basis of the “off/on” PET mechanism.35 Upon enzymatic cleavage of the substrate peptide, a signicant difference in the luminescence intensity was observed (Fig. 9). When the leucine group was removed by LAP, the aniline group acted as a quencher causing the PET process to be switched “on” and then no luminescence could be seen. It should be noted that LAP is an enzyme in association with a wide range of clinical diseases, such as of the liver and pancreas, and, therefore, LAP uorescent probes should be of practical value in clinical diagnosis. This report disclosed a novel strategy for developing PET-based probes for all proteases. 4.2.5 Carbonic anhydrase isozymes IX. Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze rapid physiological reactions including the interconversion of carbon dioxide and water to bicarbonate and protons. These enzymes are involved in various pathological and physiological processes, such as lipogenesis, gluconeogenesis and tumorigenicity. As a kind of transmembrane a-CA isoform, human carbonic anhydrase IX (hCA IX) is recognized as a target of cancer diagnosis and treatment.36 However, hitherto there has been no selective uorescent probe for CA IX. Recently, Qian and co-workers designed and synthesized a highly-sensitive PET-based uorescent probe with an ideal “off/on” switch for CA IX (Fig. 10), which could release strong uorescence when recognized.37 In this case, saccharin was employed as a recognition motif because of its high specicity for CA IX. When the saccharin group is recognized by the target enzyme, the conformation of this probe will change to suppress the PET process and, as a result, the quenched uorescence is released
Fig. 9
Reaction scheme of the LAP probe. Ln ¼ Tb, Eu.
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Fig. 10 Strategy of the fluorescent probe for CA IX with space-folded PET mechanism.
again. It is well known that hCA IX is highly overexpressed in hypoxic cells; in the meantime, SiHa cells were singled out for cell imaging on account of their relatively high expression of endogenous CA IX. Therefore, in cell imaging, two parallel SiHa cells were treated under normoxic and hypoxic conditions at 37 C before incubation with the probe. The imaging results obviously exemplied stronger uorescence in hypoxic cells than in normoxic; in other words, these outputs claried a much higher selectivity towards CA IX than other CA enzymes. In conclusion, the designed probe could be a protable uorescent probe for the detection of CA IX in vitro or in cellulo. In our opinion, this successful strategy could afford a new avenue for the design of PET-based probes through changing the conformation of the probe. 4.2.6 Arylamine N-acetyltransferase 2. Arylamine N-acetyltransferases (NATs) are metabolism enzymes that catalyze the acetylation of aromatic amines or arylhydroxylamines through acetyl-CoA.38 NATs oen play an imperative role in various signicant pharmacological and toxicological reactions, including the detoxication of arylamines by N-acetylation and bioactivation reactions of arylamine carcinogens.39 NAT1 and NAT2 have been reported to be related to several diseases.40 Therefore, the detection of NAT activities is of importance in clinical diagnosis. Because NAT1 is ubiquitously expressed, and NAT2 is located mainly in liver and colorectal tissues, the detection of NAT2 is urgently required for biological study. Nagano et al. introduced a lanthanide complex “off/on” probe that showed high specicity for NAT2 over NAT1.41 They initially synthesized the probe as a product which was obtained from catalysis by LAP35 that we discussed above. The aniline group functioned as the quencher of the probe, but the quenched luminescence by PET could be released aer Nacetylation by NAT (Fig. 11). Hereto, a reasonable probe with an “on/off” switch was established for NAT. With the switch, the probe provided high SNRs. Meanwhile, considering that NATs are imperative metabolism enzymes, the probe is expected to be a useful tool for drug discovery or in clinical diagnosis. The product of one enzyme can be a substrate for another enzyme. Therefore, this strategy can be used to develop two-step continuous uorescent probes for two different enzymes. Xu and co-workers designed and synthesized a near-infrared “off/on” uorescent probe for NAT2 with high sensitivity and selectivity.42 Because of superior tissue penetration and weak
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Fig. 11 Schematic of the novel NAT probe based on the PET strategy.
auto-uorescence interference, such a near-infrared probe has important inuence in studying biological processes, especially those in vivo.23,43 They employed Cy7 as the uorophore and the arylamines as the quencher, which are linked by a sulfur atom to form the PET system, in which the arylamines functioned as the substrates of the NATs. When the probe molecule was catalyzed by NAT to generate an acetylation product, the PET process was blocked and the uorophore released the uorescence (Fig. 12). Beneting from its high sensitivity and selectivity for NAT2, as well as its long emission wavelength, the probe could be applied for the real-time uorescence imaging of NAT2 in vivo. The results clearly demonstrated that the uorescence in the liver is much stronger than in other tissues of mice, which is in line with the high expression of NAT2 in the liver. To conrm the in vivo results, tissue homogenate assays were carried out by incubating the probe with different tissue homogenates. The results also conrmed that the intensity in the liver is the highest of all tissues. Obviously, the nearinfrared probe could be a valuable tool for clinical diagnosis and biochemical analysis. 4.2.7 Cyclooxygenase-2 (COX-2). In the past few years, uorescence imaging has provided a convenient tool for the early diagnosis of cancer because of its high signal-to-background ratio and selectivity. Cyclooxygenase-2 (COX-2) is a biomarker of almost all cancer cell lines, and can be used to distinguish cancer cells from normal ones. Recently, Peng et al. designed and synthesized a novel “off/on” uorescent probe targeted towards COX-2 and localized in the Golgi apparatus of cancer cells.44 The mechanism of the uorescent probe depends on PET: when binding to COX-2 in the Golgi apparatus of cancer cells, the probe changes its conformation from a folded to an
Fig. 12
unfolded state, which restrains the PET process from the uorophore acenaphtho[1,2-b]quinoxaline (ANQ) to the recognition group indomethacin (IMC) through release of the uorescence (Fig. 13). It needs to be noted that such a PET-quenched uorescent probe elucidated high selectivity and sensitivity to COX-2 for uorescence imaging of the Golgi apparatus through normal and two-photon uorescence microscopy. As a result, the probe could be a practical reagent for the early diagnosis of cancers. 4.2.8 Human neutrophil elastase. Human neutrophil elastase (HNE) is widely considered to be associated with several human diseases. Recently, Yang and co-workers designed and synthesized the rst non-peptide-based PET “off/on” uorescent probe which showed a highly specic response for HNE.45 They chose 7-amino-4-triuoromethyl coumarin (AFC) as the uorophore and the pentauoroethylcarbonyl group as the quencher, linked by an amide bond. When catalyzed by HNE, the pentauoroethylcarbonyl group was removed and the uorescence released (Fig. 14). This small PET-based uorescent probe can be easily synthesized and requires no particular storage, compared with peptide-based substrates. Moreover, it can be utilized to develop an efficient and cost-effective uorescent assay to determine the enzymatic activity of HNE. It should be emphasized that this assay will promote the future high-throughput discovery of HNE inhibitors as well as clinical diagnosis of elastase-related diseases. 4.2.9 Serum albumin. Nagano and co-workers discovered that the solvent-dependent change of uorescence on/off switch of BODIPY derivatives is because of the inuence of environmental polarity on the PET process. On the basis of this nding, they successfully prepared an environmentally-sensitive uorescent probe library of BODIPY derivatives.46 The probes could be used to assess the polarity of the surface of bovine serum albumin (BSA) in vitro (Fig. 15). In this case, the uorescence of the molecule is quenched by the PET process in buffer solutions, but the PET process is less favorable at the surface of BSA
Fig. 13
The strategy of the COX-2 probe.
Fig. 14
Mechanism of the fluorescent probe for HNE.
Schematic representation of PET-based NAT fluorescent
probe.
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because of the hydrophobic environment. Fluorescence switches of these probes are realized based on the polarity of the protein. Hence, the environmentally-sensitive BODIPY library can be applied to sense the hydrophobicity of the target protein surface, membranes and receptors. 4.2.10 Human NAD(P)H:quinone oxidoreductase isozyme 1. Human NAD(P)H:quinone oxidoreductase isozyme 1 (NQO1) is intimately involved with cancer and is oen present in human tumor cells.47 Moreover, NQO1 is found in the cytosol that catalyzes the reduction of quinones to hydroquinones, thus making this enzyme an ideal target for prouorophores. Recently, McCarley and co-workers designed and synthesized an “off/on” uorescent probe for hNQO1 based on the PET mechanism48 (Fig. 16). They chose 1,8naphthalimide as the uorophore and quinone propionic acid as the quencher. The pharmacophore was attached to the uorophore via an N-methylethanolamine linker through a carbamate to the amine of the naphthalimide ring. Aer extensive evaluation, they found that uorescence of the probe Q3NI is controlled by PET quenching. The uorescence signal of the Q3NI probe is quenched via oxidative electron transfer (OeT) by the covalently attached quinone propionic acid, and once the quinone is reduced to its hydroquinone (HQ3NI), the uorescence is quenched by reductive electron transfer (ReT), a common feature with naphthalimide dyes.49 Subsequently, the uorescence of NI released aer the cyclization/cleavage reaction of the hydroquinone via the gem-dialkyl effect. It needs to be noted that the whole process occurs at a high rate. In total, this prouorogenic probe provides a real-time, highly sensitive and selective human tumor cell analysis and differentiation method. In the meantime, the uorophore dequenching strategy and the entire fast and continuous
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procedure provides a new direction for us to develop brand new probes.
4.3
PET-based probes for RNA
Fluorescent imaging is considered a useful toolbox for analyzing biomolecules in vitro and in vivo. Although more and more uorescent sensors for ions and proteins are being reported,50,51 there have been few reasonable uorescent probes for sequence-specic RNA with a high signal-to-noise ratio and cell-permeability.52 As a result, there is an urgent demand for the development of a desirable uorescent sensor for sequence-specic RNA that could permeate the cell membrane and restore the uorescence only in the presence of the target RNA. Koide and Sparano developed uorescent probes for sequence-specic RNA on the basis of a PET strategy.15,53 The probe is composed of the uorophore (F) and the quencher (Q), and the quencher also plays another indispensable role of the ligand for the target RNA (Fig. 17). When the quencher binds to the target RNA sequence, the PET process is suppressed, as well as the uorescence being released. Based on this strategy, this group synthesized a series of compounds as uorescent probes of RNA. They employed 20 ,70 dichlorouorescein (DCF) as the uorophore, and 1-(4-methoxyphenyl)piperazine (MPP) or its derivatives as the quencher. A NOESY NMR assay indicates that the compounds prefer the closed conformation under natural conditions. In order to nd a sequence-specic RNA towards the probes, they immobilized MPP onto agarose resin to isolate aptamers for MPP. Fluorescence titration was used to examine the ability of the isolated
Fig. 17
Restoration of fluorescence by interaction of quencher with
RNA.
Fig. 15 BSA acts as a switch of fluorescence on/off by blocking the PET process.
Fluorescence enhancement by conformation change upon binding the RNA aptamer.
Fig. 18 Fig. 16 The utilization of PET quenching in the hNQO1 probe Q3NI.
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Table 1
Critical Review PET-based fluorescent probes mentioned in this review
Fluorescent probe
Target
lex/nm
lem/nm
Change of quantum yield
Available for in vitro test
Available for live cells
DNAFs DNAT-Me TG-Phos TG-bGal AM-TG-bGal LAP probes CA IX probe NAT probe NAT probe
GST GST ALP b-Gal b-Gal LAP CA IX NAT2 NAT2
492 490 492 492 453 330 511 330 790
514 514 509 509 518 547, 614 534 545 815
34-fold 23-fold — 419-fold 470-fold 500-fold — 479-fold 20-fold
Yes Yes Yes Yes Yes Yes Yes Yes Yes
COX-2 probe Coumarin probe BODIPY derivatives hNQO1 probe RNA probe
COX-2 HNE BSA hNQO1 RNA
457 370 500 374 505
547 495 515 490 525
28-fold 20-fold 10 to 100-fold 33-fold >7-fold
Yes Yes Yes Yes Yes
— Yes — Yes Tumor imaging — Yes — Fluorescence imaging in vivo Yes — Yes Yes —
MPP aptamers to enhance the uorescence of the probes. Cyclodextrin assays proved that aptamers such as cyclodextrin bind to MPP to make the molecule change its conformation and then suppress the PET process (Fig. 18). The researchers established a new paradigm for developing uorescent sensors for specic RNA, which makes it possible to study gene expression at the RNA level using the uorescent imaging method.
5.
Conclusions
Biomacromolecules, including enzymes, proteins and RNAs, play signicant roles in complex biological systems. In view of the fact that a broad variety of diseases are caused by abnormal expression of biomacromolecules, visualization of their localizations and activities is very important for pathological study and clinical diagnosis. Fluorescence imaging is one of the most promising techniques for the real-time monitoring of biomolecules in the physiological environment with high temporal and spatial resolution, which helps researchers to recognize the biological roles of biomolecules in such complex systems. And uorescence imaging is regarded as the future of medical imaging. Furthermore, an increasing number of uorophores are becoming available with improved properties, such as superior stability and a reasonable emission wavelength. As one of the most widely used uorescence switching strategies, the PET mechanism has provided various uorescent probes for uorescence imaging. The combination or binding of probes with targets may lower the HOMO energy level of the quencher or increase the distance between the uorophore and the quencher, so as to result in the PET process “on/off” switching. With this mechanism, we can obtain superb results with a high signal-to-noise ratio that is very prominent for in vivo imaging. So far, several small-molecule uorescent probes for detecting biomacromolecules have been well developed on the basis of the PET mechanism (Table 1). It should be emphasized that these PET-based uorescent probes will be helpful for researchers to determine the quantity of their biomacromolecule
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Ref. 26 26 29 32 33 35 37 41 42 44 45 46 48 15 and 53
targets, understand the pathogenesis of various diseases caused by abnormal expression of biomacromolecules, and be effective enough to promote the development of life sciences. Besides the characteristic advantages, PET probes are rapid, sensitive and selective. Considering that the general excitation wavelength of uorophores is under 600 nm, which may lead to tissue autouorescence, a PET-based near-infrared uorescent probe is urgently needed for deep-tissue imaging. Due to the PET-based probes being sensitive to pH changes, scientists should bear in mind the inuence of pH during the design process and the entire experimental procedure. As a result, considering that PET-based uorescent probes for biomacromolecules are fantastic tools for the study of biomacromolecule activities and looking into relevant diseases, a bright future of these uorescent probes will be expected in the elds of diagnosis, analysis and medical imaging.
Acknowledgements The present work was supported by grants from the National Program on Key Basic Research Project (no. 2013CB734002), the National Natural Science Foundation of China (no. 81370085), the Program of New Century Excellent Talents in University (no. NCET-11-0306), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13028), the Shandong Natural Science Foundation (no. JQ201019) and the Independent Innovation Foundation of Shandong University, IIFSDU (no. 2014JC008 and 2012JC002). We also thank Professor A. Prasanna de Silva (Queen's University Belfast) for his helpful comment on the PET mechanism.
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CRITICAL REVIEW
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Design strategy for photoinduced electron transfer-based small-molecule fluorescent probes of biomacromolecules Wei Zhang, Zhao Ma, Lupei Du and Minyong Li* As the cardinal support of innumerable biological processes, biomacromolecules such as proteins, nucleic acids and polysaccharides are of importance to living systems. The key to understanding biological processes is to realize the role of these biomacromolecules in thte localization, distribution, conformation and interaction with other molecules. With the current development and adaptation of fluorescent technologies in biomedical and pharmaceutical fields, the fluorescence imaging (FLI) approach of using small-molecule fluorescent probes is becoming an up-to-the-minute method for the detection and monitoring of these imperative biomolecules in life sciences. However, conventional small-molecule fluorescent probes may provide undesirable results because of their intrinsic deficiencies such as low signal-to-noise ratio (SNR) and false-positive errors. Recently, small-molecule fluorescent
Received 26th December 2013 Accepted 3rd March 2014
probes with a photoinduced electron transfer (PET) “on/off” switch for biomacromolecules have been thoroughly considered. When recognized by the biomacromolecules, these probes turn on/off the PET
DOI: 10.1039/c3an02379f
switch and change the fluorescence intensity to present a high SNR result. It should be emphasized that these PET-based fluorescent probes could be advantageous for understanding the pathogenesis of
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various diseases caused by abnormal expression of biomacromolecules. The discussion of this successful
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology of Natural Products (MOE), School of Pharmacy, Shandong University, Jinan, Shandong
250012, China. E-mail: [email protected]; Fax: +86-531-8838-2076; Tel: +86-5318838-2076
Zhao Ma is currently studying for a PhD in the Department of Medicinal Chemistry, School of Pharmacy, Shandong University since 2011. His research area involves design, synthesis and bioactivity study of novel uorescent probes for a1-adrenoceptor and reactive oxygen species (ROS). Lupei Du is currently an associate professor at the School of Pharmacy, Shandong University. She obtained her PhD degree from China Pharmaceutical University in 2006, and got her postdoctoral training at the Department of Chemistry, Georgia State University (2006–2009). She joined Shandong University in 2009. Her main research interests include the rational design and synthesis of medicinal molecules and bioactive probes. Minyong Li is a professor at the School of Pharmacy, Shandong University. He received his PhD degree from China Pharmaceutical University in 2005. He began his academic career in 2005 with Dr Binghe Wang at the Department of Chemistry, Georgia State University as a postdoctoral research associate. From 2007 to 2009, he was a research assistant professor at the Department of Chemistry, Georgia State University. In 2009, he moved to his current institution as a professor. His research interests are in the general areas of medicinal chemistry and chemical biology. Wei Zhang received his bachelor's degree at the School of Pharmacy, Shandong University in 2011. He stayed at Shandong University to pursue his master's degree in the Li lab. His research interests mainly focus on the design, synthesis and bioactivity study of uorescent probes for a1-adrenoceptor. This journal is © The Royal Society of Chemistry 2014
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Critical Review strategy involved in this review will be a valuable guide for the further development of new PET-based small-molecule fluorescent probes for biomacromolecules.
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1. Introduction Biomacromolecules that include proteins, nucleic acids and polysaccharides oen play pivotal roles in living systems, which cause a wide variety of diseases in the case of abnormal expression. Although the structures of many biomacromolecules are successfully analyzed, it remains difficult to understand their complicated positions in biological processes. In view of the vivid visualization of biomolecules, uorescence imaging (FLI) technology has been a practical and ideal method for the detection of biological substances.1 With considerable attempts by scientists, an obvious development of small-molecule uorescent probes for biosystems has been achieved within the past few decades.2,3 FLI can reveal the localization and distribution of intracellular macromolecules, sometimes at the single-molecule level,4–6 which allows us to understand the roles of biomacromolecules in living systems. Fluorescent probes are indispensable tools for bioimaging, and a large number of uorescent probes have been well designed and synthesized so far. Nevertheless, conventional techniques of imaging biomacromolecules with organic dyes or uorescent probes regularly consist of a number of painstaking steps. In the concept to design small-molecule uorescent probes for bioimaging, the photoinduced electron transfer (PET) effect is one of the most widely used sensing mechanisms.7–9 Recently, numerous PET-based uorescent probes have been developed for biomacromolecule imaging. In general, this type of probe is structurally divided into two parts: a uorophore and a quencher. The uorescence of the probe is suppressed by the quencher under free conditions and can then be released upon binding by blocking the PET process when the quencher reacts with the target substrate. By using these PET-based uorescent probes, the localization, distribution and conformational change of target molecules can be conveniently unveiled. Compared with those regular probes without uorescence switches, PET-based probes can easily achieve a high signal-tonoise ratio (SNR), which is a tremendously decisive factor for in vitro and in vivo uorescence imaging. The present review article summarizes the PET-based uorescent probes for biomacromolecules and their applications in bioimaging. A brief discussion on the advantages of uorescence imaging will be presented herein, as well.
in emission intensity is called uorescence quenching. As one of the mechanisms that can result in uorescence quenching, the PET effect is a widely used method to diminish the uorescence of uorescent probes.7,8,10,11 A typical PET system consists of an aromatic uorophore, an aliphatic amine and a short methylene chain as the linker.12 In other words, a PET-based uorescent sensor for biomacromolecules oen includes three parts: a receptor that serves as an electron donor or a quencher, a uorophore that acts as the electron acceptor, and a spacer that links these two parts. In this system, the intramolecular electron transfer from the receptor to the uorophore will lead to uorescence quenching. Nevertheless, when the receptor binds upon its targets, this PET process is restricted or totally suppressed, and as a result, the sensor regains its uorescence13,14 (Fig. 1). The mechanism of PET-based uorescent probes can be explained by frontier orbital theory (Fig. 2).12,15–18 When the uorophore is excited by an appropriate light wavelength, the electron of the highest occupied molecular orbital (HOMO) is transferred to the lowest unoccupied molecular orbital (LUMO). Because the receptor is adjacent to the uorophore and the HOMO energy level of the receptor is between the LUMO and HOMO levels of the uorophore, the electron transfers from the HOMO of the receptor to the HOMO of the uorophore which prevents the electron in the LUMO of the uorophore from returning to the HOMO, and then the PET effect occurs to quench the emitted uorescence. When the receptor binds upon the target, the HOMO energy of the receptor changes to be lower than the HOMO energy level of the uorophore. Therefore, the PET process is blocked, and the uorescence is recovered.
Fig. 1
The mechanism of PET-based fluorescent probes.
Fig. 2
Frontier orbital theory for the PET effect.
2. Photoinduced electron transfer (PET) effect When irradiated by ultraviolet or visible light, certain substances can emit light with different wavelengths and intensities, and if the irradiation is stopped, the emitted light will cease instantaneously. This emitted light is known as uorescence. The intensity of the uorescence emission can be weakened by a large number of methods and such a reduction
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3. The advantage of PET-based fluorescence imaging (FLI) FLI is one of the most powerful techniques for medical imaging. Compared to other imaging methods, such as X-ray radiography, computer tomography (CT), magnetic resonance imaging (MRI) and ultrasonography (US), FLI is more convenient, specic, sensitive, faster-responding and is more easily handled. In addition, FLI has been successfully implemented in endoscopy19 and surgery.20,21 However, overall, FLI has some inadequacies, such as a low SNR and the tendency for false-positive errors. These defects can be overcome by using “switchable” uorescent probes, for example, PET-based probes. Switchable uorescent probes can be turned on only when specically bound to the targets of interest, otherwise the probe uorescence is quenched. Consequently, the imaging result with a particularly high targetto-background ratio will be presented. PET-based FLI can monitor biomolecules of interest noninvasively in living systems with high spatial and temporal resolution, as opposed to other imaging techniques. Moreover, this PET-based FLI has a high potential for no-wash live-cell imaging, thus enabling the rapid identication of biomacromolecules in living cells with a high SNR.22 The combination of these advantages would contribute to conveniently revealing the biological roles that the biomolecules of interest perform within complex organisms. As a result, PET-based FLI is effective enough to promote the development of life sciences, which will be generally considered in the future of medical imaging.
4. PET-based small-molecule fluorescent probes 4.1
The uorophores in PET-based probes
A wide range of small-molecule uorophores with several core structures, including BODIPY, uorescein, rhodamine, cyanine, naphthalimide and coumarin (Fig. 3), ranging from nearinfrared (NIR) light to blue light, can be applied in designing a PET-based uorescent probe. These commercially available uorophores are hydrophilic and contain no net charge, so as to be reasonable candidates for in vivo imaging.
Fig. 3 Small-molecule fluorophores including BODIPY, fluorescein, rhodamine, cyanine (Cy7), naphthalimide and coumarin.
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A successful PET-based uorescent probe not only depends on a high sensitivity and selectivity towards the target, but also relies upon the appropriate choice of uorophore. The appropriate uorophore is bonded to the selected recognition group through chemical reactions to form a PET-based uorescent probe. For example, Tang and co-workers designed a PET-based probe for copper(II).23 N-Hydroxyacetamide was chosen as the Cu2+ receptor and tricarbocyanine (Cy) as the NIR uorophore. This was the rst PET-based NIR uorescent probe for Cu2+ and was successfully applied to living cells and tissues to visualize Cu2+.
4.2
PET-based probes for enzymes
PET-based uorescent probes are particularly protable for the sensitive and selective monitoring of target enzyme activities. In a typical case, a PET-based probe can be recognized by a specic enzyme to cleave the PET quencher, which leads to the disappearance of the PET effect. As a result, this system recovers to release the uorescence. Based on this PET mechanism, so far various uorescent probes have been well developed for manifesting the functional role of enzymes in the physiological environment. 4.2.1 Glutathione S-transferase. As a kind of the dimeric enzyme related to detoxication reactions,24 glutathione Stransferase (GST) is involved in a large number of biological processes, such as drug metabolism, protecting our body against endogenous reactive oxygen species (ROS) and xenobiotic biotransformation.25 In other words, GST can catalyze the reaction of reduced glutathione (GSH) with the targets, including both exogenous and endogenous molecules. Recently, Nagano et al. designed and synthesized an “off/on” uorescent probe for GST based on the PET mechanism.26 On the basis of 3,4-dinitrobenzanilide (NNBA) as a specic substrate for GST, they prepared some uorogenic substrate probes (DNAFs). DNAFs have excellent kinetic parameters on releasing uorescence catalyzed by GST. However, these DNAFs were membrane-impermeable which is inappropriate for living cells. Therefore, the authors designed and synthesized another uorogenic substrate, DNAT-Me, which expressed a dramatic uorescence surge aer GST-catalyzed glutathionylation and subsequent denitration (Fig. 4). This was available for imaging GST activities in living cells (Fig. 5). These results indicated that DNAT-Me was a suitable uorogenic substrate for uorescence imaging of the intracellular distribution of GST and also for research on GST activities, including drug resistance in cancer cells and the detection of GST-overexpressing pre-neoplastic foci in diagnosis. This design strategy would serve as a direction for developing probes on the structural basis of enzyme substrates in living cells. 4.2.2 Alkaline phosphatase. Alkaline phosphatase (ALP) is a hydrolase with high sensitivity towards numerous types of target molecules by removal of the phosphate groups.27 A conventional utilization of ALP is used to conjugate with a secondary antibody to identify the target protein in Western blot immunoanalysis. The combination of ALP with a uorescent probe can achieve the discernable signal amplication.28
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Fig. 4 DNAT-Me catalyzed by GST to release fluorescence.
Fig. 5 Confocal microscopic imaging of DNAT-Me and nuclearspecific dye SYTO-loaded HuCCT1 cells. The results displayed the imaging efficacy of DNAT-Me to visualize the GST activity in HuCCT1 cells. (Reprinted with permission from ref. 26. Copyright 2008, American Chemical Society.)
However, the application of available uorescent probes to Western blot analysis is limited at the present time. Recently, Nagano et al. designed and synthesized an “off/on” uorescent probe for ALP based on the PET mechanism that is suitable for Western blot analysis.29 They obtained almost non-uorescent TG-Phos by linking the phosphate group to the phenolic group of 2-Me-4-OMe TG, which could be hydrolysed by ALP to release strong uorescence as well as conrm its high affinity for Western blots (Fig. 6). It should be underlined that this new probe TG-Phos provided suitable sensitivity to ALP for multicolor labeling. In our opinion, this approach may provide a rapid and practical identication of proteins other than ALP on Western blotting analysis. 4.2.3 b-Galactosidase. As an exoglycosidase that catalyzes the hydrolysis of b-galactosides to generate monosaccharides, b-galactosidase (b-Gal) is widely recognized as a marker enzyme,
Fig. 6 Reaction of TG-Phos with ALP to produce highly fluorescent hydrolyzed products by breaking the PET process.
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in particular as a gene expression marker.30 Though there have been many uorescent substrates for b-Gal,31 most of them have been applied with in vitro systems and only a few in vivo. Recently, Nagano, Urano and colleagues developed a unique, sensitive and membrane-permeable PET-based “off/on” uorescent probe, TG-bGal, for b-Gal.32 TG-bGal has an O-alkylated xanthene as the uorophore and a 3-methoxytoluene moiety as the PET donor. When hydrolyzed by b-Gal, the b-galactoside group was released, and the probe released the uorescence (Fig. 7). In view of its reasonable membrane-permeability, this probe enables imaging of b-Gal activities in living cells in a realtime manner. It needs to be noted that the “off/on” switch of such a probe gives results with a high SNR, which is crucial for cell imaging. Based on these results, this group worked torward examining whether the probe, TG-bGal, was applicable for targeted tumor imaging. However, the results were unsatisfactory because the uorescent hydrolysis product of TG-bGal was hydrophobic, which made the probe easily washed out from cells thus weakening the uorescence. In other words, the probe TG-bGal was not suitable for targeted tumor imaging. Based on the photochemical and photophysical experiments, they modied the previous probe by adding an ester moiety. The new probe, AM-TG-bGal, also based on the PET mechanism, manifests a great uorescence enhancement when hydrolyzed by b-Gal and the ester moiety is subsequently hydrolyzed by widespread intracellular esterases33 (Fig. 8). The nal hydrolysis product is hydrophilic and can be well retained in the cells even if being washed, thus avoiding the loss of uorescence. In tumor imaging experiments, the mouse tumor model was labeled with an avidin–b-galactosidase conjugate and followed by incubation with AM-TG-bGal. Aerwards, the intraperitoneal tumors were clearly visualized in the uorescence imaging. These results indicated the bright prospect for applications of the new probe,
Fig. 7 A highly sensitive fluorescent probe based on the PET mechanism for b-galactosidase.
Fig. 8 Reaction of AM-TG-bGal as a b-Gal and esterase-sensitive probe based on PET.
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such as in video-assisted laparoscopic tumor resection. The design strategy provided a unique method for other researchers on tumor imaging by using a two-step procedure: rstly the localization on target cancer cells of a target enzyme and subsequently its recognition with a highly sensitive uorescent probe, which results in a high tumor-to-background ratio. 4.2.4 Protease. A protease, oen related to a number of disease processes, functions as an enzyme for the removal of a specic peptide from a substrate peptide or protein. Among the proteases, microsomal leucine aminopeptidase (LAP), an exopeptidase to eliminate the N-terminal amino acids of unsubstituted oligopeptides, plays an essential role in tumor metastasis.34 Nagano et al. designed and synthesized some lanthanide-based functional probes for LAP on the basis of the “off/on” PET mechanism.35 Upon enzymatic cleavage of the substrate peptide, a signicant difference in the luminescence intensity was observed (Fig. 9). When the leucine group was removed by LAP, the aniline group acted as a quencher causing the PET process to be switched “on” and then no luminescence could be seen. It should be noted that LAP is an enzyme in association with a wide range of clinical diseases, such as of the liver and pancreas, and, therefore, LAP uorescent probes should be of practical value in clinical diagnosis. This report disclosed a novel strategy for developing PET-based probes for all proteases. 4.2.5 Carbonic anhydrase isozymes IX. Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze rapid physiological reactions including the interconversion of carbon dioxide and water to bicarbonate and protons. These enzymes are involved in various pathological and physiological processes, such as lipogenesis, gluconeogenesis and tumorigenicity. As a kind of transmembrane a-CA isoform, human carbonic anhydrase IX (hCA IX) is recognized as a target of cancer diagnosis and treatment.36 However, hitherto there has been no selective uorescent probe for CA IX. Recently, Qian and co-workers designed and synthesized a highly-sensitive PET-based uorescent probe with an ideal “off/on” switch for CA IX (Fig. 10), which could release strong uorescence when recognized.37 In this case, saccharin was employed as a recognition motif because of its high specicity for CA IX. When the saccharin group is recognized by the target enzyme, the conformation of this probe will change to suppress the PET process and, as a result, the quenched uorescence is released
Fig. 9
Reaction scheme of the LAP probe. Ln ¼ Tb, Eu.
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Fig. 10 Strategy of the fluorescent probe for CA IX with space-folded PET mechanism.
again. It is well known that hCA IX is highly overexpressed in hypoxic cells; in the meantime, SiHa cells were singled out for cell imaging on account of their relatively high expression of endogenous CA IX. Therefore, in cell imaging, two parallel SiHa cells were treated under normoxic and hypoxic conditions at 37 C before incubation with the probe. The imaging results obviously exemplied stronger uorescence in hypoxic cells than in normoxic; in other words, these outputs claried a much higher selectivity towards CA IX than other CA enzymes. In conclusion, the designed probe could be a protable uorescent probe for the detection of CA IX in vitro or in cellulo. In our opinion, this successful strategy could afford a new avenue for the design of PET-based probes through changing the conformation of the probe. 4.2.6 Arylamine N-acetyltransferase 2. Arylamine N-acetyltransferases (NATs) are metabolism enzymes that catalyze the acetylation of aromatic amines or arylhydroxylamines through acetyl-CoA.38 NATs oen play an imperative role in various signicant pharmacological and toxicological reactions, including the detoxication of arylamines by N-acetylation and bioactivation reactions of arylamine carcinogens.39 NAT1 and NAT2 have been reported to be related to several diseases.40 Therefore, the detection of NAT activities is of importance in clinical diagnosis. Because NAT1 is ubiquitously expressed, and NAT2 is located mainly in liver and colorectal tissues, the detection of NAT2 is urgently required for biological study. Nagano et al. introduced a lanthanide complex “off/on” probe that showed high specicity for NAT2 over NAT1.41 They initially synthesized the probe as a product which was obtained from catalysis by LAP35 that we discussed above. The aniline group functioned as the quencher of the probe, but the quenched luminescence by PET could be released aer Nacetylation by NAT (Fig. 11). Hereto, a reasonable probe with an “on/off” switch was established for NAT. With the switch, the probe provided high SNRs. Meanwhile, considering that NATs are imperative metabolism enzymes, the probe is expected to be a useful tool for drug discovery or in clinical diagnosis. The product of one enzyme can be a substrate for another enzyme. Therefore, this strategy can be used to develop two-step continuous uorescent probes for two different enzymes. Xu and co-workers designed and synthesized a near-infrared “off/on” uorescent probe for NAT2 with high sensitivity and selectivity.42 Because of superior tissue penetration and weak
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Fig. 11 Schematic of the novel NAT probe based on the PET strategy.
auto-uorescence interference, such a near-infrared probe has important inuence in studying biological processes, especially those in vivo.23,43 They employed Cy7 as the uorophore and the arylamines as the quencher, which are linked by a sulfur atom to form the PET system, in which the arylamines functioned as the substrates of the NATs. When the probe molecule was catalyzed by NAT to generate an acetylation product, the PET process was blocked and the uorophore released the uorescence (Fig. 12). Beneting from its high sensitivity and selectivity for NAT2, as well as its long emission wavelength, the probe could be applied for the real-time uorescence imaging of NAT2 in vivo. The results clearly demonstrated that the uorescence in the liver is much stronger than in other tissues of mice, which is in line with the high expression of NAT2 in the liver. To conrm the in vivo results, tissue homogenate assays were carried out by incubating the probe with different tissue homogenates. The results also conrmed that the intensity in the liver is the highest of all tissues. Obviously, the nearinfrared probe could be a valuable tool for clinical diagnosis and biochemical analysis. 4.2.7 Cyclooxygenase-2 (COX-2). In the past few years, uorescence imaging has provided a convenient tool for the early diagnosis of cancer because of its high signal-to-background ratio and selectivity. Cyclooxygenase-2 (COX-2) is a biomarker of almost all cancer cell lines, and can be used to distinguish cancer cells from normal ones. Recently, Peng et al. designed and synthesized a novel “off/on” uorescent probe targeted towards COX-2 and localized in the Golgi apparatus of cancer cells.44 The mechanism of the uorescent probe depends on PET: when binding to COX-2 in the Golgi apparatus of cancer cells, the probe changes its conformation from a folded to an
Fig. 12
unfolded state, which restrains the PET process from the uorophore acenaphtho[1,2-b]quinoxaline (ANQ) to the recognition group indomethacin (IMC) through release of the uorescence (Fig. 13). It needs to be noted that such a PET-quenched uorescent probe elucidated high selectivity and sensitivity to COX-2 for uorescence imaging of the Golgi apparatus through normal and two-photon uorescence microscopy. As a result, the probe could be a practical reagent for the early diagnosis of cancers. 4.2.8 Human neutrophil elastase. Human neutrophil elastase (HNE) is widely considered to be associated with several human diseases. Recently, Yang and co-workers designed and synthesized the rst non-peptide-based PET “off/on” uorescent probe which showed a highly specic response for HNE.45 They chose 7-amino-4-triuoromethyl coumarin (AFC) as the uorophore and the pentauoroethylcarbonyl group as the quencher, linked by an amide bond. When catalyzed by HNE, the pentauoroethylcarbonyl group was removed and the uorescence released (Fig. 14). This small PET-based uorescent probe can be easily synthesized and requires no particular storage, compared with peptide-based substrates. Moreover, it can be utilized to develop an efficient and cost-effective uorescent assay to determine the enzymatic activity of HNE. It should be emphasized that this assay will promote the future high-throughput discovery of HNE inhibitors as well as clinical diagnosis of elastase-related diseases. 4.2.9 Serum albumin. Nagano and co-workers discovered that the solvent-dependent change of uorescence on/off switch of BODIPY derivatives is because of the inuence of environmental polarity on the PET process. On the basis of this nding, they successfully prepared an environmentally-sensitive uorescent probe library of BODIPY derivatives.46 The probes could be used to assess the polarity of the surface of bovine serum albumin (BSA) in vitro (Fig. 15). In this case, the uorescence of the molecule is quenched by the PET process in buffer solutions, but the PET process is less favorable at the surface of BSA
Fig. 13
The strategy of the COX-2 probe.
Fig. 14
Mechanism of the fluorescent probe for HNE.
Schematic representation of PET-based NAT fluorescent
probe.
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Critical Review
because of the hydrophobic environment. Fluorescence switches of these probes are realized based on the polarity of the protein. Hence, the environmentally-sensitive BODIPY library can be applied to sense the hydrophobicity of the target protein surface, membranes and receptors. 4.2.10 Human NAD(P)H:quinone oxidoreductase isozyme 1. Human NAD(P)H:quinone oxidoreductase isozyme 1 (NQO1) is intimately involved with cancer and is oen present in human tumor cells.47 Moreover, NQO1 is found in the cytosol that catalyzes the reduction of quinones to hydroquinones, thus making this enzyme an ideal target for prouorophores. Recently, McCarley and co-workers designed and synthesized an “off/on” uorescent probe for hNQO1 based on the PET mechanism48 (Fig. 16). They chose 1,8naphthalimide as the uorophore and quinone propionic acid as the quencher. The pharmacophore was attached to the uorophore via an N-methylethanolamine linker through a carbamate to the amine of the naphthalimide ring. Aer extensive evaluation, they found that uorescence of the probe Q3NI is controlled by PET quenching. The uorescence signal of the Q3NI probe is quenched via oxidative electron transfer (OeT) by the covalently attached quinone propionic acid, and once the quinone is reduced to its hydroquinone (HQ3NI), the uorescence is quenched by reductive electron transfer (ReT), a common feature with naphthalimide dyes.49 Subsequently, the uorescence of NI released aer the cyclization/cleavage reaction of the hydroquinone via the gem-dialkyl effect. It needs to be noted that the whole process occurs at a high rate. In total, this prouorogenic probe provides a real-time, highly sensitive and selective human tumor cell analysis and differentiation method. In the meantime, the uorophore dequenching strategy and the entire fast and continuous
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procedure provides a new direction for us to develop brand new probes.
4.3
PET-based probes for RNA
Fluorescent imaging is considered a useful toolbox for analyzing biomolecules in vitro and in vivo. Although more and more uorescent sensors for ions and proteins are being reported,50,51 there have been few reasonable uorescent probes for sequence-specic RNA with a high signal-to-noise ratio and cell-permeability.52 As a result, there is an urgent demand for the development of a desirable uorescent sensor for sequence-specic RNA that could permeate the cell membrane and restore the uorescence only in the presence of the target RNA. Koide and Sparano developed uorescent probes for sequence-specic RNA on the basis of a PET strategy.15,53 The probe is composed of the uorophore (F) and the quencher (Q), and the quencher also plays another indispensable role of the ligand for the target RNA (Fig. 17). When the quencher binds to the target RNA sequence, the PET process is suppressed, as well as the uorescence being released. Based on this strategy, this group synthesized a series of compounds as uorescent probes of RNA. They employed 20 ,70 dichlorouorescein (DCF) as the uorophore, and 1-(4-methoxyphenyl)piperazine (MPP) or its derivatives as the quencher. A NOESY NMR assay indicates that the compounds prefer the closed conformation under natural conditions. In order to nd a sequence-specic RNA towards the probes, they immobilized MPP onto agarose resin to isolate aptamers for MPP. Fluorescence titration was used to examine the ability of the isolated
Fig. 17
Restoration of fluorescence by interaction of quencher with
RNA.
Fig. 15 BSA acts as a switch of fluorescence on/off by blocking the PET process.
Fluorescence enhancement by conformation change upon binding the RNA aptamer.
Fig. 18 Fig. 16 The utilization of PET quenching in the hNQO1 probe Q3NI.
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Table 1
Critical Review PET-based fluorescent probes mentioned in this review
Fluorescent probe
Target
lex/nm
lem/nm
Change of quantum yield
Available for in vitro test
Available for live cells
DNAFs DNAT-Me TG-Phos TG-bGal AM-TG-bGal LAP probes CA IX probe NAT probe NAT probe
GST GST ALP b-Gal b-Gal LAP CA IX NAT2 NAT2
492 490 492 492 453 330 511 330 790
514 514 509 509 518 547, 614 534 545 815
34-fold 23-fold — 419-fold 470-fold 500-fold — 479-fold 20-fold
Yes Yes Yes Yes Yes Yes Yes Yes Yes
COX-2 probe Coumarin probe BODIPY derivatives hNQO1 probe RNA probe
COX-2 HNE BSA hNQO1 RNA
457 370 500 374 505
547 495 515 490 525
28-fold 20-fold 10 to 100-fold 33-fold >7-fold
Yes Yes Yes Yes Yes
— Yes — Yes Tumor imaging — Yes — Fluorescence imaging in vivo Yes — Yes Yes —
MPP aptamers to enhance the uorescence of the probes. Cyclodextrin assays proved that aptamers such as cyclodextrin bind to MPP to make the molecule change its conformation and then suppress the PET process (Fig. 18). The researchers established a new paradigm for developing uorescent sensors for specic RNA, which makes it possible to study gene expression at the RNA level using the uorescent imaging method.
5.
Conclusions
Biomacromolecules, including enzymes, proteins and RNAs, play signicant roles in complex biological systems. In view of the fact that a broad variety of diseases are caused by abnormal expression of biomacromolecules, visualization of their localizations and activities is very important for pathological study and clinical diagnosis. Fluorescence imaging is one of the most promising techniques for the real-time monitoring of biomolecules in the physiological environment with high temporal and spatial resolution, which helps researchers to recognize the biological roles of biomolecules in such complex systems. And uorescence imaging is regarded as the future of medical imaging. Furthermore, an increasing number of uorophores are becoming available with improved properties, such as superior stability and a reasonable emission wavelength. As one of the most widely used uorescence switching strategies, the PET mechanism has provided various uorescent probes for uorescence imaging. The combination or binding of probes with targets may lower the HOMO energy level of the quencher or increase the distance between the uorophore and the quencher, so as to result in the PET process “on/off” switching. With this mechanism, we can obtain superb results with a high signal-to-noise ratio that is very prominent for in vivo imaging. So far, several small-molecule uorescent probes for detecting biomacromolecules have been well developed on the basis of the PET mechanism (Table 1). It should be emphasized that these PET-based uorescent probes will be helpful for researchers to determine the quantity of their biomacromolecule
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Ref. 26 26 29 32 33 35 37 41 42 44 45 46 48 15 and 53
targets, understand the pathogenesis of various diseases caused by abnormal expression of biomacromolecules, and be effective enough to promote the development of life sciences. Besides the characteristic advantages, PET probes are rapid, sensitive and selective. Considering that the general excitation wavelength of uorophores is under 600 nm, which may lead to tissue autouorescence, a PET-based near-infrared uorescent probe is urgently needed for deep-tissue imaging. Due to the PET-based probes being sensitive to pH changes, scientists should bear in mind the inuence of pH during the design process and the entire experimental procedure. As a result, considering that PET-based uorescent probes for biomacromolecules are fantastic tools for the study of biomacromolecule activities and looking into relevant diseases, a bright future of these uorescent probes will be expected in the elds of diagnosis, analysis and medical imaging.
Acknowledgements The present work was supported by grants from the National Program on Key Basic Research Project (no. 2013CB734002), the National Natural Science Foundation of China (no. 81370085), the Program of New Century Excellent Talents in University (no. NCET-11-0306), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13028), the Shandong Natural Science Foundation (no. JQ201019) and the Independent Innovation Foundation of Shandong University, IIFSDU (no. 2014JC008 and 2012JC002). We also thank Professor A. Prasanna de Silva (Queen's University Belfast) for his helpful comment on the PET mechanism.
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