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Small organic molecules as fluorescent probes for nucleotides and their derivatives
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Trends in Analytical Chemistry, Vol. 29, No. 4, 2010
Small organic molecules as fluorescent probes for nucleotides and their derivatives Xi Juan Zhao, Cheng Zhi Huang Nucleoside monophosphates, diphosphates, triphosphates, and stringent alarmone {i.e. guanosine 3 0 -diphosphate-5 0 -di(tri)phosphate [(p)ppGpp]} are important to organisms. There are detection methods for these anions (e.g., based on absorption, fluorescence emission and chemiluminescence). We review the rise in fluorescent probes using small organic molecules for detecting these anions. The requirements of fluorescence real-time monitoring and imaging analysis make it certain that more economic, more specific, and more environmentally and biologically benign fluorescent probes will be designed in the near future. ª 2010 Elsevier Ltd. All rights reserved. Keywords: Adenosine mono (di or tri) phosphate; Cytidine triphosphate; Fluorescence; Fluorescent probe; Guanosine mono (di or tri) phosphate; Nucleotide; Nucleotide derivative; Small organic molecule; Stringent alarmone; Uridine di(tri) phosphate
1. Introduction Xi Juan Zhao, Cheng Zhi Huang* Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China Cheng Zhi Huang College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, PR China
*
Corresponding author. Tel.: +86 23 68254659; E-mail: [email protected]
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As integral parts of nucleic acids, nucleotides are very significant units in life. Also, nucleotide derivatives exist widely in organisms. Both nucleotides and their derivatives participate in biological and chemical processes. The importance of these phosphate-containing anions is therefore self evident, so that detection or identification of these anions remains a critical focus of research. Detection of these phosphate-containing anions started from detection of inorganic phosphate or pyrophosphate with the signal response of molecular absorption [1,2], fluorescence [3–16] and chemiluminescence [17]. There is also detection of nucleotides and their derivatives [18–22] that can be considered biophosphates based on HPLC. With the development and the maturity of aptamer science, aptamers have been applied to detect nucleotides due to the high affinity and high specificity of aptamers for targets [23]. The aptamer-based ATP assay has good selectivity based on
fluorescence [24–29], color [30] or electrochemistry [31,32]. Fluorescence-based techniques including quantitative detection or real-time monitoring have good sensitivity and selectivity [33], which are related to the design of specific, small organic molecules (SOMs) acting as fluorescence probes. Generally, fluorescent SOMs should have two function groups: the fluorophore that gives fluorescence emission and acts as signal reporter; and, the ligand that is the molecular recognition element and can bind specifically to the analyte. The two function groups are assembled together as a unit or separated with a spacer that can permit transfer of electron or charge. Regardless of environmental factors, the yield of the fluorescence emission of the fluorophore group determines the sensitivity, and the design of the ligand determines the selectivity of the fluorescent probe or the reagent. Ideallydesigned SOMs as fluorescent probes therefore have the advantages of the good sensitivity of fluorescence-based techniques and the selectivity of the ligand. Also, the response for the recognition between the probe and the analyte should be specific. For these advantages, SOMs have been well exploited as fluorescent probes in chemistry, biology and medicine. There have been several reviews relating to the recognition or sensing of phosphate anions from different aspects [34– 38]. For example, the Hong and Yoon group covered chemosensors for pyrophosphate in terms of topological and structural classification, using signal
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Trends in Analytical Chemistry, Vol. 29, No. 4, 2010
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Figure 1. Structures of nucleotides and their derivatives.
changes in fluorescence and absorption [38], while Wolfbeis et al. comprehensively summed up fluorescent probes for inorganic phosphates and biophosphates [37]. However, few reviews have been reported to probe nucleotides and their derivatives based on the fluorescence signals. In this article, we deal with the past 30 years of progress in fluorescent probes for nucleotides and their derivatives (Fig. 1). We confine ourselves to fluorescent probes that are SOMs, and will not cover those (e.g., aptamers) where fluorescent SOMs are employed as fluorophore tags. To understand the application of the fluorescent SOM probes, we first summarize the three approaches to interaction shown in Fig. 2, where F1 and F2 denote the signal intensities of fluorescence emission before and after the interaction between the SOMs and the nucleotides or their derivatives. The fluorescent probes bind the
nucleoside phosphates directly or indirectly through an intermediate fluorescent indicator. In either case, the properties of the probe or the fluorescent indicator change to give rise to variations in fluorescent signals. 2. Probes for adenosine phosphate, diphosphate and triphosphate Adenosine phosphate, diphosphate and triphosphate are key players in organisms. It is well-known that ATP, as an energy donor, takes part in the energy metabolism in living cells, and also participates in the biosynthesis process as a reactant [39]. The concentration of ATP can be used as a quality standard of the viability of blood cells for transfusion [40]. It is therefore very significant theoretically and practically to detect adenosine phosphates.
Figure 2. Three types of approaches to the interaction for probes and nucleoside phosphates.
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Since lanthanide ions exhibit large coordination numbers and high coordination flexibility, they can coordinate with some SOMs to form complexes. Since the coordination numbers of lanthanide ions are not full, electronegative phosphate of nucleotides can combine with lanthanide ions, and the spectral properties of the lanthanide-ion complexes then change. Chang et al. reported that Tb3+ could form a complex with 1, 10-phenanthroline (1) and ATP can increase the fluorescence emission of the complex at pH 7.0 [41]. Regrettably, the selectivity of this probe is not good because ADP and AMP can also increase the fluorescence intensity to some extent so that their interference cannot be ignored.
Zhao et al. used the Tb3+-tiron (2) complex as a fluorescent probe to determine nucleotides and polynucleotides based on fluorescence quenching [42]. The fluorescence intensity of Tb3+-tiron at 546 nm decreased significantly in the presence of ATP, ADP or AMP when excited at 317 nm. The extent of quenching depended on the amount of phosphate available for binding to Tb3+. Similarly, the Tb3+-tiron system could be used only to detect solutions of pure substance and its selectivity is unsatisfactory.
Tb3+ forms a complex with norfloxacin (3) and ATP can remarkably enhance the emission at 545 nm [43]. This probe can be successfully applied to the determination of ATP in injection and tablet. Scha¨ferling et al. tested the selectivity of the fluorescent probe and found cAMP did not interfere, but pyrophosphate caused little fluorescence quenching compared to ATP [44]. They therefore used Tb-norfloxacin as a probe for the determination of adenylyl-cyclase activity.
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Some Eu3+-based complexes also have been used as fluorescent probes for adenosine phosphate, diphosphate and triphosphate. Both oxytetracycline (4) and doxycycline (5) containing b-diketonate configuration were reported to form complexes with Eu3+, which could be used to probe ATP [45,46]. The coordination number of Eu3+ with oxytetracycline and doxycycline is 8, which is not satisfied, so H2O molecules fill up the vacancy. ATP can replace H2O and compensate for the energy loss through the O-H vibration of water. The fluorescence of Eu3+-oxytetracycline and Eu3+-doxycycline at 612 nm can therefore be greatly enhanced by ATP. Regrettably, the authors did not consider whether or not ADP and AMP had such an effect.
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Scha¨ferling et al. further established a method to determine the activity of creatin kinase based on the different effects of ATP and ADP on the fluorescence emission of Eu3+-tetracycline (6) [47]. ATP strongly quenches the fluorescence intensity, whereas ADP has no significant effect. The authors also studied the effect of AMP, cAMP, GTP, GDP and pyrophosphate on the fluorescence intensity of the Eu3+-tetracycline complex. Most important is the application in the determination of the activity of a creatin kinase, since there is little interference.
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fluorescence emission of the probe could be quenched by nucleotides and responded very selectively toward AMP and GMP [49]. They supposed the mechanism involved hydrogen-atom abstraction or exciplex formation, so they predicted that DBO may become a useful fluorophore to detect nucleotides.
The above compounds (1–6) are easily available, but poor selectivity makes researchers attempt to synthesize
Inouye et al. synthesized a series of pyrenophanes and found that bis(diazoniacrown)-substituted pyrenophane (8) can recognize the nucleoside triphosphates [50]. The ability to recognize decreases in the order of triphosphate > diphosphate > monophosphate. With ATP as an example, fluorescence emission of 8 gets reduced with adding ATP. The interaction mechanism is p-stacking between nucleobase and the pyrene fluorophores and the electrostatic interactions of the positive diazoniacrowns with phosphate moieties. The drawback is that this kind of probe cannot specifically recognize a certain nucleoside triphosphate.
new compounds that could act as probes for adenosine nucleotides with improved selectivity. In this case, Anslyn et al. reported that the use of the guanidinium entities, along with the tripeptide arms, Ser-Tyr-Ser, could produce resin-bound sensors with excellent selectivity for ATP [48]. The sensor designed gives the largest fluorescence increase upon addition of ATP, whereas AMP and GTP have little effect on the change in fluorescence of the sensor, suggesting that both triphosphates and nucleotide bases play important roles in the recognition process. The Nau group designed a new probe of 2, 3-diazabicyclo [2.2.2] oct-2-ene (DBO, 7), and found that the
Dumy et al. designed a cyclic miniature esterase that can hydrolyze compound 9 to compound 10 [51]. They then tested the inhibitory effect of orthophosphate (Pi), AMP, D-myoinositol-1,4,5-triphosphate (IP3), citric acid, ATP and ADP on the miniature esterase and found only ADP was effective. Pi, AMP, IP3, ATP, or citric acid turn the solution yellow because of the formation of 10, whereas the solution containing ADP remains colorless and no fluorescence signal of 10 can be detected in this solution with the time increasing. This work is attractive because it has a good selectivity for ADP against ATP.
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The complex 13-Cd2+ contains a fluorophore of 7-amino-4-trifluoromethylcoumarin and Cd2+-cyclen (1,4,7,10-tetraazacyclododecane) as an anion receptor [54]. When AMP is added to a solution of 13-Cd2+, the excitation wavelength at 500 nm shows red shift. Because ADP and ATP had higher affinities for 13-Cd2+ than AMP, the authors did not follow traditional ideas by using this complex to distinguish AMP, ADP and ATP, but smartly applied the different signal of AMP and cAMP to 13-Cd2+ for monitoring phosphodiesterase activity with their finding that adding cAMP hardly changes the excitation spectrum. Phosphodiesterase can cleave cAMP to AMP, and, by monitoring the increase of AMP, can attain the expected purpose.
Ligands 11 and 12 can form some complexes with Eu3+, and then phosphate-containing anions will occupy the vacant coordination sites of Eu3+ [52,53]. Under appropriate conditions, the conformation of 11 and Eu3+-12 will change, following the change of the corresponding absorption or fluorescence spectra. The fluorescence intensity of the Eu3+-11 complex can be enhanced by adding ATP, and ADP or AMP induces a smaller fluorescence enhancement. Similarly, ATP, ADP or AMP also enhances the fluorescence emission of the Eu3+-12 complex. The selectivity of both probes for ATP is poor because the influence of ADP and AMP on the fluorescence is not ignored. Hamachi et al. recently attached a dipicolylamine (DPA)-2Zn2+ system to a fluorophore [55–57]. Complex 14 has good selectivity among ATP, CTP and GTP in fluorescence sensing. ATP can enhance the fluorescence emission of 14, and CTP induces a weaker enhancement than ATP, whereas GTP gradually decreases the fluorescence intensity. The affinity of 14 for ADP and AMP is smaller than that for ATP, while cAMP has no signal fluorometrically. However, the selectivity for the detection of ATP is still not good [55].
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Bis-Zn/DPA-anthracene receptor 15 embedded in the hydrogel 17 can distinguish ATP from other anionic species based on fluorescence, but it cannot discriminate a specific phosphate-containing species among the phosphate anion derivatives. After receptor 16 is incorporated in supramolecular hydrogel 17, the fluorescence emission of 16 (at 512 nm) is enhanced following the wavelength blue-shift for phenyl phosphate, and gets quenched for ATP with the emission red-shift, whereas it has no change for non-phosphate anions. It is noteworthy that there are no such phenomena in aqueous solution or agarose gel, which lacks hydrophobic domains, indicating that the medium (e.g., 17) is very important for these findings [56].
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Dual-emission probes (18 and 19) can be employed to detect ATP in a ratiometric manner – the first example of ratiometric fluorescence-sensing system for ATP [57]. Addition of ATP to 18 and 19 reduces the emission intensities and the blue shift of the emission wavelength. This unique emission-wavelength shift is due to dissociation of the first Zn2+ from the acridine fluorophore following formation of the binuclear Zn2+ complex. Since ADP has the same effect as ATP, whereas phosphate (Pi) and AMP cause no change in the fluorescence intensity of 19, probe 19 is applied in monitoring apyrase-catalyzed hydrolysis of ADP.
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(P2 O4 7 , F/Fo = 54) caused a large increase in the fluorescence of 21, whereas HPO2 4 , AMP, cGMP induced negligible fluorescence change, even under a high concentration. It is easy to understand that the selectivity for a single substance is not bad. Ingeniously, the author applied probe 21 to fluorescence visualization of ATP-particulate stores in living cells, combining with quinacrine, a widely-used probe to locate the ATP store.
Das et al. investigated the interactions between a new chromogenic complex (20) and some biologically important anions {e.g., ATP, ADP, AMP, pyrophosphate (PPi) and phosphate [58]}, and they found that only the addition of ATP made the color of 20 change from pale yellow to light pink in aqueous solution, while ADP, AMP, PPi and phosphate had no such effect. In order to further confirm the efficient, selective binding of ATP to 20, the authors studied the fluorescence changes of 20 after adding these anions. The fluorescence emission was found to be partially quenched by ATP, whereas no such quenching was observed in the excess of ADP, AMP, PPi or H2 PO 4 . Remarkably, complex 20 can be used as a colorimetric staining agent for yeast cells, and staining can be detected under a simple light microscopy.
A new xanthene-based Zn2+ complex, reported by Hamachi in 2008, can sense nucleoside polyphosphates (e.g., ATP) with a ‘‘turn-on’’ fluorescence signal [59]. The coordination of Zn2+ to the DPA sites of 21 induced the formation of a non-fluorescent deconjugated xanthene ring, while adding nucleoside polyphosphates recovered the conjugated structure of the xanthene fluorophore, leading to the ‘‘turn-on’’ fluorescence signal. The selectivity of 21 towards various nucleoside polyphosphates was evaluated. XTP (X = A, G, C, F/ Fo > 30), XDP (X = A, U, F/Fo > 15), and pyrophosphate 360
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Jolliffe et al. synthesized a new molecular receptor (22) based on a diketopiperazine ring with dipicolylamine (DPA)-2Zn2+ arms [60]. Compound 22 can quench the fluorescence of 23, which can be used as an indicator in displacement assays to evaluate anionbinding capability. When various monophosphate, diphosphate, triphosphate and non-phosphate anions are added to the solution with a 1: 1 ratio of receptor 22 and indicator 23, only ATP, ADP, GTP, PPi and citrate can recover the fluorescence emission of 23, meaning that these ions show significant indicator displacement. However, none of the monophosphate derivatives (e.g., AMP, or cAMP) has this signal. Although 22 shows high affinity and selectivity for diphosphate and triphosphate in aqueous solution at pH 7.4, except for non-phosphate anion and citrate, it is obvious that the probe lacks selectivity for a single diphosphate or triphosphate anion.
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AMP or GMP has no such effect in the excitation spectra in aqueous solution. Furthermore, the interaction of 25 with ATP in mitochondria is also tested and the same phenomena are observed, indicating that the specificity of 25 for ATP is good in the presence of cell organelles, so it is possible for probe 25 to determine ATP in vivo.
A new phenanthroline-containing macrocycle (24), designed by Bencini et al., can selectively recognize ATP among GTP, TTP, CTP as well as ADP and AMP at pH 6 based on fluorescence quenching [61]. Compound 24 could be easily protonated in aqueous solutions and has six positive charges at pH 6 7, and the hexaprotonated 24 can form stable complexes with nucleoside phosphate, diphosphate and polyphosphate. Only addition of ATP to the hexaprotonated 24 (1:1 molar ratio) can completely quench the fluorescence of 24, while ADP, AMP or the other triphosphate nucleosides induces a much smaller decrease of the fluorescence intensity. The fluorescence quenching is attributed to the charge-charge and hydrogen-bonding interactions between the anionic phosphate moiety and the ammonium groups of 24 and p-stacking between the nucleobases and the phenanthroline unit of 24. The selectivity of this probe for ATP is very good compared with the previous researches.
Yoon et al. designed a unique ratiometric fluorescent sensor (26) for ATP based on a transition of excimer vs. monomer pyrene fluorescence [63]. Usually, the monomeric emission of pyrene moieties is at 375 nm, while the emission of its excimer is at 487 nm. Upon addition of ATP, GTP, TTP, UTP, CTP, Pi or PPi, the excimer-pyrene fluorescence of 26 is quenched to different extents. Surprisingly, only ATP makes the fluorescence of monomeric pyrene increase. In view of this unique change, compound 26 can be used to discriminate ATP from the other three nucleoside triphosphates. Furthermore, the selectivity for ATP over ADP and AMP is also tested. The result is that ADP induces correspondingly smaller changes in the fluorescence of 26 than ATP, but the effects of AMP on the monomeric and excimer emissions are negligible.
3-Hydroxy-4 0 -(dimethylamino) flavone (FME probe 25) can form a complex with ATP, leading to increase of fluorescence-emission intensity and an accompanying considerable red shift in the excitation wavelength of the FME probe [62]. Whereas inosine-5 0 -triphosphate (ITP), bnicotinamide adenine dinucleotide (NAD), GTP, UTP, ADP,
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When the ratiometric fluorescent technique is introduced, the monomer vs. excimer pyrene fluorescence (F375/F487) for 26 upon addition of ATP is much larger than that upon addition of ADP and AMP, which is large enough to distinguish ATP from ADP and AMP. According to the authors, adenine can sit between the two pyrenes in 26 and thus separate pyrene moieties resulting in the monomer-pyrene fluorescence, while the other three bases interact outside the pyrene-pyrene dimmer, leading to the quenching of the excimer fluorescence. Depending on these findings, probe 26 can be applied to ATP-staining experiments and monitor apyrase activity in real time. Compound 26 is a very selective, specific fluorescent probe for ATP among other nucleoside triphosphates, and ADP and AMP at physiological pH. By the way, polythiophene derivatives as polymers can also act as fluorescent probes for adenosine phosphate, diphosphate and triphosphate. Shinkai et al. established a method of detecting ATP based on polythiophene derivative 27. Fluorescence quenching of 27 at 529 nm is much more effective in the presence of ATP (kex = 445 nm) than AMP (kex = 435 nm) or ADP (kex = 435 nm) and the detection limit can be extended to 108 mol/L [64]. The Bazan group chose polythiophene derivative 27 as a probe to visualize glucose phosphorylation directly [65].
From all the above descriptions, we can see that the following two types of probes, fluorophore-DPA-Zn2+ and lanthanide ion-ligand, play dominant roles in probing adenosine phosphate, diphosphate and triphosphate, especially adenosine triphosphate. Besides the two types of probes, most probes for adenosine phosphates involve metal-ion coordination events, which might be accompanied by electrostatic or p–p stacking interactions. 3. Probes for guanosine phosphate, diphosphate and triphosphate The effects of guanosine phosphates on the biological systems are vital. Some are involved in the carbohydrate metabolism and the stringent response [66,67], so 362
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detection of these anions makes sense. One thing to be noted is that the imidazole group is a very valuable ligand in the design of fluorescent probes for GMP and its derivatives. Yoon et al. developed a new fluorescent anthracene and imidazolium compound (28) that could effectively and selectively recognize GTP among ATP, ADP, AMP, pyrophosphate, H2 PO 4 , F and Cl [68]. Compound 28 shows a distinct chelation-enhanced fluorescence quenching (CHEQ) effect with a slight red shift after adding GTP. The quenching mainly attaches to the guanine base in GTP, while the other anions induce chelation-enhanced fluorescence (CHEF) effects on 28 at different levels. This probe can work in aqueous solution of physiological pH. It is a pity that the effects of GDP and GMP on the fluorescence of 28 are not considered in the paper [68]. Subsequently, Yoon and Kim et al. synthesized four fluorescent imidazolium chemosensors for recognizing pyrophosphate, and three of them were anthracene-bearing imidazolium receptors [14].
The Yoon group again designed a new fluorescent cavitand derivative with four imidazolium groups and four pyrene groups for recognizing GTP [69]. The addition of GTP gradually quenched the fluorescence emission of the cavitand derivative, and ATP, CTP or ADP caused a relatively small CHEQ effect, whereas excessive pyrophosphate and H2 PO 4 had no such effect. According to the authors, the selectivity for GTP over ATP and CTP was over 5 times and 10 times, respectively. The fluorescence quenching was due to the (C–H)+–X hydrogen bond formation (i.e. the interactions between the positive imidazolium groups and the negative phosphate groups in the nucleotides). However, the influence of the other nucleoside phosphates, diphosphates and triphosphates (e.g., GDP and GMP) on the fluorescence of the cavitand derivative was not examined. Ramaiah et al. synthesized a novel cyclophane (29) that can selectively recognize ATP based on the decrease
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in absorption of 29 after adding ATP [70]. Regrettably, the fluorescence yields of 29 are negligible, which limits its application as a sensitive fluorescence probe. In order to make up for the limitation, Ramaiah group used an indicator-displacement scheme in 2006 and developed a ‘‘turn-on’’ fluorescence assay for GTP [71]. It should be noted that the compound HPTS (10) has high fluorescence yields and can act as a fluorescence indicator. Compound 29 forms a complex with 10 and then quenches the fluorescence emission of 10. When various nucleosides and nucleoside-phosphate derivatives are added to the solution of the complex formed [29-HPTS], a competitive displacement process may happen {i.e. HPTS may be released from [29-HPTS]}. GTP induced the maximum displacement, resulting in about 150-fold recovery in fluorescence intensity of HPTS at 512 nm, while ATP and ITP caused 45-fold and 50-fold enhancement, respectively, and adenosine, AMP, ADP, CTP or UTP induced negligible changes. According to the authors, the whole process of the assay involved electron transfer and the synergistic effects of p-stacking and electrostatic interactions. Compound 29 combined with 10 can successfully discriminate GTP from ATP, ITP, AMP, ADP, CTP, UTP and adenosine in not only buffer but also biological fluids through a visual ‘‘ONOFF-ON’’ fluorescence mechanism. However, whether GDP and GMP can displace the HPTS was not considered.
electrostatic interactions between Cu2+ of 31 and the suitably placed phosphate group of GMP; (b) coordination between Cu2+ ions and N7 of the guanine base; and, (c) p-stacking between the anthracene moieties and the aromatic unit of GMP. The selectivity of the metallocyclophane 31 for GMP is favorable. The combinatorial benzimidazolium dye 32, synthesized by Chang et al., is a very good ‘‘turn-on’’ fluorescent sensor for GTP [73]. In order to evaluate the selectivity of 32 for GTP, the effects of all the nucleosides (adenosine, uridine, cytidine and guanosine) and nucleotides (XNP, where X = A, U, C, G, and N = mono, di, tri) were tested on the fluorescence of 32. When excited at 480 nm, GTP caused the maximum fluorescence enhancement, whereas the other analytes induced less change in fluorescence. Visual distinction is also obvious under 365 nm UV lamp light because only the GTP-containing solution shows green fluorescence, the others being colorless. So far, this is the first probe that can selectively recognize GTP among all the other nucleosides (adenosine, uridine, cytidine, and guanosine) and nucleoside phosphates, diphosphates and triphosphates. (a)
In 2009, the Ramaiah group again developed a supramolecular Cu(II) metallocyclophane (31), which could selectively recognize GMP based on fluorescence quenching [72]. Metallocyclophane 31 is formed from ligand 30 and CuCl2 in a 2:2 stoichiometry, and they found that GMP results in 90% quenching of the fluorescence intensity of 31, whereas adenosine, guanosine, AMP, ADP, ATP, GDP, GTP and 3 0 ,5 0 -cGMP show negligible changes in the fluorescence emission of 31. The quenching mechanism includes:
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Besides the above SOMs as fluorescent probes for GMP and its derivatives, as a fluorescent probe, Tb3+ can also be used for GTP detection [74]. The problem is that the selectivity of the Tb3+ probe is not good, especially in vivo, mainly due to the interference of GDP. In 2009, Vuojola et al. developed a homogeneous competitive Fo¨rster resonance energy transfer (FRET) assay for GTP based on energy transfer from lanthanidechelate donors to Rab21 GTPase-joined fluorescent protein acceptors [75]. Compared with the other assays for GTP, the innovation and the selectivity of this paper were excellent. So far, there have been not many SOMs as probes for GMP and its derivatives compared to AMP and its derivatives. Interestingly, these probes mostly have the imidazole subunit, involving coordination, (C–H)+–X hydrogen bond and electrostatic interactions, which indicates that, in the design of probes for GMP and its derivatives, we should pay sufficient attention to the imidazole group.
survive persistently. If bacteria are put in different living environments, (p)ppGpp exhibits various functions {e.g., (p)ppGpp can influence the production of antibiotics, virulence, symbiosis, porulation and colony morphology [79]}. Here, guanosine tetraphosphate (ppGpp) or guanosine pentaphosphate (pppGpp), as stringent alarmone, is considered to be another derivative of guanosine monophosphate. In view of its importance, detecting it becomes significant. Hong et al. reported the first fluorescent chemosensor, PyDPA (33), which can selectively detect (p)ppGpp, from among other nucleotides and their derivatives or pyrophosphate in water based on pyrene-excimer fluorescence [80]. When ATP, GTP, CTP, TTP, UTP, cAMP, cGMP or inorganic pyrophosphate (PPi) is added to the aqueous solution of PyDPA, only the pyrene-monomer fluorescence at 380 nm is enhanced. With addition of (p)ppGpp, strong pyrene-excimer fluorescence of PyDPA at 470 nm is observed, indicating that PyDPA can recognize (p)ppGpp from all the other analytes. Furthermore, PyDPA was applied to detect in real time the synthesis of ppGpp in vitro through extracted E. coli ribosomal complexes and could also detect ppGpp produced in the starved bacterial cells. The design of probe PyDPA is artful due to the pyrene-excimer formation upon adding (p)ppGpp to PyDPA. Moreover, the selectivity and the sensitivity are good.
4. Probes for stringent alarmone [(p)ppGpp] Stringent response is considered to be a mechanism for bacteria to survive in the face of adversity. When bacteria are in stress circumstances (e.g., amino-acid starvation), an anomalous reaction of the ribosome will happen due to lack of aminoacylated tRNA. In this process, guanosine 3 0 -diphosphate-5 0 -di(tri)phosphate [(p)ppGpp] is synthesized by RelA through the transfer of a pyrophosphate from ATP to the 3 0 -OH of GDP (or GTP) [66,76–78]. At this time, (p)ppGpp can make bacteria 364
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This is the only specific fluorescent probe for (p)ppGpp at present. Compared with the other nucleotides, the difference of the structure of (p)ppGpp lies in the 3 0 -diphosphate. If we want to design fluorescent probes for it, we must consider both 3 0 -diphosphate and 5 0 -di(tri)phosphate simultaneously, just as in the design of PyDPA (33), or else the selectivity of the probe is poor
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because of the interference of GTP, GDP and GMP. There are therefore some difficulties in designing fluorescent probes for (p)ppGpp. Research is in progress.
5. Probes for cytidine triphosphate and uridine diphosphate and triphosphate Uridine phosphates and cytidine phosphates widespread in living cells also play vital roles in the biological process. For example, UTP is a basal part in synthesis of RNA and glycosylation processes. Regrettably, there are few selective, specific probes for uridine or cytidine phosphates, so it is urgent to establish such fluorescent probes. Tb3+ can form a strongly fluorescent complex with Tiron (2), which can also be used to determine CTP and UTP besides ATP, ADP and AMP [42]. The detection limits of Tb3+-Tiron for CTP and UTP are 0.2 ng/mL and 0.3 ng/mL, respectively. Compound 8 prefers the nucleoside triphosphates to diphosphates or phosphates [50]. ATP can quench the fluorescence intensity of 8, while GTP, CTP and UTP have the similar effects to ATP on changes in the fluorescence of 8. Obviously, the specificity and the selectivity of the above two probes for CTP and UTP are bad. Yoon et al. designed a selective fluorescent probe (34) for UTP/UDP based on a perylene-dipicolylamine (DPA)2Zn2+ structure, which works under physiological conditions [81]. Compound 34 shows fluorescence enhancement upon addition of UTP and UDP. CTP also induces a negligible increase in fluorescence, whereas Pi, PPi, ATP, GTP, ADP, AMP or UMP has little quenching effect. In view of this phenomenon, compound 34 can recognize UTP and UDP selectively, and the association constants of UTP-34 and UDP-34 are calculated as 6.0 · 103/M and 1.1 · 104/M, respectively. According to the authors, the mechanism of this probe for UTP/ UDP may ascribe cooperative binding of the Zn-centers in 34 with the uridine and phosphate moieties of UTP/ UDP and an optimum spacer template binding offered by the perylene moiety. Furthermore, the probe was successfully applied to monitor the glycosylation processes involved in UTP and UDP.
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In designing probes for UMP and CMP and their derivatives, perhaps we should still think how to design the ligands, because the ligand is the binding site determining the selectivity of the probe. At the same time, the signal reporter should also be carefully chosen. We expect that more selective and more specific probes for uridine or cytidine phosphates might be designed in the near future.
6. Concluding remarks SOMs as fluorescent probes for nucleotides and their derivatives are obviously important. These SOM probes should usually have a fluorescence-signal transducer (fluorophore) and the ability to bind with analytes (the ligands). The two critical requirements might make the SOMs be a single unit or comprise two parts in a structure. For the latter case, the connection of the fluorophores with ligands is important, and electron or charge transfer might be involved. For fluorophores, the SOMs should involve pyrene, anthracene, xanthene and perylene, so that electron or charge transfer may occur easily. To bind with analytes, the ligands as recognition units generally include the dipicolylamine (DPA)-Zn2+ system, imidazole, amido groups and so on. For example, probe 34 has two very clear structural components – perylene acts as fluorophore, while dipicolylamine (DPA)-2Zn2+ as ligand – so that it can realise the specific recognition of UTP/UDP against the other phosphatecontaining anions. The interaction process between SOM probes and analytes might involve coordination, hydrogen bond, p– p stacking, electrostatic interaction and so on, wherein fluorescent signal changes are essential. Regrettably, there are several limitations in these reported methods (e.g., some probes cannot work under physiological conditions, while some others cannot give distinct signal changes, or the selectivity is not ideal). All these drawbacks restrict practical applications in biological systems. Since nucleotides and their derivatives are of significance to organisms, it is necessary to design more specific probes that can be applied to monitor certain biological events associated with nucleotides. It is worth
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noting that an attractive, promising field is the design of fluorescence SOMs for real-time detection or imaging of stringent alarmone. The development of these fluorescent probes is progressing gradually, so we are convinced that more selective, more specific and more practical probes for nucleoside phosphates could be designed in the near future. Acknowledgements The authors are extremely grateful for the financial support by the National Natural Science Foundation of China (No. 90813019), and express deep thanks to the scholars whose names are in the references.
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Small organic molecules as fluorescent probes for nucleotides and their derivatives Xi Juan Zhao, Cheng Zhi Huang Nucleoside monophosphates, diphosphates, triphosphates, and stringent alarmone {i.e. guanosine 3 0 -diphosphate-5 0 -di(tri)phosphate [(p)ppGpp]} are important to organisms. There are detection methods for these anions (e.g., based on absorption, fluorescence emission and chemiluminescence). We review the rise in fluorescent probes using small organic molecules for detecting these anions. The requirements of fluorescence real-time monitoring and imaging analysis make it certain that more economic, more specific, and more environmentally and biologically benign fluorescent probes will be designed in the near future. ª 2010 Elsevier Ltd. All rights reserved. Keywords: Adenosine mono (di or tri) phosphate; Cytidine triphosphate; Fluorescence; Fluorescent probe; Guanosine mono (di or tri) phosphate; Nucleotide; Nucleotide derivative; Small organic molecule; Stringent alarmone; Uridine di(tri) phosphate
1. Introduction Xi Juan Zhao, Cheng Zhi Huang* Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China Cheng Zhi Huang College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, PR China
*
Corresponding author. Tel.: +86 23 68254659; E-mail: [email protected]
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As integral parts of nucleic acids, nucleotides are very significant units in life. Also, nucleotide derivatives exist widely in organisms. Both nucleotides and their derivatives participate in biological and chemical processes. The importance of these phosphate-containing anions is therefore self evident, so that detection or identification of these anions remains a critical focus of research. Detection of these phosphate-containing anions started from detection of inorganic phosphate or pyrophosphate with the signal response of molecular absorption [1,2], fluorescence [3–16] and chemiluminescence [17]. There is also detection of nucleotides and their derivatives [18–22] that can be considered biophosphates based on HPLC. With the development and the maturity of aptamer science, aptamers have been applied to detect nucleotides due to the high affinity and high specificity of aptamers for targets [23]. The aptamer-based ATP assay has good selectivity based on
fluorescence [24–29], color [30] or electrochemistry [31,32]. Fluorescence-based techniques including quantitative detection or real-time monitoring have good sensitivity and selectivity [33], which are related to the design of specific, small organic molecules (SOMs) acting as fluorescence probes. Generally, fluorescent SOMs should have two function groups: the fluorophore that gives fluorescence emission and acts as signal reporter; and, the ligand that is the molecular recognition element and can bind specifically to the analyte. The two function groups are assembled together as a unit or separated with a spacer that can permit transfer of electron or charge. Regardless of environmental factors, the yield of the fluorescence emission of the fluorophore group determines the sensitivity, and the design of the ligand determines the selectivity of the fluorescent probe or the reagent. Ideallydesigned SOMs as fluorescent probes therefore have the advantages of the good sensitivity of fluorescence-based techniques and the selectivity of the ligand. Also, the response for the recognition between the probe and the analyte should be specific. For these advantages, SOMs have been well exploited as fluorescent probes in chemistry, biology and medicine. There have been several reviews relating to the recognition or sensing of phosphate anions from different aspects [34– 38]. For example, the Hong and Yoon group covered chemosensors for pyrophosphate in terms of topological and structural classification, using signal
0165-9936/$ - see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2009.12.011
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Figure 1. Structures of nucleotides and their derivatives.
changes in fluorescence and absorption [38], while Wolfbeis et al. comprehensively summed up fluorescent probes for inorganic phosphates and biophosphates [37]. However, few reviews have been reported to probe nucleotides and their derivatives based on the fluorescence signals. In this article, we deal with the past 30 years of progress in fluorescent probes for nucleotides and their derivatives (Fig. 1). We confine ourselves to fluorescent probes that are SOMs, and will not cover those (e.g., aptamers) where fluorescent SOMs are employed as fluorophore tags. To understand the application of the fluorescent SOM probes, we first summarize the three approaches to interaction shown in Fig. 2, where F1 and F2 denote the signal intensities of fluorescence emission before and after the interaction between the SOMs and the nucleotides or their derivatives. The fluorescent probes bind the
nucleoside phosphates directly or indirectly through an intermediate fluorescent indicator. In either case, the properties of the probe or the fluorescent indicator change to give rise to variations in fluorescent signals. 2. Probes for adenosine phosphate, diphosphate and triphosphate Adenosine phosphate, diphosphate and triphosphate are key players in organisms. It is well-known that ATP, as an energy donor, takes part in the energy metabolism in living cells, and also participates in the biosynthesis process as a reactant [39]. The concentration of ATP can be used as a quality standard of the viability of blood cells for transfusion [40]. It is therefore very significant theoretically and practically to detect adenosine phosphates.
Figure 2. Three types of approaches to the interaction for probes and nucleoside phosphates.
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Since lanthanide ions exhibit large coordination numbers and high coordination flexibility, they can coordinate with some SOMs to form complexes. Since the coordination numbers of lanthanide ions are not full, electronegative phosphate of nucleotides can combine with lanthanide ions, and the spectral properties of the lanthanide-ion complexes then change. Chang et al. reported that Tb3+ could form a complex with 1, 10-phenanthroline (1) and ATP can increase the fluorescence emission of the complex at pH 7.0 [41]. Regrettably, the selectivity of this probe is not good because ADP and AMP can also increase the fluorescence intensity to some extent so that their interference cannot be ignored.
Zhao et al. used the Tb3+-tiron (2) complex as a fluorescent probe to determine nucleotides and polynucleotides based on fluorescence quenching [42]. The fluorescence intensity of Tb3+-tiron at 546 nm decreased significantly in the presence of ATP, ADP or AMP when excited at 317 nm. The extent of quenching depended on the amount of phosphate available for binding to Tb3+. Similarly, the Tb3+-tiron system could be used only to detect solutions of pure substance and its selectivity is unsatisfactory.
Tb3+ forms a complex with norfloxacin (3) and ATP can remarkably enhance the emission at 545 nm [43]. This probe can be successfully applied to the determination of ATP in injection and tablet. Scha¨ferling et al. tested the selectivity of the fluorescent probe and found cAMP did not interfere, but pyrophosphate caused little fluorescence quenching compared to ATP [44]. They therefore used Tb-norfloxacin as a probe for the determination of adenylyl-cyclase activity.
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Some Eu3+-based complexes also have been used as fluorescent probes for adenosine phosphate, diphosphate and triphosphate. Both oxytetracycline (4) and doxycycline (5) containing b-diketonate configuration were reported to form complexes with Eu3+, which could be used to probe ATP [45,46]. The coordination number of Eu3+ with oxytetracycline and doxycycline is 8, which is not satisfied, so H2O molecules fill up the vacancy. ATP can replace H2O and compensate for the energy loss through the O-H vibration of water. The fluorescence of Eu3+-oxytetracycline and Eu3+-doxycycline at 612 nm can therefore be greatly enhanced by ATP. Regrettably, the authors did not consider whether or not ADP and AMP had such an effect.
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Scha¨ferling et al. further established a method to determine the activity of creatin kinase based on the different effects of ATP and ADP on the fluorescence emission of Eu3+-tetracycline (6) [47]. ATP strongly quenches the fluorescence intensity, whereas ADP has no significant effect. The authors also studied the effect of AMP, cAMP, GTP, GDP and pyrophosphate on the fluorescence intensity of the Eu3+-tetracycline complex. Most important is the application in the determination of the activity of a creatin kinase, since there is little interference.
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fluorescence emission of the probe could be quenched by nucleotides and responded very selectively toward AMP and GMP [49]. They supposed the mechanism involved hydrogen-atom abstraction or exciplex formation, so they predicted that DBO may become a useful fluorophore to detect nucleotides.
The above compounds (1–6) are easily available, but poor selectivity makes researchers attempt to synthesize
Inouye et al. synthesized a series of pyrenophanes and found that bis(diazoniacrown)-substituted pyrenophane (8) can recognize the nucleoside triphosphates [50]. The ability to recognize decreases in the order of triphosphate > diphosphate > monophosphate. With ATP as an example, fluorescence emission of 8 gets reduced with adding ATP. The interaction mechanism is p-stacking between nucleobase and the pyrene fluorophores and the electrostatic interactions of the positive diazoniacrowns with phosphate moieties. The drawback is that this kind of probe cannot specifically recognize a certain nucleoside triphosphate.
new compounds that could act as probes for adenosine nucleotides with improved selectivity. In this case, Anslyn et al. reported that the use of the guanidinium entities, along with the tripeptide arms, Ser-Tyr-Ser, could produce resin-bound sensors with excellent selectivity for ATP [48]. The sensor designed gives the largest fluorescence increase upon addition of ATP, whereas AMP and GTP have little effect on the change in fluorescence of the sensor, suggesting that both triphosphates and nucleotide bases play important roles in the recognition process. The Nau group designed a new probe of 2, 3-diazabicyclo [2.2.2] oct-2-ene (DBO, 7), and found that the
Dumy et al. designed a cyclic miniature esterase that can hydrolyze compound 9 to compound 10 [51]. They then tested the inhibitory effect of orthophosphate (Pi), AMP, D-myoinositol-1,4,5-triphosphate (IP3), citric acid, ATP and ADP on the miniature esterase and found only ADP was effective. Pi, AMP, IP3, ATP, or citric acid turn the solution yellow because of the formation of 10, whereas the solution containing ADP remains colorless and no fluorescence signal of 10 can be detected in this solution with the time increasing. This work is attractive because it has a good selectivity for ADP against ATP.
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The complex 13-Cd2+ contains a fluorophore of 7-amino-4-trifluoromethylcoumarin and Cd2+-cyclen (1,4,7,10-tetraazacyclododecane) as an anion receptor [54]. When AMP is added to a solution of 13-Cd2+, the excitation wavelength at 500 nm shows red shift. Because ADP and ATP had higher affinities for 13-Cd2+ than AMP, the authors did not follow traditional ideas by using this complex to distinguish AMP, ADP and ATP, but smartly applied the different signal of AMP and cAMP to 13-Cd2+ for monitoring phosphodiesterase activity with their finding that adding cAMP hardly changes the excitation spectrum. Phosphodiesterase can cleave cAMP to AMP, and, by monitoring the increase of AMP, can attain the expected purpose.
Ligands 11 and 12 can form some complexes with Eu3+, and then phosphate-containing anions will occupy the vacant coordination sites of Eu3+ [52,53]. Under appropriate conditions, the conformation of 11 and Eu3+-12 will change, following the change of the corresponding absorption or fluorescence spectra. The fluorescence intensity of the Eu3+-11 complex can be enhanced by adding ATP, and ADP or AMP induces a smaller fluorescence enhancement. Similarly, ATP, ADP or AMP also enhances the fluorescence emission of the Eu3+-12 complex. The selectivity of both probes for ATP is poor because the influence of ADP and AMP on the fluorescence is not ignored. Hamachi et al. recently attached a dipicolylamine (DPA)-2Zn2+ system to a fluorophore [55–57]. Complex 14 has good selectivity among ATP, CTP and GTP in fluorescence sensing. ATP can enhance the fluorescence emission of 14, and CTP induces a weaker enhancement than ATP, whereas GTP gradually decreases the fluorescence intensity. The affinity of 14 for ADP and AMP is smaller than that for ATP, while cAMP has no signal fluorometrically. However, the selectivity for the detection of ATP is still not good [55].
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Bis-Zn/DPA-anthracene receptor 15 embedded in the hydrogel 17 can distinguish ATP from other anionic species based on fluorescence, but it cannot discriminate a specific phosphate-containing species among the phosphate anion derivatives. After receptor 16 is incorporated in supramolecular hydrogel 17, the fluorescence emission of 16 (at 512 nm) is enhanced following the wavelength blue-shift for phenyl phosphate, and gets quenched for ATP with the emission red-shift, whereas it has no change for non-phosphate anions. It is noteworthy that there are no such phenomena in aqueous solution or agarose gel, which lacks hydrophobic domains, indicating that the medium (e.g., 17) is very important for these findings [56].
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Dual-emission probes (18 and 19) can be employed to detect ATP in a ratiometric manner – the first example of ratiometric fluorescence-sensing system for ATP [57]. Addition of ATP to 18 and 19 reduces the emission intensities and the blue shift of the emission wavelength. This unique emission-wavelength shift is due to dissociation of the first Zn2+ from the acridine fluorophore following formation of the binuclear Zn2+ complex. Since ADP has the same effect as ATP, whereas phosphate (Pi) and AMP cause no change in the fluorescence intensity of 19, probe 19 is applied in monitoring apyrase-catalyzed hydrolysis of ADP.
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(P2 O4 7 , F/Fo = 54) caused a large increase in the fluorescence of 21, whereas HPO2 4 , AMP, cGMP induced negligible fluorescence change, even under a high concentration. It is easy to understand that the selectivity for a single substance is not bad. Ingeniously, the author applied probe 21 to fluorescence visualization of ATP-particulate stores in living cells, combining with quinacrine, a widely-used probe to locate the ATP store.
Das et al. investigated the interactions between a new chromogenic complex (20) and some biologically important anions {e.g., ATP, ADP, AMP, pyrophosphate (PPi) and phosphate [58]}, and they found that only the addition of ATP made the color of 20 change from pale yellow to light pink in aqueous solution, while ADP, AMP, PPi and phosphate had no such effect. In order to further confirm the efficient, selective binding of ATP to 20, the authors studied the fluorescence changes of 20 after adding these anions. The fluorescence emission was found to be partially quenched by ATP, whereas no such quenching was observed in the excess of ADP, AMP, PPi or H2 PO 4 . Remarkably, complex 20 can be used as a colorimetric staining agent for yeast cells, and staining can be detected under a simple light microscopy.
A new xanthene-based Zn2+ complex, reported by Hamachi in 2008, can sense nucleoside polyphosphates (e.g., ATP) with a ‘‘turn-on’’ fluorescence signal [59]. The coordination of Zn2+ to the DPA sites of 21 induced the formation of a non-fluorescent deconjugated xanthene ring, while adding nucleoside polyphosphates recovered the conjugated structure of the xanthene fluorophore, leading to the ‘‘turn-on’’ fluorescence signal. The selectivity of 21 towards various nucleoside polyphosphates was evaluated. XTP (X = A, G, C, F/ Fo > 30), XDP (X = A, U, F/Fo > 15), and pyrophosphate 360
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Jolliffe et al. synthesized a new molecular receptor (22) based on a diketopiperazine ring with dipicolylamine (DPA)-2Zn2+ arms [60]. Compound 22 can quench the fluorescence of 23, which can be used as an indicator in displacement assays to evaluate anionbinding capability. When various monophosphate, diphosphate, triphosphate and non-phosphate anions are added to the solution with a 1: 1 ratio of receptor 22 and indicator 23, only ATP, ADP, GTP, PPi and citrate can recover the fluorescence emission of 23, meaning that these ions show significant indicator displacement. However, none of the monophosphate derivatives (e.g., AMP, or cAMP) has this signal. Although 22 shows high affinity and selectivity for diphosphate and triphosphate in aqueous solution at pH 7.4, except for non-phosphate anion and citrate, it is obvious that the probe lacks selectivity for a single diphosphate or triphosphate anion.
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AMP or GMP has no such effect in the excitation spectra in aqueous solution. Furthermore, the interaction of 25 with ATP in mitochondria is also tested and the same phenomena are observed, indicating that the specificity of 25 for ATP is good in the presence of cell organelles, so it is possible for probe 25 to determine ATP in vivo.
A new phenanthroline-containing macrocycle (24), designed by Bencini et al., can selectively recognize ATP among GTP, TTP, CTP as well as ADP and AMP at pH 6 based on fluorescence quenching [61]. Compound 24 could be easily protonated in aqueous solutions and has six positive charges at pH 6 7, and the hexaprotonated 24 can form stable complexes with nucleoside phosphate, diphosphate and polyphosphate. Only addition of ATP to the hexaprotonated 24 (1:1 molar ratio) can completely quench the fluorescence of 24, while ADP, AMP or the other triphosphate nucleosides induces a much smaller decrease of the fluorescence intensity. The fluorescence quenching is attributed to the charge-charge and hydrogen-bonding interactions between the anionic phosphate moiety and the ammonium groups of 24 and p-stacking between the nucleobases and the phenanthroline unit of 24. The selectivity of this probe for ATP is very good compared with the previous researches.
Yoon et al. designed a unique ratiometric fluorescent sensor (26) for ATP based on a transition of excimer vs. monomer pyrene fluorescence [63]. Usually, the monomeric emission of pyrene moieties is at 375 nm, while the emission of its excimer is at 487 nm. Upon addition of ATP, GTP, TTP, UTP, CTP, Pi or PPi, the excimer-pyrene fluorescence of 26 is quenched to different extents. Surprisingly, only ATP makes the fluorescence of monomeric pyrene increase. In view of this unique change, compound 26 can be used to discriminate ATP from the other three nucleoside triphosphates. Furthermore, the selectivity for ATP over ADP and AMP is also tested. The result is that ADP induces correspondingly smaller changes in the fluorescence of 26 than ATP, but the effects of AMP on the monomeric and excimer emissions are negligible.
3-Hydroxy-4 0 -(dimethylamino) flavone (FME probe 25) can form a complex with ATP, leading to increase of fluorescence-emission intensity and an accompanying considerable red shift in the excitation wavelength of the FME probe [62]. Whereas inosine-5 0 -triphosphate (ITP), bnicotinamide adenine dinucleotide (NAD), GTP, UTP, ADP,
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When the ratiometric fluorescent technique is introduced, the monomer vs. excimer pyrene fluorescence (F375/F487) for 26 upon addition of ATP is much larger than that upon addition of ADP and AMP, which is large enough to distinguish ATP from ADP and AMP. According to the authors, adenine can sit between the two pyrenes in 26 and thus separate pyrene moieties resulting in the monomer-pyrene fluorescence, while the other three bases interact outside the pyrene-pyrene dimmer, leading to the quenching of the excimer fluorescence. Depending on these findings, probe 26 can be applied to ATP-staining experiments and monitor apyrase activity in real time. Compound 26 is a very selective, specific fluorescent probe for ATP among other nucleoside triphosphates, and ADP and AMP at physiological pH. By the way, polythiophene derivatives as polymers can also act as fluorescent probes for adenosine phosphate, diphosphate and triphosphate. Shinkai et al. established a method of detecting ATP based on polythiophene derivative 27. Fluorescence quenching of 27 at 529 nm is much more effective in the presence of ATP (kex = 445 nm) than AMP (kex = 435 nm) or ADP (kex = 435 nm) and the detection limit can be extended to 108 mol/L [64]. The Bazan group chose polythiophene derivative 27 as a probe to visualize glucose phosphorylation directly [65].
From all the above descriptions, we can see that the following two types of probes, fluorophore-DPA-Zn2+ and lanthanide ion-ligand, play dominant roles in probing adenosine phosphate, diphosphate and triphosphate, especially adenosine triphosphate. Besides the two types of probes, most probes for adenosine phosphates involve metal-ion coordination events, which might be accompanied by electrostatic or p–p stacking interactions. 3. Probes for guanosine phosphate, diphosphate and triphosphate The effects of guanosine phosphates on the biological systems are vital. Some are involved in the carbohydrate metabolism and the stringent response [66,67], so 362
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detection of these anions makes sense. One thing to be noted is that the imidazole group is a very valuable ligand in the design of fluorescent probes for GMP and its derivatives. Yoon et al. developed a new fluorescent anthracene and imidazolium compound (28) that could effectively and selectively recognize GTP among ATP, ADP, AMP, pyrophosphate, H2 PO 4 , F and Cl [68]. Compound 28 shows a distinct chelation-enhanced fluorescence quenching (CHEQ) effect with a slight red shift after adding GTP. The quenching mainly attaches to the guanine base in GTP, while the other anions induce chelation-enhanced fluorescence (CHEF) effects on 28 at different levels. This probe can work in aqueous solution of physiological pH. It is a pity that the effects of GDP and GMP on the fluorescence of 28 are not considered in the paper [68]. Subsequently, Yoon and Kim et al. synthesized four fluorescent imidazolium chemosensors for recognizing pyrophosphate, and three of them were anthracene-bearing imidazolium receptors [14].
The Yoon group again designed a new fluorescent cavitand derivative with four imidazolium groups and four pyrene groups for recognizing GTP [69]. The addition of GTP gradually quenched the fluorescence emission of the cavitand derivative, and ATP, CTP or ADP caused a relatively small CHEQ effect, whereas excessive pyrophosphate and H2 PO 4 had no such effect. According to the authors, the selectivity for GTP over ATP and CTP was over 5 times and 10 times, respectively. The fluorescence quenching was due to the (C–H)+–X hydrogen bond formation (i.e. the interactions between the positive imidazolium groups and the negative phosphate groups in the nucleotides). However, the influence of the other nucleoside phosphates, diphosphates and triphosphates (e.g., GDP and GMP) on the fluorescence of the cavitand derivative was not examined. Ramaiah et al. synthesized a novel cyclophane (29) that can selectively recognize ATP based on the decrease
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in absorption of 29 after adding ATP [70]. Regrettably, the fluorescence yields of 29 are negligible, which limits its application as a sensitive fluorescence probe. In order to make up for the limitation, Ramaiah group used an indicator-displacement scheme in 2006 and developed a ‘‘turn-on’’ fluorescence assay for GTP [71]. It should be noted that the compound HPTS (10) has high fluorescence yields and can act as a fluorescence indicator. Compound 29 forms a complex with 10 and then quenches the fluorescence emission of 10. When various nucleosides and nucleoside-phosphate derivatives are added to the solution of the complex formed [29-HPTS], a competitive displacement process may happen {i.e. HPTS may be released from [29-HPTS]}. GTP induced the maximum displacement, resulting in about 150-fold recovery in fluorescence intensity of HPTS at 512 nm, while ATP and ITP caused 45-fold and 50-fold enhancement, respectively, and adenosine, AMP, ADP, CTP or UTP induced negligible changes. According to the authors, the whole process of the assay involved electron transfer and the synergistic effects of p-stacking and electrostatic interactions. Compound 29 combined with 10 can successfully discriminate GTP from ATP, ITP, AMP, ADP, CTP, UTP and adenosine in not only buffer but also biological fluids through a visual ‘‘ONOFF-ON’’ fluorescence mechanism. However, whether GDP and GMP can displace the HPTS was not considered.
electrostatic interactions between Cu2+ of 31 and the suitably placed phosphate group of GMP; (b) coordination between Cu2+ ions and N7 of the guanine base; and, (c) p-stacking between the anthracene moieties and the aromatic unit of GMP. The selectivity of the metallocyclophane 31 for GMP is favorable. The combinatorial benzimidazolium dye 32, synthesized by Chang et al., is a very good ‘‘turn-on’’ fluorescent sensor for GTP [73]. In order to evaluate the selectivity of 32 for GTP, the effects of all the nucleosides (adenosine, uridine, cytidine and guanosine) and nucleotides (XNP, where X = A, U, C, G, and N = mono, di, tri) were tested on the fluorescence of 32. When excited at 480 nm, GTP caused the maximum fluorescence enhancement, whereas the other analytes induced less change in fluorescence. Visual distinction is also obvious under 365 nm UV lamp light because only the GTP-containing solution shows green fluorescence, the others being colorless. So far, this is the first probe that can selectively recognize GTP among all the other nucleosides (adenosine, uridine, cytidine, and guanosine) and nucleoside phosphates, diphosphates and triphosphates. (a)
In 2009, the Ramaiah group again developed a supramolecular Cu(II) metallocyclophane (31), which could selectively recognize GMP based on fluorescence quenching [72]. Metallocyclophane 31 is formed from ligand 30 and CuCl2 in a 2:2 stoichiometry, and they found that GMP results in 90% quenching of the fluorescence intensity of 31, whereas adenosine, guanosine, AMP, ADP, ATP, GDP, GTP and 3 0 ,5 0 -cGMP show negligible changes in the fluorescence emission of 31. The quenching mechanism includes:
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Besides the above SOMs as fluorescent probes for GMP and its derivatives, as a fluorescent probe, Tb3+ can also be used for GTP detection [74]. The problem is that the selectivity of the Tb3+ probe is not good, especially in vivo, mainly due to the interference of GDP. In 2009, Vuojola et al. developed a homogeneous competitive Fo¨rster resonance energy transfer (FRET) assay for GTP based on energy transfer from lanthanidechelate donors to Rab21 GTPase-joined fluorescent protein acceptors [75]. Compared with the other assays for GTP, the innovation and the selectivity of this paper were excellent. So far, there have been not many SOMs as probes for GMP and its derivatives compared to AMP and its derivatives. Interestingly, these probes mostly have the imidazole subunit, involving coordination, (C–H)+–X hydrogen bond and electrostatic interactions, which indicates that, in the design of probes for GMP and its derivatives, we should pay sufficient attention to the imidazole group.
survive persistently. If bacteria are put in different living environments, (p)ppGpp exhibits various functions {e.g., (p)ppGpp can influence the production of antibiotics, virulence, symbiosis, porulation and colony morphology [79]}. Here, guanosine tetraphosphate (ppGpp) or guanosine pentaphosphate (pppGpp), as stringent alarmone, is considered to be another derivative of guanosine monophosphate. In view of its importance, detecting it becomes significant. Hong et al. reported the first fluorescent chemosensor, PyDPA (33), which can selectively detect (p)ppGpp, from among other nucleotides and their derivatives or pyrophosphate in water based on pyrene-excimer fluorescence [80]. When ATP, GTP, CTP, TTP, UTP, cAMP, cGMP or inorganic pyrophosphate (PPi) is added to the aqueous solution of PyDPA, only the pyrene-monomer fluorescence at 380 nm is enhanced. With addition of (p)ppGpp, strong pyrene-excimer fluorescence of PyDPA at 470 nm is observed, indicating that PyDPA can recognize (p)ppGpp from all the other analytes. Furthermore, PyDPA was applied to detect in real time the synthesis of ppGpp in vitro through extracted E. coli ribosomal complexes and could also detect ppGpp produced in the starved bacterial cells. The design of probe PyDPA is artful due to the pyrene-excimer formation upon adding (p)ppGpp to PyDPA. Moreover, the selectivity and the sensitivity are good.
4. Probes for stringent alarmone [(p)ppGpp] Stringent response is considered to be a mechanism for bacteria to survive in the face of adversity. When bacteria are in stress circumstances (e.g., amino-acid starvation), an anomalous reaction of the ribosome will happen due to lack of aminoacylated tRNA. In this process, guanosine 3 0 -diphosphate-5 0 -di(tri)phosphate [(p)ppGpp] is synthesized by RelA through the transfer of a pyrophosphate from ATP to the 3 0 -OH of GDP (or GTP) [66,76–78]. At this time, (p)ppGpp can make bacteria 364
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This is the only specific fluorescent probe for (p)ppGpp at present. Compared with the other nucleotides, the difference of the structure of (p)ppGpp lies in the 3 0 -diphosphate. If we want to design fluorescent probes for it, we must consider both 3 0 -diphosphate and 5 0 -di(tri)phosphate simultaneously, just as in the design of PyDPA (33), or else the selectivity of the probe is poor
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because of the interference of GTP, GDP and GMP. There are therefore some difficulties in designing fluorescent probes for (p)ppGpp. Research is in progress.
5. Probes for cytidine triphosphate and uridine diphosphate and triphosphate Uridine phosphates and cytidine phosphates widespread in living cells also play vital roles in the biological process. For example, UTP is a basal part in synthesis of RNA and glycosylation processes. Regrettably, there are few selective, specific probes for uridine or cytidine phosphates, so it is urgent to establish such fluorescent probes. Tb3+ can form a strongly fluorescent complex with Tiron (2), which can also be used to determine CTP and UTP besides ATP, ADP and AMP [42]. The detection limits of Tb3+-Tiron for CTP and UTP are 0.2 ng/mL and 0.3 ng/mL, respectively. Compound 8 prefers the nucleoside triphosphates to diphosphates or phosphates [50]. ATP can quench the fluorescence intensity of 8, while GTP, CTP and UTP have the similar effects to ATP on changes in the fluorescence of 8. Obviously, the specificity and the selectivity of the above two probes for CTP and UTP are bad. Yoon et al. designed a selective fluorescent probe (34) for UTP/UDP based on a perylene-dipicolylamine (DPA)2Zn2+ structure, which works under physiological conditions [81]. Compound 34 shows fluorescence enhancement upon addition of UTP and UDP. CTP also induces a negligible increase in fluorescence, whereas Pi, PPi, ATP, GTP, ADP, AMP or UMP has little quenching effect. In view of this phenomenon, compound 34 can recognize UTP and UDP selectively, and the association constants of UTP-34 and UDP-34 are calculated as 6.0 · 103/M and 1.1 · 104/M, respectively. According to the authors, the mechanism of this probe for UTP/ UDP may ascribe cooperative binding of the Zn-centers in 34 with the uridine and phosphate moieties of UTP/ UDP and an optimum spacer template binding offered by the perylene moiety. Furthermore, the probe was successfully applied to monitor the glycosylation processes involved in UTP and UDP.
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In designing probes for UMP and CMP and their derivatives, perhaps we should still think how to design the ligands, because the ligand is the binding site determining the selectivity of the probe. At the same time, the signal reporter should also be carefully chosen. We expect that more selective and more specific probes for uridine or cytidine phosphates might be designed in the near future.
6. Concluding remarks SOMs as fluorescent probes for nucleotides and their derivatives are obviously important. These SOM probes should usually have a fluorescence-signal transducer (fluorophore) and the ability to bind with analytes (the ligands). The two critical requirements might make the SOMs be a single unit or comprise two parts in a structure. For the latter case, the connection of the fluorophores with ligands is important, and electron or charge transfer might be involved. For fluorophores, the SOMs should involve pyrene, anthracene, xanthene and perylene, so that electron or charge transfer may occur easily. To bind with analytes, the ligands as recognition units generally include the dipicolylamine (DPA)-Zn2+ system, imidazole, amido groups and so on. For example, probe 34 has two very clear structural components – perylene acts as fluorophore, while dipicolylamine (DPA)-2Zn2+ as ligand – so that it can realise the specific recognition of UTP/UDP against the other phosphatecontaining anions. The interaction process between SOM probes and analytes might involve coordination, hydrogen bond, p– p stacking, electrostatic interaction and so on, wherein fluorescent signal changes are essential. Regrettably, there are several limitations in these reported methods (e.g., some probes cannot work under physiological conditions, while some others cannot give distinct signal changes, or the selectivity is not ideal). All these drawbacks restrict practical applications in biological systems. Since nucleotides and their derivatives are of significance to organisms, it is necessary to design more specific probes that can be applied to monitor certain biological events associated with nucleotides. It is worth
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noting that an attractive, promising field is the design of fluorescence SOMs for real-time detection or imaging of stringent alarmone. The development of these fluorescent probes is progressing gradually, so we are convinced that more selective, more specific and more practical probes for nucleoside phosphates could be designed in the near future. Acknowledgements The authors are extremely grateful for the financial support by the National Natural Science Foundation of China (No. 90813019), and express deep thanks to the scholars whose names are in the references.
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