JP4150064B2 - Fluorescence sensor for anion detection - Google Patents

Description

  The present invention particularly relates to a sensor compound capable of detecting an anion in an aqueous solution and various measurement methods using the compound.

Fluorescent sensors are useful for analyzing and elucidating the role of biomolecules in biological systems. For example, several functions of intracellular Ca ^II have been elucidated using fluorescent Ca ^II indicators ¹ . Many other metal sensors and pH sensors have been developed ² .

There is now an increasing interest in anion recognition ³ and anion sensor detection ⁴ because numerous organic and inorganic anions play an important role in living organisms. The development of an anion sensor will lead to the development of a new sensor that can detect biological organic molecules having an anion group in the molecule. However, most previously known sensors only function in organic media and are difficult to use for biochemical or physiological experiments.
In general, anions are larger than cations such as metal ions, and thus are more susceptible to solventization restrictions than cations. In organic solvents, solvation energy is relatively small and electrostatic interactions work effectively, so it is not difficult to capture and detect anions. However, in aqueous solvents suitable for biological applications, it is very difficult to recognize anions due to strong hydration. To date, although many are known in the organic environment, few fluorescent anion sensors that operate in aqueous solutions have been developed.

A fluorescent anion sensor for use in aqueous solution must meet two requirements. One is a sufficiently strong affinity for anions in water, and the other is the ability to convert anion recognition into a fluorescent signal. Most known anion sensors meet the latter requirement, but do not have sufficient affinity for anions in water. Some anion hosts can trap anions in an aqueous solvent, but they are simply host molecules, not sensor molecules. It is difficult to meet both requirements simultaneously.

Crazuik et al successfully developed a fluorescent anion sensor that operates in aqueous solution ⁵ . Such sensors detection mechanism is based on changing by the pK _a of the amino group adjacent to the fluorophore anion binding. However, this sensor can measure only in a specific pH range and is sensitive to pH change. Therefore, it is difficult to apply this sensor detection mechanism to biological experiments.

  Accordingly, an object of the present invention is to provide a sensor compound that can quantitatively measure anions in an aqueous solution that can be used in various biological experimental systems.

As a result of intensive studies to solve the above problems, the present inventor has developed a new means for detecting anions and completed the present invention.
That is, the present invention relates to a compound having an anion host composed of a metal complex of a cyclic polyamine and a fluorescent dye, and in a coordinate bond with the metal ion of the metal complex, between a substituent on the fluorescent dye and the anion. According to the compound, the spectrum of the fluorescent dye is changed by the ligand exchange function.
More specifically, the present invention relates to a compound having an anion host composed of a metal complex of a cyclic polyamine and a fluorescent dye, and the electron donation on the fluorescent dye involved in the coordinate bond with the metal ion of the metal complex The electron-donating property on the fluorescent dye is increased by the ligand exchange function between the functional substituent and the anion, and as a result, the spectrum of the fluorescent dye is changed.
The ligand exchange function between the electron-donating substituent on the fluorescent dye and the anion is based on the fluorescent dye in an aqueous solution system in which an anionic species having a strong coordinating power with respect to the anion host (metal complex) does not exist. The electron-donating substituent is coordinated to the metal ion, but by adding an anionic species with strong coordinating power to the system, the electron-donating substituent is removed from the coordinate bond with the metal ion, and the As a result, the change in the electronic state generated in the electron-donating substituent is transmitted to the fluorescent dye and changes its fluorescence spectrum. More specifically, the absorption (excitation) spectrum of the fluorescent dye is shifted to the longer wavelength side. .
Therefore, as long as it is a compound that causes a change in the spectrum of the fluorescent dye based on such a principle, the structure and type of the cyclic polyamine, the metal ion, and the fluorescent dye, which are the constituent parts of the compound of the present invention, are not particularly limited.
In order to efficiently cause such a change in the fluorescence spectrum, it is preferable that the anion host and the fluorescent dye are bonded via a linker.

Furthermore, the present invention relates to a fluorescent sensor comprising such a compound, particularly a fluorescent sensor capable of detecting an anion in an aqueous solution.
Examples of anions that can be quantitatively measured by the sensor of the present invention include inorganic anions such as pyrophosphate ions, phosphate ions, and halogen ions, organic anions such as citrate ions, and various nucleotide groups. 1 type or multiple types of anion selected from can be mentioned.

The present invention further relates to a method for measuring the concentration of anions in an aqueous solution, for example, based on the degree of shift of the absorption (excitation) spectrum of the fluorescent dye to the longer wavelength side using the compound of the present invention as a sensor.
Furthermore, the present invention uses the compound of the present invention as a sensor, and measures, for example, increase / decrease in anionic residues in an aqueous solution based on the degree of shift of the absorption (excitation) spectrum of the fluorescent dye toward the longer wavelength side. Concerning.
The present invention further relates to a method for measuring the activity of an enzyme involved in a reaction involving an increase or decrease of an anionic residue based on a change in fluorescence intensity using the compound of the present invention as a sensor. Examples of such enzymes include phosphodiesterases such as cyclic mononucleotide-specific phosphodiesterases (phosphodiesterase 3 ′, 5′-cyclic mononucleotide).

In the compound of the present invention, the number of nitrogen atoms and carbon atoms constituting the cyclic polyamine and the total number thereof are not particularly limited, but preferably) to 12, more preferably 12, and the number of nitrogen atoms is preferably 3 or 4, more preferably 4.
Suitable cyclic polyamines specifically, 1,4,7,10-tetraazacyclododecane (cyclen) and the like.
The electron donating substituent on the fluorescent dye of the compound of the present invention is not particularly limited, but can be appropriately selected by those skilled in the art depending on the kind of the fluorescent dye, cyclic polyamine, metal ion, etc. An amino group can be mentioned .
Fluorescent dyes can also be appropriately selected by those skilled in the art depending on other components, and preferred examples include 7- substituted coumarin derivatives such as 7-amino-trifluoromethylcoumarin.
As the linker, for example, an alkylene group such as an ethylene group and a methylene group can be used.
Preferable examples of the metal constituting the metal complex include transition metals such as CdII.

  The compound of the present invention can be easily prepared by those skilled in the art, for example, by a synthetic method as specifically shown in the following examples. Furthermore, the method for obtaining each substance, the method for synthesizing each compound, and the property analysis data regarding the synthesized compound (PDF) are available free of charge via the Internet http://pubs.acs.org. .

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

Example 1
Preparation of Compound Cyclone-conjugated coumarin (1) was synthesized using 7-amino-4-trifluoromethylcoumarin as a starting material in 4 steps shown in the following chemical reaction formula, and NH-silica gel (Fuji Silysia Chemical). And purified by column chromatography. The CdII complex of the compound (1) synthesized as described above, that is, the complex 1-CdII, obtained as a perchlorate, was recrystallized from ₂ -propanol / H ₂ O.

The reaction reagents used in the synthesis route of the cyclen-conjugated coumarin (1) and complex 1-CdII shown above are as follows. (A) TsCl pyridine,
(b) BrCH ₂ CH ₂ Br, Cs ₂ CO ₃ , (c) concentrated H ₂ SO ₄ , 90 ° C. (d) cyclen,
(e) Cd (ClO ₄ ) ₂ 60 ° C.
It should be noted that other compounds belonging to the present invention can be appropriately synthesized by those skilled in the art according to the above synthetic route.
The compound of the present invention thus synthesized functions as a fluorescence sensor for detecting anions, and the mechanism for detecting anions is shown in FIG. Here, M ^{n +} is a metal ion that can be stably chelated by the cyclen.

Example 2
In order to find out which metal best matches our design concept, we have complexed compound (1) with ZnII, CdII, and CuII, and compounds without metal. The absorption spectrum and fluorescence spectrum of (1) were measured. The concentration of compound (1) was 5 mM, and 1.2 equivalents of all metals were added based on compound (1). The solution was buffered with 100 mM HEPES (pH 7.4). The measurement was carried out using a UV-1600 UV-visible spectrometer (Japan, Kyoto, Shimadzu) at a concentration of 5 mM at 25 ° C. Further, F-4500 (Tokyo, Japan, Hitachi) was used as the fluorescence spectrometer.
The obtained absorption spectrum and excitation spectrum are shown in FIG. It is well known that these metals are strongly coordinated by cyclens. As shown in FIG. 1, the anion sensor detection requires coordination of the 7-amino group to the metal, and the involvement of a single electron pair of nitrogen in the metal coordination induces such a shift in wavelength. The desired spectral change was a blue shift in the absorption and excitation spectra.
The spectra were measured after sufficient equilibration (greater than 3 hours) due to the very slow formation of complexes with ZnII, CdII and CuII. The peak of the absorption spectrum of Compound (1) was 382 nm, and those of Complex 1-ZnII, Complex 1-CdII and Complex 1-CuII were 388 nm, 342 nm and 342 nm, respectively. That is, when CdII and CuII were added to the solution of compound (1), blue shifts in the respective absorption spectra were observed. This supports the idea that the 7-amino group coordinates to CdII and CuII. The absorption spectrum of complex 1-ZnII shifted slightly red. This red shift indicates that there is some interaction between ZnII and 7-amino-4-trifluoromethylcoumarin, but the 7-amino group is probably coordinated to ZnII for the reasons described above. There seems to be nothing.
The excitation spectra of Compound (1), Complex 1-ZnII, and Complex 1-CdII were similar to their absorption spectra, although the fluorescence intensity of Complex 1-ZnII was slightly increased. On the other hand, the fluorescence of complex 1-CuII was significantly reduced due to the nature of CuII (CuII ions are generally known to deactivate the fluorescence of various fluorescent compounds).
From the principle of anion sensor detection shown in FIG. 1, the central metal ion needs to be coordinated by five nitrogen atoms containing a 7-amino group. Therefore, complex 1-ZnII cannot act as an anion sensor unless ZnII is coordinated by the 7-amino group of compound (1) in a neutral aqueous solution containing 100 mM HEPES. Furthermore, since the fluorescence of the complex 1-CuII is strongly deactivated, this is also not suitable as a fluorescence sensor.
Thus, based on the absorption and excitation spectra, we have concluded that complex 1-CdII is the best fluorescent anion sensor based on our design concept as shown in FIG.

Example 3
Potentiometric pH titration Next, according to the procedure described in detail in reference ^12, 1: 1 metal - Compound (1) (0.17 mM and 0.5 mM) complex systems (Compound (1) · 4HCO _4, Compound (1 ) · 4HClO ₄ + Zn (ClO ₄ ) ₂ and compound (1) · 4HClO ₄ + Cd (ClO ₄ ) ₂ ), ionic strength I = 0.1M (Et ₄ NClO ₄ ), potential difference pH titration experiment at 25 ° C Went. Ligand protonation constants, metal complex formation constants and distribution diagrams were calculated using the computer programs SUPERQUAD ¹³ and MINIQUAD ¹⁴ .
The results are shown in Table 1. The protonation equilibrium constants of compound (1) were 9.67 (logK ₁ = logb ₀₁₁ ) and 8.39 (logK ₂ = logb ₀₁₂ -logb ₀₁₁ ). Other more low pK _a could not be calculated.

The stability constant of compound (1) with respect to ZnII and CdII shown as logb _{110 in} Table 1 was calculated, and logK (complex 1-ZnII) and logK (complex 1-CdII) were 10.80 and 11.38, respectively. The reported logK (cyclen ZnII) and logK (cyclen CdII) are 16.2 and 14.3, respectively. If the coordination formation of compound (1) -metal is the same as that of cyclen, logK (complex 1-ZnII) is expected to be larger than logK (1-CdII), but not That is, the relative stability order of the compound (1) -metal complex was reversed compared to the cyclen metal complex. This result suggests that the coordination formation of complex 1-CdII is different from that of complex 1-ZnII. We consider that water molecules may prevent the aromatic amino group from coordinating to ZnII even in the absence of an anion that binds strongly to ZnII, and that ZnII is not coordinated by the aromatic amino group. Yes. On the other hand, the results are consistent with the idea that CdII is chelated by five nitrogen atoms, ie, the four amino groups of the cyclene and the aromatic amino group. Therefore, the pH titration results also suggest that the complex 1-CdII satisfies the design principle in FIG.

Example 4
Fluorescence detection of anions using 1-Cd ^II Next, in order to investigate how various anions affect the excitation spectrum, various anions were added to the synthesized complex 1-CdII solution (5 mM). . All spectra were measured at 25 ° C. in the presence of 100 mM HEPES buffer (pH 7.4).
When the sodium pyrophosphate (PPi) solution was added to the complex 1-CdII solution, the excitation spectrum shifted towards longer wavelengths in a dose-dependent manner. As shown in FIG. 3, after adding 10 mM PPi, λ _max shifted from 342 nm to 383 nm. The excitation spectrum of the complex 1-CdII solution + 10 mM PPi was similar to that of the free base (FIG. 2) (eg, the peak wavelength of both was approximately 380 nm). As described in the design concept section, in the case of 7-aminocoumarin, a deep color shift in the excitation spectrum indicates an increase in the electron density of the 7-amino group ⁷ . These results indicate that, as we expected, the unstable fifth ligand (aromatic amino group) replaced the PPi anion in a dose-dependent manner (FIG. 1). In short, it was shown that PPi has a greater affinity for CdII-cyclene than an aromatic amino group even if the amino group is present in the molecule in a neutral aqueous solution.

In addition, the effect of other anions was investigated using similar methods, the fluorescence measurements at the peaks of the excitation spectrum were plotted against the concentration of anions, and a sensor consisting of complex 1-CdII for each anion according to the following equation: The apparent dissociation constant (K _d ) was calculated.
_{Formula: F = (F 0 + F} max [A] / K d) / (1 + [A] / K d)
In the above equation, F ₀ is the first F value without anion, F _max is the maximum value of F, and [A] is the final concentration of anion added to the solution. All anions were added as sodium salts or potassium salts. All K _d values were calculated under conditions of 100 mM HEPES buffer (pH 7.4).
The results are shown in Table 2 below. * _Kd was too large to calculate.

As is clear from the results in Table 2, citrate showed a strong affinity. Phosphate has a much lower affinity than PPi, probably due to its protonated form, and HPO ₄ ²⁻ and H ₂ PO ₄ ⁻ are the dominant species in aqueous solutions of pH 7.4, which are more likely than PPi There are few negative charges. Perchlorate did not change at 100 mM. For oxoacids, the polyvalent anion bound to complex 1-CdII more strongly than the monovalent or divalent anion. Such data suggests that the anion interacts with complex 1-CdII, possibly via both metal and the NH proton of the cyclene ring, through multipoint recognition.
The halogen ion, spectral ^{changes, I -> Br -> Cl} - >> F - occur in order, reflecting the degree of soft basicity. This is consistent with the fact that Cd ^II is a soft acid and has a high affinity for soft bases.

Now, some nucleotides are negatively charged organic compounds that play an important role in living organisms, especially cyclic nucleotides like cAMP and cGMP act as intracellular second messengers, so sensor detection of various nucleotides Very important. Then, next, these nucleotides were examined.
First, four nucleoside monophosphates, AMP, GMP, CMP and UMP were added to a 5 mM complex 1-CdII solution. Interestingly, only GMP deactivated the fluorescence, and the other three nucleotides elicited a red shift in the excitation spectrum like the inorganic anion (FIG. 4 [a]). The apparent dissociation constants are shown in Table 2. These results suggest that the shape of the excitation spectrum and the dissociation constant depend on the nucleotide base. One possible explanation is that coumarin interacts directly with nucleotide bases.
Two cyclic nucleotides, cAMP and cGMP, showed lower affinity for complex 1-CdII than their hydrolyzed products, AMP and GMP, respectively. The spectrum data of cAMP and AMP are shown in FIG. 4 [b]. Even when 10 mM cAMP was added, the change in the excitation spectrum of 1 in the complex 1-CdII was small. This spectrum was similar to the spectrum when 100 mM AMP was added. The addition of cAMP less than 1 mM hardly changed the excitation spectrum of complex 1-CdII. Considering this result, the difference in binding strength between AMP and cAMP was probably based on the number of anion sites.

Example 5
Next, the reversibility of anion sensor detection was examined. If the sensor detection system is reversible, a decrease in the anion coordinated to CdII will cause a blue shift in the excitation spectrum, which should recover to the original spectrum. To bind PPi and prevent PPi from coordinating to CdII, an excess amount of MgII was added to the solution containing PPi. When 10 mg of Mg (ClO ₄ ) ₂ was added to a solution of complex 1-CdII (5 mM) containing 1 mM PPi, the excitation spectrum actually shifted to blue and was λmax 345 nm (FIG. 5 [a]). . This spectrum almost exactly coincided with the spectrum of complex 1-CdII containing no PPi.
Furthermore, the influence of pyrophosphate hydrolysis was investigated. Inorganic pyrophosphatases are known to convert one pyrophosphate into two phosphate ions ⁹ . Since phosphate is weaker than PPi and coordinates to complex 1-CdII, cleavage of PPi should induce a spectral change similar to that seen with large amounts of MgII ions. When 0.33 U / ml inorganic pyrophosphatase and 0.5 mM Mg (ClO ₄ ) ₂ are added to a complex 1-CdII solution containing 1 mM pyrophosphate at pH 7.4, the peak of the excitation spectrum becomes shorter. It shifted quickly (FIG. 5 [b]). These results also indicate that the sensor detection mechanism is reversible.

Example 6
Measurement of phosphodiesterase To investigate the potential of this system for biochemical applications, we attempted to monitor the activity of phosphodiesterase (PDE). We use phosphodiesterase 3 ′, 5′-cyclic mononucleotide ¹⁰ which catalyzes the conversion of cyclic nucleotides to nucleoside monophosphates, for example, conversion of cAMP to AMP (Chemical Formula 2) by cleavage of the phosphodiester of the cyclic nucleotide. It was. Since such an enzyme is involved in intracellular signal transduction, it is extremely important to develop a system for detecting its activity ¹¹ with high sensitivity.

As shown in Example 5, the fluorescent sensor of the present invention consisting of the complex 1-CdII recognized AMP much more strongly than cAMP. Therefore, we next attempted to detect PDE activity in real time by monitoring the rise in AMP.
That is, after adding 2 U / ml (final concentration) of PDE to 5 mM complex 1-CdII containing 100 mM HEPES, 10 mM cAMP, 10 mM CaCl ₂ and 0.02 U / ml PDE activator at pH 7.4. The excitation spectrum (radiation wavelength is 500 nm) was measured several times. As a result, as shown in FIGS. 6 [a] and [b], the fluorescence intensity (excitation wavelength: 380 nm / emission wavelength: 500 nm) gradually increased. This spectral change indicates the conversion from cAMP to AMP. From these results, it was revealed that the complex 1-CdII, which is the compound of the present invention, is useful for real-time fluorescence measurement of phosphodiesterase 3 ′, 5′-cyclic nucleotide activity. This approach is also expected to be applicable to the measurement of other enzyme activities.

  Based on spectral and pH potential data, the inventor has weakly coordinated CdII by the 7-amino group of the 7-substituted coumarin derivative, so that some of the anion can replace it, It was confirmed that the fluorescence sensor for anion detection composed of such a compound could be coordinated to CdII ions and was reversible. Furthermore, the present inventor has demonstrated that the phosphodiesterase activity that hydrolyzes the phosphodiester bond of a cyclic nucleotide can be monitored in an aqueous solution using the complex 1-CdII which is the compound of the present invention. A sensor having such characteristics has not been known so far, and the complex 1-CdII, which is a compound of the present invention, can be a preferred anion sensor in many biological and analytical applications.

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FIG. 1 shows a mechanism in which the compound of the present invention functions as a fluorescent sensor for detecting anions and detects anions. FIG. 2 shows (a) absorption spectra and (b) excitation spectra (radiation wavelength is 500 nm) of (a) various metals and compounds (1). Here, the vertical axis: (a) fluorescence intensity, (b) absorbance, and horizontal axis: wavelength. FIG. 3 shows 5 mM complex 1-sodium pyrophosphate (0, 0.003, 0.01, 0.03, 0.1, 0.3, 3, 10 mM) added at 25 ° C. in 100 mM HEPES buffer (pH 7.4). The excitation spectrum of CdII is shown. The emission wavelength is 500 nm, the vertical axis is “fluorescence intensity”, and the horizontal axis is “wavelength”. FIG. 4 shows a comparison of excitation spectra for (a) AMP, GMP, CMP and UMP, and (b) 5 mM complex 1-CdII when AMP and cAMP are added. The concentration of each nucleotide is 10 mM. FIG. 5 shows (a) Complex 1 with 1 mM Na ₄ P ₂ O ₇ (no symbol) and 1 mM Na ₄ P ₂ O ₇ +10 mM Mg ₂ (ClO ₄ ) ₂ (open circles). Excitation spectrum of CdII and (b) Complex 1-CdII with 1 mM Na ₂ P ₄ O ₇ before addition of inorganic pyrophosphate (no symbol) and 20 minutes after addition (open circle) An excitation spectrum is shown. The emission wavelength is 500 nm. FIG. 6 shows (a) change in excitation spectrum of complex 1-CdII (emission wavelength is 500 nm) and (b) plot of fluorescence intensity-time (excitation / emission wavelength is 380 nm / 500 nm) in the measurement of phosphodiesterase activity. Show.

Claims (3)

A method for detecting fluorescence of pyrophosphate in a dose-dependent manner, comprising measuring a shift of an excitation spectrum of a fluorescent dye of a compound represented by the following formula 1.
Formula 1
The method according to claim 1, wherein a shift of the excitation spectrum of the fluorescent dye of the compound toward the long wavelength side is detected. The method according to claim 1 or 2, wherein the transition metal is cadmium.