JP2015101570A - Novel compound and oxygen concentration measurement reagent using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a light emission reagent to quantify the oxygen concentration of micro environment such as a cell from a change in light emission spectrum with high sensitivity in real time.SOLUTION: This invention provides a compound that comprises a linker, an oxygen concentration responsive phosphorophore comprising a cationic iridium complex that binds to a first end of the linker, and a fluorophore that binds to a second end of the linker. Specifically, the following compound is used to measure oxygen concentration.

Description

  The present invention relates to a novel compound and an oxygen concentration measuring reagent using the same.

  The luminescent probe method is very effective as a method for non-invasively and highly sensitively measuring the oxygen concentration of a specific part of a micro structure such as a cell. In general, an oxygen concentration measurement method using a luminescent probe utilizes the fact that the luminescence of the probe molecule is quenched by collision with the oxygen molecule, that is, the luminescence intensity of the luminescent probe changes depending on the oxygen concentration. The method for obtaining the oxygen concentration from the change in emission intensity gives an accurate value when the probe molecule concentration and excitation light intensity distribution are uniform, but the probe molecule concentration distribution is uniform, as in intracellular oxygen concentration measurement. If not, it will be affected by the concentration, making analysis difficult. Therefore, as a method not affected by the concentration, a method using a change in the light emission lifetime is considered. However, in general, the measurement of the light emission lifetime requires an expensive light source such as a pulse laser and an advanced optical measurement technique, so that the apparatus becomes large.

As a prior art (Patent Document 1: WO 2010/044465), the present inventor has developed an oxygen concentration measurement reagent based on the above principle (the following compound C343-Pro ₄ -BTP), and the oxygen concentration in the solution and in the lipid membrane Quantification was performed. In addition, measurement of oxygen concentration in cultured cells was attempted using C343-Pro ₄ -BTP. However, it was found that C343-Pro ₄ -BTP has low lipid solubility and thus has low ability to migrate to cells, and only qualitative evaluation is possible.

WO 2010/044465

  An object of the present invention is to provide a luminescent reagent for quantifying oxygen concentration in a microenvironment such as a cell in real time with high sensitivity from a change in emission spectrum.

  The present inventor has intensively studied to solve the above problems. As a result, in a compound comprising a linker, an oxygen concentration-responsive phosphorophore bound to the first end of the linker, and a fluorophore bound to the second end of the linker, an oxygen concentration-responsive phosphorphor As a result, it was found that the intracellular oxygen concentration can be efficiently measured by using a cationic iridium complex, and the present invention has been completed based on this.

That is, the present invention is as follows.
[1] A compound comprising a linker, an oxygen concentration-responsive phosphorophore bonded to the first end of the linker, and a fluorophore bonded to the second end of the linker, the oxygen concentration-responsive phosphor A compound wherein the photogroup is a group comprising a cationic iridium complex.
[2] The compound according to [1], wherein the triplet level of the oxygen concentration-responsive phosphorescent group is smaller than the triplet level of the fluorophore.
[3] The compound according to [1] or [2], wherein the cationic iridium complex has the following structure (1), (2) or (3).
[4] The compound according to any one of [1] to [3], wherein the fluorophore contains any of the following groups.
[5] The compound according to any one of [1] to [4], wherein the linker comprises the following structure.
One or more of the carbon-carbon bonds of each ring may be a double bond.
[6] The compound according to any one of [1] to [4], wherein the linker is polyproline.
[7] The compound according to any one of [1] to [6], which is the following compound.
[8] An oxygen concentration measuring reagent containing the compound according to any one of [1] to [7].

The reagent for measuring oxygen concentration using the compound of the present invention has a fluorophore that emits short-lived (nanosecond order) fluorescence and a phosphorescent group that emits long-lived (microsecond order) phosphorescence in one molecule. Is a feature. Since fluorescence has a short lifetime, it is hardly affected by dissolved oxygen. On the other hand, since phosphorescence has a long lifetime, it collides with oxygen molecules within the excitation lifetime and undergoes significant quenching. Thus, for example, if a molecule that gives green light emission to the fluorophore and a molecule that gives red light emission to the phosphorophore are used, when there is no oxygen, the light emission of both luminophores is mixed, giving yellow light emission. In the presence of oxygen, red phosphorescence is quenched, resulting in an intelligent luminescent probe that emits green light. Further, if the emission spectrum is measured using this probe, the oxygen concentration can be easily quantified by the ratio method taking the ratio of the fluorescence intensity and the phosphorescence intensity.
Since the compound of the present invention has good cell permeability, it can be suitably used for measurement of intracellular oxygen concentration.
An oxygen concentration imaging image can be obtained by adding a reagent developed in the present invention to a cell culture medium and acquiring a luminescence image with a fluorescence microscope. In addition, the oxygen concentration can be measured in real time by using a microplate reader.

Compound absorption in acetonitrile _{(C343-Pro 4 -BTQ +,} C343-Pro 8 -BTQ +), shows emission spectra. _{C343-Pro 4 -BTQ +, C343} -Pro 8 -BTQ +, shows the results of the observation C343-Pro ₄ HeLa cells was added -BTP or MCF-7 cell fluorescence microscope (photograph). In HeLa cells, C343-Pro ₄ -BTQ + was added and cultured for 20 hours under 20% and 2.5% oxygen partial pressure, followed by C343 fluorescence (excitation wavelength: 400-410 nm, observation wavelength:> 460-510) nm), BTQ + phosphorescence (excitation wavelength: 400-410 nm, observation wavelength:> 610 nm), a luminescence microscopic image and a ratio imaging image (phosphorescence image / fluorescence image) are shown (photograph). In HeLa cells, C343-Pro ₈ -BTQ + was added and cultured for 20 hours under 20% and 2.5% partial pressure of oxygen, then C343 fluorescence (excitation wavelength: 400-410 nm, observation wavelength:> 460-510) nm), BTQ + phosphorescence (excitation wavelength: 400-410 nm, observation wavelength:> 610 nm), a luminescence microscopic image and a ratio imaging image (phosphorescence image / fluorescence image) are shown (photograph). Shows an emission spectrum after incubation by adding the HeLa or MCF-7 cells C343-Pro ₄ -BTQ + or C343-Pro ₈ -BTQ + 20 hours in a 96-well black microplate. HeLa cells or MCF-7 cells was added to C343-Pro ₈ -BTQ +, ratio (phosphorescence intensity / fluorescence intensity) when cultured by changing the oxygen partial pressure diagram showing the measurement results.

  The present invention is described in detail below.

In the compound of the present invention, an oxygen concentration-responsive phosphorophore and an oxygen-insensitive fluorophore are bonded and linked to the first and second ends of the linker, respectively. If the triplet level of the fluorophore is lower than the triplet level of the phosphorescent group, energy transfer occurs from the phosphorescent group to the fluorophore, which may cause a significant decrease in phosphorescence intensity. As shown in the figure below, the excited triplet (T ₁ ) level of the fluorophore is changed to the excited triplet (T ₁ ') level of the phosphorescent group, as shown in the figure below. It is preferable to combine the luminophores so as to be higher than that.

The oxygen concentration-responsive phosphorescent group is a group that emits phosphorescence depending on the oxygen concentration, specifically, a group containing a cationic iridium complex.
A cationic iridium complex is a metal complex having Ir (III) as a central metal and an aromatic molecule as a ligand and means a cationic one. For example, the following (1) or (2) The iridium complex of these is mentioned.
The cationic iridium complex may form a salt with the anion. Examples of the anion include hexafluorophosphate ion (PF _6- ), chloride ion (Cl-), bromide ion (Br-), and trifluoroacetate ion (CF ₃ COO-).
The excited triplet (T ₁ ') levels of these phosphors are 181.5 kJ / mol, 175.1 kJ / mo1, and 167.8 kJ / mol, respectively.

The fluorophore can be appropriately selected according to the above phosphorescent group, such as NBD (4-Nitrobenzo-2-oxa-1,3-diazole), FITC, or coumarin dyes, rhodamines, BODIPY, cyanine dyes, etc. The following NBD, FITC, or C343 is preferable.
The excited triplet (T ₁ ) levels of these fluorophores are 181, 197 and 206 kJ / mol, respectively.

  The linker that connects the fluorophore and the phosphorescent group is not particularly limited as long as it chemically bonds the two, but in order to avoid the proximity of the fluorophore and the phosphorescent group, the linker that connects the fluorophore and the phosphorescent group. Is preferably as rigid as possible. Further, the length is preferably 20 mm or more. The upper limit of the length is not particularly limited, but is preferably 30 mm or less. The molecular weight of the linker moiety is preferably 4,000 or less.

  Steroids and polypeptides can be suitably used as linkers because they can be bound to luminophores relatively easily. In addition, if a peptide containing a water-soluble amino acid such as aspartic acid or lysine is used as a peptide residue, the compound can be made water-soluble. The polypeptide is preferably a polypeptide having 4 to 20 amino acid residues (more preferably 4 to 12), and examples thereof include polyproline.

For example, a linker containing the following cholesterol skeleton can also be used as a linker.
Note that one or more of the carbon-carbon bonds in each ring may be a double bond.

More specifically, the following compounds are mentioned. However, the compounds of the present invention are not limited to the following compounds as long as they exhibit color development depending on the oxygen concentration.
This compound satisfies the above energy relation T ₁ > T ₁ ′.

  The compound of the present invention can be obtained by reacting a phosphorophore compound and a fluorophore compound with a linker compound having a reactive group at both ends. Specifically, according to the method described in the examples below. Can be synthesized.

  In addition, other fluorophores can be used in place of C343 in the above compound.

Since the emission color of the compound of the present invention changes depending on the oxygen concentration, it can be used as an oxygen concentration measuring reagent for measuring the oxygen concentration based on the color development. For example, in the case of compound C343-Pro _4-12 -BTQ +, it can be determined that the oxygen concentration is low when it is purple, and the oxygen concentration is high when it is blue.

It is also possible to quantitatively measure the oxygen concentration by obtaining the relationship between the oxygen concentration and the phosphorescence intensity / fluorescence intensity ratio (Ip / If) in advance.

  When the oxygen concentration measurement reagent of the present invention is used to detect the oxygen concentration in a sample, after adding the oxygen concentration measurement reagent of the present invention to the sample and incubating, the fluorescence is such that the compound can be excited and phosphorescence can be observed. Phosphorescence and fluorescence can be observed using a microscope, a fluorescence measuring device, a fluorescence imaging device, a microplate reader, and the like.

  The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.

<Synthesis of compounds>
Synthesis of C343-Pro ₄ -BTQ ⁺
Synthesis of C343-Pro ₄ -COOH
Scheme 1 shows the procedure for deprotecting C343-Pro ₄ -OtBu. Dioxane hydrochloride (10 mL) was added to C343-Pro ₄ -OtBu (72.4 mg, 0.10 mmol) and allowed to stand overnight. After completion of the reaction, dehydrated tetrahydrofuran (THF) was added and concentrated under reduced pressure to obtain C343-Pro ₄ -COOH.
¹ H NMR (400 MHz, DMSO) δ: 7.09 (s, 1H), 6.31 (s, 1H), 4.93-4.52 (m, 4H), 4.23-4.11 (m, 2H), 3.87-3.80 (m, 1H ), 3.49-3.42 (m, 1H), 3.34-3.25 (m, 4H), 2.71-2.64 (m, 3H),
2.38-1.68 (m, 23H)

C343-Pro ₄ - Synthesis of (phen-Pipe)
Scheme 2 shows the condensation reaction between C343-Pro ₄ -COOH and phen-Pipe. To C343-Pro ₄ —COOH (˜0.3 mmol), phen-Pipe (˜0.3 mmol) was added, and dehydrated dimethylformamide (DMF) (5 mL) was added. HATU (200 mg, 0.52 mmol) and HOBt (75.0 mg, 0.49 mmol) were added. Further, dehydrated DMF (5 mL) and DIEA (200 μL) were added and reacted overnight under a nitrogen atmosphere.
After the reaction, distilled water (50 mL) and brine (50 mL) were added to the reaction solution, and the product was extracted with chloroform (200 mL × 2). The organic layer was dried by adding Na ₂ SO ₄ and then concentrated under reduced pressure. After concentration, purification was performed with an alumina column (CHCl ₃ : MeOH = 98: 2). Thereafter, chloroform was subjected to reprecipitation with hexane, the yellow crystals C343-Pro ₄ - was obtained (phen-Pipe) (97.8 mg , 0.11 mmol, 35.4% yield).
ESI-MS (positive): calcd.for C ₅₂ H ₅₇ N ₉ O ₇ ⁺ : 919.44, found: m / z = 942.6 ([M + Na] ⁺ )

Synthesis of C343-Pro ₄ -BTQ ⁺
Scheme 3 shows the synthesis of C343-Pro ₄ -BTQ ⁺ . _{C343-Pro 4 - (phen-} Pipe) (~0.05mmol) with chlorine two bridge complex (0.025 mmol) in dichloromethane BTQ (CH ₂ Cl ₂₎ and methanol (MeOH) was added,
The mixture was refluxed for 4 hours under a nitrogen atmosphere. Thereafter, a KPF ₆ was added to warm to room temperature and allowed to react for 1 hour. All purifications were performed on an amine column (CHCl ₃ : MeOH = 98: 2) to obtain red crystals of C343-Pro ₄ -BTQ ⁺ .
ESI-MS (positive): calcd.for C ₈₆ H ₇₇ F ₆ IrN ₁₁ O ₇ PS ₂ : 1777.47, found: m / z = 827.7 ([M-PF ₆ + H + Na] ²⁺ )

In Scheme 3, the compound of the present invention can also be synthesized by using the following iridium complex chlorine bibridge complex instead of BTQ chlorine bibridge complex. The same applies to Scheme 5 described later.

The compound (II) can be synthesized as follows.

Synthesis of C343-Pro ₈ -BTQ ⁺
Synthesis of H-Pro ₄ -OtBu
Scheme ₄ shows the deprotection procedure for Z-Pro ₄ -OtBu. Z-Pro ₄ -OtBu (1.8 g, 3.0 mmol) to Pd
/ C and MeOH were added and the Z group was deprotected by hydrogenation reaction. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain H-Pro ₄ -OtBu as white crystals.
¹ H NMR (400 MHz, CDCl ₃ ) δ: 4.77-4.67 (m, 2H), 4.44-4.41 (m, 1H), 3.80-3.5 (m, 7H),
3.16-3.10 (m, 1H), 2.81-2.75 (m, 1H), 2.22-1.70 (m, 16H), 1.41 (s, 9H)

Synthesis of Z-Pro ₄ -OH
Scheme ₄ shows the deprotection procedure for Z-Pro ₄ -OtBu. Dioxane hydrochloride (15 mL) was added to Z-Pro ₄ -OtBu (1.8 g, 3.0 mmol) and allowed to stand overnight to deprotect tBu ether. After completion of the reaction, dehydrated THF was added, and the mixture was concentrated under reduced pressure to obtain Z-Pro ₄ —OH as white crystals.
¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.40-7.27 (m, 4H), 5.19-4.96 (m, 2H), 4.76-4.41 (m, 4H), 3.84-3.45 (m, 8H), 2.31- 1.80 (m, 16H)

Synthesis of Z-Pro ₈ -OtBu
Scheme 5 shows the condensation reaction between H-Pro ₄ -OtBu and Z-Pro ₄ -OH. HOBt (505 mg, 3.3 mmol) was added to H-Pro ₄ -OtBu (~ 3 mmol) and Z-Pro ₄ -OH (~ 3 mmol). Furthermore, EDC hydrochloride (690 mg, 3.6
mmol) and dehydrated dichloromethane (50 mL) were added and allowed to react overnight under a nitrogen atmosphere. After the reaction is complete, concentrate under reduced pressure, dichloromethane (50 mL), 10% aqueous citric acid solution (50 mL × 2), aqueous sodium bicarbonate solution (50 mL × 2), water (50 mL), brine (50 mL) To extract the product. The organic layer was dried by adding Na ₂ SO ₄ and then concentrated under reduced pressure. Z-Pro ₈ -OtB of white crystal
u (2.38 g, 2.41 mmol, yield 80.3%) was obtained.
¹ H NMR (300 MHz, CDCl ₃ ) δ: 7.37-7.27 (m, 5H), 5.19-4.96 (m, 2H), 4.78-4.71 (m, 6H), 4.58-4.40 (m, 2H), 3.78- 3.36 (m, 16H), 2.13-1.81 (m, 32H), 1.41 (s, 9H)
ESI-MS (positive): calcd.for C ₅₂ H ₇₂ N ₈ O ₁₁ ⁺ : 984.53, found: m / z = 1007.1 ([M + Na] ⁺ )

Synthesis of H-Pro ₈ -OtBu
Scheme 6 shows the deprotection procedure for Z-Pro ₈ -OtBu. Pd / C and methanol were added to Z-Pro ₈ -OtBu (2.38 g, 2.41 mmol), and the Z group was deprotected by hydrogenation reaction. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. Gray crystals of H-Pro ₈ -OtBu (2.04 g, 2.40 mmol, 99.6% yield) were obtained.
¹ H NMR (400 MHz, CDCl ₃ ) δ: 4.76-4.66 (m, 6H), 4.43-4.40 (m, 1H), 3.92-3.49 (m, 15H), 3.18-3.12 (m, 1H), 2.90- 2.84 (m, 1H), 2.19-1.70 (m, 31H), 1.41 (s, 9H)

Synthesis of C343-Pro ₈ -OtBu
Scheme 7 shows the condensation reaction between H-Pro ₈ -OtBu and C343. HATU (494.3 mg, 1.3 mmol) was added to H-Pro ₈ -OtBu (936 mg, 1.1 mmol) and C343 (285.3 mg, 1.0 mmol). DIEA (1 mL) and dehydrated DMF (30 mL) were further added, and the reaction was allowed to proceed overnight under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and chloroform (50 mL) and water (50 mL × 2) were added to extract the product. The organic layer was dried by adding Na ₂ SO ₄ and then concentrated under reduced pressure. Purification was performed by silica gel column chromatography (CHCl ₃ : MeOH = 19: 1). Hexane was added and the mixture was concentrated under reduced pressure to obtain yellow crystals C343-Pro ₈ -OtBu, which was identified by NMR.
¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.79 (s, 1H), 6.81 (s, 1H), 4.80-4.71 (m, 7H), 4.44-4.41 (m, 1H), 3.92-3.26 (m, 16H), 2.94-2.71 (m, 4H), 2.25-1.65 (m, 40H), 1.41 (s, 9H)

Synthesis of C343-Pro ₈ -OH
Scheme 8 shows the procedure for deprotecting C343-Pro ₈ -OtBu. Dioxane hydrochloride (30 mL) was added to Z-Pro ₄ -OtBu (871 mg, 0.78 mmol) and allowed to stand overnight to deprotect tBu ether. After completion of the reaction, dehydrated THF was added and concentrated under reduced pressure to obtain C343-Pro ₈ -OH.
¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.81 (s, 1H), 6.84 (s, 1H), 4.81-4.59 (m, 7H), 4.41-4.32 (m, 1H), 3.75-3.47 (m, 16H), 3.30-3.26 (m, 2H), 2.95-2.73 (m, 2H), 2.37-1.82 (m, 40H)

C343-Pro ₈ - Synthesis of (phen-Pipe)
Scheme 9 shows the condensation reaction between C343-Pro ₈ -OH and phen-Pipe. HATU (494 mg, 1.01 mmol) was added to C343-Pro ₈ -OtBu (871 mg, 0.78 mmol) and phen-Pipe (463 mg, 1.75 mmol). DIEA (2 mL) and dehydrated DMF (30 mL) were further added, and the reaction was allowed to proceed overnight under a nitrogen atmosphere. After the reaction is complete
The reaction mixture was concentrated under reduced pressure, and the product was extracted by adding chloroform (50 mL) and water (50 mL × 2). The organic layer was dried by adding Na ₂ SO ₄ and then concentrated under reduced pressure. Purification was performed by amine column chromatography (CHCl ₃ ). And concentrated under reduced pressure addition of hexane, yellow crystals _{C343-Pro 8 - (phen-} Pipe) (184.5 mg, 0.14 mmol, 12.7% yield).
¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.19-9.18 (m, 1H), 9.08-9.01 (m, 1H), 8.57-8.54 (m, 1H), 8.13-8.11 (m, 1H), 7.79 ( s, 1H), 7.71-7.37 (m, 3H), 6.81 (s, 1H), 5.00-4.71 (m, 8H), 3.91-3.52 (m, 18H), 3.29-3.25 (m, 4H), 2.90- 2.72 (m, 2H), 2.22-1.87 (m, 38H), 1.29-1.21 (m, 4H), 0.916-0.853 (m, 2H)
ESI-MS (positive): calcd.for C ₇₂ H ₈₅ N ₁₃ O ₁₁ : 1307.65, found: m / z = 665.6 ([M + H + Na] ²⁺ )

Synthesis of C343-Pro ₈ -BTQ ⁺
Scheme 10. shows the synthesis of C343-Pro ₈ -BTQ ⁺ . _{C343-Pro 4 - (phen-} Pipe) (~0.1 mmol) and chlorine two bridge complex (0.05 mmol) of _{BTQ CH 2 Cl 2 (20 mL} ) and MeOH the (15 mL) was added, under nitrogen atmosphere, 50 ° C. At reflux for 4 hours. Thereafter, a KPF ₆ was added to warm to room temperature and allowed to react for 1 hour. After completion of the reaction, it was concentrated under reduced pressure. Purification is performed by amine column chromatography (CHCl ₃ : MeOH = 19
: I went in 1). Hexane was added and the mixture was concentrated under reduced pressure to obtain orange crystals of C343-Pro ₈ -BTQ ⁺ .
ESI-MS (positive): calcd.for C ₁₀₆ H ₁₀₅ F ₆ IrN ₁₅ O ₁₁ PS ₂ : 2165.68, found: m / z = 1021.8 ([M-PF ₆ + H + Na] ²⁺ )

<Spectral data>
Synthesis of C343-Pro ₄ -BTQ ⁺
C343-Pro ₄ -COOH
¹ H NMR (400 MHz, DMSO) δ: 7.09 (s, 1H), 6.31 (s, 1H), 4.93-4.52 (m, 4H), 4.23-4.11 (m, 2H), 3.87-3.80 (m, 1H ), 3.49-3.42 (m, 1H), 3.34-3.25 (m, 4H), 2.71-2.64 (m, 3H),
2.38-1.68 (m, 23H)

_{C343-Pro 4 - (phen-} Pipe)
ESI-MS (positive): calcd.for C ₅₂ H ₅₇ N ₉ O ₇ : 919.44, found: m / z = 942.6 ([M + Na] ²⁺
)

C343-Pro ₄ -BTQ ⁺
ESI-MS (positive): calcd.for C ₈₆ H ₇₇ F ₆ IrN ₁₁ O ₇ PS ₂ : 1777.47, found: m / z = 827.7 ([M-PF ₆ + H + Na] ²⁺ )

Synthesis of C343-Pro ₈ -BTQ ⁺
H-Pro ₄ -OtBu
¹ H NMR (400 MHz, CDCl ₃ ) δ: 4.77-4.67 (m, 2H), 4.44-4.41 (m, 1H), 3.80-3.5 (m, 7H),
3.16-3.10 (m, 1H), 2.81-2.75 (m, 1H), 2.22-1.70 (m, 16H), 1.41 (s, 9H)

Z-Pro ₄ -OH
¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.40-7.27 (m, 4H), 5.19-4.96 (m, 2H), 4.76-4.41 (m, 4H), 3.84-3.45 (m, 8H), 2.31- 1.80 (m, 16H)

Z-Pro ₈ -OtBu
¹ H NMR (300 MHz, CDCl ₃ ) δ: 7.37-7.27 (m, 5H), 5.19-4.96 (m, 2H), 4.78-4.71 (m, 6H), 4.58-4.40 (m, 2H), 3.78- 3.36 (m, 16H), 2.13-1.81 (m, 32H), 1.41 (s, 9H)
ESI-MS (positive): calcd.for C ₅₂ H ₇₂ N ₈ O ₁₁ : 984.53, found: m / z = 1007.1 ([M + Na] ⁺
)

H-Pro ₈ -OtBu
¹ H NMR (400 MHz, CDCl ₃ ) δ: 4.76-4.66 (m, 6H), 4.43-4.40 (m, 1H), 3.92-3.49 (m, 15H), 3.18-3.12 (m, 1H), 2.90- 2.84 (m, 1H), 2.19-1.70 (m, 31H), 1.41 (s, 9H)

C343-Pro ₈ -OtBu
¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.79 (s, 1H), 6.81 (s, 1H), 4.80-4.71 (m, 7H), 4.44-4.41 (m, 1H), 3.92-3.26 (m, 16H), 2.94-2.71 (m, 4H), 2.25-1.65 (m, 40H), 1.41 (s, 9H)

C343-Pro ₈ -OH
¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.81 (s, 1H), 6.84 (s, 1H), 4.81-4.59 (m, 7H), 4.41-4.32 (m, 1H), 3.75-3.47 (m, 16H), 3.30-3.26 (m, 2H), 2.95-2.73 (m, 2H), 2.37-1.82 (m, 40H)

_{C343-Pro 8 - (phen-} Pipe)
¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.19-9.18 (m, 1H), 9.08-9.01 (m, 1H), 8.57-8.54 (m, 1H), 8.13-8.11 (m, 1H), 7.79 ( s, 1H), 7.71-7.37 (m, 3H), 6.81 (s, 1H), 5.00-4.71 (m, 8H), 3.91-3.52 (m, 18H), 3.29-3.25 (m, 4H), 2.90- 2.72 (m, 2H), 2.22-1.87 (m, 38H), 1.29-1.21 (m, 4H), 0.916-0.853 (m, 2H)
ESI-MS (positive): calcd.for C ₇₂ H ₈₅ N ₁₃ O ₁₁ : 1307.65, found: m / z = 665.6 ([M + H + Na] ²⁺ )

C343-Pro ₈ -BTQ ⁺
ESI-MS (positive): calcd.for C ₁₀₆ H ₁₀₅ F ₆ IrN ₁₅ O ₁₁ PS ₂ : 2165.68, found: m / z = 1021.8 ([M-PF ₆ + H + Na] ²⁺ )

<Evaluation of compound>
FIG. 1 shows absorption and emission spectra of the compounds (C343-Pro ₄ -BTQ +, C343-Pro ₈ -BTQ +) in acetonitrile. The emission spectrum was measured using 405 nm as the excitation wavelength.
Fluorescence derived from and phosphorescence derived from BTQ + were observed. In addition, it was confirmed that the phosphorescence intensity of BTQ + significantly decreased under air saturation compared with deoxygenated conditions.

Figure 2 shows the C343-Pro ₄ -BTQ +, C343-Pro ₈ -BTQ +, or C343-Pro ₄ -BTP solution added to the culture solution of HeLa cells or MCF-7 cells to a final concentration of 5 μM, and cultured for 20 hours. Thereafter, it is washed, and a microscopic image (excitation wavelength: 400-410 nm, observation wavelength:> 455 nm) observed with a fluorescence microscope is shown. As a result, clear emission images were obtained only with C343-Pro ₄ -BTQ + and C343-Pro ₈ -BTQ +. Since C343-Pro ₄ -BTP is extremely high in lipid solubility, it has been a problem that it aggregates in the culture medium and has low cell migration. In the present invention, since the iridium complex is made cationic, aggregation in the culture solution is suppressed, and it is considered that cell migration is greatly increased.

Next, C343-Pro ₄ -BTQ + (Fig. 3) and C343-Pro ₈ -BTQ + (Fig. 4) solutions were added to the HeLa cell culture solution to a final concentration of 5 μM, and under 20% and 2.5% oxygen partial pressure. After cultivating for 20 hours, washing, and fluorescence fluorescence using C343 fluorescence (excitation wavelength: 400-410 nm, observation wavelength:> 460-510 nm), BTQ + phosphorescence (excitation wavelength: 400-410 nm, observation wavelength:> 610 nm) was observed. A luminescence microscopic image and a ratio imaging image (phosphorescence image / fluorescence image) are shown in FIGS. As a result, C343 fluorescence and BTQ + phosphorescence were observed for both C343-Pro ₄ -BTQ + and C343-Pro ₈ -BTQ +, and the fluorescence intensity did not change under 20% or 2.5% oxygen partial pressure, whereas the phosphorescence intensity. Is 2.5 compared to 20%
It was found to increase under% oxygen partial pressure. In the ratio imaging image, it was also found that the ratio increased under 20% oxygen partial pressure than 20%. Since the image ratio is almost constant in the cell, it can be seen that the oxygen concentration in the cell is almost uniform.

Furthermore, an experiment was conducted using a microplate reader to determine the oxygen concentration. Cultivate HeLa cells or MCF-7 cells in a 96-well black microplate, and use C343-Pro ₄ -BTQ +
C343-Pro ₈ -BTQ + solution was added to a final concentration of 5 μM, incubated for 20 hours, washed, and measured with a microplate reader. FIG. 5 shows the emission spectrum obtained. As a result, almost no C343 fluorescence was observed in C343-Pro ₄ -BTQ +, whereas C343 fluorescence and BTQ + phosphorescence were observed in C343-Pro ₈ -BTQ +. From this result, it was confirmed that C343-Pro ₈ -BTQ + is useful in experiments using a microplate reader. The phosphorescence intensity of BTQ + increased under 2.5% oxygen partial pressure, which is consistent with the experiment using a microscope.

FIG. 6 shows the results of measuring the ratio (phosphorescence intensity / fluorescence intensity) while changing the culture oxygen partial pressure. At 20% oxygen partial pressure, it was 6.4 for HeLa cells and 7.4 for MCF-7 cells, whereas at 2.5% oxygen partial pressure, it was 8.5 for HeLa cells and 9.9 for MCF-7 cells. A calibration curve can be created by conducting similar experiments at other oxygen partial pressures, and the intracellular oxygen concentration can be quantified in real time based on the calibration curve.

  The oxygen concentration measuring reagent of the present invention can be used in fields such as analytical chemistry, life science, bioimaging field, medical diagnosis, cell biology, and environmental measurement. Specifically, it can be used as an oxygen concentration determination reagent, a hypoxic cell imaging reagent, a hypoxic tumor diagnostic reagent, and the like.

Claims (8)

A compound comprising a linker, an oxygen concentration-responsive phosphorescent group bonded to the first end of the linker, and a fluorophore bonded to the second end of the linker, wherein the oxygen concentration-responsive phosphorophore is A compound which is a group containing a cationic iridium complex. The compound according to claim 1, wherein the triplet level of the oxygen concentration-responsive phosphorescent group is smaller than the triplet level of the fluorophore. The compound according to claim 1 or 2, wherein the cationic iridium complex has the following structure (1), (2) or (3).
The compound according to any one of claims 1 to 3, wherein the fluorophore contains any of the following groups.
The compound according to any one of claims 1 to 4, wherein the linker comprises the following structure.
One or more of the carbon-carbon bonds of each ring may be a double bond. The compound according to any one of claims 1 to 4, wherein the linker is polyproline. The compound according to any one of claims 1 to 6, which is the following compound.
The oxygen concentration measuring reagent containing the compound as described in any one of Claims 1-7.