Detection of glucose in solutions also containing an alpha-hydroxy acid or a beta-diketone

Detection of glucose in solutions that also contain alpha-hydroxy acid or beta-diketone

Figure 1 illustrates the standardized fluorescence emission (I/Io @ 420 nm) of the marker as described in Example 1.

Figure 2 illustrates the standardized fluorescence emission of the marker as described in Example 2 (I/Io@428nm)

Figure 3 illustrates the standardized fluorescence emission of the marker as described in Example 3 (I/Io@428nm)

Figure 4 illustrates the standardized fluorescence emission of the marker described in Example 4 (I/Io@427nm)

Figure 5 illustrates the standardized fluorescence emission of the marker as described in Example 5 (I/Io@540 nm)

Figure 6 illustrates the absorption spectrum of the label as described in Example 6.

Figures 7-8 illustrate the absorbance ratio (450nm/530nm) of the marker as described in Example 6.

Figure 9 illustrates the standardized fluorescence emission (I/Io at 550 nm) of the marker as described in Example 6.

Figure 10 illustrates the fluorescence spectra of the marker described in Example 6 in the absence of glucose and in the presence of 100 milligrams of molecular weight glucose

Figure 11 illustrates the standardized fluorescence emission (I/Io at 550 nanometers) of the label described in Example 6 in the presence of glucose and lactate.

Figure 12 illustrates the standardized fluorescence emission of a label exposed to glucose as described in Example 10 (I/Io at 525 nm)

Figure 13 illustrates the standardized fluorescence emission (I/Io at 530 nm) of a label exposed to lactate as described in Example 10

Figure 14 shows the relative fluorescence emission (I@430 nm) of the label exposed to glucose and lactate as described in Example 11.

Figure 15 shows the relative fluorescence emission (I@430 nm) of the label exposed to glucose and lactate as described in Example 12.

Figure 16 illustrates the fluorescence of markers exposed to glucose and lactate as described in Example 13.

References before and after related applications

The present invention is a partial continuation of the application preamble No. 10/029, 184 filed on December 28, 2001, and a partial continuation of the preamble application No. 09/754,217 filed on January 5, 2001. Application, and claimed that the preface of application No. 60/363,885 filed on March 14, 2002, the preface of Application No. 60/329,746 filed on October 18, 2001, and the preface filed on February 21, 2001 Apply for the benefit of Prologue No. 60/269,887.

Technical field

The present invention relates to the detection of glucose in samples that also contain potential interfering compounds (such as alfa-hydroxy acid or beta-diketone)

Prior art

The compounding effect of carbohydrates (including glucose) and phenylboronic acid has been known for a long time, and the reversibility of this interaction has been used as the benchmark for chromatographic separation of carbohydrates. In particular, Lorand and Edwards proposed in 1959 the association constant of phenylboronic acid and many saturated polyols in aqueous associations; the binding interaction is very weak (for example, ethylene glycol, K_d= 360 mg molecular weight) arranged to medium strength (for example, glucose, K_d= 9.1 mg molecular weight). Refer to J. Yoon et al. Bioorganic and Medicinal Chemistry 1(4): 267-71 (1993). It is believed that the binding mechanism occurs through the bonding between the hydroxyl group adjacent to the glucose and the hydroxyl group on the borate moiety.

US Patent No. 5,503,770 (James et al.) describes fluorescent boric acid-containing compounds that emit high-intensity fluorescence when combined with sugars (including glucose). The fluorescent compound has a molecular structure containing a fluorophore, at least one phenylboronic acid moiety, and at least one amine providing nitrogen atom. The nitrogen atom is arranged near the phenylboronic acid moiety, so it interacts with boric acid in the molecule. . Once combined with sugars, this interaction therefore causes the compound to emit fluorescence. Also refer to T. James et al. J. Am. Chem. Soc. 117(35):8982-87 (l995).

In addition, it is known in the art to use anthracenyl boronic acid-containing compounds to detect blood glucose as a fluorescent sensor. For example, J. Yin et al. J. Am. Chem. Soc. 114: 5874-5875 (1992) shows that anthracenyl boronic acid can be used as a signal for carbohydrate binding (including the combination of glucose and fructose) for the fluorescence chemistry. Test agent.

Unfortunately, the compounds that interact with glucose in the above-mentioned manner also have a tendency to interact with other compounds with hydroxyl groups, thus reducing the specificity of the glucose test, especially when the test may include interfering amounts of lactate, acetyl acetate, etc. When the body sample. For example, some diabetic patients also suffer from lactic acidosis, where the blood lactate value is greater than 5 millimoles/liter. Therefore, there is still a great demand for glucose assays that are relatively insensitive to potential interference with hydroxyl compounds (such as lactate).

Summary of the invention

In one aspect of the present invention, it relates to a method for detecting glucose or its concentration in a sample that also contains alpha-hydroxy acid or beta-diketone, which comprises: a) exposing the sample to at least two The compound of the distinguishing element of glucose is oriented so that the interaction between the compound and glucose is more stable than the interaction between the compound and alpha-hydroxy acid or beta-diketone. The compound also contains detectable properties. For the detectable part, when the compound is exposed to glucose in the sample, the characteristic changes according to the concentration method; and b) any changes in the characteristic are measured to determine the presence of glucose in the sample or its The concentration in which the presence of alpha-hydroxy acid or beta-diketone does not substantially interfere with the determination.

In another aspect of the present invention, it relates to a compound having the following structure

<chemistry general="n"><img file="TW200401034A_D0001.tif" /></chemistry>

Where: -R₁With R₂Same or different, and selected from the following: i) hydrogen; ii) change R₈Part of the pKa and hydrolytically stable substituents, iii) a detectable part or iv) a linking group capable of attaching to a solid support or polymer matrix, the support or matrix optionally includes a detectable part; -R3 is hydrogen or a linking base that can be attached to a solid support or a polymer matrix. The support or matrix includes a detectable part if necessary; -R₄With R₅Same or different, and selected from the following: i) hydrogen; ii) change R₈Part of the pKa and hydrolytically stable substituents, iii) the detectable part or iV) the linking base that can be attached to the solid support or the polymer matrix, the support or the matrix optionally includes the detectable part; -Each Z is independently carbon or nitrogen; -R6 and R7 are the same or different, and are i) a linking group with 0 or 10 adjacent or branched carbon and/or heteroatoms or ii) a solid support Or a polymer matrix attached to the linking group, the support or the matrix optionally includes a detectable part; -R is selected from the following: i) aliphatic and/or aromatic spacers, which include from 1 to 10 Adjacent atoms of the group consisting of free carbon, oxygen, nitrogen, sulfur and phosphorus; ii) detectable part or iii) a linking base capable of attaching to a solid support or a polymer matrix, the support or matrix as required Including detectable parts;-every R₈The same or different, and the part that needs to be protected, when it is not protected, it can interact with the glycol group existing near the glucose; and -R₉With R_l0The same or different, and are i) hydrogen; ii) detectable moiety or iii) group, which is a) a linking base that can be attached to a solid support or a polymer matrix, the support or matrix optionally includes The detection part, and/or b) includes a functional group that can modify the physical properties of the compound; the proviso-labeled compound includes at least one detectable part that directly associates with it or becomes a part of a solid support or polymer. In another aspect of the present invention, the present invention relates to a detection system containing the above-mentioned compounds.

Implementation

In one aspect of the present invention, it provides a way to detect glucose or its concentration in a sample that also contains interfering compounds (such as alfa-hydroxy acid or beta-diketone). These potentially interfering compounds include lactate, acetyl acetate, β-hydroxybutyric acid and so on.

The present invention is carried out using a marker compound that can distinguish glucose in the sample, but is relatively impossible to distinguish the interfering compound in the sample. The marker compound has at least 2 discriminating elements of glucose, which are oriented so that the interaction between the marker compound and glucose is more stable than the interaction between the marker compound and the interfering compound.

Suitable discriminating elements include parts that can better reverse the interaction with glucose, especially the glycol groups present in glucose. Many of these distinguishing elements are known, and preferably include boric acid, borate ion, quinquerine, quinquerite, telluric acid, tellurite ion, germanic acid, germanate ion, and the like. The best distinguishing element is boron. Of course, the distinguishing element can be blocked with a protective group until it is used. These are well-known protecting groups and include neopentyl alcohol, pinacol and the like. In a specific embodiment, the blocked discriminating element is deblocked in the medium of the compound to be used (see, for example, Example 5).

Preferably, the discriminating elements are spaced at a suitable distance from each other on the label compound, allowing at least two discriminating elements to interact with glucose, resulting in an increase in specificity. Generally, distinguishing elements can have up to 30 atom spacers between each other. It is preferable to orient the discriminating elements so that they can be separated by about 6 angstroms when interacting with glucose.

The marker compound of the present invention can detect a characteristic, and the characteristic changes in a concentration manner when the compound is exposed to a sample containing glucose. Many of these qualities are known and can be used in the present invention. For example, the marker compound may include luminescent (fluorescent or phosphorescent) or chemiluminescent moieties, absorption-based moieties, and the like. The label compound may include an energy-donating part and an energy receptor part, with separate intervals so that there is a detectable change when the label compound interacts with glucose. The label compound may include a fluorophore and a photoinhibitor, and is structured such that the photoinhibitor inhibits the configuration of the fluorophore in the absence of glucose. In this case, in the presence of glucose, the label undergoes a configuration change, which causes the photoinhibitor to move to a sufficient distance from the fluorophore, so fluorescence is emitted. Conversely, the fluorophore and the photoinhibitor can be structured such that they are fully isolated when there is no glucose and the configuration of the fluorophore to emit fluorescence; once interacting with glucose, the fluorophore and photoinhibition The agent moves to a distance close enough to cause photoinhibition. On January 5, 2001, the Prologue No. 09/754,219 of the joint trial application with the title of "Detection of Analytes" was proposed to explain the concept of configuration change in more detail. It is incorporated into this article for reference.

Another selection-based marker may include parts such as fluorophores that can interact with the discriminating element or another part of the discriminating element in a spatial arrangement, so that the fluorophore emits in the absence of glucose Fluorescent. When glucose is added, the glucose competes with the interaction between the fluorophore and the discriminating element, or with the interaction between the fluorophore and other parts of the discriminating element spatially arranged, causing a decrease in fluorescence. Example 6 is an example that illustrates this concept. It is also recognized that when the fluorophore interacts with the distinguishing element or the other part of the spatial arrangement of the element in the absence of glucose, the fluorophore does not emit fluorescence or emits a relatively low fluorescence value. The markers. Once glucose is added, the glucose will compete with the interaction between the fluorophore and the discriminating element, or with the interaction between the fluorophore and other parts of the discriminating element arranged in space, causing an increase in fluorescence. .

Other detectable parts include those whose fluorescence is affected by glucose interaction through light-induced electron transfer or induced effects. These include the lanthanide chelates disclosed in the U.S. Preliminary Application No. 09/265,979 filed on March 11, 1999 (and as published in PCT International Application WO99/46600 on September 16, 1999) (It is incorporated herein for reference), polyaromatic hydrocarbons and their derivatives, coumarin, BoDiPy, danSyl, catechol, etc. The other part of the category includes those whose absorption spectrum changes when the labeled compound interacts with glucose, including Alizarin Red. The other part of the category includes those parts that adjust their fluorescence by proximity effect, for example, energy donor/receptor pairs, such as dansyl/dabSyl and so on.

Preferably, detectable characteristics are detectable spectral changes, such as absorption characteristics (for example, absorbance and/or spectral drift); fluorescence disappearance time (measured in the measurement time domain or frequency domain), fluorescence intensity , The change of fluorescence anisotropy or polarization; the spectral drift of the emission spectrum; the change of the disappearance of the time-resolved heterogeneity (measured in the time domain or the frequency domain), etc.

If necessary, the marker compound of the present invention assumed to be soluble can be used directly in the solution. On the other hand, assuming that the expected application has such requirements, the marker compound can be immobilized (such as mechanical traps, or covalently or ionic attachment) on or in insoluble surfaces or substrates, such as glass, plastic, Polymeric substance, etc. When the labeling compound is trapped in, for example, another polymer, the trap should preferably penetrate into the glucose sufficiently, allowing a suitable interaction between the glucose and the labeling compound.

If the marker compound is not very or insoluble in water, it is undesirable to detect in the aqueous medium. The marker compound can be copolymerized with hydrophilic monomers to form hydrophilic macromolecules, as proposed on August 4, 2000. As described in the pending US application No. 09/632,624, the content of which is incorporated herein for reference.

The preferred marker compound has the following structure:

<chemistry general="n"><img file="TW200401034A_D0002.tif" /></chemistry>

Where: -R_lWith R₂Same or different, and selected from the following: i) hydrogen; ii) change R₈Part of the pKa and hydrolytically stable substituents, iii) a detectable part or iv) a linking group capable of attaching to a solid support or polymer matrix, the support or matrix optionally includes a detectable part; -R₃Hydrogen or a linking base that can be attached to a solid support or a polymer matrix. The support or matrix includes a detectable part if necessary; -R₄With R₅Same or different, and selected from the following: i) hydrogen; ii) change R₈Part of the pKa and hydrolytically stable substituents, iii) the detectable part or iV) the linking group that can be attached to the solid support or the polymer matrix, the support or the matrix optionally includes the detectable part; -Each Z series is independently carbon or nitrogen; -R₆With R₇The same or different, and are i) a linking group with 0 or 10 adjacent or branched carbon and/or heteroatoms or ii) a linking group that can be attached to a solid support or a polymer matrix, the support or matrix depends on Need to include a detectable part; -R is selected from the following: i) aliphatic and/or aromatic spacer, which includes from 1 to 10 selected from the group consisting of carbon, oxygen, nitrogen, sulfur and phosphorus Adjacent atoms; ii) a detectable part or iii) a linking group that can be attached to a solid support or a polymer matrix, the support or matrix optionally includes a detectable part;-each R₈The same or different, and the part that needs to be protected, when it is not protected, it can interact with the glycol group existing near the glucose; and -R9 and R_l0The same or different, and are i) hydrogen; ii) detectable moiety or iii) group, which is a) a linking base capable of attaching to a solid support or a polymer matrix, the support or matrix optionally includes The detection part, and/or b) includes a functional group capable of modifying the physical properties of the compound; the proviso marking compound includes at least one detectable part that directly associates with it or becomes a part of a solid carrier or polymer.

Generally, people familiar with the art can easily understand that it is suitable for changing R₈Part of the pKa and hydrolytic stability groups, including halogen, nitro, amino, alkyl substituted with halogen, optionally substituted carboxy, ketone, ketone, nitrile, amide, ester, alkoxy Bases such as bases.

The linking group suitable for any substituent may include from about 1 to about 20 adjacent atoms, which may be branched or substituted, and may include one or more heteroatoms, which can be further reacted or reacted with the polymer or The functional group attached to the support is terminated. Examples of suitable linking groups include alkyl groups, aryl groups, amide groups, polyamides, polyethers, all optionally substituted groups and combinations thereof.

R₉And R_l0It can further include functional groups that can modify the physical properties of the compound, such as solubility and pKa. For example, these include optionally substituted carboxylic acid esters, amine groups, quaternary ammonium groups, sulfonate esters, PEG and the like.

Of course, when any substituent is a detectable part, it can also include a suitable linking group to connect the detectable part with the rest of the label compound. Suitable linking bases include those listed above. Suitable detectable parts include those defined above.

R₈It is preferably selected from the group consisting of boric acid, borate ion, quinquerine, quinquerite, telluric acid, tellurate ion, germanic acid, germanate ion, and combinations thereof.

It is also understood from the above definition that the compound and detection system of the present invention can have a polymer form. Therefore, the whole compound (including the distinguishing element and the detectable part) can be connected with the existing polymer, or the whole compound in the form of a monomer can be polymerized or copolymerized with another suitable monomer to form a polymer. Another option is to copolymerize two separate monomer components (one including the distinguishing element and the other including the detectable part), so the resulting polymer includes all the elements necessary for the system (refer to Example 6).

The marker compound of the present invention has many uses, including use as a marker in the fields of energy, medicine and agriculture. For example, label compounds can be used to detect low levels in physiological buffers or physiological fluids (such as blood, plasma, serum, tissue fluid, cerebrospinal fluid, urine, saliva, crystalline fluid, lymph, tears, or sweat). Or too high glucose levels, so it provides valuable information for diagnosing or monitoring diseases such as diabetes or adrenal insufficiency.

There is a need to monitor and control glucose used in medical/agricultural production for human therapeutic applications.

The application of the present invention in agriculture includes detecting the glucose value in soybeans and other agricultural products. Care must be taken to monitor the glucose levels of these high-value products (such as wine grapes) during critical harvest decisions. When glucose is the most expensive carbon source and raw material for the fermentation process, it is important to monitor the most appropriate glucose reactor feed rate control when authoritative wine production. The control of reactor mixing and glucose concentration is also the key to the quality control of beverages and fermented beverages, which consume a large amount of glucose and fermentable sugars (the nearby glycol).

When the label compound incorporates a fluorescent label substituent, various detection techniques are also known in the art. For example, the compounds of the present invention (for example, US Patent No. 5,517,313) can be used in fluorescent sensing devices or can be combined with polymer materials, such as test paper for visual inspection. This latter technique may allow, for example, the measurement of glucose in a manner similar to the determination of pH with litmus paper. The compounds described herein can also be used as simple reagents for standard desktop analytical instruments, such as fluorometers or clinical fluorometers manufactured by Shimadzu, Hitachi, Jasco, Beckman and others. Analyzer. These molecules can also provide optical fiber-based analyte-specific chemical/optical signal transmission made by Ocean Optics (Dunedin, Florida) or Oriel Optics.

US Patent No. 5,517,313 (this disclosure is incorporated herein by reference) describes a fluorescent sensing device in which the compound of the present invention is used to determine the presence of glucose or its concentration in a liquid medium. The sensing device includes a layered array of a matrix containing fluorescent label molecules (hereinafter referred to as "fluorescent matrix"), a high-pass filter, and a photodetector. In this device, the light source (preferably a light-emitting diode ("LED")) is at least partially located within the marker or in the waveguide. When a marker matrix is configured, the incident light from the light source causes the marker The molecules fluoresce. The high-pass filter allows the emitted light to reach the light detector, while filtering the scattered incident light from the light source. The regional presence of glucose modulates (eg, reduces or enhances) the fluorescence of the marker molecule used in the device described in US Patent No. 5,517,313.

In the sensor described in US Patent No. 5,517,313, the substance containing the marker molecule can penetrate the analyte. Therefore, the analyte can diffuse into the substance from the surroundings of the test medium, thereby affecting the fluorescence emitted by the labeled compound. The structure composed of a light source, a substance containing a marker compound, a high-pass filter and a photodetector makes at least part of the fluorescent light emitted by the marker compound collide with the photodetector to generate an electrical signal, which is the glucose surrounding the medium Concentration indication.

According to other possible specific embodiments of using the marker compound of the present invention, the sensing device is also described in U.S. Patent Nos. 5,910,661, 5,917,605, and 5,894,351, all of which are incorporated herein for reference. For example, the compound of the present invention is used in an implantable device that continuously monitors blood glucose levels in vivo. Suitable devices are described in, for example, the United States Preamble No. 09/383,148 and U.S. Patent Nos. 5,833,603, 6,002,954, and 6,011,984 filed for joint trial on August 26, 1999, all of which are incorporated herein to for reference.

Those familiar with the art can use easily known reaction mechanisms and reagents (for example, including reaction mechanisms that comply with the general procedures described below) to prepare the compounds of the present invention without undue experimental amounts.

Example 1 Water-soluble copolymer of anthracene derivative and MAPTAC

I. Synthesis of mono-borate-anthracene marker copolymerized in water-soluble polymer: A.9-[3-(methacrylamido)propylamino]methylanthracene and DIEA (18.5g, 25.0 Milliliters, 144 millimoles, 6.5 equivalents) was added dropwise over 20 minutes in 250 milliliters of CHCl at 0°C₃N-(3-aminopropyl) methacrylamide hydrochloride (11.82 g, 66.0 millimoles, 3.0 equivalents) and DBMP (10 mg, as a marker) in a suspension. The mixture was allowed to warm to 25°C and then cooled to 0°C again. Will be in CHCl₃A solution of 9-chloromethylanthracene (5.0 g, 22 mmol) in (100 ml) was added dropwise to the cooled mixture over 1 hour. The mixture was then stirred at 25°C for 1 hour, at 50°C for 12 hours and then at 70°C for 2 hours. At this time, the mixture was washed with 4×60 ml portions of water, and the combined aqueous layer was washed with CH₂Cl₂extraction. The combined organic extracts were subjected to anhydrous Na₂SO₄Dried, decanted and concentrated in vacuo. The crude material is separated by silica gel chromatography (flashing silica gel, 2-5% CH₃OH/CH₂Cl₂) Purification, yielding 2.44 g (33%) solid product. TLC: Merck (Merck) silicone 60 flat plate, Rf 0.39 to 90/10 CH₂Cl₂/CH₃OH, observed with UV (254/366), nindantrione stain. B.9-N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0003.tif" />-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene DiEA (2.85 g, 3.84 ml, 22.0 millimoles, 3.0 equivalents) was added in batches over 10 minutes Add 200 ml CHCl at 0°C₃9-[3-(methacrylamido)propylamino]methylanthracene (2.44 g, 7.34 mmol) and DBMP (10 mg, as a marker) in 9-[3-(methacrylamido) Add (2-bromomethylphenyl) neopentyl borate (2.49 grams, 8.81 millimoles 1.2 equivalents) solution. The mixture was then stirred at 25°C for 20 hours. At this time, the mixture was washed with water, and the combined water layer was washed with CH₂Cl₂extraction. The combined organic extracts were subjected to anhydrous Na₂SO₄Dried, decanted and concentrated in vacuo. The crude material is separated by silica gel chromatography (flashing silica gel, 2-5% CH₃OH/CH₂Cl₂) Purification, yielding 2.50 g (76%) of pale yellow crystalline solid.

Melting point: 72-73°C TLC: Mok Silicone 60 flat plate, Rf 0.36 to 90/10 CH₂Cl₂/CH₃OH, observed with UV (254/366), nindantrione stain. C.9-[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0004.tif" />-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene and MAPTAC (1:20 molar ratio) water-soluble copolymer of 4,4'- Azobis(cyanovaleric acid) (0.008 g, 0.03 millimoles, 1,4 mole% of total monomer) was added to 9-[N-[2-(5,5) in 1.5 ml of ethylene glycol -Dimethylboron<img file="TW200401034A_D0005.tif" />-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene (0.0490 g, 0.105 mmol) and [3-(methacrylamido)propyl Base] trimethylammonium chloride (MAPTAC, 50% by weight aqueous solution, 0.48 g, 0.90 ml, 2.1 millimoles, 20 equivalents) in solution. The solution was flushed with argon for 5 minutes and then heated to 60°C in the dark for 18 hours. At this time, the viscous solution was cooled to 25°C, diluted with 5 ml of water and dialyzed with 3x4 litres of water through a cellulose acetate membrane (MWCO3500). The dialysate was concentrated to dryness, yielding 0.339 g (68%) of a yellow glassy solid.

II. The fluorescence adjustment effect of glucose and lactate is measured by the fluorescence adjustment effect of glucose and lactate of the copolymer prepared in this example (which includes a single discriminating element). Figure 1 shows the normalized fluorescence of a 0.5 mg/ml copolymer solution (1:20 molar ratio) in PBS containing 0-20 mg molecular weight glucose; b) 0-20 mg molecular weight lactate Launch (I/Io@420 nm). The spectrum was recorded using Shimadzu RF-5301 fluorescence photometer, which was excited by @365nm, excitation aperture of 1.5nm, emission aperture of 5nm, room temperature. The error bar is the standard deviation of the repeated value of each data point. The fluorescence of the copolymer is affected by the presence of glucose and acetate.

Example 2 Bi-borate-label covalently attached to water-soluble polymer to regulate glucose and potential physical interference

I. Synthesis of single methacrylate monomer of bis-borate-anthracene label

<chemistry general="n"><img file="TW200401034A_D0006.tif" /></chemistry>

A. 9, 10-bis[[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene 9, 10-bis(chloromethyl)anthracene (3.94 g, 14.3 millimoles) Add 40 ml CHCl at 23°C₃2-(2-aminoethoxy)ethanol (31.4 g, 30.0 ml, 299 millimoles, 20.9 equivalents) in the solution. The solution was stirred in the dark for 67 hours. Add 100 ml CH2Cl at this time₂, And take 1X50ml and 2X100ml NaHCO₃(Saturated aqueous solution) cleaning. The organic extract is subjected to anhydrous Na₂SO₄Dry, filter, and concentrate, yielding 4.67 g (79%) of yellow powder. Continue the reaction as the product is (~85% purity by RP-HPLC). HPLC conditions: HP1100 HPLC chromatography, Vydac 201TP 10_X250mm column, 0.100ml injection, 2ml/min, detection at 370nm, A^＝Water (0.1% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, and 100% B in 2 minutes; The stay time is 15.6 minutes.

<chemistry general="n"><img file="TW200401034A_D0007.tif" /></chemistry>

B.9,10-Bis[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0008.tif" />-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene will be 9,l0-bis[[2-(2-hydroxyl Ethoxy) ethylamino] methyl] anthracene (4.02 g, 9.75 millimoles), DIEA (12.6 g, 17.0 ml, 97.5 millimoles, 10.0 equivalent) and (2-bromomethylphenyl) boronic acid A solution of amyl ester (13.7 g, 48 millimoles, 4.9 equivalents) was stirred at 23°C in the dark for 46 hours. At this point, the reaction mixture was first concentrated by rotary evaporation and then vacuum pump to remove DIEA. The residue was separated by alumina column chromatography (150 grams of activated neutral alumina, 0-3% CH₃OH/CH₂Cl₂) Purified to produce 5.67 grams (70%) of viscous oil, which solidifies on standing. Continue the reaction as the product is (~85% purity by RP-HPLC). TLC: Mok's basic alumina plate, Rf 0.33 to 95/5 of CH₂Cl₂/CH₃OH, observed by UV (254/366). HPLC conditions: HP1100 HPLC chromatography, Vydac 201TP 10x250 mm column, 0.100 ml injection, 2 ml/min, detection at 370 nanometers, A = water (0.1% HFBA) and B = MeCN (0.1% HFBA) , Gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; residence time 18.8 minutes.

<chemistry general="n"><img file="TW200401034A_D0009.tif" /></chemistry>

C. 9-[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0010.tif" />-2-yl)benzyl]-N-[2-(2-methylpropanoxyethoxy)ethylamino]methyl-10-[N-[2-(5,5-dimethyl boron<img file="TW200401034A_D0011.tif" />-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene (single methacrylate monomer) will be in 15ml CH₂Cl₂9,10-bis[N-[2-(5,5-dimethylboron<img file="TW200401034A_D0012.tif" />-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene (0.298 g, 0.359 mmol), methacrylic acid (0.304 g, 0.300 ml, A solution of 3.53 millimoles, 9.84 equivalents), DCC (0.965 grams, 4.68 millimoles, 13.0 equivalents) and N,N-dimethylaminopyridine (0.020 grams, 0.16 millimoles, 0.46 equivalents) at 23°C Stir in the dark for 4 hours. At this time the reaction mixture was filtered and concentrated by rotary evaporation. The residue was separated by alumina column chromatography (50 grams of activated neutral alumina, 0-4% CH₃OH/CH₂Cl₂) To yield 0.150 g (47%) of a yellow solid substance. FABMS: Calculate C₅₂H₆₆B₂N₂O₉[M]^ten855; experimental value [M+1]+886. TLC: Mok's basic alumina plate, Rf 0.45 with 95/5 CH2Cl2/CH3OH, and UV (254/366) observation. HPLC: HP 1100 HPLC chromatography, Vydac201TP10x250 mm column, 0.100 ml injection, 2 ml/min, detection at 370 nm, A=water (0.1% HFBA) and B=MeCN (0.1% HFBA) , Gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; residence time 21 minutes. D.9-[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0013.tif" />-0-yl)benzyl]-N-[2-(2-methylpropanoxyethoxy)ethylamino]methyl-10-[N-[2-(5,5-dimethyl boron<img file="TW200401034A_D0014.tif" />-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene and TMAMA (1:50 molar ratio) water-soluble copolymer will be 3.00ml 9-[N-[2-(5,5-Dimethylboron in MeOH<img file="TW200401034A_D0015.tif" />-2-yl)benzyl]-N-[2-(2-methylpropanoxyethoxy)ethylamino]methyl-10-[N-[2-(5,5-dimethyl Boron-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene (0.0024 g, 0.0033 mmol) solution was added to 0.600 ml of water [ 2-(Methacryloxy)ethyl]trimethylammonium chloride (TMAMA, 70% by weight in water, 0.344 g monomer, 1.66 millimoles, 50 equivalents) in solution. 4,4,-Azobis(4-cyanovaleric acid) (0.0075 g, 0.027 millimoles, 1.6 mole% of total monomer) was added to the mixture. The solution was filtered through 0.45 micron membrane filter paper, flushed with nitrogen and then heated at 55°C for 16 hours in the dark. At this point the viscous solution was cooled to 25°C and concentrated in vacuo. The residue was diluted with 20 ml of water and filtered through 0.2 micron membrane filter paper. The polymer solution was dialyzed with 2×4 liters of water through a cellulose acetate membrane (MWCO3500). 38.5 ml of polymer solution was obtained by dialysis. Part of the solution was concentrated to dryness, showing 0.0075 g of polymer per 1.0 ml of solution. Total production of 0.289 grams (77%) of polymer. II. Determination of the fluorescence adjustment effect of glucose, lactate and acetyl acetate in the copolymer prepared in this example (which includes two discriminating elements) with glucose, lactate and acetyl acetate . Figure 2 shows the 1.5 mg/ml anthracene bisborate-TMAMA in PBS containing a) 0-20 mg molecular weight glucose; b) 0-2 mg molecular weight lactate; c) 0-20 mg molecular weight lithium acetate in PBS The standardized fluorescence emission (I/Io@428 nm) of the solution (molar ratio of 1:50). The spectrum was recorded using Shimadzu RF-5301 Fluorometer, which was excited by @365nm, excitation aperture of 1.5nm, emission aperture of 1.5nm, room temperature. The fluorescence of the copolymer is affected by the presence of glucose, but not by the presence of lactate or acetyl acetate.

Example 3

The dose-response effect of lactate in solution on glucose is the fluorescence effect of bis-borate-anthracene label

<chemistry general="n"><img file="TW200401034A_D0016.tif" /></chemistry>

A.9,10-Bis[[2-(terbutoxycarbonyl)ethylamino]methyl]anthracene will be in 75ml CHCl₃Β-alanine tert-butyl hydrochloride (3.06 grams, 16.8 millimoles, 5.09 equivalents), DIEA (4.27 grams, 5.75 ml, 33.0 millimoles, 10.00 equivalents) and 9,10 -A solution of bis(chloromethyl)anthracene (0.9l0 g, 3.31 mmol) was stirred at 23°C in the dark for 93 hours. At this time, the solution was filtered, and NaHCO was used in 1x40ml and 2x60ml portions₃(Saturated aqueous solution) cleaning. The organic extract is subjected to anhydrous Na₂SO₄Dry, filter, and concentrate to produce a crude yellow solid. The residue was separated by silica gel column chromatography (30 g gravity grade gel, 0-3% CH₃OH/CH₂Cl₂) Purified to produce 1.06 grams (65%) of viscous yellow-orange product. The reaction continues as it is. TLC: Mork Silicone 60 Tablet, Rf 0.33 to 95/5 CH₂Cl₂/CH₃OH, observed by UV (254/366).

<chemistry general="n"><img file="TW200401034A_D0017.tif" /></chemistry>

B.9,10-Bis[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0018.tif" />-2-yl)benzyl]-N-[2-(terbutoxycarbonyl)ethylamino]methyl]anthracene will be in 30ml CHCl₃The 9,10-bis[[2-(terbutoxycarbonyl)ethylamino]methyl]anthracene (1.60 g, 3.25 mmol), DIEA (4.45 g, 6.00 ml, 34.4 mmol) , 10.6 equivalents) and (2-bromomethylphenyl) neopentyl borate (4.80 g, 17.0 millimoles, 5.22 equivalents) were stirred at 23°C in the dark for 4.5 days. At this time 45 ml of CHCl₃Add to the mixture, and add the mixture to 2×25 mL portions of NaHCO₃(Saturated aqueous solution) Qingxian. The organic extract is subjected to anhydrous Na₂SO₄Dry, filter and concentrate to produce crude red oil. The residue was separated by alumina column chromatography (100 grams of activated neutral alumina, 0-3% CH₃OH/CH₂Cl₂) Purified to produce ~3.5 grams of orange solid. The product was dissolved, and then a white precipitate (DIEA-HBr salt) formed. The solution was filtered and the filter was concentrated, yielding 2.72 g (93%) of an orange solid. Continue the reaction as the product is (&gt;80% approx. degree of RP-HPLC). TLC: Mok's basic alumina plate, Rf0.66 to 95/5 of CH₂Cl₂/CH₃OH, observed by UV (254/366). HPLC conditions: HP1100 HPLC chromatography spectrum, Vydac20lTP10×250 mm column, 0.100 ml injection, 2 ml/min, detection at 370 nm, A = water (0.1% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B over 2 minutes, 10-80% B over 18 minutes, 80-100% B over 2 minutes, and 100% B over 2 minutes; residence time 23.9 minutes.

<chemistry general="n"><img file="TW200401034A_D0019.tif" /></chemistry>

C. 9,10-Bis[N-(2-Dihydroxyboronylbenzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene will be in 5ml 20% TFA/CH₂Cl₂9,l0-bis[N-[2-(5,5-dimethylboron<img file="TW200401034A_D0020.tif" />2-yl)benzyl]-N-[2-(terbutoxycarbonyl)ethylamino]methyl]anthracene (0.556 g, 0:620 millimoles) in¹Stir in the dark at 23°C for 25 hours. At this point, the reaction mixture was concentrated under a stream of N2. The residue was wet triturated with 3 x 10 ml portions of ether. The residual solid was dried in vacuum, yielding 0.351 g (87%) of a fluffy yellow powder. FAB MS: Glycerin base; calculated C₄₂H₄₆B₂N₂O_l0(Diglycerin adduct) [M]^＋760; experimental value [M]^＋760. HPLC: HP 1100 HPLC chromatography, Waters 5x100 mm NovaPak HR C18 column, 0.025 ml injection, 0.75 ml/min, 1.5 ml injection circle, detection at 360 nm, A = water (0.1% HFBA) And B=MeCN (0.1%HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; stay time 16.7 minutes . D. Measure the fluorescence regulation effect of glucose and lactate on the fluorescence regulation effect of glucose and lactate in the marker compound prepared in this example (which includes two distinguishing elements). Figure 3 shows that a) 0-10 mg molecular weight glucose, 0 mg molecular weight lactate; b) 0-10 mg molecular weight glucose, 2 mg molecular weight lactate; c) 0-10 mg molecular weight glucose, 5 mg molecular weight lactate Fluorescence (at 428 nanometers) of a 0.75 microgram molecular weight dicarboxylate bis-borate-anthracene marker solution in PBS. The spectrum was recorded with Shimadzu RF-530l fluorescence photometer, which was excited by @365nm, excitation aperture of 1.5nm, emission aperture of 1.5nm, room temperature. All points are measured three times, including ±1 SD error bars. The presence of lactate does not substantially affect the fluorescence modulation effect of glucose on the label.

Example 4

When the label is covalently immobilized in the hydrogel, the selectivity of the bis-borate glucose label to glucose over the lactate and acetyl acetate I. Double-methacrylamide monomer Preparative effect

<chemistry general="n"><img file="TW200401034A_D0021.tif" /></chemistry>

A. 9,10-Bis[3-(methacrylamido)propylamino]methylanthracene will be 9,10-bis(chloromethyl)anthracene (1.5g, 5.45ml) in 200ml CHCl3 Mol), DIEA (28.l7 grams, 38.00 ml, 218 millimoles, 40 equivalents), N-(3-aminopropyl) methacrylamide hydrochloride (9.76 grams, 54.5 millimoles, 10.0 equivalent) and ~5 mg of BHT suspension was stirred at 23°C in the dark at 40°C for 4 days. At this time, the temperature was increased to 45°C, and the mixture was stirred for more than 3 days. A precipitate formed at this time. Filter the mixture and dissolve the solid in the minimum amount of CH₂Cl₂middle. A yellow crystalline solid formed overnight, which is the expected product of dihydrochloride (3.15 g, quantitative). TLC: Mok's basic alumina plate, Rf 0.31 with 90/10 of CH2Cl2/CH₃OH, observed by UV (254/366). HPLC: HP ll00 HPLC chromatography, WaterS 5x100 mm NovaPak HR C18 column, 0.100 ml injection, 0.75 ml/min, detection at 360 nm, A = water (0.1% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B over 2 minutes, 10-80% B over 18 minutes, 80-100% B over 2 minutes, 100% B over 2 minutes; residence time 15.0 minutes.

<chemistry general="n"><img file="TW200401034A_D0022.tif" /></chemistry>

B.9,10-Bis[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0023.tif" />-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene (double-methacrylamido monomer) will be 9,l0 in 20 ml of CHCl3 -Bis[3-(methacrylamido)propylamino]methylanthracene (0.650 g, 1.34 millimoles of free amine), DIEA (0.6l2 g, 0.825 ml, 4.74 millimoles, 3.55 equivalents ), (2-bromomethylphenyl) neopentyl borate (1.34 g, 4.74 millimoles, 3.55 equivalents) and BHT (5 mg, as a marker) were stirred at 23°C in the dark for 5 days. At this time, the reaction mixture was concentrated in vacuum and the residue was subjected to alumina chromatography (200 grams of activated neutral alumina, 0-2% CH₃OH/CH₂C1₂) Purified to produce 0.465 g (39%) of very viscous yellow oil. TLC: Mok basic alumina plate, Rf 0.59 with 90/10 of CH2Cl2/CH₃OH, observe with UV (254/366). HPLC: HP l100 HPLC chromatography, WaterS 5×100 mm NovaPak HR Cl8 column, 0.050 ml injection, 0.75 ml/min, detection at 360 nm, A = water (0.1% HFBA) and B=MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; residence time 16.9 minutes. C. Preparation of N,N-Dimethacrylamide Hydrogel with Glucose Marker Preparation of N,N-Dimethacrylamide (40% by weight) and N,N' in ethylene glycol -A solution of methylene bisacrylamide (0.8% by weight). Add 9,10-bis[N-[2-(5,5-dimethylboron<img file="TW200401034A_D0024.tif" />-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methylanthracene (17.8 mg, 2x10^-5Mol) and 40 microliters of aqueous ammonium persulfate (5% by weight) are combined with 1 ml of ethylene glycol monomer solution. Place the resulting solution in a glove box flushed with nitrogen. The N,N,N',N'-tetramethylethylenediamine aqueous solution (80 μl, 5 wt%) was added to the monomer formulation to accelerate the polymerization. The resulting formulation was poured into a model constructed with a microscope lens and 100-micron stainless steel spacers. After maintaining for 8 hours under nitrogen, put the model in phosphate buffered saline (PBS) (100 mg molecular weight PBS, pH=7.4), separate the microscope slide and take out the hydrogel. Wash the hydrogel with 100 ml PBS containing 1 mg molecular weight sodium lauryl sulfate and 1 mg molecular weight EDTA sodium salt for 3 days, change the solution every day, and then wash with DMF/PBS (10/90 by volume, 3x100 ml) And finally washed with PBS (pH=7.4, 3x100ml). The obtained hydrogel polymer was stored in PBS containing 0.2% by weight of sodium azide and 1 mg molecular weight EDTA sodium salt (10 mg molecular weight PBS, pH=7.4). II. Measure the fluorescence adjustment of glucose, lactate and acetyl acetate for the marker compound prepared in this example (which includes two discriminating elements) to adjust the fluorescence of glucose, lactate and acetyl acetate effect. Figure 4 shows the glucose discriminating molecule including this example in 10 mg PBS (pH 7.4) including 0.2% NaN3 and 1 mg molecular weight EDTA (including various amounts of L-sodium lactate, lithium acetate or α-D-glucose) The standardized fluorescence emission of the hydrogel (I/Io@427nm). Use Shimadzu RF-5301 fluorescence photometer to record data, which is excited by @365 nm (pore = 3 nm) and emitted at 427 nm (pore = 3 nm), with low selectivity, at 37°C Next, use a temperature-controlled sample holder. Before the measurement, the tank tube containing 3 ml of the expected solution was equilibrated at 37°C for 15 minutes. Each hydrogel sample was measured with 4 independent samples. Error bars are the standard deviation of the four replicates for each data point. The hydrogel including glucose discriminating molecules was prepared as described previously. Load the hydrogel at 45^。The glass sheet in the PMMA trough tube of incident light is covered with polyester mesh. Include 0.2%NaN₃1, 5, 10 and 20 mg molecular weight L-sodium lactate solution [Aldrich] prepared in 10 mg molecular weight PBS (pH 7.4) with 1 mg molecular weight EDTA; 5, 10 and 20 mg molecular weight acetyl Lithium acetate solution [Adretril] and 1, 2, 4, 5, 10 and 20 mg molecular weight α-D-glucose solutions. The fluorescence of the copolymer is affected by the presence of glucose, but not by the presence of lactate or acetyl acetate.

Example 5 Selectivity of glucose to lactate using bis-borate discrimination and nearby inhibition of signal generation

AN-(2,2-diethoxyethyl)-4-bromo-1,8-naphthalenedimethamide will be 4-bromo-1,8-naphthoic anhydride (10.0 g , 36.1 millimoles) and aminoacetaldehyde diethyl acetal (4.81 grams, 5.26 ml, 36.1 millimoles, 1 equivalent) suspension was stirred at 45°C for 3 days. At this time, the resulting suspension was filtered, washed with EtOH, and the residue was dried, yielding 13.3 g (94%) of light brown solid product TLC: Mork Silica 60 plate, Rf0.17 with 98/2 CH₂Cl₂/CH₃OH was observed by UV (254/366). HPLC: HP 1l00 HPLC chromatography, WaterS5X100mm NOVaPak HR C18 column, 0.050ml injection, 0.75ml/min, 1.5ml injection circle, detection at 360nm, A=water (0.1%HFBA) and B= MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, and 100% B in 2 minutes; sojourn time 24.2 minutes. BN-(2,2-diethoxyethyl)-4-butylamino-1,8-naphthalenedimethylamide will be N-(2,2-diethoxyethyl) in 8 ml of NMP )-4-bromo-1,8-naphthamide (0.797 g, 2.03 millimoles) and n-butylamine (1.48 g, 2.00 ml, 20.2 millimoles, 9.96 equivalents) in a solution Stir at 45°C for 66 hours. At this time the resulting suspension was allowed to cool to 25°C, followed by filtration. The residue was dissolved in 50 ml ether and extracted with 3×50 ml water. The organic extract is subjected to anhydrous Na₂SO₄Dry, filter and concentrate to produce a crude yellow powder. The crude material was separated by silica gel chromatography (25 g gravity grade gel, 01% CH₃OH/CH₂Cl₂) Purified, yielding 0.639 g (82%) of yellow powder. TLC: Mork Silicone 60 flat plate, Rf0.71 to 95/5 CH₂Cl₂/CH₃OH, observed by UV (254/366). HPLC: HP 1100 HPLC chromatography, Waters 5X100 mm NOVaPak HR C18 column, 0.050 ml injection, 0.75 ml/min, l. 5 ml injection circle, detected at 450 nm, A = water (0.1% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B for 2 minutes, 10-80% B for 18 minutes, 80- 100% B takes 2 minutes, 100% B takes 2 minutes; the stay time is 23.5 minutes. CN-(2-oxyethyl)-4-butylamino-l,8-naphthalenedimethamide will be N-(2,2-diethoxyethyl)-4-butyl in 25 ml of acetone A solution of 1,8-naphthalene dimethylamide (0.622 g, 1.62 millimoles) and p-toluenesulfonic acid monohydrate (0.010 g, 0.053 millimoles, 0.032 equivalents) was stirred at 25°C for 18 Hour. At this time, the solution was filtered and the residue was separated by silica gel chromatography (25 g gravity grade gel, 0-l% CH₃OH/CH₂Cl₂) Purification, yielding 0.470 g (94%) orange solid. TLC: Mork Silicone 60 flat plate, Rf 0.61 to 95/5 CH₂Cl₂/CH₃OH, observed by UV (254/366).^lH NMR(400MHz, CDCl₃);δ1.03(t, 3H,J=7.3HZ), 1.53(m,2H), 1.78(m,2H), 3.38(t,2H,J=7.2Hz), 5.02(S,2H) ,6.64(d,1H,J=8.6HZ), 7.52(dd, lH, J=7.4, 8.3HZ), 8.08(dd, 1H,J=1HZ, 8.5HZ), 8.38(d, lH, J=8.3 HZ), 8.46 (dd, 1H, J=1.0, 7.3HZ), 9.75 (S, 1H). HPLC: HP 1100 HPLC chromatography, WaterS 5x100 mm NovaPak HR C18 column, 0.050 ml injection, 0.75 ml/min, 1.5 ml injection circle, detection at 450 nm, A = water (0.1% HFBA) And B=MeCN (0.1% HFBA), gradient: 10% B over 2 minutes, 10-80% B over 18 minutes, 80-100% B over 2 minutes, 100% B over 2 minutes; residence time 19.6 minutes. DN-(4-dimethylaminobenzyl)-1,6-diaminohexane will mix 4-dimethylaminobenzaldehyde (1.00 g, 6.70 millimoles) in 20 ml of anhydrous EtOH, Na₂SO₄A suspension of (6.70 g, 47.2 millimoles, 7.04 equivalents) and 1,6-diaminohexane (3.89 grams, 33.5 millimoles, 5.00 equivalents) was stirred for 18 hours in the dark under nitrogen at 25°C. At this time the solution was filtered and NaBH₄(L.73 grams, 45.8 millimoles, 6.84 equivalents) was added to the filter. The suspension was stirred at 25°C for 5 hours. At this time, the reaction mixture was concentrated, and the residue was dissolved in 50 ml of water and_XExtract with 50 ml of ether. The combined organic extracts were washed with 2×50 ml water. The combined aqueous extracts were extracted with 2 x 50 mL ether. The combined organic extracts were subjected to Na₂SO₄Dry, filter and concentrate to produce 1.35 grams (81%) of viscous oil. TLC: Mork Silicone 60 tablets, Rf 0.58 to 80/15/5 of CH₂Cl₂/CH₃OH/iPrNH₂, Observed with ninhydrin staining agent, UV (254/366). HPLC: HP 1100 HPLC chromatography, WaterS 5×100 mm NovaPak HR Cl8 column, 0.050 ml injection, 0.75 ml/min, 1.5 ml injection circle, detection at 280 nm, A = water (0.1% HFBA) And B=MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; residence time 13.3 minutes. EN-2-[6-N-(N-4-dimethylaminobenzyl)aminohexyl]aminoethyl)-4-butylamino-1,8-naphthalenedimethylamide will be in 20ml anhydrous N-(4-dimethylaminobenzyl)-1,6-diaminohexane (0.554 g, 2.22 millimoles, 2.00 equivalents) and acetic acid (0.067 g, 1.1 millimoles) in MeOH , 1.0 equivalent) was added to the suspension of N-(2-oxyethyl)-4-butylamino-1,8-naphthalenedimethylamide (0.346 g, 1.11 mmol) in 25 ml of anhydrous MeOH In the liquid. Add NaCNBH in 5 ml of anhydrous MeOH₃(0.070 g, 1.1 millimoles, 1.0 equivalent) solution was added to the mixture. The reaction mixture was stirred at 25°C for 15 hours. At this time the MeOH was removed by rotary evaporation, and the residue was dissolved in 30 ml of water. The solution was adjusted to pH 2 with 1 equivalent of HCl and then stirred at 25°C for 1 hour. At this time, the solution was adjusted to pH 12 with 1 equivalent of NaOH and then 3x50 ml of CH₂Cl₂extraction. The combined organic extracts were washed with 3x50 ml of water, and subjected to anhydrous Na₂SO₄Dry, filter and concentrate to produce a crude brown oil. The crude material is separated by silica gel chromatography (35 grams of gravity grade gel, 0-50% CH₃OH/CH₂Cl₂, Then 45/50/5 CH₃OH/CH₂C1₂/iPrNH₂) Purified to produce 0.190 g (32%) of the diamine product FABMS: Calculate C₃₃H₄₅N₅O₂[M]^＋544; experimental value[M]^＋544. TLC: Mork Silicone 60 flat plate, Rf 0.42 to 80/20 CH₂Cl₂/CH₃OH, observed with ninhydrin stain and UV (254/366). HPLC: HP 1100 HPLC chromatography, Waters 5×100 mm NovaPak HR Cl8 column, 0.050 ml injection, 0.75 ml/min, 1.5 ml injection circle, detection at 450 nm, A = water (0. l%HFBA) and B=MeCN (0.1%HFBA), gradient: 10%B in 2 minutes, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B in 2 minutes; The stay time is 17.6 minutes. FN-2-[6-N-(N-4-dimethylaminobenzyl)-6-N-[2-(5,5-dimethylboron<img file="TW200401034A_D0025.tif" />2-yl)benzyl]aminohexyl]-[2-(5,5-dimethylboron<img file="TW200401034A_D0026.tif" />-2-yl)benzyl]aminoethyl-4-butylamino-1,8-naphthalenedimethamide will be in 2ml CHCl₃A solution of (2-bromomethylphenyl) neopentyl borate (0.390 g, 1.38 millimoles, 5.00 equivalents) in 5 ml CHCl₃N-2-[6-N-(N-4-dimethylaminobenzyl)aminohexyl]aminoethyl)-4-butylamino-1,8-naphthalenedimethylamide (0.150 g, 0.276 millimolar) and DIEA (0.355 g, 0.478 ml, 2.81 millimolar, 10.0 equivalent). The solution was then stirred at 25°C for 27 hours. At this time, the mixture was concentrated and the residue was subjected to alumina column chromatography (100 grams of activated neutral alumina, 0-5% CH₃OH/CH₂Cl₂) Purified to produce 0.024 grams (19%) of viscous brown oil. FAB MS (Glycerin Base): Calculate C₅₃H₆₇B₂N₅O₈[M]^＋924 (using diglycerin adduct instead of di-neopentyl boric acid); experimental value [M]^＋924. TLC: Mok neutral alumina plate, Rf 0.62 with 80/20 CH2Cl₂/CH₃OH, observed by UV (254/366). HPLC: HP l100 HPLC chromatography, Waters 5x100 mm NovaPak HR C18 column, 0.050 ml injection, 0.75 ml/min, 1.5 ml injection circle, detection at 450 nm, A = water (0.1% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; sojourn time 20.7 minutes . GN-2-[6-N-(N-4-dimethylaminobenzyl)-6-N-[2-(dihydroxyboron)benzyl]aminohexyl]-[2-(dihydroxyboron Benzyl]aminoethyl-4-butylamino-1,8-naphthalenedimethylamide (nBuF-hexyl-Q-bisborate) The free bisboronic acid product used in glucose research is derived from N -2-[6-N-(N-4-dimethylaminobenzyl)-6-N-[2-(5,5-dimethylboron<img file="TW200401034A_D0027.tif" />-2-yl)benzyl]aminohexyl]-[2-(5,5-dimethylboron<img file="TW200401034A_D0028.tif" />-2-yl)benzyl]aminoethyl-4-butylamino-1,8-naphthalene dimethylamide in MeOH/PBS buffer system dissolution. H. Measure the fluorescence regulation effect of glucose and lactate on the fluorescence regulation effect of glucose and lactate in the marker compound prepared in this example (which includes two distinguishing elements). Figure 5 shows the normalized fluorescence emission (I/ Io@535 nm). The spectrum was recorded using Shimadzu RF-5301 fluorescence photometer, which was excited by @450nm, excitation aperture of 1.5nm, emission aperture of 1.5nm, room temperature. The error bar is the standard deviation of the 3 replicates for each data point. The fluorescence of the label is affected by the presence of glucose, but is not substantially affected by the presence of lactate.

Example 6

Glucose or lactate pair contains N-[3-(methacrylamido)propyl]-3,,4-hydroxy-9,10-dioxy-2-anthracenesulfonamide (alizarin red S monomer ) And α,α'-bis[N-[2-(5,5-dimethylboron<img file="TW200401034A_D0029.tif" />-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene (bis-boronic acid monomer) acrylamide gel effect: A. 3,4-Dihydroxy-9,10-dioxy-2-anthracenesulfonic acid chloride: 3,4-dihydroxy-9,10-dioxy-2-anthracenesulfonic acid sodium salt (1.4 g, 3 9 millimoles) and 30 ml of chlorosulfonic acid, and heated to 90 °C for 5 hours, then the solution was cooled to 0 °C, and poured into 100 grams of ice. In a solution melted with ice, with CH₂Cl₂(3X100ml) After extraction, the dichloromethane extracts were combined and used Na₂SO₄Drying and evaporation yielded 0.87 grams of solids (66% yield). BN-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxy-2 anthracenesulfonyl chloride: 3,4-dihydroxy-9,l0- Dioxy-2-anthracene sulfonyl chloride (96 mg, 0.28 mmol) and N-(3-aminopropyl) methacrylamide hydrochloride (108 mg, 0.6 mmol) and 20 ml CH₂Cl₂merge. Will Et₃N (303 mg, 3 mmol) was added to the suspension. The mixture was stirred at room temperature for 24 hours, filtered and the solution was evaporated. The obtained solid is in CH₂Cl₂/MeOH(90/10) SiO as a dissolving agent₂(10 grams) for column chromatography. The product was obtained as a red solid (80 mg, 64% yield). FAB MS: Calculate C_2lH_2oN₂O₇SM⁺445; experimental value M⁺445. HPLC: HP 1100 HPLC chromatography, Waters 5×100 mm NovaPak HR Cl8 column, 0.100 ml injection, 0.75 ml/min, 2 ml injection circle, detection at 370 nm, A = water (0.1% HFBA ) And B = MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; sojourn time 17.67 minute. C.α,α'-bis[3-(methacrylamido)propylamino]-1,4-xylene will be N-(3-aminopropyl)methacrylic acid in 75ml of dry MeOH Amine hydrochloride (3.00 grams, 16.8 millimoles, 2.21 equivalents), DIEA (6.5 grams, 8.8 ml, 50 millimoles, 6.6 equivalents), terephthalaldehyde (1.02 grams, 7.60 milliliters) Mol) and Na₂SO₄(10.7 grams, 75.3 millimoles, 9.91 equivalents) of the solution was stirred at 25°C in the dark for 18 hours. Add more Na at this time₂SO₄(10.7 grams, 75.3 millimoles, 9.91 equivalents) and continue stirring for more than 6 hours. At this time the solution was filtered and NaBH₄(1.73 grams, 45.7 millimoles, 6.01 equivalents) was added to the filter in batches and then stirred at 25°C for 21 hours. The suspension was filtered through Celite and the filtrate was concentrated. Dissolve the residue in 100 ml CH₂Cl₂Neutralize with l×25ml saturated aqueous NaHCO₃Clean. The organic extract is subjected to anhydrous Na₂SO₄Dry, filter and concentrate to produce viscous oil. The reaction continues as it is. HPLC: HP 1100 HPLC chromatography, Vydac 201TP l0×250 mm column, 0.100 ml injection, 2.00 ml/min, detection at 260 nm, A = water (0.1% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B over 2 minutes, 10-80% B over 18 minutes, 80-100% B over 2 minutes, 100% B over 2 minutes; residence time 15.8 minutes. D.α,α'-Bis[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0030.tif" />-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene will be in 75ml CH₂Cl₂Α,α'-bis[3-(methacrylamido)propylamino]-l,4-xylene (2.94 g, 7.61 mmol), DIEA (2.97 g, 4.00 ml, 23.0 mmol) Ears, 3.02 equivalents), (2-bromomethylphenyl) neopentyl borate (6.50 g, 23.0 millimoles, 3.02 equivalents) and BHT (5 mg, as a marker) in the dark at 25°C Stir for 28 hours. At this time, the mixture is divided into 1×25 ml saturated aqueous NaHCO₃Clean. The organic extract is subjected to anhydrous Na₂SO₄Dry, filter and concentrate. 200 ml of ether was added to the residue, and the suspension was stirred for 18 hours. Filter the suspension and dissolve the residue in CH₂Cl₂, Filter and concentrate the filtrate. 150 ml of ether was added to the solid residue, and the suspension was stirred for 18 hours. At this point the suspension was filtered, yielding 1.98 g (33%) of a fluffy pink powder. FAB MS: Calculate C₄₆H₆₄B₂N₄O₆[M]^＋790 ; Experimental value [M+l]⁺791. HPLC: HP 1100 HPLC chromatography, Waters 5×100 mm NovaPak HR C18 column, 0.050 ml injection, 0.75 ml/min, detection at 280 nm, A = water (0.1% HFBA) and B = MeCN ( 0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; residence time 13.4 minutes. E. Containing N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxy-2-anthracenesulfonamide (alizarin red S monomer) and α,α^,-DoubleN-2-(5,5-dimethylboron<img file="TW200401034A_D0031.tif" />2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-l,4-xylene acrylamide gel preparation: preparation of acrylamide containing 30% by weight And 0.8% by weight of N, N^，-A glycol solution of methylene bisacrylamide. The N-[3-(methacrylamido)propyl]-3,4-dihydroxy-9,10-dioxy-2-anthracenesulfonamide (1.5 mg, 3.38x10^-6Mol) and α,α^，-DoubleN-2-(5,5-dimethylboron<img file="TW200401034A_D0032.tif" />-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-1,4-xylene (28 mg, 3.54x10^-5Mol) and 800 microliters of ethylene glycol monomer solution and 40 microliters of 5 wt% aqueous ammonium persulfate. The formulation was put into a glove box flushed with nitrogen together with a model constructed with a glass microscope lens and a 100-micron stainless steel spacer. N, N, N^,,N^，-Tetramethylethylenediamine aqueous solution (40 microliters, 5 wt%) was added to the monomer solution to accelerate the polymerization, and the final formulation was poured into the glass mold. The model was left under nitrogen for 16 hours, and then it was inserted into PBS (pH=7.4), and the glass lens was separated to supply the hydrogel polymer in the form of a film. The resulting hydrogel film was washed with 100 ml of sodium lauryl sulfate with a molecular weight of 1 mg and washed with phosphate buffered saline for 3 days. The solution was changed every day, followed by MeOH/PBS (20/80 by volume, 3x100 ml) Wash and finally wash with PBS (pH=7.4, 3x100 ml). The hydrogel polymer was stored in PBS (10 mg molecular weight PBS, pH=7.4) containing 0.2% by weight of sodium azide and 1 mg molecular weight EDTA sodium salt. F. The absorption value adjustment effect of glucose and lactate is used to determine the absorption value adjustment effect of the marker hydrogel prepared in this example (which includes two distinguishing elements) with glucose and lactate. The vinyl amine gel was loaded in the PMMA tank in the same manner as described in Example 4. Heat phosphate-buffered saline (PBSpH7.4) including a predetermined amount of glucose or sodium lactate in a water bath to 37°C, and put it into the PMMA tank containing the gel, and then allow the PMMA tank to pass through at 37°C. Reach equilibrium in 15 minutes. Each absorption value measurement of glucose or lactate concentration is performed 3 times. Each measurement uses the absorbance value at 650 nm as the blank value, and subtracts A (650 nm) from all A values (450 nm) and A values (530 nm). Figure 6 shows including with and without glucose The absorption spectrum of acrylamide gel (30%) of 4 mg molecular weight Alizarin S and 44 mg molecular weight diboronic acid monomer. Figure 7 shows the effect of glucose on the absorption value of acrylamide gel (30%) including 4 mg molecular weight Alizarin S and 44 mg molecular weight diboronic acid monomer. Figure 8 shows the effect of sodium lactate on the absorption value of acrylamide gel (30%) including 4 mg molecular weight Alizarin S and 44 mg molecular weight diboronic acid monomer. The absorption value of the marker is affected by the presence of glucose, but is not substantially affected by the presence of lactate. G. Measure the fluorescence adjustment effect of glucose and lactate substantially according to the acrylamide gel synthesized in Example 6 (except for the use of 1.9 mg of N-[3-(methacrylamide) propyl]-3 , 4-Dihydroxy-9,10-dioxy-2-anthracene sulfonamide and 35 mg α, α^，-Bis[N-[2-(5,5-dimethylboron<img file="TW200401034A_D0033.tif" />2-yl)benzyl]-N-[3-(methacrylamido)propylamino]-except 1,4-xylene] for fluorescence regulation.

The experiment was performed on a Shimadzu RF-5301 PC fluorometer equipped with a variable temperature accessory (excitation at 4700 nm, 3/10 nm aperture, high sensitivity). Attach the acrylamide gel to a piece of 45^。Glue it to the PMMA fluorescent groove on the glass lens of the corner. Fill the tank with 2.5 ml PBS (pH=7.4) and heat to 37°C. Prepare glucose stock solutions (100 mg molecular weight and 500 mg molecular weight) in PBS (pH = 7.4), and heat to 37°C in a water bath. Add an aliquot of the heated glucose stock solution to the PMMA tank regularly, and monitor the fluorescence intensity with time as a factor at 550 nm (measured every 2 minutes). Use YSI model 2300 STAT with glucose analyzer to measure the glucose concentration in the PMMA tank. The results shown in Figure 9 show that added glucose reduces the fluorescence intensity of the marker hydrogel. The same effect is observed in Figure 10, which shows the effect of glucose on the fluorescence spectrum of the same type of gel.

It is believed that the occurrence of this effect is due to the following considerations. The methacrylamide monomer (acceptor molecule) of Alizarin S includes nearby glycol functionality and monomer functionality (refer to the structure below). Alizarin S and bis-borate distinguishing element monomers (refer to the structure below) can reversibly react with each other in aqueous solutions and organic solvents to form boric acid esters. The borate molecule formed in this reversible reaction is a fluorescent molecule, but the alizarin S monomer itself can not see fluorescence emission visually in aqueous solution and organic solvent (such as MeOH). Therefore, once combined with the glucose discriminating element, Alizarin S will change its optical properties, such as absorption value and fluorescence mass yield.

<chemistry general="n"><img file="TW200401034A_D0034.tif" /></chemistry>

A solution of Alizarin S with monomer functionality and glucose discriminating element with monomer functionality can be prepared together with the hydrogel monomer and crosslinking agent. The copolymerization of the mixture produces a hydrogel substance, which can diffuse to various small to medium-sized molecules, so that the analyte can be detected and quantified. For example, analytes such as glucose can diffuse in the hydrogel matrix and replace the receptor molecules previously bound to the discriminating element. This event caused a change in the optical rotation properties of the hydrogel film because it currently includes more receptor molecules that are not bound to the discriminating element.

The fluorescence modulation effect of glucose and lactate with the marker compound prepared in this example (which includes two distinguishing elements) was also determined. The experiment was performed on a Shimadzu RF-5301PC fluorometer equipped with a variable temperature accessory (excitation at 470 nm, 5/10 nm pore, low sensitivity). Attach the acrylamide gel to a piece of 45. Glue it to the PMMA fluorescent groove on the glass lens of the corner. Fill the tank with 2.5 ml PBS (pH=7.4), and heat to 37°C in a water bath. A stock solution of sodium lactate (100 mg molecular weight) in PBS (pH=7.4) was prepared and heated in a water bath. To 37°C. Prepare glucose stock solutions (100 mg molecular weight and 500 mg molecular weight) in PBS (pH = 7.4), and heat to 37°C in a water bath. Add aliquots of the heated lactate stock solution to the PMMA tank regularly, and monitor the fluorescence intensity with time as a factor at 550 nm (measured every 2 minutes) until the lactate concentration reaches 8 mg molecular weight . Then add an aliquot of the heated glucose stock solution to the PMMA tank regularly, and monitor the fluorescence intensity with time as a factor at 550 nm (measured every 2 minutes). Use YSI model 2300STAT plus glucose analyzer to measure the glucose concentration in the PMMA tank. The results shown in Figure 11 show that the added lactate has no obvious effect on the fluorescence intensity of the marker hydrogel, and the subsequent addition of glucose will reduce the fluorescence intensity of the marker hydrogel.

Example 7

The single methacrylamide monomer of bis-borate-anthracene:

<chemistry general="n"><img file="TW200401034A_D0035.tif" /></chemistry>

A. 9-Chloromethyl-10-[[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene hydrochloride salt of 2-(2-aminoethoxy)ethanol (0.495 g , 0.475 ml, 4.71 millimoles) 9 in 200 ml NMP_，l0-bis(chloromethyl)anthracene (5.18 g, 18.8 millimoles, 3.99 equivalents) in suspension. The mixture was stirred in the dark for 17 hours. At this time the reaction mixture was concentrated to ~50 ml under reduced pressure and 50°C. The residue was separated by silica gel chromatography (150 grams of gravity grade silica gel, 0-l0% CH₃OH/CH₂Cl₂) Purification, yielding 0.425 g (24%) yellow/orange solid. TLC: Mork Silicone 60 flat plate, Rf 0.72 to 70/30 CH₂Cl₂/CH₃OH, observed with UV (254/366), mulberry triketone stain. HPLC: HPl100 HPLC chromatography, Vydac20lTP10x250 mm column, 0.100 ml injection, 2 ml/min, detection at 370 nm, A = water (0.1% HFBA) and B = MeCN (0.1 %HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; residence time 16.1 minutes.

<chemistry general="n"><img file="TW200401034A_D0036.tif" /></chemistry>

B.9-[[2-(2-Hydroxyethoxy)ethylamino]methyl]-10-[[(3-methenylamino)propylamino]methyl] anthracene will be in 25ml CHCl3 9-chloromethyl-10-[[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene hydrochloride (1.56 grams, 4.10 millimoles) solution dropwise Add 125 ml CHCl at 23°C₃N-(3-aminopropyl) methacrylamide hydrochloride (3.08 grams, 17.2 millimoles, 4.2 equivalents), DIEA (5.19 grams, 7.00 milliliters, 40.1 millimoles, 9.8 equivalents) and ~3 mg of BHT in suspension. The mixture was then stirred in the dark for 92 hours. At this time, the reaction mixture was filtered and 2X40ml NaHCO₃(Saturated aqueous solution) cleaning. The organic extract is subjected to anhydrous Na₂SO₄After drying, filtering and concentrating, a viscous orange solid was produced, which was purified by alumina chromatography (50 g of activated neutral alumina, 0-5% CH3OH/CH2Cl2), yielding 0.364 g (20 %) Orange solid TLC: Mok Silicone 60 tablets, Rf0.16 at 70/30 CH₂Cl₂/CH₃OH, observed with UV (254/366), nindantrione stain. HPLC: HP1100 HPLC chromatography, Vydac 201TP 10×250 mm column, 0.100 ml injection, 2 ml/min, detection at 370 nm, A = water (0.1% HFBA) and B = MeCN (0.1 %HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; residence time 16.85 minutes.

<chemistry general="n"><img file="TW200401034A_D0037.tif" /></chemistry>

C.9-[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0038.tif" />-2-yl)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(5,5-dimethylboron<img file="TW200401034A_D0039.tif" />-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene (mono-methacrylamide monomer) will be used in 20 ml of CHCl₃9-[[2-(2-hydroxyethoxy)ethylamino]methyl]-10-[[(3-methenamido)propylamino]methyl]anthracene (0.343g, 0.763ml Mol), DIEA (0.965 g, 1.30 ml, 9.8 equivalents) and (2-bromomethylphenyl) neopentyl borate (1.09 g, 3.85 millimoles, 5.0 equivalents) in the dark at 23°C Stir for 25 hours. At this time, the reaction mixture was first concentrated by rotary evaporation and then using a vacuum pump to remove DIEA. The residue was separated by alumina column chromatography (40 grams of activated neutral alumina, 0-10% CH₃OH/CH₂Cl₂) Purification, yielding 0.299 g (46%) yellow-orange solid. The compound can be copolymerized with suitable monomers (as previously described), deprotected and used to detect glucose. FAB MS: Calculate C_5lH₆₅B₂N₃O₇[M]^＋854; experimental value[M+1]^＋855. TLC: Mok's basic alumina plate, Rf 0.35 to 95/5 of CH₂Cl₂/CH₃OH, observe with UV (254/366). HPLC: HP l100 HPLC chromatography, Vydac 201TP 10x250 mm column, 0.100 ml injection, 2 ml/min, detection at 370 nm, A = water (0.1% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B over 2 minutes, 10-80% B over 18 minutes, 80-100% B over 2 minutes, 100% B over 2 minutes; residence time 19.7 minutes.

Example 8

Double-methacrylamide monomer of bis-borate-anthracene

<chemistry general="n"><img file="TW200401034A_D0040.tif" /></chemistry>

A.9,10-Bis[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0041.tif" />-2-yl)benzyl]-N-[2-(2-methylpropanoxyethoxy)ethylamino]methyl]anthracene will be in 5ml CH₂Cl₂9,l0-bis[N-[2-(5,5-dimethylboron<img file="TW200401034A_D0042.tif" />-2-yl)benzyl]-N-[2-(2-hydroxyethoxy)ethylamino]methyl]anthracene (0.100 g, 0.120 mmol, Reference Example 2), methacrylic acid (0. ll2 grams, 0.110 ml, 1.30 millimoles, 10.8 equivalents), DCC (0.316 grams, 1.53 millimoles, 15.8 equivalents) and N,N-dimethylaminopyridine (0.014 grams, 0.11 millimoles, 0.92 equivalents) The solution of) was stirred at 0°C for 1 hour, and then at 23°C for 22 hours. At this time the reaction mixture was filtered and concentrated by rotary evaporation. The residue was separated by alumina column chromatography (30 grams of activated neutral alumina, 0-2% CH₃OH/CH₂Cl₂) Purification, yielding 0.030 g (26%) yellow solid. The intercalating compound can be copolymerized with suitable monomers (as previously described), deprotected and used to detect glucose. FAB MS: Calculate C₅₆H_7oB₂N₂O₁₀[M]⁺953; experimental value [M]⁺951 (weak molecular ion peak). TLC: Mok's basic alumina plate, Rf0.67 to 95/5 of CH₂Cl₂/CH₃OH, observed by UV (254/366). HPLC: HP 1100 HPLC chromatography, Waters 5×100mm NovaPak HR C18 column, 0.100ml injection, 0.75ml/min, 2ml injection circle, detection at 370nm, A=water (0.1% HFBA) And B=MeCN (0.1% HFBA), ladder and: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; stay time 19.6 minutes .

Example 9

Double 5-aminopentyl bis-borate-anthracene

<chemistry general="n"><img file="TW200401034A_D0043.tif" /></chemistry>

A. 9,10-bis[[5-(t-Boc)-aminopentylamino]methyl] anthracene will be 9,10-bis(chloromethyl)anthracene (0.28 g, 1 millimolar), DIEA (7.0 ml, 40 millimoles), mono-tert-butoxycarbonyl 1,5-diaminopentane (3.75 grams, 10 millimoles) and 50 milliliters of CHC1₃The suspension was stirred at 45°C in the dark for 2 days. Saturate the solution with H₂O/NaHCO₃Wash and dry the organic phase (Na₂SO₄) And evaporate the solvent. The residue was separated by alumina chromatography (40 grams of activated neutral alumina, 95/5 vol% CH₂Cl₂/MeOH) to produce 0.55 grams of viscous oil. Use this substance as it is for the following steps.

<chemistry general="n"><img file="TW200401034A_D0044.tif" /></chemistry>

B.9,10-Bis[N-[2-(5,5-Dimethylboron<img file="TW200401034A_D0045.tif" />2-yl)benzyl]-N-[5-(t-BOC)-aminopentylamino]methyl]anthracene will be in 20ml CH₂Cl₂The 9,10-bis[[5-(t-Boc)-aminopentylamino] methyl] anthracene (0.3 g, 0.49 millimoles), DIEA (0.35 ml, 2 millimoles) and (2 A solution of -bromomethylphenyl) neopentyl borate (0.566 g, 2.0 mmol) was stirred at 25°C in the dark for 2 days. At this time, the reaction mixture was concentrated in vacuum, and the residue was subjected to alumina chromatography (60 g activated neutral alumina, 98/2 vol% CH₂C_l2/MeOH) to produce 0.401 grams of yellow oil. Use this substance as it is for the following steps.

<chemistry general="n"><img file="TW200401034A_D0046.tif" /></chemistry>

C. 9,10-bis[N-(2-dihydroxyboronylbenzyl)-N-[5-aminopentylamino]methyl]anthracene trifluoroacetate will be 9,10-bis[N-[ 2-(5,5-Dimethylboron<img file="TW200401034A_D0047.tif" />-2-yl)benzyl]-N-[5-(t-BOC)-aminopentylamino]methyl]anthracene (0.4g, 0.39mmol) dissolved in 20ml CH₂Cl₂/TFA (80/20% by volume). The solution was stirred for 12 hours, the solvent was evaporated, and the residue was washed with 10 ml of ether. A total of 373 mg (72% yield) of solids was obtained. The product has ~80% purity (RP-HPLC). The compound can be copolymerized with suitable monomers (as previously described), deprotected and used to detect glucose. HPLC: HP 1100 HPLC chromatography, Waters 5x100 mm NovaPak' HR C18 column, 0.050 ml injection, 0.75 ml/min, detection at 360 nm, A = water (0.1% HFBA) and B = MeCN (0.1 %HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; residence time 16.0 minutes.

Example 10

<chemistry general="n"><img file="TW200401034A_D0048.tif" /></chemistry>

AN-2-(tert-butoxycarbonyl)aminoethyl-4-bromonaphthylene-1,8-dicarboxyimide: Nt-Boc-ethylene diamine (F1uka (F1uka), 1_.6 grams, 10 millimoles) and 4-bromo-1,8-naphthalenedicarboxylic acid anhydride (Adritril, 2_.77 g, 10 mmol) was combined with 60 ml of absolute ethanol, and the suspension was stirred at 60°C for 20 hours, cooled to room temperature and filtered. The obtained solid was washed with 30 ml of cold EtOH and dried under vacuum. 3_.84 grams (91%) yield. NMR(CDC1₃):1_.28(9H,s); 3_.52(2H,t); 4_.35(2H,t); 4_.92(1H,s); 7_.84(1H,t); 8_.04(1H,d); 8_.42(1H,d); 8_.58(1H,d); 8_.67(1H,d).

<chemistry general="n"><img file="TW200401034A_D0049.tif" /></chemistry>

BN-2-(tert-butoxycarbonyl)aminoethyl-4-(N^，-Methylaminoethylamino) naphthalene-l,8-dicarboximide: N-methylethylenediamine (1_.48 grams, 20 millimoles) was combined with 2 ml of 1-methyl-2-pyrrolidone (NMP), and then N-2-(tert-butoxycarbonyl)aminoethyl-4-bromonaphthyl-1, 8-Dicarboxyimide (0_.35 grams, 0_.845 millimoles). The resulting solution was stirred at 45°C for 40 hours, and then NMP and N-methylethylenediamine were evaporated under vacuum. The residue obtained was subjected to column chromatography (20 grams of silica gel, first with CH₂Cl₂/MeOH(90/10), followed by CH₂Cl₂/MeOH/Et₃N(75/20/5)). A yellow solid was obtained (0.311 g, 89% yield). The purity was checked by RP-HPLC.

<chemistry general="n"><img file="TW200401034A_D0050.tif" /></chemistry>

C. N-aminoethyl-4-(N^，-Aminovinyl-N"-[2-(dihydroxyboronyl)benzyl]methylamino)naphthylene-1,8-dicarboxyimidine trifluoroacetate will N-2-(tertbutyl Oxycarbonyl)aminoethyl-4-(N^，-Methylaminoethylamino) naphthalene-1,8-dicarboximide (0.3 g, 0.73 millimoles), 2-bromomethylphenylboronic acid, pinacol ester (0.6 g, 2 millimoles) Mol), N,N-diisopropyl-N-ethylamine (1.3 ml, 8 millimoles) and 10 ml CH₂Cl₂merge. The solution was stirred for 20 hours, and then 2 grams of PS-triamine resin (Argonaut Technologies, 3.38 millimoles/gram) was added. The reaction mixture was stirred with the resin for 10 hours, then the resin was removed by filtration, and the₂Cl₂(2×20ml) for cleaning. CH to be merged₂Cl₂The solution is evaporated and dried under vacuum.

<chemistry general="n"><img file="TW200401034A_D0051.tif" /></chemistry>

Dichloromethane containing 20% by volume of TFA and 5% by volume of triisopropylsilyl was added to the obtained orange residue. The resulting solution was stirred at room temperature for 10 hours, then the solvent was evaporated and the residue was wet triturated with ether, resulting in a yellow solid. The solid was filtered and dried in vacuum (580 mg yield). The purity of the material was checked by RP-HPLC. Use it as a solid for the following steps. DN-(3-Dihydroxyboron-5-nitrophenamido)ethyl-4-(N'-aminovinyl-N"-[2-(Dihydroxyboron)benzyl]methyl Amino) naphthylene-1,8-dicarboximide: N-aminoethyl-4-(N'-aminovinyl-N"-[2-(dihydroxyboron)benzyl]methan Amino) naphthalene-1,8-dicarboxyimide trifluoroacetate (0.225 g, 0.4 millimoles), 3-carboxy-5-nitrophenylboronic acid (0.085 g, 0.4 millimoles), Diphenyl azide phosphate (0.13 mL, 0.6 mmol) was combined with 2 mL of anhydrous DMF. N,N-Diisopropyl-N-ethylamine (0.7 mL, 4 mmol) was added, and the solution was stirred for 20 hours. Ether (10 ml) was added to the reaction mixture, and the insoluble residue was separated and mixed with 5 ml CH₂Cl₂Sonication produces an orange solid, which is filtered and dried under vacuum (38 mg, 15% yield). The purity of the solid was checked by RP-HPLC. NMR(dmso-d6/D2O, 90/10): δ2.32(3H,s); 2.82(2H,t); 3.58(2H,t); 3.65(2H,t); 3.70(2H,s); 6.65(1H,d);7.0-7.3(4H,m);7.68(1H,t); 8.18(1H,d); 8.42(1H,d); 8.47(1H,d); 8.l-8.35( 3H,m). E. Monitor N-(3-dihydroxyboron-5-nitrophenamido)ethyl-4-(N'-aminovinyl-N"-[2-(dihydroxyboronyl) with fluorescence )Benzyl]methylamino)naphthylene-1,8-dicarboximide interaction with glucose was tested in MeOH/phosphate buffered saline (PBS, 10 mg molecular weight, pH=7.4 ) Perform the experiment. N-(3-Hydroxyboro-5-nitrophenamido)ethyl-4-(N,-aminovinyl-) in MeOH/PBS (50/50% by volume) The concentration of N,,-[2-(dihydroxyboronyl)benzyl]methylamino)naphthalene-1,8-dicarboximide is 15 grams of molecular weight. The glucose concentration is changed from 0 milligrams to 50 milligrams. The molecular weight and the concentration of L-sodium lactate were changed from 0 milligram molecular weight to 7 milligram molecular weight. Experiment with Shimadzu RF-5301PC fluorometer: set the excitation wavelength at 430 nanometers and monitor the emission in the range of 480-650 nanometers, 3/1.5 nanometer pore width, high sensitivity PMT.

The results are shown in Figures 12 and 13, which show that the marker fluorescence of this example is affected by the presence of glucose, but not by the presence of lactate.

Example 11

6-(Cyclohexane carboxyamido)hexylamine marker monomer

<chemistry general="n"><img file="TW200401034A_D0052.tif" /></chemistry>

<chemistry general="n"><img file="TW200401034A_D0053.tif" /></chemistry>

A-9-[N-[3-(Methacrylamido)propylamino]methyl]-10-N-[(6-aminohexylamino)methyl]anthracene 9,10-bis(chloro Methyl)anthracene (0.150 grams, 0.545 millimoles) was added to 200 ml of CHCl₃3-aminopropyl methacrylamide (0.775 grams, 5.45 millimoles, 10.0 equivalents) and tert-butyl N-(6-aminohexyl) aminocarboxylate (1.18 grams, 5.45 millimoles, l0. 0 equivalent) and some BHT crystal solution. The reaction mixture was then stirred at room temperature in the dark for 4 days. At this time CHCl₃Evaporate and dissolve the residue in 100 ml ether. Take the organic layer to 8x125ml saturated aqueous NaHCO₃And 5x200ml phosphate buffer (0.4g molecular weight, pH7.0) extraction. Add Na to the pH of the combined phosphate buffer wash₂CO₃(Saturated aqueous solution) Adjust to pH 11, then add 5x300ml CH₂Cl₂extraction. The combined organic layer was concentrated, and the residue was dissolved in 5 ml in CH₂Cl₂In 20% TFA solution. The mixture was stirred at room temperature for 2 hours. At this time, the reaction mixture was mixed with 4xl0 ml of saturated aqueous NaHCO₃extraction. Add Na to the pH of the combined water layer₂CO₃(Saturated aqueous solution) Adjust to pH 11, then add 4x75ml CH₂Cl₂extraction. The combined organic extracts were subjected to anhydrous Na₂SO₄Drying, filtering and concentration in vacuo yielded 0.068 g (27%) of product. TLC: a) Mork Silicone 60 plate, before deprotection, Rf0.16 is 70/30 CH₂Cl₂/CH₃OH, observed by UV (254/366); final product, Rf0.27 with 85/14.5/0.5 CH₂Cl₂/CH₃OH/iPrNH₂, Observed by UV (254/366). HPLC: HP 1100 HPLC chromatography, WaterS 8x100 mm NovaPak HR C18 column, 0.100 ml injection, 0.75 ml/min, 0.400 ml injection circle, detection at 360 nm, A = water (0.1% HFBA) And B = MeCN (0.1% HFBA), gradient: 10% B over 2 minutes, 10-80% B dense 18 minutes, 80-100% B over 2 minutes, 100% B over 2 minutes; stay time 15.5 minutes .

<chemistry general="n"><img file="TW200401034A_D0054.tif" /></chemistry>

B. 9-[N-[3-(methacrylamido)propylamino]methyl]-l0-[N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene A solution of cyclohexanecarboxylic acid N-hydroxysuccinimidyl ester (0.845 g, 3.76 millimoles, 1.03 equivalents) was added dropwise over 1 hour at room temperature in 20 ml of CH₂Cl₂9-[N-[3-(methacrylamido)propylamino]methyl]-10-N-[(6-aminohexylamino)methyl]anthracene (1.68 grams, 3.63 millimoles) in ) And a little BHT crystal solution. The reaction was then stirred at room temperature in the dark for 16 hours. At this point the reaction mixture was concentrated in vacuo and the residue was dissolved in 105 ml ether/CH₂Cl₂(90/l5) solution. The organic layer was extracted with 4×225 ml of phosphate buffer (0.4 g molecular weight, pH 7.0). Add Na to the pH of the combined phosphate buffer wash₂CO₃(Saturated aqueous solution) Adjust to pH 11, then add 6×500ml CH₂Cl₂extraction. The combined organic layer was subjected to anhydrous Na₂SO₄Drying, filtering and concentration in vacuo yielded 1.2 grams (60%) of product. TLC: Mork Silicone 60 flat plate, Rf 0.30 to 85/14.5/0.5 CH₂Cl₂/CH₃OH/iPrNH₂, Observed by UV (254/366). HPLC: HP l100 HPLC chromatography, WaterS 8×100 mm NovaPak HR Cl8 column, 0.100 ml injection, 0.75 ml/min, 0.400 ml injection circle, detection at 360 nm, A = water (0.l% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 10o% B in 2 minutes; stay time 17.4 minutes.

<chemistry general="n"><img file="TW200401034A_D0055.tif" /></chemistry>

C.9-[N-(2-Dihydroxyboronyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-Dihydroxyboronyl) Benzyl)-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene will be in 30ml CHCl₃9-[N-[3-(methacrylamido)propylamino]methyl]-10-[N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene (1.0 g, 1.8 millimoles), DIEA (1.81 grams, 2.44 ml, 14.0 millimoles, 7.8 equivalents), 2-bromomethylphenyl borate pinacol ester (2.14 grams, 7.20 millimoles, 4.0 equivalents) ) And a little BHT crystal solution was stirred at room temperature in the dark for 60 hours. At this time the reaction mixture was concentrated, and the residue (9-[N-[2-(4,4,5,5-tetramethyl-1,3,2-dioxoboron<img file="TW200401034A_D0056.tif" />)Benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5-tetramethyl-1,3, Boron 2-dioxide<img file="TW200401034A_D0057.tif" />) Benzyl]-N-[6-(cyclohexanecarboxamido)hexylamino]methyl]anthracene) was suspended in 150 ml of ether. The organic layer was washed with 4×50 ml of phosphate buffer (0.4 g molecular weight, pH 7.0). The organic layer was concentrated and the residue was dissolved in 200 ml of ether in 0.1 equivalent of aqueous HCl. The water layer was cleaned with 3×50 ml of ether: ethyl acetate (1:1), and the pH was adjusted to add Na₂CO₃(Saturated aqueous solution) Adjust to pH 11, then add 3x150ml CH₂Cl₂extraction. The combined organic layer was subjected to anhydrous Na₂SO₄Drying, filtering, and concentration in vacuo yielded a red oily compound. The residue was dissolved in ether and concentrated in vacuo to yield 1.17 g (85%) of product as a yellow solid. TLC: Mork Silicone 60 flat plate, Rf0.59 to 80/20 CH₂C1₂/CH₃OH, observed by UV (254/366). HPLC: HP 1l00 HPLC chromatography, Waters 8x100 mm Novapak HR C18 column, 0.100 ml injection, 0.75 ml/min, 0.400 ml injection circle, detection at 360 nm, A = water (0.1% HFBA) and B =MeCN (0.1% HFBA), gradient: 10% B over 2 minutes, 10-80% B over 18 minutes, 80-100% B over 2 minutes, 100% B over 2 minutes; residence time 19.6 minutes.^lH NMR (9:1 of d₆-Acetone/D₂O): δ0.90(m,2H),l.03(m,2H),1.18-l.30(m,6H), 1.35-1.48(4H), 1.62(m,1H,O=C -CH(CH₂)CH₂),1.66-1.75(m,7H),1.77(m,2H,N-CH₂-CH₂-CH₂-N), 2.52(m,2H,N-CH₂-CH₂-), 2.63(m,2H,N-CH₂-CH₂-), 2.98(m,4H,-CH2-NH-C=O), 3.98(S,4H,benzene-CH₂-N), 4.57 (s, 2H, anthracene-CH2-N), 4.59 (S, 2H, anthracene-CH₂-N), 5.20 (t, 1H, J = 1.5HZ, C = CH2), 5.46 (S, 1H, C = CH2), 7.4-7.5 (m, 8H, Ar-H), 7.52 (m, 2H ,Ar-H), 7.95 (m, 2H, Ar-H), 8.23 (m, 4H, Ar-H) D. With 9-[N-(2-dihydroxyborylbenzyl)-N- [3-(Methacrylamido)propylamino]methyl]-10-[N-(2-dihydroxyboronylbenzyl)-N-[6-(cycloethyl-carboxamido)hexylamine N,N-dimethylacrylamide hydrogel prepared from methyl]methyl]anthracene N,N-methacrylamide (40% by weight) in phosphate buffer (pH=7.4, 200 mg molecular weight) ) And a solution of N,N'-methylenebisacrylamide (0.8% by weight). The 9-[N-(2-dihydroxyboronyl benzyl)-N-[3-(methacrylamido)propylamino]methyl]-l0-[N-(2-dihydroxyboronyl benzyl) Yl)-N-[6-(Cyclohexanecarboxamido)hexylamino]methyl]anthracene (18 mg, 2.15x10^-5Mol) and 60 mg of fructose combined with 2 ml of MeOH. The solution was sonicated until all the fructose was dissolved, and then evaporated to produce a solid. Add 1 ml of phosphate buffer solution including monomer to the solid. After 10 minutes of sonication, the solution was filtered through 0.2 micron PTFE membrane filter paper. Combine aqueous ammonium persulfate (20 microliters, 5 wt%) with the formulation. Place the resulting solution in a glove box flushed with nitrogen. The N,N,N',N'-tetramethylethylenediamine aqueous solution (40 microliters, 5 wt%) was added to the monomer formulation to accelerate the polymerization. The resulting formulation was poured into a model constructed with glass microscope slides and 100-micron stainless steel spacers. After maintaining for 8 hours under nitrogen, the model was put into phosphate-buffered saline (pH=7.4), the microscope slide was separated, and the hydrogel was taken out. The hydrogel was washed with phosphate-buffered saline (PBS) with 100 ml of 1 mg molecular weight sodium lauryl sulfate and 1 mg molecular weight tetrasodium EDTA for 3 days. The solution was changed every day, followed by EtOH/PBS (with 20/80 by volume, 3x100ml) and finally washed with PBS (pH=7.4, 3x100ml). The obtained hydrogel film was stored in PBS (pH=7.4) containing 0.02% by weight of sodium azide and 1 mg of molecular weight tetrasodium EDTA. E. The fluorescence adjustment effect of glucose was used to determine the fluorescence adjustment effect of glucose and lactate on the 6-(cyclohexanecarboxamido) hydroxylamine label/DMA hydrogel membrane prepared in this example. Figure 14 shows that in the presence of 0 to 20 mg molecular weight α-D-glucose; 0 to 10 mg molecular weight L-sodium lactate and 0-20 mg molecular weight α-D-glucose in the presence of 4 mg molecular weight sodium lactate in PBS (pH 7 .4, including 0.02% NaN₃And 1 mg molecular weight EDTA) relative fluorescence emission (I@430 nm) of the hydrogel film. Load the hydrogel membrane (100 μm thickness, 8 mm diameter disc) at 45. The corner of the PMMA grooved tube. All measurements were performed in a Shimadzu RF-5301 Fluorometer at 37°C, excitation at 370 nm (pore = 3 nm) and emission at 430 nm (pore = 3 nm) at low PMT sensitivity ). Check the concentration of glucose and L-sodium lactate using a YSI model 2300STAT plus a glucose analyzer. The error bar is the standard deviation of the 3 replicates for each data point. Fluorescence is affected by the presence of glucose, but not by the presence of lactate. Moreover, the presence of lactate (4 mg molecular weight) has no significant effect on the 0-20 mg molecular weight glucose calibration curve.

Example 122-(Hydroxyethyl) amine standard meter monomer

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Chemical name: 9-[N-(2-Dihydroxyboronyl)-N[3-(methacrylamido)propylamino]methyl]-l0-[N-(2-Dihydroxyboronyl) Benzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene (unblocked) Chemical formula: C₄₀H₄₅B₂N₃O₇MW: 701.4 Physical appearance: dark yellow powder Solubility: PBS/methanol, methanol, ethanol, chloroform, dichloromethane blocked label: 9-[N-[2-(4,4,5,5-tetramethyl- 1,3,2-Dioxyboron<img file="TW200401034A_D0059.tif" />)Benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5-methyl-l,3,2 -Boron Dioxide<img file="TW200401034A_D0060.tif" />)Benzyl]-N-[2-(carboxyethyl)amino]methyl]anthracene I. Synthesis

<chemistry general="n"><img file="TW200401034A_D0061.tif" /></chemistry>

A.9-[N-[3-(Methacrylamido)propylamino]methyl]-10-[N-[2-(tert-butoxycarbonyl)ethylamino]methyl]anthracene 9 ,l0-bis(chloromethyl)anthracene (5.00 g, 18.2 millimoles) was added to 3-aminopropylmethacrylamide (12.9 grams, 90.7 millimoles, 4.99 equivalents) in 700 ml of CHC13, β -Tert-butyl alanine (13.2 grams, 90.9 millimoles, 5.00 equivalents) and a solution of some BHT crystals. The reaction mixture was then stirred at 30°C in the dark for 88 hours. At this time CHCl₃Evaporate and dissolve the residue in 500 ml ether. The solution was stirred for 1 hour, at which time salts precipitated from the solution. The ether solution was filtered and then 10x350ml saturated aqueous NaHCO₃extraction. The ether layer was further extracted with 6×350 ml of phosphate buffer (0.2 g molecular weight, pH 6.5). Add Na to the pH of the combined phosphate buffer wash₂CO₃(Saturated aqueous solution) Adjust to pHll-12, then add 6×500ml CH₂Cl₂extraction. The combined organic layer was passed through Na₂SO₄Drying, filtering, and concentration in vacuo yielded crude oily product. The crude product was separated by silica gel chromatography (50 gm steamed silica gel, 0-5% MeOH/CH₂Cl₂Step gradient) purification, yielding 2.04 g (23%) of viscous yellow solid TLC: Mok Silica 60 plate, Rf0.29 at 90/l0 CH₂C1₂/CH₃OH, observed with UV (254/366) and nindantrione stain. HPLC conditions: HP 1100 HPLC color channel spectrum, WaterS 5×100 mm NovaPak HR C18 column, 0.100 ml injection, 0.75 ml/min, 1.500 ml injection circle, detection at 280 nm, A = water (0.1% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; stay time 17. 0 minutes.

<chemistry general="n"><img file="TW200401034A_D0062.tif" /></chemistry>

B.9-[N-[2-(4,4,5,5-tetramethyl-1,3,2-dioxoboron<img file="TW200401034A_D0063.tif" />)Benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5-tetramethyl-1,3, Boron 2-dioxide<img file="TW200401034A_D0064.tif" />) Benzyl]-N-[2-(tert-butoxycarbonyl)ethylamino]methyl]anthracene will be 9-[N-[3-(methacrylamido)propylamino in 50 ml CHCl3 ]Methyl]-10-[N-[2-(tert-butoxycarbonyl)ethylamino]methyl]anthracene (1.5g, 3.1mmol), DIEA (3.16g, 4.26ml, 24.4ml A solution of mol, 7-9 equivalents), 2-bromomethylphenylboronic acid pinacol ester (3.64 grams, 12.2 millimoles, 3.9 equivalents) and a little BHT crystals were stirred at room temperature in the dark for 16 hours. At this time the reaction mixture was concentrated, and the residue was suspended in 200 ml of ether. The ether layer was extracted with 3×125 ml phosphate buffer (0.2 g molecular weight, pH 7.0), and subjected to anhydrous Na₂SO₄Dry, filter, and concentrate in vacuo to give crude product. The residue was wet triturated with hexane, yielding 2.14 g (76%) of a white solid. HPLC: HP 1100 HPLC chromatography, WaterS 5×100 mm NovaPak HR C18 column, 0.200 ml injection, 0.75 ml/min, 1.500 ml injection circle, detection at 280 nm, A=water (0.1% HFBA) And B=MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; residence time 19.2 minutes.

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C.9-[N-(2-Dihydroxyboronyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-Dihydroxyboronyl) Benzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene, add 5 ml of 20% TFA/CH₂Cl₂The 9-[N-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaboroxin) benzyl]-N-[3-(methacrylamido )Propylamino]methyl]-10-[N-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)benzyl]-N-[2-( A solution of tert-butoxycarbonyl)ethylamino]methyl]anthracene (0.294 g, 0.319 mmol) was stirred at room temperature in the dark for 22 hours. At this time, the reaction mixture was concentrated and the residue was wet-grinded with ether. The residue was dissolved in 5 ml of acetone/water (90:10) and stirred for 2 hours. At this time, the reaction mixture was concentrated, and the residue was mixed with water and PBS (pH 7.4, including 0.02% NaN₃And 1 mg molecular weight EDTA) wet milling to obtain a recovery of 0.062 g (28%) of light yellow solid HPLC: HP 1100 HPLC chromatography, Waters 5x100 mm NoVaPak HR C18 column, 0.100 ml injection, 0.75 ml/min, 1.500 ml Injection circle, detected at 280 nm, A = water (0.1% HFBA) and B = MeCN (0.1% HFBA), gradient: 10% B for 2 minutes, 10-80% B for 18 minutes, 80-100% B is 2 minutes, 100% B is 2 minutes; stay time is 17.4 minutes. FABMS: glycerin base; calculated C₄₆H₅₃B₂N₃O₇(Diglycerin adduct) [M]^＋813; experimental value [M+2]^＋815D. With 9-[N-(2-dihydroxyboronyl benzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-dihydroxyboron Benzyl)-N-[2-(carboxyethyl)amino]methyl]anthracene N,N-dimethylacrylamide hydrogel prepared in phosphate buffer (pH=7.4, 200 mg molecular weight ) In the solution of N,N-methacrylamide (40% by weight) and N,N,-methacrylamide (0.8% by weight). 9-[N-(2-Dihydroxyborobenzyl)-N-[3-(methacrylamido)propylamino]methyl]-10-[N-(2-dihydroxyborobenzyl) Group)-N-[2-(carboxyethyl)amino]methyl]anthracene (14 mg, 2.0×10^-5Mol) and 60 The milligrams of fructose was combined with 2 milliliters of MeOH. The solution is sonicated until all the fructose is dissolved and evaporated, producing a solid. Add 1 ml of phosphate buffer solution including monomer to the solid. After 10 minutes of sonication, the solution was filtered through 0.2 micron PTFE filter paper. Combine aqueous ammonium persulfate (20 microliters, 5 wt%) with the formulation. Place the resulting solution in a glove box flushed with nitrogen. The N,N,N',N'-tetramethylethylenediamine aqueous solution (40 microliters, 5 wt%) was added to the monomer formulation to accelerate the polymerization. The obtained formulation was poured into a model constructed with a microscope lens and a 100-micron stainless steel spacer. After maintaining for 8 hours under nitrogen, the model was placed in phosphate-buffered saline (10 mg molecular weight, pH=7.4), the microscope slide was separated, and the hydrogel was taken out. The hydrogel was washed with phosphate buffered saline (PBS) with 100 ml of 1 mg molecular weight sodium lauryl sulfate and 1 mg molecular weight tetrasodium EDTA for 3 days. The solution was changed every day, followed by EtOH/PBS (with 20/80 by volume, 3x100ml) and finally washed with PBS (pH=7.4, 3x100ml). The obtained hydrogel film was stored in PBS (10 mg molecular weight, pH=7.4) containing 0.02 wt% sodium azide and 1 mg molecular weight tetrasodium EDTA. II. Determination of the fluorescence adjustment effect of glucose and lactate in the 2-(carboxyethyl)amine label/DMA hydrogel film prepared in this example by the fluorescence adjustment effect of glucose. Figure 15 shows in PBS containing 0 to 20 mg molecular weight α-D-glucose; 0 to 10 mg molecular weight L-sodium lactate and 0-20 mg molecular weight glucose in the presence of 3 mg molecular weight L-sodium lactate (pH 7.4, including 0.02%NaN₃And 1 mg molecular weight EDTA) relative fluorescence emission (I@430 nm) of the hydrogel film. Load the hydrogel membrane (100 microns thickness, 8 mm diameter disc) in a PMMA trough tube with an angle of 45°. All measurements were performed in a Shimadzu RF-5301 Fluorometer at 37°C, with 370 nm excitation (pore = 3 nm) and 430 nm emission (pore = 3 nm) at low PMT sensitivity . Use YSI type 2300 STAT plus glucose analyzer to check the concentration of glucose and L-sodium lactate. Plot the data as the average of 3 replicates for each data point. Fluorescence is affected by the presence of glucose, but not by the presence of lactate. Moreover, the presence of lactate (4 mg molecular weight) has no significant effect on the 0-20 mg molecular weight glucose calibration curve

Example 13 includes two detectable portions of fluorescent glucose markers:

<chemistry general="n"><img file="TW200401034A_D0066.tif" /></chemistry>

Chemical name: 9-[N-(2-Dihydroxyboronbenzyl)-N-[3-(Methacrylamido)propylamino]methyl]-10-[N-(2-Dihydroxyboron Benzyl)-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonyl)ethylamino]methyl]anthracene (unblocked) Chemical formula: C₇₃H₈₇B₂N₅O₇MW: 1168 Physical appearance: dark yellow powder Solubility: PBS/methanol, methanol, ethanol, chloroform, dichloromethane. Compounds blocked with pinacol: 9-[N-[2-(4,4,5,5-tetra Methyl-1,3,2-dioxoboron<img file="TW200401034A_D0067.tif" />)Benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5-tetramethyl-1,3, Boron 2-dioxide<img file="TW200401034A_D0068.tif" />)Benzyl]-N-[3-(N-6-(9-anthracenecarboxamido)hexylaminocarbonylethylaminomethyl)anthracene I. Synthesis

<chemistry general="n"><img file="TW200401034A_D0069.tif" /></chemistry>

A. 9-Anthracene Chlorine Anthracene-9-carboxylic acid (1.2 g, 5.4×10-³Mol) and 15 ml of sulfite chloride. The solution was refluxed for 2 hours, and then the volatile components were evaporated. The obtained solid was dried under high vacuum for 24 hours, yielding 1.3 g of substance (quantitative yield). Use this substance as it is for the following steps.

<chemistry general="n"><img file="TW200401034A_D0070.tif" /></chemistry>

B. N-(6-aminohexyl) anthracene-9-carboxamide hydrochloride will be in 50 ml of anhydrous CH₂Cl₂The 9-anthracene chloride (1.3 g, 5.4 mmol) in 9-anthracene chloride (1.3 g, 5.4 mmol) was added dropwise at 0°C in 100 ml CH₂Cl₂In 11.6 grams (100 millimoles) of hexamethylene diamine. The solution was stirred at 0°C for 1 hour, then allowed to warm to room temperature and stirred overnight. The solvent was evaporated, and 200 ml of water was added to the residue. The mixture was sonicated and stirred for 1 hour and then filtered. The filtered solid was dried under vacuum for 24 hours. MeOH (50 mL) and 2 mL concentrated HCl were added to the solid, then the MeOH was evaporated. The resulting solid was washed with hot CH2Cl2/MeOH (90/10 vol%) and recrystallized from MeOH, yielding 0.51 g (26%) of product. The purity of the product was checked by HPLC. HPLC: HP 1100 HPLC chromatography, WaterS 5X100 mm NOVaPak HR C18 column, 0.1 ml injection, 0.75 ml/min, 2 ml injection circle, detection at 280 nm, A = water (0.1% HFBA) and B =MeCN (0.1%HFBA), gradient: 10%B in 2 minutes, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B in 2 minutes; residence time 16.5 minutes .

<chemistry general="n"><img file="TW200401034A_D0071.tif" /></chemistry>

C.9-[N-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)benzyl]-N-[3-(methacrylamido )Propylamino]methyl]-10-[N-[2-(4,4,5,5-tetramethyll,3,2-dioxaboron flavor)benzyl]-N-[3-(N -6-(9-anthracene carboxyamido)hexylaminocarbonylethylaminomethyl)anthracene: 9-[N-[2-(4,4,5,5-tetramethyl-1,3, 2-Dioxyboron)benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5-tetramethyl 1,3,2-Dioxyboron)benzyl]-N-[2-carboxyethylamino]methyl]anthracene (40 mg, 4.5×10^-5Mol) and N-(6-aminohexyl)anthracene-9-carboxamide hydrochloride (20 mg, 5.6×10^-5Mol), diphenyl azide phosphate (15.4 mg, 5.6×10^-5Mol) and 2 ml DMF. Diisopropylethylamine (20 μl, 2.44×10^-4Mol) was added to the mixture, and the solution was stirred at room temperature for 24 hours. The DMF was evaporated under high vacuum, the residue was dissolved in 50 mL EtOAc and washed with water (3×10 mL). The EtOAc solution was separated and dried (Na₂SO₄) And evaporation, yielding 46 mg (87% yield) of solids. The purity of the material was checked by HPLC. HPLC: HP l100 HPLC chromatography, Waters 5×100 mm NovaPak HR Cl8 column, 0.1 ml injection, 0.75 ml/min, 2 ml injection circle, detection at 280 nm, A = water (0.1% HFBA ) And B = MeCN (0.1% HFBA), gradient: 10% B in 2 minutes, 10-80% B in 18 minutes, 80-100% B in 2 minutes, 100% B in 2 minutes; sojourn time 20.42 minute. FAB mass spectrum: glycerol matrix; calculated C6₇H₇₅B₂N₅O₉(Diglycerin adduct) [M]^＋=1l16; experimental value [M+1]^＋=l117D. The fluorescent effect of glucose on the markers immobilized in the hydrogel membrane. Preparation of HEMA/methacrylic acid hydrogels with glucose markers: prepared in phosphate buffer (pH = 7.4, 200 mg molecular weight ) Is a 50% by weight solution of 2-hydroxyethyl methacrylate (4.75 g) and methacrylic acid (0.25 g). The glucose marker (11 mg, 1.0x10^-5Mol) and 60 mg of fructose combined with 2 ml of MeOH. The solution is sonicated until all the fructose is dissolved and evaporated, producing a solid. Add 1 ml of phosphate buffer solution including monomer to the solid. After 10 minutes of sonication, the solution was filtered through 0.2 micron PTFE filter paper. Combine aqueous ammonium persulfate (20 microliters, 5 wt%) with the formulation. Place the resulting solution in a glove box flushed with nitrogen. The N,N,N',N'-tetramethylethylenediamine aqueous solution (40 microliters, 5 wt%) was added to the monomer formulation to accelerate the polymerization. The resulting formulation was poured into a model constructed with a microscope lens and 100-micron stainless steel spacers. After maintaining for 8 hours under nitrogen, the model was placed in phosphate buffered saline (10 mg molecular weight, pH=7.4), the microscope slide was separated, and the hydrogel was taken out. The hydrogel was washed with phosphate buffered saline (PBS) with 100 ml of 1 mg molecular weight sodium lauryl sulfate salt and 1 mg molecular weight tetrasodium EDTA for 3 days. The solution was changed every day, followed by EtOH/PBS (with The volume is 20/80, 3x100ml) and finally washed with PBS (pH=7.4, 3x100ml). The obtained hydrogel film was stored in PBS (10 mg molecular weight, pH=7.4) containing 0.02 wt% sodium azide and 1 mg molecular weight tetrasodium EDTA. The effects of glucose and L-sodium lactate on the hydrogel film containing glucose markers were tested on a Shimadzu RF-5301 PC fluorometer equipped with a variable temperature accessory. Set the excitation wavelength at 370 nm, 3/3 nm aperture, low PMT sensitivity, and scan emission from 400 to 600 nm. Check the concentration of glucose and L-sodium lactate using a YSI model 2300STAT plus a glucose analyzer.

Load the hydrogel membrane (100 microns thickness, 8 mm diameter circle) at 45^。The corner of the PMMA grooved tube. Will include the expected amount of glucose, L-sodium lactate and glucose with L-sodium lactate in phosphate buffered saline (PBS_，pH=7.4) Heat to 37°C in a water bath, and put it into the PMMA tank containing the hydrogel. After each addition, allow the PMMA tank to reach equilibrium at 37°C for 45 minutes. Perform fluorescence intensity measurements for each glucose/lactate concentration on two different samples and use the average value in the calibration curve. Obtain the calibration curve of glucose, L-sodium lactate and glucose in the presence of 3 mg molecular weight L-sodium lactate (fluorescence intensity at 430 nm vs. concentration). The results are shown in Figure 16.

Example 146-(3-Carboxylanylamino)hexylamino label monomer

<chemistry general="n"><img file="TW200401034A_D0072.tif" /></chemistry>

Compounds blocked with pinacol: 9-N-2-(4,4,5,5-tetramethyl-1,3,2-dioxoboron<img file="TW200401034A_D0073.tif" />)Benzyl]-N-[3-(methacrylamido)propylamino]methyl]-10-[N-[2-(4,4,5,5-tetramethyl-1,3, Boron 2-dioxide<img file="TW200401034A_D0074.tif" />) Benzyl]-N-[6-(3-carboxypropylamino)hexylamino]methyl]anthracene unblocked compound: 9-[N-(2-dihydroxyborylbenzyl)-N- [3-(Methacrylamido)propylamino]methyl]-10-[N-(2-dihydroxyboronylbenzyl)-N-[6-(3-carboxypropylamino)hexylamine The synthesis of methyl]methyl]anthracene can be carried out in a similar manner to Example 11, using 9-[N-[3-(methacrylamido)propylamino]methyl]-10-N-[ 6-(hexylamino)methyl]anthracene is used as a raw material. In contrast, the amine raw material was reacted with the N-hydroxysuccinimidyl (NHS) ester of the monomethyl ester of succinic acid (instead of the NHS ester of cyclohexanecarboxylic acid used in Example 11). An additional alkaline hydrolysis step is required to complete the synthesis.

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Example 15 Glucose marker/monomer excited by visible light

<chemistry general="n"><img file="TW200401034A_D0076.tif" /></chemistry>

Chemical name: N-(3-methacrylamidopropyl)-4-[2-N-[[2-(dihydroxyboronyl)benzyl]-[6-(N-[2-(twoBoronyl)benzyl]-6-N-(3-carboxypropylaminoethyl)aminohexyl)]aminoethylamino]naphthylene-1,8-dicarboxyamide Chemical formula: C₄₇H₆₀B₂N₆O_loMW:890 can be synthesized as shown below:

<chemistry general="n"><img file="TW200401034A_D0077.tif" /></chemistry>

Example 16 Visible light excitation of alternative glucose markers/monomers

<chemistry general="n"><img file="TW200401034A_D0078.tif" /></chemistry>

Chemical name: N-butyl-4-[2-N-[;[2-(dihydroxyboronyl)benzyl]-[6-(N-[2-(dihydroxyboronyl)benzyl]-6 -N-(2-Methylacrylamidoethyl)aminohexyl)]aminoethylamino]naphthylene-1,8-dicarboxamide Chemical formula: C₄₄H₅₇B₂N₅O₇With MW:789.5, the compound can be synthesized as shown below:

<chemistry general="n"><img file="TW200401034A_D0079.tif" /></chemistry>

<chemistry general="n"><img file="TW200401034A_D0080.tif" /></chemistry>

Schematic description

Figure 1 illustrates the standardized fluorescence emission (I/Io @ 420 nm) of the marker as described in Example 1.

Figure 2 illustrates the standardized fluorescence emission of the marker as described in Example 2 (I/Io@428nm)

Figure 3 illustrates the standardized fluorescence emission of the marker as described in Example 3 (I/Io@428nm)

Figure 4 illustrates the standardized fluorescence emission of the marker described in Example 4 (I/Io@427nm)

Figure 5 illustrates the standardized fluorescence emission of the marker as described in Example 5 (I/Io@540 nm)

Figure 6 illustrates the absorption spectrum of the label as described in Example 6.

Figures 7-8 illustrate the absorbance ratio (450nm/530nm) of the marker as described in Example 6.

Figure 9 illustrates the standardized fluorescence emission (I/Io at 550 nm) of the marker as described in Example 6.

Figure 10 illustrates the fluorescence spectra of the marker described in Example 6 in the absence of glucose and in the presence of 100 milligrams of molecular weight glucose

Figure 11 illustrates the standardized fluorescence emission (I/Io at 550 nanometers) of the label described in Example 6 in the presence of glucose and lactate.

Figure 12 illustrates the standardized fluorescence emission of a label exposed to glucose as described in Example 10 (I/Io at 525 nm)

Figure 13 illustrates the standardized fluorescence emission (I/Io at 530 nm) of a label exposed to lactate as described in Example 10

Figure 14 shows the relative fluorescence emission (I@430 nm) of the label exposed to glucose and lactate as described in Example 11.

Figure 15 shows the relative fluorescence emission (I@430 nm) of the label exposed to glucose and lactate as described in Example 12.

Figure 16 illustrates the fluorescence of markers exposed to glucose and lactate as described in Example 13.