Fluorescent molecule with quinolinium ion as framework, polypeptide or protein marked by fluorescent molecule and preparation method of fluorescent molecule
1. A fluorescent molecule with a quinolinium ion as a framework is characterized in that the general formula of the fluorescent molecule is shown as formula I-a, formula I-b or formula I-c:
wherein R is1Selected from hydrogen or phenyl; r2Selected from hydrogen, trifluoromethyl or methoxy; x is selected from oxygen or nitrogen hydrogen.
2. The quinolinium ion-based fluorescent molecule of claim 1, wherein said fluorescent molecule is selected from one of the following structural formulas:
3. a method for preparing a quinolinium ion-based fluorescent molecule according to any one of claims 1 to 2, comprising the steps of:
dissolving a compound III in an organic solvent, adding a compound II-a and a monovalent gold catalyst, and reacting under the conditions of illumination and room temperature to generate a target compound I-a, namely the fluorescent molecule;
or dissolving the compound III in an organic solvent, adding the compound II-b or the compound II-c and a monovalent gold catalyst, and reacting under the conditions of illumination and room temperature to generate a target compound I-b, namely the fluorescent molecule;
or dissolving the compound III-c in an organic solvent, adding the compound II-b and a monovalent gold catalyst, and reacting under the conditions of illumination and room temperature to generate a target compound I-c, namely the fluorescent molecule;
the above reaction schemes are respectively as follows:
4. the method according to claim 3, wherein the molar ratio of the compound II-a to the compound III to the monovalent gold catalyst is 1.0: 1.5-2: 0.05-0.1;
alternatively, the molar ratio of the compound II-b or compound II-c, compound III and monovalent gold catalyst is 1.0: 1.5-2: 0.05-0.1;
or the mole ratio of the compound II-b, the compound III-c and the monovalent gold catalyst is 1.0: 1.5-2: 0.05-0.1.
5. The method according to claim 3, wherein the target compound I-a, the target compound I-b or the target compound I-c are reacted for 10 to 24 hours.
6. The method of claim 3, wherein the monovalent gold catalyst is tris (4-trifluoromethylphenyl) gold chloride or triphenylphosphine gold chloride.
7. A polypeptide or protein labeled with a fluorescent molecule, each of which contains cysteine, wherein the fluorescent molecule is the quinolinium ion-based fluorescent molecule according to any one of claims 1 to 2.
8. The fluorescent molecule-tagged polypeptide or protein of claim 7, wherein said polypeptide is selected from the group consisting of CSKFR, KSTFC, ASCGTN, STSSCNLSK, AYEWMWCFHQK, and AYEWMWCFHQR;
alternatively, the protein is selected from one of bovine serum albumin and human serum albumin.
9. The fluorescent molecule-labeled polypeptide or protein of claim 7, wherein the molar ratio of the fluorescent molecule to the polypeptide is 1: 1 to 100: 1;
or, the molar ratio of the fluorescent molecule to the protein is 1: 1 to 100: 1.
10. a method for preparing a polypeptide or protein labeled with a fluorescent molecule according to any one of claims 7 to 9, comprising the steps of: and mixing the polypeptide solution or the protein solution, the fluorescent molecule solution, the buffer solution and the solvent, and reacting to obtain the polypeptide or the protein marked by the fluorescent molecule.
Background
The protein is an organic macromolecule formed by combining more than 20 amino acids according to different proportions, and is a material basis for forming all lives. Coupling fluorescent molecules to proteins for tracking the mechanism of action of protein molecules or identifying specific protein molecules has become an important research means in the professions of biochemistry, medicinal chemistry, etc. For the present time, the techniques for modifying proteins that have been commercialized mainly use N-hydroxysuccinimide groups to modify the nitrogen terminus on the lysine branch and maleimide groups to modify the thiol terminus on the cysteine branch. However, because the surface of the protein is often widely distributed with lysine with hydrophilic groups, the coupling sites and the number of the lysine are difficult to control, the fluorescence change cannot be quantitatively monitored, and the lysine can not be used for researching the space structure change of the protein. The maleimide-cysteine conjugate product has been reported to undergo hydrolytic cleavage in a buffer solution. Meanwhile, fluorescent molecules which can be used for labeling have great limitation, and the fluorescent groups which are connected with the protein at present are still limited to structures such as fluorescein, rhodamine, coumarin, fluoroboric fluorescent and anthocyanin due to the limitation of synthesis technology. Except fluorescein and rhodamine which take oxygen anthracene as a structural basis, the modified structures have weak water solubility and poor quantum yield in aqueous solution, and protein denaturation is easily caused after modification, so that the application range is narrow. The luminescent properties of fluorescein and rhodamine are affected by pH, so the hydrophilicity and hydrophobicity of molecules can affect the structure, function and photophysical properties of the modified protein. Therefore, the development of new fluorescent molecules and the application of the fluorescent molecules to selective markers in protein or polypeptide molecules have important value.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a fluorescent molecule with a quinolinium ion as a skeleton, a polypeptide or protein labeled with the same, and a preparation method thereof.
The technical scheme of the invention is as follows:
in a first aspect of the present invention, a fluorescent molecule with a quinolinium ion as a skeleton is provided, wherein the general formula of the fluorescent molecule is represented by formula I-a, formula I-b, or formula I-c:
wherein R is1Selected from hydrogen or phenyl; r2Selected from hydrogen, trifluoromethyl or methoxy; x is selected from oxygen or nitrogen hydrogen.
The fluorescent molecule comprises a quinolinium ion skeleton, trimethylsilyl which is bonded with the quinolinium ion skeleton through a carbon-silicon single bond, and p-propinylphenyl which is connected with the quinolinium ion skeleton through a carbon-carbon single bond. Compared with the commonly used fluorescent molecules such as fluorescein, rhodamine, coumarin, fluoroboro fluorescence, anthocyanin and the like, the fluorescent molecule disclosed by the invention is simple to synthesize, and the substituent can be simply and conveniently changed to obtain the fluorescent molecules with different luminescent properties. In addition, the fluorescent molecule with the quinolinium ion as the framework can be specifically and covalently combined with thiol on cysteine, so that the fluorescent molecule can be used for high-selectivity biomarkers for active sulfur-based proteins or polypeptides.
Optionally, the fluorescent molecule is selected from one of the following structural formulas:
in a second aspect of the present invention, there is provided a method for preparing a fluorescent molecule having a quinolinium ion as a skeleton, comprising the steps of:
dissolving a compound III (quinoline diazonium salt) in an organic solvent, adding a compound II-a (trimethylsilyl alkyne derivative) and a monovalent gold catalyst (monovalent gold complex), and reacting under the conditions of illumination and room temperature (20-30 ℃) to generate a target compound I-a, namely the fluorescent molecule;
or dissolving a compound III (quinoline diazonium salt) in an organic solvent, adding a compound II-b or a compound II-c (trimethylsilyl alkyne derivative) and a monovalent gold catalyst, and reacting under the conditions of illumination and room temperature (20-30 ℃) to generate a target compound I-b, namely the fluorescent molecule;
or dissolving a compound III-c (quinoline diazonium salt) in an organic solvent, adding a compound II-b (trimethylsilyl alkyne derivative) and a monovalent gold catalyst, and reacting under the conditions of illumination and room temperature (20-30 ℃) to generate a target compound I-c, namely the fluorescent molecule;
the above reaction schemes are respectively as follows:
alternatively, the mole ratio of compound II-a, compound III, and monovalent gold catalyst is 1.0: 1.5-2: 0.05-0.1;
alternatively, the molar ratio of the compound II-b or compound II-c, compound III and monovalent gold catalyst is 1.0: 1.5-2: 0.05-0.1;
or the mole ratio of the compound II-b, the compound III-c and the monovalent gold catalyst is 1.0: 1.5-2: 0.05-0.1.
Alternatively, the target compound I-a, the target compound I-b or the target compound I-c may be reacted for 10 to 24 hours, which may be determined according to TLC plate analysis whether the reaction of the starting materials is completed.
Optionally, the monovalent gold catalyst is tris (4-trifluoromethylphenyl) phosphine gold chloride, of the formula (4-CF)3C6H4)3PAuCl. The gold monovalent catalyst may also be triphenylphosphine gold chloride, of the formula Ph3PAuCl。
In a third aspect of the present invention, there is provided a polypeptide or protein labeled with a fluorescent molecule, each of the polypeptide or protein containing cysteine, wherein the fluorescent molecule is the fluorescent molecule having a quinolinium ion as a skeleton according to the present invention.
The fluorescent molecule comprises a quinolinium ion skeleton and p-propiophenoylphenyl connected with the quinolinium ion skeleton through a carbon-carbon single bond, and the specific structure is shown in the following formula. The terminal alkynyl on the molecule is electron-deficient and can perform nucleophilic substitution reaction with sulfhydryl on polypeptide or protein containing cysteine, so that the fluorescent molecule can be connected to the polypeptide or protein with high selectivity, and the coupling yield is moderate to good. The fluorescent molecules are utilized to successfully realize the fluorescent labeling of various polypeptides and proteins.
Optionally, the polypeptide is selected from one of CSKFR, KSTFC, ASCGTN, stsscnlsk, AYEMWCFHQK, AYEMWCFHQR;
alternatively, the protein is selected from one of Bovine Serum Albumin (BSA), Human Serum Albumin (HSA).
Optionally, the molar ratio of the fluorescent molecule to the polypeptide is 1: 1 to 100: 1;
or, the molar ratio of the fluorescent molecule to the protein is 1: 1 to 100: 1.
in a fourth aspect of the present invention, there is provided a method for preparing the polypeptide or protein labeled with the fluorescent molecule of the present invention, which comprises the steps of: and mixing the polypeptide solution or the protein solution, the fluorescent molecule solution, the buffer solution and the solvent, and reacting to obtain the polypeptide or the protein marked by the fluorescent molecule.
Optionally, the concentration of the polypeptide solution or the protein solution is 0.01-10mmol/L, wherein water or buffer is used as a solvent.
Optionally, the concentration of the fluorescent molecule solution is 2-200mmol/L, wherein acetonitrile is used as a solvent.
Optionally, the buffer is phosphate buffer with pH of 5.8-8.0, and the concentration is 0-500 mmol/L.
In the invention, the obtained polypeptide or protein marked by the fluorescent molecule is analyzed, and the analysis is mainly mass spectrometry. Further, the analysis of the fluorescent molecule-labeled polypeptide includes LC-MS and LC-MS/MS analysis; the analysis of the fluorescent molecule-labeled protein includes LC-MS analysis.
Drawings
FIG. 1a is an LC-MS spectrum of CSKFR-I-a1 in example 10 of the present invention;
FIG. 1b is the LC-MS/MS spectrum of CSKFR-I-a1 in example 10 of the present invention
FIG. 2a is an LC-MS spectrum of CSKFR-I-b1 in example 11 of the present invention;
FIG. 2b is the LC-MS/MS spectrum of CSKFR-I-b1 in example 11 of the present invention
FIG. 3a is the LC-MS spectrum of CSKFR-I-c in example 12 of the present invention;
FIG. 3b is the LC-MS/MS spectrum of CSKFR-I-c in example 12 of the present invention
FIG. 4 is an LC-MS spectrum (m/z 66433 → m/z 66875) of BSA-I-b1 in example 35 of the present invention;
FIG. 5 is an LC-MS/MS spectrum of BSA-I-b1 in example 35 of the present invention.
Detailed Description
The present invention provides a fluorescent molecule using a quinolinium ion as a framework, a polypeptide or protein labeled by the same, and a preparation method thereof, and the present invention is further described in detail below in order to make the objects, technical schemes, and effects of the present invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1: synthesis of fluorescent molecule I-a1
(1) 4- (2-trimethylsilylethynyl) benzaldehyde (2.02g,10mmol) was dissolved in anhydrous tetrahydrofuran THF (10mL) under a nitrogen atmosphere, the reaction mixture was cooled to 0 ℃ and an ethynylmagnesium bromide solution (30mL, 0.5M) was slowly added dropwise thereto, and after completion of the addition, stirring was continued at room temperature overnight. After completion of the reaction, the reaction was quenched with 50mL of saturated aqueous ammonium chloride solution, extracted with dichloromethane, and the organic extracts were combined and washed once with saturated brine, dried over magnesium sulfate, and concentrated. Purifying by column chromatography to obtain white solid IV; (2) adding a mixture of 1: 5 and Dichloromethane (DCM), 5 equivalents of manganese dioxide as the oxidant are added portionwise with stirring, and after the addition is complete stirring is continued at room temperature until the reaction is complete. After the reaction was completed, the reaction solution was diluted with dichloromethane, and excess manganese dioxide was removed by filtration through a celite pad. Washing the filtrate with water and brine in sequence, drying with magnesium sulfate, and spin-drying to obtain a product II-a; (3) under the protection of inert gas nitrogen, compound II-a and compound III-a, and univalent gold complex (4-CF)3C6H4)3PAuCl, with 5mL of acetonitrile (CH) solvent3CN) was dissolved in a 30mL reaction tube and subjected to catalytic cycloaddition reaction under a 16 watt blue LED lamp at room temperature. After 16 hours of reaction, removing the organic solvent by rotary evaporation, passing the obtained residue through a silica gel column, performing gradient elution by using a mixed solution of dichloromethane and methanol, combining the rotary evaporation to remove the solvent, adding dichloromethane, extracting for multiple times by using water, combining the water phases, and finally removing the water by rotary evaporation to obtain the compound I-a 1.
Characterization analytical data for compound I-a 1: orange solid, yield 49%;
1H NMR(400MHz,CDCl3)δ9.13(d,J＝9.0Hz,1H),8.92(d,J＝4.4Hz,2H),8.27(t,J＝9.0Hz,3H),8.14(d,J＝7.9Hz,1H),8.05(d,J＝7.7Hz,1H),7.98(d,J＝7.7Hz,1H),7.67–7.60(m,3H),7.50(d,J＝9.0Hz,1H),7.30(s,1H),3.60(s,1H),0.10(s,9H)；
HRMS(ESI)C29H24NOSi+theoretical calculation 430.1622, found 430.1623.
Example 2: synthesis of fluorescent molecule I-a2
The specific operation is basically the same as that of the embodiment 1, except that: compound III-a is replaced with compound III-b.
Characterization analytical data for compound I-a 2: yellow solid, yield 25%;
1H NMR(400MHz,CDCl3)δ9.35(d,J＝8.4Hz,1H),9.27(s,1H),8.59(d,J＝8.0Hz,1H),8.35(d,J＝8.6Hz,2H),8.34–8.25(m,1H),8.21–8.12(m,2H),8.07–7.99(m,4H),7.90(d,J＝8.9Hz,1H),7.83–7.75(m,4H),7.56–7.47(m,1H),4.61(s,1H),0.17(s,9H)；
HRMS(ESI)C35H28NOSi+theoretical calculation 506.1935, found 506.1951.
Example 3: synthesis of fluorescent molecule I-a3
The specific operation is basically the same as that of the embodiment 1, except that: compound III-a is replaced with compound III-c.
Characterization analytical data for compound I-a 3: brown solid, yield 17%;
1H NMR(400MHz,CDCl3)δ9.00(d,J＝8.3Hz,1H),8.93(d,J＝9.0Hz,1H),8.75(d,J＝7.9Hz,1H),8.27(d,J＝7.2Hz,2H),8.07(d,J＝7.8Hz,1H),7.66–7.54(m,5H),7.44(d,J＝8.6Hz,1H),7.24(t,J＝7.8Hz,1H),4.12(s,3H),3.62(s,1H),0.13(s,9H)；
HRMS(ESI)C30H26NO2Si+theoretical calculation of 460.1727, actual measurementValue 460.1719.
Example 4: synthesis of fluorescent molecule I-b1
(1) Dicyclohexylcarbodiimide (DCC) (2.48g,12mmol) was dissolved in Dichloromethane (DCM) under nitrogen and stirred for 15 minutes at-15 ℃, then propiolic acid (759mg,11mmol) was added dropwise, the clear solution became cloudy, after stirring for 1 hour was continued, a solution of aniline (1.89g,10mmol) in dichloromethane was added dropwise, after which stirring was continued overnight at-15 ℃, and then the next day at room temperature for the whole day. Putting a layer of absorbent cotton into a cloth-type funnel, pressing tightly, putting silica gel, pouring the reaction liquid, remaining the DCC byproduct solid on the silica gel layer, washing three times by using an eluent with PE (polyethylene) EA (2: 1) until the DCC byproduct layer is white, and performing spin-drying on the filtrate to obtain a brown solid II-b by direct column chromatography. (2) The specific operation is basically the same as that in the step (3) in the embodiment 1, except that: the compound II-a is replaced by the compound II-b.
Characterization analytical data for compound I-b 1: yellow solid, yield 41%;
1H NMR(400MHz,DMSO-d6)δ11.16(s,1H),9.31(d,J＝9.0Hz,1H),9.12(d,J＝8.4Hz,1H),9.07(d,J＝8.9Hz,1H),8.40(d,J＝8.1Hz,1H),8.32(dd,J＝8.0,1.2Hz,1H),8.23–8.17(m,1H),8.08(t,J＝7.8Hz,1H),7.75–7.70(m,3H),7.62(d,J＝9.0Hz,1H),7.51(d,J＝8.5Hz,2H),7.47–7.37(m,1H),4.52(s,1H),0.07(s,9H)；
HRMS(ESI)C29H25N2OSi+theoretical calculation 445.1731, found 445.1790.
Example 5: synthesis of fluorescent molecule I-b2
The specific operation is basically the same as that of example 4, except that: compound III-a is replaced by compound III-d.
Characterization analytical data for compound I-b 2: yellow solid, yield 43%;
1H NMR(400MHz,DMSO-d6)δ11.17(s,1H),9.46(d,J＝9.0Hz,1H),9.37(d,J＝8.8Hz,1H),9.24(d,J＝8.9Hz,1H),8.50(s,1H),8.44–8.35(m,2H),7.83–7.71(m,3H),7.68(d,J＝9.0Hz,1H),7.51(d,J＝8.6Hz,2H),4.54(s,1H),0.09(s,9H)；
HRMS(ESI)C30H24F3N2OSiNa+theoretical calculation 536.1508, found 536.1504.
Example 6: synthesis of fluorescent molecule I-b3
The specific operation is basically the same as that of example 4, except that: compound III-a is replaced with compound III-b.
Characterization analytical data for compound I-b 3: orange solid, yield 45%;
1H NMR(400MHz,DMSO-d6)δ11.16(s,1H),9.30(d,J＝8.5Hz,1H),9.21(s,1H),8.40(d,J＝8.3Hz,1H),8.20(t,J＝7.7Hz,1H),8.06(t,J＝7.7Hz,1H),7.98(dd,J＝7.6,1.7Hz,3H),7.76(d,J＝6.7Hz,4H),7.68(t,J＝8.5Hz,3H),7.61(d,J＝8.3Hz,2H),7.46–7.40(m,1H),4.55(s,1H),0.08(s,9H)；
HRMS(ESI)C35H29N2OSi+theoretical calculation 521.2044, found 521.2052.
Example 7: synthesis of fluorescent molecule I-b4
The specific operation is basically the same as that of example 4, except that: compound III-a is replaced with compound III-c.
Characterization analytical data for compound I-b 4: yellow solid, yield 48%;
1H NMR(400MHz,DMSO-d6)δ11.16(s,1H),9.15(d,J＝9.1Hz,1H),9.08(d,J＝9.4Hz,1H),8.91(d,J＝9.0Hz,1H),8.25(d,J＝8.0Hz,1H),7.71–7.74(m,3H),7.66(t,J＝7.5Hz,1H),7.58–7.51(m,2H),7.51(d,J＝8.2Hz,2H),7.37(t,J＝8.2Hz,1H),4.51(s,1H),4.13(s,3H),0.09(s,9H)；
HRMS(ESI)C30H27N2O2Si+theoretical calculation 475.1836, found 475.1847.
Example 8: synthesis of fluorescent molecule I-c
The specific operation is basically the same as that of example 4, except that: compound III-a is replaced with compound III-c.
Characterization of compounds I-c analytical data: yellow solid, yield 10%;
1h NMR (400MHz, acetone-d6)δ10.37(s,1H),9.20(d,J＝8.5Hz,1H),9.14(d,J＝8.5Hz,1H),8.69(d,J＝8.4Hz,1H),8.62(d,J＝7.4Hz,1H),8.47(dd,J＝8.0,1.2Hz,1H),8.36–8.29(m,3H),8.26–8.20(m,1H),8.18–8.13(m,1H),8.11(d,J＝8.2Hz,2H),8.03(s,1H),7.80(d,J＝8.6Hz,2H),3.90(s,1H),0.25(s,9H)；
HRMS(ESI)C29H25N2OSi+Theoretical calculation 445.1731, found 445.1790.
Example 9: optical Properties of fluorescent molecules I-a, I-b and I-c
The fluorescent molecules I-a, I-b and I-c synthesized according to the above experimental steps are excited in the purple visible light band and emit fluorescence from blue light to red light. Specific optical parameters include the wavelength of maximum absorption (λ)Absorption of) Maximum emission wavelength (λ)Launching) Stokes shift and quantum yield, as shown in table 1 below.
TABLE 1
Example 10: selective marker of fluorescent molecule I-a1 of quinolinium framework for polypeptide CSKFR
The method comprises the following specific steps: mu.L of CSKFR solution (water as solvent, concentration of 1mmol/L), 1. mu.L of fluorescent molecule I-a1 solution (acetonitrile as solvent, concentration of 10mmol/L), and 80. mu.L of phosphate buffer pH 7.4 (concentration of 50mmol/L) and 9. mu.L of acetonitrile were mixed in a 1.5mL centrifuge tube and left to react at 25 ℃ for 2 hours at room temperature. The modified polypeptide product CSKFR-I-a1 was characterized and sequenced after 2 hours by LC-MS and LC-MS/MS analysis, and the results are shown in FIGS. 1a and 1 b.
Example 11: selective marker of fluorescent molecule I-b1 of quinolinium framework for polypeptide CSKFR
The specific operation is basically the same as that of example 10, except that: i-a1 was replaced by I-b 1. The modified polypeptide product CSKFR-I-b1 was characterized and sequenced by LC-MS and LC-MS/MS analysis, and the results are shown in FIGS. 2a and 2 b.
Example 12: selective marker of fluorescent molecule I-c of quinolinium framework on polypeptide CSKFR
The specific operation is basically the same as that of example 10, except that: i-a1 is replaced by I-c. The modified polypeptide product CSKFR-I-c was characterized and sequenced by LC-MS and LC-MS/MS analysis, and the results are shown in FIG. 3a and FIG. 3 b.
Example 13: selectable marker of fluorescent molecule I-a1 of quinolinium framework for polypeptide KSTFC
The specific operation is basically the same as that of example 10, except that: the CSKFR is replaced with a KSTFC.
Example 14: selectable marker of fluorescent molecule I-b1 of quinolinium framework for polypeptide KSTFC
The specific operation is basically the same as that of example 11, except that: the CSKFR is replaced with a KSTFC.
Example 15: selectable marker of fluorescent molecule I-c of quinolinium framework for polypeptide KSTFC
The specific operation is basically the same as that of example 12, except that: the CSKFR is replaced with a KSTFC.
Example 16: selective marker of fluorescent molecule I-a1 with quinolinium skeleton for polypeptide ASCGTN
The specific operation is basically the same as that of example 10, except that: the CSKFR was replaced with ASCGTN.
Example 17: selective marker of fluorescent molecule I-b1 with quinolinium skeleton for polypeptide ASCGTN
The specific operation is basically the same as that of example 11, except that: the CSKFR was replaced with ASCGTN.
Example 18: selective marker of fluorescent molecule I-c of quinolinium framework on polypeptide ASCGTN
The specific operation is basically the same as that of example 12, except that: the CSKFR was replaced with ASCGTN.
Example 19: selective marker of polypeptide STSSSCNLSK by quinolinium-skeleton fluorescent molecule I-a1
The specific operation is basically the same as that of example 10, except that: the CSKFR was replaced with STSSSCNLSK.
Example 20: selective marker of polypeptide STSSSCNLSK by quinolinium-skeleton fluorescent molecule I-a2
The specific operation is basically the same as that of example 19, except that: i-a1 was replaced by I-a 2.
Example 21: selective marker of polypeptide STSSSCNLSK by quinolinium-skeleton fluorescent molecule I-a3
The specific operation is basically the same as that of example 19, except that: i-a1 was replaced by I-a 3.
Example 22: selectable marker of polypeptide STSSSCNLSK by quinolinium-skeleton fluorescent molecule I-b1
The specific operation is basically the same as that of example 11, except that: the CSKFR was replaced with STSSSCNLSK.
Example 23: selectable marker of polypeptide STSSSCNLSK by quinolinium-skeleton fluorescent molecule I-b2
The specific operation is substantially the same as in example 22, except that: i-b1 was replaced by I-b 2.
Example 24: selective marker of polypeptide STSSSCNLSK by fluorescent molecule I-c of quinolinium skeleton
The specific operation is substantially the same as in example 22, except that: i-b1 was replaced by I-c.
Example 25: selective marker of polypeptide AYEMWCFHQK by quinolinium-skeleton fluorescent molecule I-a1
The specific operation is basically the same as that of example 10, except that: the CSKFR was replaced with AYEMWCFHQK.
Example 26: selectable marker of polypeptide AYEMWCFHQK by quinolinium-skeleton fluorescent molecule I-b1
The specific operation is basically the same as that of example 11, except that: the CSKFR was replaced with AYEMWCFHQK.
Example 27: selective marker of polypeptide AYEMWCFHQK by fluorescent molecule I-c of quinolinium skeleton
The specific operation is basically the same as that of example 12, except that: the CSKFR was replaced with AYEMWCFHQK.
Example 28: fluorescent molecule I-a1 of quinolinium skeleton for selective marker of polypeptide AYEMWCFHQR
The specific operation is basically the same as that of example 10, except that: the CSKFR was replaced with AYEMWCFHQR.
Example 29: fluorescent molecule I-b1 of quinolinium backbone for selectable marker of polypeptide AYEMWCFHQR
The specific operation is basically the same as that of example 10, except that: the CSKFR was replaced with AYEMWCFHQR.
Example 30: fluorescent molecule I-b2 of quinolinium backbone for selectable marker of polypeptide AYEMWCFHQR
The specific operation is substantially the same as in example 29, except that: i-b1 was replaced by I-b 2.
Example 31: fluorescent molecule I-b3 of quinolinium backbone for selectable marker of polypeptide AYEMWCFHQR
The specific operation is basically the same as that of example 31, except that: i-b1 was replaced by I-b 3.
Example 32: fluorescent molecule I-b4 of quinolinium backbone for selectable marker of polypeptide AYEMWCFHQR
The specific operation is basically the same as that of example 31, except that: i-b1 was replaced by I-b 4.
Example 33: polypeptide AYEMWCFHQR selectable marker by quinolinium skeleton fluorescent molecule I-c
The specific operation is basically the same as that of example 12, except that: the CSKFR was replaced with AYEMWCFHQR.
Example 34: the fluorescence-labeled polypeptide compounds obtained by the above experimental operations can be analyzed by LC-MS to obtain conversion rates, and specific conversion rate parameters are shown in Table 2 below.
TABLE 2
Example 35: selective marker of quinolinium-backbone fluorescent molecule I-b1 for Bovine Serum Albumin (Bovine Serum Albumin)
The specific operation is as follows: mu.L of bovine serum albumin solution (BSA solution with water as solvent at a concentration of 1mmol/L), 10. mu.L of fluorescent molecule I-b1 solution (acetonitrile as solvent at a concentration of 10mmol/L), and 80. mu.L of phosphate buffer solution (pH 7.4 at a concentration of 50mmol/L) were mixed in a 1.5mL centrifuge tube. The reaction mixture was reacted at 25 ℃ for 16 hours. After the end, the bovine serum albumin labeled by the fluorescent molecule is purified from Bio-Spin P6, photographed, analyzed by LC-MS and digested by trypsin for LC-MS/MS analysis. In addition, LYSOZYME containing no cysteine was used as a control protein and the above procedure was repeated.
The test results of the LC-MS analysis are shown in FIG. 4.
The test results of the LC-MS/MS analysis are shown in FIG. 5.
Example 36: selective marker of fluorescent molecule I-c of quinolinium framework for bovine serum albumin
The specific operation is substantially the same as in example 35, except that: i-b1 was replaced by I-c.
Example 37: selective marker of quinolinium-backbone fluorescent molecule I-b1 for Human Serum Albumin (HSA)
The specific operation is substantially the same as in example 35, except that: BSA was replaced with HSA.
The specific operation is substantially the same as in example 35, except that: i-b1 was replaced by I-c, and BSA was replaced by HSA.
Example 38: the conversion rates of the fluorescence-labeled protein compounds obtained by the above experimental operations can be obtained by LC-MS analysis, and the specific conversion rate parameters are shown in Table 3 below.
TABLE 3
Example 39: the fluorescent-labeled protein compounds obtained by the above experimental procedures were compared with Lysozyme (Lysozyme) containing no cysteine residue as a control protein by photographing with naked eyes and under an ultraviolet lamp, as shown in table 4 below. The results of the experiments show that the Lysozyme (Lysozyme) solution without cysteine residues is unchanged before and after the addition of the fluorescent molecules I-b1 and I-c, and does not show fluorescence, and only the bovine serum albumin BSA and the human serum albumin HAS containing cysteine residues show strong fluorescence after the addition of the fluorescent molecules I-b1 and I-c. The fluorescent molecule pair can highly selectively label the protein of the active sulfur group.
TABLE 4
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
