Preparation and application of live bacteria fluorescent probe

Abstract

The present application relates to the field of biological detection, and more particularly to compounds, probes, kits and their applications. The compounds provided by the present application include: modified rhodamine series fluorescent dyes and D-type amino acids, intermediate groups for regulating the luminescence properties of the dyes. Through the optimization of the dye part, we obtain a series of compounds that can be detected in different fluorescent color channels (400-820 nm).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to the preparation and application of a fluorescent probe for living bacteria. BACKGROUND

[0002] It is of great significance to detect the metabolism and other biological indicators of bacteria by fluorescent labeling of bacterial cell walls. In 2012, Kuru et al. invented fluorescent dye-coupled D-amino acid (FDAA) probes that can fluorescently label bacteria, which have been widely used in the field of microbiology. These probes can specifically recognize bacteria but not other eukaryotes such as plant and animal cells. However, these probes require additional cell fixation and washing after labeling, and cannot directly observe the morphology and growth of living bacteria. In 2019, Hsu et al. developed rotor-based fluorescent dye-coupled D-amino acid probes. These probes emit light after labeling bacteria, but do not emit light in solution. Therefore, real-time cell observation without rinsing can be achieved. However, the fluorescence intensity of this type of probe is low and cannot be used for super-resolution fluorescence imaging. SUMMARY

[0003] Therefore, the present application provides a compound, a probe, a kit and an application thereof for super-resolution fluorescence imaging of living bacteria. The present inventors found that by connecting a fluorescent dye molecule and an active reaction group through a polarity regulating group, the fluorescence brightness of the fluorescent probe molecule after labeling the bacterial cell wall can be greatly improved, thereby obtaining a new structure of fluorescent probe, which has the characteristics of polarity response, can be used for specific labeling of bacterial cell walls, has fast labeling speed, significant fluorescence activation, strong anti-photobleaching ability, wide application range, and can be effectively used for labeling, tracking and quantitative research of various bacteria.

[0004] To achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0005] The present application provides a compound, the structural formula of which is shown in formula 1 or formula 2:

[0006]

[0007] Among them:

[0008] A is the residue of a fluorescent labeling signal molecule without a carboxyl group;

[0009] L is a 3-6 membered heterocyclic ring or an aromatic ring;

[0010] R1 is a polarity regulating group;

[0011] R is the residue of a D-amino acid or a D-amino acid amide without an amino group or a carboxyl group.

[0012] In some embodiments of the present application, the above-mentioned compound, the structural formula of A is shown in any of formula 3 to formula 12.

[0013]

[0014]

[0015] In some embodiments of the present application, the above-mentioned compound, the structural formula of L is shown in formula 13 or formula 14.

[0016]

[0017] wherein: R2 is F; X is S.

[0018] In some embodiments of the present application, the above-mentioned compound, the polar regulating group includes any of methyl, allyloxy, allylthiol and isobutylthiol.

[0019] In some embodiments of the present application, the above-mentioned compound, the structural formula of R is shown in formula 15 or formula 16.

[0020]

[0021] In some embodiments of the present application, the above-mentioned compound has a structural formula shown in any of formula 17 to formula 35:

[0022]

[0023]

[0024] In some embodiments of the present application, the above-mentioned compound, the fluorescent probe with self-aggregation effect is shown in formula 1. Such fluorescent probe refers to a dye molecule which can have self-aggregation phenomenon in aqueous solution, so that the fluorescence intensity is significantly weakened. Alternatively, the probe with self-aggregation behavior is a probe molecule whose maximum fluorescence emission intensity in the same concentration and excitation wavelength conditions at 25℃ is greater than 2, preferably greater than 5, more preferably greater than 10 in the ratio of HEPES buffer containing 0.2% sodium dodecyl sulfate (SDS) to HEPES buffer without SDS. It can effectively label the bacterial cell wall and achieve the no-wash fluorescent labeling with a signal-to-noise ratio greater than 5. The concentration range of the probe molecule is 1x10 -7 ~1x10 -4 M.

[0025] In some embodiments of the present application, the above-mentioned compound is a fluorescent probe dependent on the efficiency of bacterial labeling, as shown in Formula 2; such a fluorescent probe has no obvious fluorescence change in an in vitro assay, but due to its significantly faster labeling rate than traditional probes, it can effectively label the bacterial cell wall and achieve a no-washing fluorescent labeling with a signal-to-noise ratio greater than 10. The concentration range of the probe molecule is 1x10 -7 ~1x10 -4 M.

[0026] The present application also provides a preparation method of the above-mentioned compound, which reacts raw material A, raw material L and raw material R in Formula 1 or Formula 2 to obtain the compound.

[0027] The present application also provides a probe, which comprises the above-mentioned compound and / or the compound obtained by the above-mentioned preparation method.

[0028] The present application also provides a detection reagent, which comprises the above-mentioned compound, the compound obtained by the above-mentioned preparation method and / or the above-mentioned probe, and an acceptable adjuvant.

[0029] The present application also provides a kit, which comprises the above-mentioned compound, the compound obtained by the above-mentioned preparation method, the above-mentioned probe and / or the above-mentioned detection reagent, and an acceptable carrier and / or device.

[0030] In some embodiments of the present application, the above-mentioned kit further comprises a biocompatible medium; optionally, the biocompatible medium is selected from at least one of dimethyl sulfoxide, a buffer, and physiological saline; optionally, the buffer includes but is not limited to a phosphate buffer.

[0031] The present application also provides applications of the above-mentioned compound, the compound obtained by the above-mentioned preparation method, the above-mentioned probe, the above-mentioned detection reagent and / or the above-mentioned kit in any of the following items:

[0032] (I), fluorescent labeling;

[0033] (II), fluorescent labeling of bacterial cell wall

[0034] (III), fluorescent imaging;

[0035] (IV), fluorescent detection;

[0036] (V), bacterial labeling;

[0037] (VI), detection of bacterial growth;

[0038] (VII), screening of antibacterial drugs for cell wall synthesis enzymes.

[0039] The present application also provides a method for fluorescent labeling of bacterial cell wall, which co-cultures the above-mentioned probe with the bacterial cell wall to obtain fluorescently labeled bacterial cell wall.

[0040] The present application introduces rhodamine-based dyes into D-amino acid probes, which have better fluorescence properties and can meet the requirements of super-resolution imaging. At the same time, we rationally design and modify these dye molecules, so that these probes do not emit fluorescence without labeling, but produce strong fluorescence after labeling the bacterial cell wall. Thus, it realizes real-time labeling without washing and can be used for super-resolution fluorescence imaging. This kind of probe can well meet the needs of bacterial cell biology research, and can be used to detect the growth characteristics of bacteria isolated from the environment and the host body. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows.

[0042] Figure 1 Figure 2 shows the fluorescence emission spectra of probes 6, 8, 9, 10, 11, 12, 13, 16, 17 in HEPES solution;

[0043] Figure 2 Figure 3 shows the fluorescence emission intensity of probes 1-19 in HEPES solution (white) and in HEPES solution containing 0.4% SDS (gray);

[0044] Figure 3 Figure 4 shows the live cell labeling imaging process of the probe;

[0045] Figure 4 Figure 5 shows the wash-free confocal imaging of probe 6, 8, 9, 10, 11, 12, 13, 16 labeling live Bacillus subtilis bacteria;

[0046] Figure 5 Figure 6 shows the wash-free and super-resolution STED imaging of probe 6 labeling live cells of different bacteria. DETAILED DESCRIPTION

[0047] The present application discloses compounds, probes, kits and their applications.

[0048] It should be understood that the expression "one or more of" includes each object recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0049] The use of the terms "including," "containing," or "comprising" and variations thereof, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items not specifically listed, unless otherwise specified or limited by context.

[0050] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0051] The use of any and all examples, or exemplary language herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0052] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are only used to convey generally understood precision. Numerical parameters are only approximations of numerical values, due to, for example, varying and / or standard conditions, inherent errors, measurement errors, measurement of different quantities, etc. Unless otherwise indicated, all ranges or numerical values included in this disclosure are approximations as though the terms "about" and "substantially" were also included before each term. As used herein, "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range.

[0053] The present application provides a fluorescent probe, comprising: a dye moiety A, a polarity regulating group R1, and a target active reaction group R of bacterial cell wall, the fluorescent dye is rhodamine derivative, which is a polarity sensitive dye; the polarity of the molecule can be regulated by modification of the substituent group on the polarity regulating group R1; the recognition group can be recognized by bacterial transpeptidase and labeled with bacterial cell wall through covalent bond in the process of bacterial cell wall synthesis. The dye moiety A is covalently connected to the polarity regulating group R1 through the linker L, and the recognition group R is covalently connected to the polarity regulating group R1 through amide bond. Optionally, the fluorescent probe has the structural characteristics as shown in formula 1 or formula 2,

[0054]

[0055] Wherein, the dye moiety is rhodamine derivative (formula 3-7), coumarin derivative (formula 8 and formula 10), nitrobenzoxadiazole (NBD) (formula 9), sulfonic acid-based cyanin Cy3 (formula 11) and IR-775 (formula 12):

[0056]

[0057]

[0058] The linker L is connected to the carboxyl of the rhodamine spirocyclic group through a sulfonamide group, and is connected to the polarity regulating group through an amide group, as shown in formula 13 or formula 14.

[0059]

[0060] The polarity regulating group is 1,4-dicarboxyl-substituted benzoic acid as a mother group, and the polarity of the molecule is regulated by modifying different polar substituents at the ortho position of the carboxyl group, which can be methyl, allyloxy, allylthiol, and isobutylthiol; and the two carboxyl groups form an amide bond with L and R, as shown in the formula.

[0061] The active reaction group D-type amino acid or D-type amino amide, and the polarity regulating group forms a covalent bond with the side chain amino group through a carboxylic acid thereon, which can be a structure as shown in formula 15 or formula 16:

[0062]

[0063] Optionally, the above-mentioned fluorescent probe is selected from the following formula:

[0064]

[0065]

[0066] In another aspect, a method for preparing the above-mentioned fluorescent probe is also provided, which comprises a chemical reaction step of the fluorescent dye shown in formula 1 or formula 2 with the active reaction group and the optional linker.

[0067] The present application provides a fluorescent enhanced bacterial labeling method, which comprises the following steps: co-culturing the fluorescent probe with bacteria, wherein the active reaction group of the fluorescent probe participates in the synthesis of the bacterial cell wall, and the fluorescent dye is labeled on the cell wall; optionally, the fluorescent probe is covalently labeled on the cell wall.

[0068] The present application also provides the use of the fluorescent probe in protein fluorescence labeling, bacterial growth curve kinetic study, and cell and tissue in vivo imaging.

[0069] The present application also provides a probe kit comprising the fluorescent probe. Optionally, the probe kit further comprises a biocompatible medium; optionally, the biocompatible medium is selected from at least one of dimethyl sulfoxide, a buffer, and physiological saline; and optionally, the buffer includes but is not limited to a phosphate buffer.

[0070] The fluorescent probe with self-aggregation effect according to the present application is shown in formula 1. Such fluorescent probe refers to dye molecules that can undergo self-aggregation phenomenon in aqueous solution, thus the fluorescence intensity is significantly weakened. Alternatively, the probe with self-aggregation behavior refers to a probe molecule with a ratio of maximum fluorescence emission intensity of the probe molecule in HEPES buffer containing 0.2% sodium dodecyl sulfate (SDS) to that in HEPES buffer without SDS greater than 2, preferably greater than 5, and more preferably greater than 10 under the conditions of equal concentration and excitation wavelength at 25°C. The probe can effectively label the bacterial cell wall and achieve a no-wash fluorescent labeling with a signal-to-noise ratio greater than 5. The concentration of the probe molecule ranges from 1x10 -7 to 1x10 -4 M.

[0071] The fluorescent probe depending on the labeling efficiency of bacteria according to the present application is shown in formula 2. Such fluorescent probe has no obvious fluorescence change in in vitro determination, but due to its labeling rate with bacteria is significantly faster than that of traditional probes, it can effectively label the bacterial cell wall and achieve a no-wash fluorescent labeling with a signal-to-noise ratio greater than 10. The concentration of the probe molecule ranges from 1x10 -7 to 1x10 -4 M.

[0072] The person skilled in the art can select different kinds or emission wavelengths of dyes.

[0073] According to an embodiment of the present application, the fluorescent probe has a wide range of fluorescence emission wavelength.

[0074] According to an embodiment of the present application, the fluorescence intensity of the fluorescent probe increases with the increase of the polarity of the microenvironment, and is sensitive to the polarity.

[0075] According to an embodiment of the present application, after the fluorescent probe labels the bacterial cell wall, the polarity increases due to the labeling of biological macromolecules, and the fluorescence brightness increases, thus the fluorescent probe has good fluorescence molecule activation property.

[0076] According to an embodiment of the present application, the fluorescent probe has high specificity and can be used as a powerful tool for labeling the bacterial cell wall.

[0077] According to an embodiment of the present application, the fluorescent probe has a fast labeling speed for the bacterial cell wall.

[0078] According to an embodiment of the present application, the fluorescent probe can be used for labeling live bacteria, can be used for real-time observation of bacterial growth activities, and can track the kinetic process of the labeling reaction.

[0079] According to an embodiment of the present application, the different fluorescent probes involved in the present application have spectra that do not interfere with each other, and can be used for multicolor labeling of samples, and can be used for orthogonal labeling imaging with other dyes.

[0080] According to an embodiment of the aspect, the fluorescent probe molecule has high brightness, excellent light bleaching resistance and good light stability, and can be used as a powerful tool for super-resolution imaging of bacterial cell walls.

[0081] According to an embodiment of the aspect, the fluorescent probe can detect the survival and metabolic state of various bacteria, and can be used to detect environmental and host interaction bacteria and their viability.

[0082] According to an embodiment of the aspect, the fluorescent probe can detect the activity of bacterial cell wall synthesis enzymes, and can be used to screen antibacterial drugs targeting cell wall synthesis enzymes.

[0083] In the examples 1-21 and verification examples of the present application, the raw materials and reagents used can be purchased from the market.

[0084] The present application will be further described below in conjunction with examples:

[0085] Example 1

[0086] Compound 1: In a solution of compound 3-aminoazetidine dihydrochloride (1.0 eq, 1.72 mmol, 0.25 g) in 3 mL N,N-Dimethylformamide (DMF), 1.5 mL N,N-diisopropylethylamine (DIPEA, 5.0 eq, 8.6 mmol, 1.5 mL) was added, and after stirring for 2 min, 5 mL of 9-fluorenylmethyl-N-succinimidyl carbonate (1.0 eq, 1.72 mmol, 0.58 g) dissolved in DMF was slowly added. After stirring at room temperature for 20 min, the reaction was quenched by adding 2.0 mL glacial acetic acid. The product was separated and purified by reverse-phase high-performance liquid chromatography, and freeze-dried to obtain compound 1 (white powder, 365.0 mg, yield 72%). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min), retention time 18.1 min. 1 H NMR (400 MHz, DMSO) δ 8.404 (s, 2H), 7.897 (m, 2H), 7.637 (d, J = 7.4 Hz, 2H), 7.423 (q, J = 6.8 Hz, 2H), 7.375 - 7.301 (m, 2H), 4.419 (m, 1H), 4.281 (s, 1H), 4.247 (m, 1H), 4.148 (s, 2H), 4.038 - 3.986 (m, 1H), 3.918 (t, J = 8.7 Hz, 2H). HRMS (ESI, pos. mode) m / z calc. for C 18 H 19 N2O2 + 295.1447, found 295.1440 [M+H] +.

[0087]

[0088] Example 2

[0089] Compound 2: To a solution of compound chlorosulfonyl isocyanate (1.5 eq, 1.86 mmol, 146 μί) in 2 mL of dichloromethane, tert-butanol (3.0 eq, 3.72 mmol, 356 μί) was added, and the reaction was stirred at 0 °C for 2 min and then gradually warmed to room temperature for 1 h. To a solution of compound 1 (1.0 eq, 1.24 mmol, 0.36 g) in 2 mL of dichloromethane, DIPEA (5.0 eq, 6.2 mmol, 1 mL) was added, and the reaction was stirred at 0 °C for 2 min and then the above solution was added dropwise. After the addition was completed, the reaction was stirred at 0 °C for 1 h and then gradually warmed to room temperature for 7 h. The reaction was quenched by the addition of 1.5 mL of glacial acetic acid. The product was purified by reverse-phase HPLC and lyophilized to give compound 2 (white powder, 463.4 mg, 80% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min), retention time 25.2 min. 1 H NMR (400 MHz, DMSO) δ 11.024 (s, 1H), 8.480 (d, J = 7.8 Hz, 1H), 7.892 (d, J = 7.5 Hz, 2H), 7.620 (d, J = 7.4 Hz, 2H), 7.423 (td, J = 7.5, 1.2 Hz, 2H), 7.329 (td, J = 7.4, 1.2 Hz, 2H), 4.438 - 4.204 (m, 3H), 4.132 (d, J = 4.9 Hz, 3H), 3.826 (s, 2H), 1.439 (s, 9H). HRMS (ESI, pos. mode) m / z calc. for C 23 H 27 O6N3NaS + 496.1513, found 496.1510 [M + Na] + .

[0090] Compound 3: Compound 2 (0.98 mmol, 462.5 mg) was dissolved in 3.0 mL of DCM / TFA (30%) mixed solution, and the reaction was stirred at room temperature for 1 h. The solvent was removed under vacuum, and the product was purified by reverse-phase HPLC and lyophilized to give compound 3 (white powder, 321.1 mg, 88% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 19 min. 1H NMR (400 MHz, DMSO) δ 7.899 (d, J = 7.5 Hz, 2H), 7.634 (d, J = 7.4 Hz, 2H), 7.427 (dd, J = 7.9, 6.7 Hz, 2H), 7.344 (td, J = 7.4, 1.1 Hz, 3H), 6.705 (s, 2H), 4.410 - 3.992 (m, 6H), 3.861 (s, 2H). HRMS (ESI, pos. mode) m / z calc. for C 18 H 20 N3O4S + 374.1169, found 374.1160 [M+H] + .

[0091]

[0092] Example 3

[0093] Compound 4: To a mixture of Rhodamine B (1.0 eq, 0.41 mmol, 198.3 mg), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 2.0 eq, 0.82 mmol, 430.9 mg), 1-hydroxybenzotriazole (HOBt) (2.0 eq, 0.82 mmol, 111.9 mg), 4-dimethylaminopyridine (0.2 eq, 0.082 mmol, 10.1 mg), compound 3 (1.2 eq, 0.50 mmol, 186.5 mg) in 5 mL of acetonitrile, DIPEA (10.0 eq, 4.2 mmol, 721.1 μL) was added, after 3 hours of reaction at room temperature, the reaction was quenched by the addition of 1.0 mL of glacial acetic acid. The product was isolated and purified by reverse phase high performance liquid chromatography and lyophilized to give compound 7 (red powder, 303.6 mg, 85% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 28.0 min. 1H NMR (400 MHz, CDC13) δ 8.092 (m, 1H), 7.754 (d, J = 7.5 Hz, 2H), 7.699 (m, 2H), 7.521 (d, J = 7.5 Hz, 2H), 7.393 (t, J = 7.4 Hz, 2H), 7.296 (t, J = 7.5, 2H), 7.213 (m, 1H), 7.117 (m, 2H), 6.895 - 6.790 (m, 4H), 4.258 (d, J = 7.3 Hz, 2H), 4.167 (t, J = 7.3 Hz, 3H), 3.706 (br, 2H), 3.527 (q, J = 7.1 Hz, 8H), 3.361 (br, 1H), 1.251 (t, J = 7.1 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 46 H 48 N5O6S + 798.3320, found 798.3322 [M+H] + .

[0094] Compound 5: Compound 3 (303.6 mg) was dissolved in 2.0 mL DMF / Piperidine (25%) mixed solution, after stirring at room temperature for half an hour, 1.0 mL glacial acetic acid was added to quench the reaction, the product was separated and purified by reverse phase high performance liquid chromatography, freeze-dried to obtain compound 5 (red powder, 201.6 mg, yield 92%). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time was 19.3 min. 1 H NMR (400 MHz, DMSO) δ 8.999 (s, 1H), 8.767 (s, 1H), 8.602 (s, 1H), 7.918 (d, J = 7.6 Hz, 1H), 7.763 - 7.382 (m, 2H), 7.030 (d, J = 7.5 Hz, 1H), 6.633 - 6.119 (m, 6H), 3.926 - 3.756 (m, 5H), 3.345 (d, J = 7.7 Hz, 8H), 1.105 (t, J = 6.9 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 31 H 38 O4N5S + 576.2639, found 576.2647 [M+H] + .

[0095]

[0096] Example 4

[0097] Compound 6: To a solution of compound Fmoc-D-Glu-OAll (3.0 eq, 0.261 mmol, 106.9 mg) in 0.5 mL DMF, DIPEA (5.0 eq, 0.435 mmol, 75.8 μί) was added and the reaction was stirred for 2 min before slowly adding N,N,N',N'-tetramethyl-O-(N-succinimidyl)urea tetrafluoroborate (TSTU, 2.0 eq, 0.174 mmol, 52.4 mg) in 0.5 mL DMF. The resulting mixture was stirred at room temperature for 10 min. The above solution was slowly added to a solution of compound 5 (1.0 eq, 0.087 mmol, 50.0 mg) and DIPEA (5.0 eq, 0.435 mmol, 75.8 μί) in 0.2 mL DMF. The mixture was stirred at room temperature for 1 h before the reaction was quenched by the addition of 0.5 mL glacial acetic acid. The product was purified by reverse phase HPLC and lyophilized to give compound 6 (red powder, 67.1 mg, 80% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 22.8 min. 1 H NMR (400 MHz, CDC13) δ 8.054 (s, 1H), 7.745 (d, J = 8.1 Hz, 2H), 7.670 - 7.585 (m, 4H), 7.398 - 7.361 (m, 2H), 7.349 - 7.253 (m, 2H), 7.178 (d, J = 6.4 Hz, 1H), 7.052 - 6.889 (m, 2H), 6.753 (s, 4H), 5.933 - 5.863 (m, 2H), 5.350 - 5.238 (m, 3H), 4.636 (d, J = 5.7 Hz, 2H), 4.446 - 4.281 (m, 2H), 4.213 (q, J = 7.0 Hz, 1H), 4.070 (s, 2H), 3.890 (s, 1H), 3.778 (s, 1H), 3.480 (d, J = 10.9 Hz, 8H), 2.216 - 1.933 (m, 4H), 1.339 - 0.998 (m, 12H). HRMS (ESI, pos. mode) m / z calc. for C 54 H 59 N6O9S + 967.4059, found 967.4055 [M+H] + .

[0098] Compound 7: In a solution of compound 6 (1.0 eq, 0.067 mmol, 65 mg) in 1 mL of acetonitrile, 1,3-dimethylbarbituric acid (3.0 eq, 0.201 mmol, 31.4 mg) and tetrakis(triphenylphosphine)palladium (0.3 eq, 0.0201 mmol, 23.2 mg), the mixture was stirred at room temperature for 3 hours. The product was isolated and purified by reverse-phase high-performance liquid chromatography, freeze-dried to obtain compound 7 red powder (56.1 mg, yield 90%). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 21.8 min. 1 H NMR (400 MHz, DMSO) δ 8.935 (s, 1H), 8.003 - 7.842 (m, 3H), 7.756 - 7.536 (m, 5H), 7.402 - 7.379 (m, 2H), 7.333 - 7.286 (m, 2H), 7.057 (d, J = 7.7 Hz, 1H), 6.438 (s, 5H), 4.305 - 4.139 (m, 3H), 3.976 - 3.889 (m, 2H), 3.813 - 3.707 (m, 2H), 3.613 (s, 2H), 3.327 (d, J = 8.7 Hz, 8H), 2.065 - 1.616 (m, 4H), 1.063 (s, 12H). HRMS (ESI, pos. mode) m / z calc. for C 51 H 55 O9N6S + 927.3746, found 927.3718[M+H] + .

[0099] Probe 1 : Compound 7 (0.059 mmol, 55.0 mg) was dissolved in 0.5 mL DMF / Piperidine (25%) mixed solution, after stirring at room temperature for half an hour, 0.2 mL glacial acetic acid was added to quench the reaction, the product was isolated and purified by reverse-phase high-performance liquid chromatography, freeze-dried to obtain probe 1 (red powder, 36.8 mg, yield 88%). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 21.2 min. 1H NMR (400 MHz, DMSO) δ 7.925 (d, J = 7.5 Hz, 1H), 7.680 (t, J = 7.6 Hz, 1H), 7.606 (t, J = 7.5 Hz, 1H), 7.056 (d, J = 7.7 Hz, 1H), 6.561 - 6.283 (m, 6H), 4.130 - 3.895 (m, 2H), 3.864 - 3.741 (m, 2H), 3.629 (m, 2H), 3.341 (d, J = 10.0 Hz, 8H), 2.205 - 2.072 (m, 2H), 1.976 - 1.866 (m, 2H), 1.087 (t, J = 6.9 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 36 H 45 O7N6S + 705.3052, found 705.3052 [M+H] + .

[0100]

[0101] Example 5

[0102] General synthesis of compounds 8-10: To a solution of p-mercaptoterephthalic acid (1.0 eq, 0.43 mmol, 100.0 mg) in 1 mL of DMF, NaOH (5.0 eq, 2.15 mmol, 86 mg) was added and reacted for 2 min at room temperature, then haloalkane (3.0 eq, 1.29 mmol) was added dropwise slowly and reacted for 45 min at room temperature, then 2 M HC1 was added to adjust pH to 2.0 and continued to react for 2 h at room temperature. The product was isolated and purified by reverse phase HPLC (0.1% TFA water / acetonitrile 20-95% in 35 min), lyophilized to give compound 8-10.

[0103] Compound 8:

[0104] Retention time: 25.7, yield: 80%. HRMS (ESI, neg. mode) m / z calc. for C 14 H 13 O6 - 277.0718, 277.0732 [M-H] - .

[0105] Compound 9:

[0106] Retention time: 16.1 min. Yield: 74%. 1H NMR (400MHz, DMSO) δ13.434(s,1H),7.803(s,1H),5.890–5.789(m,1H),5.291(dd,J=17. 2,1.8Hz,1H),5.151(d,J=10.1Hz,1H),3.656(d,J=6.7Hz,2H).HRMS(ESI,neg.mode)m / z calc.for C 14 H 13 O4S2-309.0261,found309.0253[M–H] – .

[0107] Compound 10:

[0108] Retention time: 21.6 minutes. Yield: 82%. 1 H NMR (400MHz, DMSO) δ13.418 (s, 2H), 7.744 (s, 2H), 2.805 (d, J = 6.7Hz, 4H), 1.836 (hept, J = 6.7Hz, 2H), 1.015 (d, J = 6.6Hz, 12H). HRMS (ESI, neg.mode) m / z calc.for C 16 H 21 O4S2 - 341.0887,found 341.0869[M–H] – .

[0109]

[0110] Example 6

[0111] General synthesis method for compounds 11-14: To a 0.5 mL DMF solution of compound 2,5-disubstituted-1,4-benzenedicarboxylic acid (3.0 eq, 0.417 mmol), DIPEA (5.0 eq, 0.695 mmol, 121.1 μL) was added. After 2 minutes of reaction, TSTU (2.0 eq, 0.278 mmol, 83.7 mg) dissolved in 0.2 mL DMF was slowly added dropwise. The resulting mixture was stirred at room temperature for 10 minutes. The above solution was slowly added dropwise to a 1.0 mL DMF solution of compound 5 (1.0 eq, 0.139 mmol, 80 mg) and DIPEA (5.0 eq, 0.695 mmol, 121.1 μL). After the mixture was stirred at room temperature for 1 hour, 0.5 mL of glacial acetic acid was added to quench the reaction. The product was separated and purified by reverse-phase high-performance liquid chromatography and freeze-dried to obtain compound 11-14 (red powder).

[0112] Compound 11:

[0113] Retention time: 23.9 min. Yield: 82 %. 1 H NMR (400 MHz, DMSO) d 8.968 (s, 1H), 7.913 (d, J = 7.5 Hz, 1H), 7.666 (s, 2H), 7.588 (t, J = 7.5 Hz, 1H), 7.054 (s, 2H), 6.534 - 6.339 (m, 5H), 3.823 - 3.808 (m, 1H), 3.690 - 3.586 (m, 3H), 3.478 (m, 1H), 3.364 - 3.292 (m, 8H), 2.454 (s, 3H), 2.218 (s, 3H), 1.069 (dt, J = 14.3, 7.0 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 41 H 46 O7N5S + 752.3113, found 752.3106 [M+H] + .

[0114] Compound 12:

[0115] Retention time: 20.5, yield: 83 %. HRMS (ESI, pos. mode) m / z calc. for C 45 H50N5O9S + 836.3324, 836.3346 [M+H] + .

[0116] Compound 13:

[0117] Retention time: 21.5 min. Yield: 80 %. 1H NMR (400MHz, DMSO) δ9.006(d,J=6.7Hz,1H),7.907(d,J=7.5Hz,1H),7.808(s,1H),7.659(t,J=7.5Hz,1H),7.586(t,J=7 .5Hz,1H),7.136(s,1H),7.040(d,J=7.7Hz,1H),6.534–6.305(m,5H),5.961–5.652(m,2H),5.280(dd,J=17.0,1.7Hz,1H ),5.194–5.078(m,2H),5.044(dd,J=10.0,1.6Hz,1H),3.833–3.821(m,1H),3.748–3.728(m,2H),3.643(d,J=6.6Hz,3H) ,3.598(d,J=6.9Hz,2H),3.552(s,1H),3.327(t,J=7.8Hz,8H),1.071(dt,J=13.8,6.9Hz,12H).HRMS(ESI,pos.mode)m / z calc.forC 45 H 50 O7N5S3 + 868.2867, found 868.2874[M+H] + ..

[0118] Compound 14:

[0119] Retention time: 23.8 minutes. Yield: 85%. 1 H NMR (400MHz, DMSO) δ7.972–7.865(m,1H),7.772(s,1H),7.700–7.550(m,2H),7. 118(s,1H),7.048–6.911(m,2H),6.571–6.292(m,5H),3.963(s,1H),3.828(s,1H ),3.742–3.689(m,3H),3.336(s,8H),2.786(dd,J=6.8,3.0Hz,4H),1.923–1.60 6(m,2H),1.200–0.982(m,18H),0.953(d,J=6.6Hz,6H).HRMS(ESI,pos.mode)m / z calc.forC 47 H 58 O7N5S3 + 900.3498,found 900.3494[M+H] + .

[0120] General synthetic procedure for compounds 15-18: To a solution of compound 11-14 (1.0 eq, 0.112 mmol) in 0.5 mL DMF, DIPEA (5.0 eq, 0.56 mmol, 97.5 μί) was added and the reaction was allowed to stir for 2 min before slowly adding 0.2 mL of TSTU (1.0 eq, 0.112 mmol, 33.7 mg) dissolved in DMF. The resulting mixture was stirred at room temperature for 10 min. To a solution of compound Boc-D-Dap-OH (3.0 eq, 0.336 mmol, 68.6 mg) and DIPEA (5.0 eq, 0.56 mmol, 97.5 μί) in 0.5 mL DMF, the above solution was slowly added and the mixture was stirred at room temperature for 1 h before quenching the reaction with 0.5 mL glacial acetic acid. The product was purified by reverse phase HPLC and lyophilized to yield compound 15-18 (red powder). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min).

[0121] Compound 15:

[0122] Retention time: 17.9 min. Yield: 81%. 1 H NMR (400 MHz, DMSO) δ 8.973 (d, J = 6.9 Hz, 1H), 8.312 (s, 1H), 7.916 (d, J = 7.5 Hz, 1H), 7.745 - 7.433 (m, 2H), 7.152 (s, 1H), 7.076 - 6.864 (m, 3H), 6.549 - 6.260 (m, 5H), 4.207 (q, J = 6.8 Hz, 1H), 3.941 (d, J = 9.0 Hz, 1H), 3.859 - 3.699 (m, 2H), 3.627 (s, 2H), 3.511 (d, J = 6.1 Hz, 2H), 3.344 (s, 8H), 2.254 (s, 3H), 2.197 (s, 3H), 1.381 (s, 9H), 1.079 (q, J = 7.1 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 49 H 60 N7O 10 S + 938.4122, found 938.4113 [M+H] + .

[0123] Compound 16:

[0124] Retention time: 21.4, yield: 86%. HRMS (ESI, pos. mode) m / z calc. for C 53 H 64 N7O12 S + 1022.4328,1022.4301[M+H] + .

[0125] Compound 17:

[0126] Retention time: 21.2 min. Yield: 85%. 1 H NMR (400 MHz, DMSO) d 7.916 (d, J = 7.4 Hz, 1H), 7.714 - 7.551 (m, 2H), 7.367 (s, 1H), 7.155 (s, 1H), 7.050 - 6.949 (m, 2H), 6.438 (s, 5H), 5.855 - 5.722 (m, 2H), 5.171 (t, J = 16.4 Hz, 2H), 5.046 (t, J = 11.2 Hz, 2H), 4.161 (t, J = 6.3 Hz, 1H), 3.944 (s, 1H), 3.805 (s, 2H), 3.728 (s, 1H), 3.623 - 3.544 (m, 7H), 3.351 (s, 8H), 1.379 (s, 9H), 1.081 (d, J = 7.6 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 53 H 64 N7O 10 S3 + 1054.3877, found 1054.3879[M+H] + .

[0127] Compound 18:

[0128] Retention time: 24.0 min. Yield: 83%. 1H NMR (400 MHz, CD3CN) δ 7.962 (d, J = 7.7 Hz, 1H), 7.826 - 7.660 (m, 2H), 7.384 (s, 1H), 7.291 (s, 1H), 7.200 (t, J = 6.5 Hz, 1H), 7.170 (s, 1H), 7.013 - 6.730 (m, 6H), 6.571 (s, 1H), 5.922 (d, J = 7.7 Hz, 1H), 4.321 (s, 1H), 3.946 (s, 2H), 3.852 - 3.716 (m, 2H), 3.678 - 3.465 (m, 11H), 2.792 (t, J = 7.4 Hz, 4H), 1.852 - 1.616 (m, 2H), 1.207 (d, J = 6.9 Hz, 12H), 0.988 (dd, J = 6.6, 2.5 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 55 H 72 N7O 10 S3 + 1086.4497, found 1086.4482 [M+H] + 。

[0129] Probe 2-5 General Synthesis Procedure: Compound 15-18 (0.09 mmol) was dissolved in 1.0 mL DCM / TFA (30%) mixture and stirred at room temperature for 1 hour. The solvent was removed in vacuo and the product was purified by reverse phase HPLC and lyophilized to give probe 2-5 (red powder). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min).

[0130] Probe 2:

[0131] Retention time: 19.2 min. Yield: 88%.1H NMR (400 MHz, DMSO) δ 7.915 (d, J = 7.6 Hz, 1H), 7.672 (t, J = 7.6 Hz, 1H), 7.596 (t, J = 7.6 Hz, 1H), 7.295 (s, 1H), 7.041 (d, J = 12.0 Hz, 2H), 6.579 - 6.272 (m, 6H), 4.097 (t, J = 5.7 Hz, 1H), 3.964 (t, J = 9.2 Hz, 1H), 3.809 - 3.724 (m, 2H), 3.693 - 3.679 (d, J = 6.5 Hz, 2H), 3.643 - 3.608 (m, 2H), 3.335 (d, J = 7.4 Hz, 8H), 2.292 (s, 3H), 2.220 (s, 3H), 1.084 (q, J = 6.9 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 44 H 52 N7O8S + 838.3598, found 838.3582 [M+H] + .

[0132] Probe 3:

[0133] Retention time: 21.9, yield: 82%. HRMS (ESI, pos. mode) m / z calc. for C 53 H 64 N7O 12 S + 1022.4328, 1022.4357 [M+H] + .

[0134] Probe 4:

[0135] Retention time: 22.6 min. Yield: 85%. 1H NMR (400 MHz, DMSO) δ 9.006 (d, J = 7.1 Hz, 1H), 8.662 (t, J = 5.9 Hz, 1H), 8.324 (t, J = 9.6 Hz, 2H), 7.904 (d, J = 7.6 Hz, 1H), 7.662 (t, J = 7.4 Hz, 1H), 7.587 (t, J = 7.4 Hz, 1H), 7.511 (s, 1H), 7.175 (s, 1H), 7.041 (d, J = 7.7 Hz, 1H), 6.515 - 6.235 (m, 6H), 5.894 - 5.693 (m, 2H), 5.180 (d, J = 16.9 Hz, 2H), 5.058 (d, J = 10.0 Hz, 2H), 4.077 (s, 1H), 3.960 (t, J = 9.1 Hz, 2H), 3.636 - 3.581 (m, 4H), 3.546 (d, J = 6.6 Hz, 1H), 3.334 (d, J = 6.7 Hz, 8H), 1.085 (q, J = 6.7 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 48 H 56 N7O8S3 + 954.3352, found 954.3354 [M+H] + 。

[0136] Probe 5:

[0137] Retention time: 24.1 min. Yield: 90%. 1 H NMR (400 MHz, CD3CN) δ 8.127 (s, 1H), 7.947 (d, J = 7.7 Hz, 1H), 7.730 (t, J = 7.5 Hz, 1H), 7.661 (t, J = 7.6 Hz, 1H), 7.430 (s, 1H), 7.171 (s, 2H), 6.827 - 6.574 (m, 7H), 4.260 (s, 1H), 3.958 (s, 2H), 3.821 (s, 3H), 3.722 (s, 2H), 3.479 (s, 8H), 2.823 (d, J = 6.8 Hz, 2H), 2.773 (d, J = 6.7 Hz, 2H), 1.851 - 1.655 (m, 2H), 1.169 (q, J = 8.2 Hz, 12H), 0.989 (d, J = 6.6 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 50 H 64 N7O8S3 + 986.3973, found 986.3973 [M+H] + .

[0138]

[0139] Example 7

[0140] Compound 19: To a solution of compound 17 (1.0 eq, 0.06 mmol) in 0.5 mL DMF, DIPEA (5.0 eq, 0.3 mmol, 52.3 μί) was added and the reaction was stirred for 2 min before slowly adding TSTU (1.0 eq, 0.06 mmol, 18.1 mg) dissolved in 0.5 mL DMF. The resulting mixture was stirred at room temperature for 10 min. The above solution was slowly added to a solution of ammonia in DMF (5.0 eq, 0.3 mmol) and stirred at room temperature for 1 h before quenching the reaction with 0.5 mL glacial acetic acid. The product was purified by reverse phase HPLC and lyophilized to give compound 19 (red powder, 53.9 mg, 83% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 19.6 min. 1 H NMR (400 MHz, CDC13) δ 8.059 (s, 1H), 7.669 (dd, J = 6.6, 3.2 Hz, 2H), 7.489 (d, J = 17.5 Hz, 2H), 7.179 (d, J = 4.5 Hz, 2H), 7.092 - 6.927 (m, 3H), 6.808 (d, J = 7.9 Hz, 4H), 6.167 (d, J = 6.5 Hz, 1H), 5.849 - 5.747 (m, 2H), 5.180 (d, J = 16.9 Hz, 1H), 5.115 - 4.950 (m, 3H), 4.364 (s, 1H), 4.128 (s, 2H), 4.028 - 3.845 (m, 5H), 3.821 - 3.632 (m, 3H), 3.555 - 3.486 (m, 12H), 1.448 (s, 9H), 1.338 - 1.166 (m, 12H). HRMS (ESI, pos. mode) m / z calc. for C 53 H 65 N8O9S3 + 1053.4031, found 1053.4030 [M+H] + .

[0141] Probe 6: Compound 19 (0.049 mmol, 53.2 mg) was dissolved in 1.0 mL of a mixture of DCM / TFA (30%) and the mixture was stirred at room temperature for 1 hour. The solvent was removed under vacuum and the product was purified by reverse phase HPLC and lyophilized to give probe 6 (red powder, 43.5 mg, 90% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 22.0 min. 1 H NMR (400 MHz, CD3CN) δ 8.436 (s, 1H), 7.947 (d, J = 7.6 Hz, 1H), 7.723 (t, J = 7.5 Hz, 1H), 7.643 (t, J = 7.7 Hz, 1H), 7.521 (s, 1H), 7.176 (s, 2H), 7.051 (s, 1H), 6.650 (s, 6H), 6.332 (s, 1H), 5.943 - 5.750 (m, 2H), 5.363 - 5.108 (m, 2H), 5.061 (d, J = 10.1 Hz, 2H), 4.235 (dd, J = 6.4, 3.1 Hz, 1H), 4.025 - 3.710 (m, 7H), 3.637 (d, J = 6.9 Hz, 2H), 3.553 (d, J = 6.8 Hz, 2H), 3.469 (s, 8H), 1.167 (d, J = 7.5 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 48 H 57 N8O7S3 + 953.3507, found 953.3497 [M+H] + .

[0142]

[0143] Example 8

[0144] The synthesis of compounds 20 and 21 was performed according to the procedure described for compounds 4 and 5.

[0145] Compound 20: yellow powder (163.7 mg, 35% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 26.3 min. 1H NMR (400 MHz, CD3CN) δ 7.959 (d, J = 7.6 Hz, 1H), 7.824 (d, J = 7.5 Hz, 2H), 7.745 - 7.638 (m, 2H), 7.601 (d, J = 7.5 Hz, 2H), 7.413 (t, J = 7.4 Hz, 2H), 7.337 (td, J = 7.5, 1.2 Hz, 2H), 7.144 (s, 1H), 6.510 (s, 6H), 4.293 (d, J = 6.7 Hz, 2H), 4.223 (t, J = 6.8 Hz, 1H), 3.878 (t, J = 8.3 Hz, 2H), 3.657 (s, 3H). HRMS (ESI, pos. mode) m / z calc. for C 38 H 32 O6N5S + 686.2068, found 686.2062 [M+H] + .

[0146] Compound 21 : yellow powder (98.9 mg, 90% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 15.0 min. 1 H NMR (400 MHz, CD3CN) δ 7.949 (d, J = 7.6 Hz, 1H), 7.787 - 7.595 (m, 2H), 7.323 - 7.167 (m, 2H), 6.859 - 6.685 (m, 2H), 6.632 - 6.493 (m, 3H), 3.955 (m 3H), 3.801 (s, 2H). HRMS (ESI, pos. mode) m / z calc. for C 23 H 22 O4N5S + 464.1387, found 464.1385 [M+H] + .

[0147]

[0148] Example 9

[0149] The synthesis of Probe 7 and Probe 8 was carried out according to the procedure described for the synthesis of Probes 2-5.

[0150] Compound 22: yellow powder (135.0 mg, 83% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 21.4 min. 1H NMR (400 MHz, CD3CN) δ 7.931 - 7.881 (m, 2H), 7.740 - 7.608 (m, 2H), 7.165 (s, 1H), 6.710 - 6.459 (m, 4H), 6.069 - 5.694 (m, 2H), 5.284 (dd, J = 17.1, 1.5 Hz, 1H), 5.181 - 5.095 (m, 2H), 5.041 (dd, J = 10.0, 1.4 Hz, 1H), 4.079 (t, J = 9.2 Hz, 1H), 3.892 - 3.721 (m, 2H), 3.693 - 3.559 (m, 6H). HRMS (ESI, pos. mode) m / z calc. for C 37 H 34 N5O7S3 + 756.1615, found 756.1613 [M+H] + .

[0151] Compound 23: yellow powder (138.1 mg, 84% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 21.3 min. 1 H NMR (400 MHz, DMSO) δ 7.903 (d, J = 7.7 Hz, 1H), 7.765 (s, 1H), 7.692 - 7.580 (m, 2H), 7.158 (s, 1H), 7.003 (s, 1H), 6.566 - 6.317 (m, 5H), 3.989 (t, J = 9.0 Hz, 1H), 3.926 - 3.685 (m, 3H), 2.799 (dd, J = 6.8, 4.3 Hz, 4H), 1.970 - 1.623 (m, 2H), 0.999 (d, J = 6.7 Hz, 6H), 0.951 (d, J = 6.6 Hz, 6H). HRMS (ESI, pos. mode) m / z calc. for C 39 H 42 N5O7S3 + 788.2241, found 788.2242 [M+H] + .

[0152] Compound 24: yellow powder (63.9 mg, 80% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 21.1 min. 1H NMR (400 MHz, DMSO) δ 7.908 (d, J = 7.6 Hz, 1H), 7.669 (d, J = 7.8 Hz, 1H), 7.608 (d, J = 7.7 Hz, 1H), 7.371 (s, 1H), 7.206 (s, 1H), 7.018 (d, J = 7.7 Hz, 1H), 6.435 (d, J = 9.9 Hz, 2H), 6.363 (d, J = 8.4 Hz, 3H), 5.874 - 5.735 (m, 2H), 5.181 (t, J = 16.9 Hz, 2H), 5.111 - 4.937 (m, 2H), 4.165 (t, J = 6.3 Hz, 1H), 3.988 (t, J = 9.1 Hz, 1H), 3.897 (s, 1H), 3.829 (s, 1H), 3.741 (s, 1H), 3.649 - 3.537 (m, 7H), 1.385 (s, 9H). HRMS (ESI, pos. mode) m / z calc. for C 45 H 48 N7O 10 S3 + : 942.2619, found 942.2622 [M+H] + 。

[0153] Compound 25: yellow powder (63.9 mg, yield 80%). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 23.8 min. 1 H NMR (400 MHz, DMSO) δ 7.914 (d, J = 7.5 Hz, 1H), 7.712 - 7.556 (m, 2H), 7.309 (s, 1H), 7.204 (s, 1H), 7.015 (s, 1H), 6.744 (s, 1H), 6.484 (d, J = 11.8 Hz, 2H), 6.394 (s, 3H), 4.159 (t, J = 6.2 Hz, 1H), 3.991 (t, J = 9.0 Hz, 1H), 3.925 - 3.702 (m, 3H), 3.552 (m, 3H), 2.814 (d, J = 6.8 Hz, 2H), 2.780 (d, J = 6.7 Hz, 2H), 1.906 - 1.637 (m, 2H), 1.379 (s, 9H), 1.197 - 0.757 (m, 12H). HRMS (ESI, pos. mode) m / z calc. for C 47 H 56 N7O 10 S3 + 974.3245, found 974.3232 [M+H] + .

[0154] Compound 26: yellow powder (63.9 mg, 80% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 13.9 min. 1 H NMR (400 MHz, CD3CN) δ 7.914 (d, J = 7.5 Hz, 1H), 7.782 - 7.573 (m, 2H), 7.467 (s, 1H), 7.241 - 7.13 (s, 3H), 6.839 - 6.211 (m, 8H), 5.990 (s, 2H), 5.894 - 5.788 (m, 2H), 5.212 - 5.026 (m, 4H), 4.222 (s, 1H), 4.081 (d, J = 9.2 Hz, 1H), 3.915 - 3.651 (m, 4H), 3.641 - 3.538 (m, 6H), 1.414 (s, 9H). HRMS (ESI, pos. mode) m / z calc. for C 45 H 49 N8O9S3 + 941.2779, found 941.2786 [M+H] + .

[0155] Compound 27: yellow powder (63.9 mg, 80% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 25.2 min. 1 H NMR (400 MHz, DMSO) δ 7.899 (d, J = 7.6 Hz, 1H), 7.842 - 7.543 (m, 2H), 7.342 (s, 1H), 7.195 (s, 1H), 7.009 (d, J = 7.8 Hz, 1H), 6.870 - 6.633 (m, 1H), 6.535 - 6.265 (m, 5H), 4.091 (t, J = 6.4 Hz, 1H), 3.985 (t, J = 9.1 Hz, 1H), 3.942 - 3.539 (m, 6H), 2.823 (d, J = 6.7 Hz, 2H), 2.779 (d, J = 6.7 Hz, 2H), 1.904 - 1.627 (m, 2H), 1.372 (s, 9H), 1.030 - 0.886 (m, 12H). HRMS (ESI, pos. mode) m / z calc. for C 47 H 57 N8O9S3 + 973.3405, found 973.3400 [M+H] + .

[0156] Probe 7: yellow powder (39 mg, 87% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 18.8 min. 1 H NMR (400 MHz, CD3CN) δ 8.158 (s, 1H), 7.906 (d, J = 7.6 Hz, 1H), 7.662 (t, J = 7.5 Hz, 1H), 7.591 (t, J = 7.5 Hz, 1H), 7.497 (s, 1H), 7.212 (s, 1H), 7.068 (d, J = 7.5 Hz, 1H), 6.953 (s, 1H), 6.728 (s, 1H), 6.524 - 6.260 (m, 6H), 5.914 - 5.793 (m, 2H), 5.282 - 5.107 (m, 2H), 5.067 (dd, J = 10.0, 1.0 Hz, 2H), 4.226 (dd, J = 6.3, 2.9 Hz, 1H), 4.074 (t, J = 9.0 Hz, 1H), 3.963 - 3.709 (m, 6H), 3.635 (d, J = 6.9 Hz, 2H), 3.576 (d, J = 6.8 Hz, 2H). HRMS (ESI, pos. mode) m / z calc. for C 40 H 41 N8O7S3 + 841.2255, found 841.2255 [M+H] + .

[0157] Probe 8: yellow powder (41.8 mg, 88% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 18.8 min. 1H NMR (400 MHz, CD3CN) δ 8.238 (s, 1H), 7.931 (d, J = 7.6 Hz, 1H), 7.764 - 7.645 (m, 1H), 7.608 (t, J = 7.4 Hz, 1H), 7.473 (s, 1H), 7.237 (s, 1H), 7.076 (d, J = 7.7 Hz, 1H), 7.008 (s, 1H), 6.739 (s, 1H), 6.544 - 6.215 (m, 6H), 4.259 (dd, J = 6.4, 3.0 Hz, 1H), 4.122 - 4.080 (m, 1H), 4.011 - 3.847 (m, 2H), 3.831 - 3.619 (m, 4H), 2.875 (d, J = 6.9 Hz, 2H), 2.834 (dd, J = 6.8, 2.2 Hz, 2H), 1.834 (dq, J = 13.4, 6.7 Hz, 2H), 1.032 (dd, J = 6.7, 3.0 Hz, 12H). HRMS (ESI, pos. mode) m / z calc. for C 42 H 49 N8O7S3 + 873.2881, 873.2880 [M+H] + .

[0158]

[0159] Example 10

[0160] Compound 28: 2,3,4,5,6-Pentafluorobenzenesulfonamide (1.0 eq, 4.05 mmol, 1.0 g), DIPEA (3.0 eq, 12.15 mmol, 2116.3 μί), and N-Boc aminoethanethiol (2.0 eq, 8.1 mmol, 1237.1 μί) were dissolved in 30 mL of methanol, and the mixture was refluxed for 4 hours. After the reaction was completed, the solvent was removed by evaporation under vacuum. The residue was purified by reverse phase high performance liquid chromatography (RP-HPLC) (0.1% TFA in water / acetonitrile 10-95%, 35 min), and lyophilized to give Compound 28 as a white solid. Retention time 27.3 min, yield 80%. 1 H NMR (400 MHz, DMSO) δ 8.388 (s, 2H), 6.887 (d, J = 5.6 Hz, 1H), 3.453 (s, 2H), 3.123 (s, 2H), 1.342 (s, 9H). HRMS (ESI, pos. mode) m / z calc. for C 13 H 16 F4N2O4S2 +427.0380, found 427.0377 [M+Na] + .

[0161]

[0162] Example 11

[0163] Compound 29: To a mixture of silicon-based rhodamine dye (1.0 eq, 0.41 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 2.0 eq, 0.82 mmol, 430.9 mg), 1-hydroxybenzotriazole (HOBt) (2.0 eq, 0.82 mmol, 111.9 mg), 4-dimethylaminopyridine (0.2 eq, 0.082 mmol, 10.1 mg), compound 28 (1.2 eq, 0.50 mmol, 186.5 mg) in 5 mL of acetonitrile, DIPEA (10.0 eq, 4.2 mmol, 721.1 μL) was added, after 3 hours of reaction at room temperature, 1.0 mL of glacial acetic acid was added to quench the reaction. The product was isolated and purified by reverse-phase high-performance liquid chromatography, and freeze-dried to give compound 28, yield 6%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 30.9 min. 1 H NMR (400 MHz, DMSO) δ 7.942 (d, J = 7.5 Hz, 1H), 7.614 - 7.495 (m, 2H), 6.900 - 6.845 (m, 3H), 6.748 (d, J = 7.6 Hz, 1H), 6.508 (dd, J = 9.1, 2.8 Hz, 2H), 6.436 (d, J = 9.0 Hz, 2H), 3.114 (s, 4H), 2.904 (s, 12H), 1.299 (s, 9H), 0.534 (s, 3H), 0.494 (s, 3H). HRMS (ESI, pos. mode) m / z calc. for C 39 H 43 F4N4O5S2Si + 815.2375, found 815.2370 [M+H] + .

[0164] Compound 30, 31 were synthesized according to the procedure described for compound 3 and 6, respectively.

[0165] Compound 30: blue powder (yield 90%). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 21.1 min. 1H NMR (400 MHz, DMSO) δ 7.947 (d, J = 7.8 Hz, 1H), 7.876 (s, 2H), 7.599 (t, J = 7.1 Hz, 1H), 7.533 (dd, J = 8.8, 6.4 Hz, 1H), 6.859 (d, J = 2.9 Hz, 2H), 6.782 - 6.740 (m, 1H), 6.572 - 6.380 (m, 4H) 3.232 - 3.187 (m, 2H), 2.967 (d, J = 7.9 Hz, 2H), 2.896 (ds, 12H), 0.536 (s, 3H), 0.491 (s, 3H). HRMS (ESI, pos. mode) m / z calc. for C 34 H 35 F4N4O3S2Si + 715.1850, found 715.1845 [M+H] + .

[0166] Compound 31 : blue powder (58% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 28 min. 1 H NMR (400 MHz, DMSO) δ 8.018 (t, J = 7.2 Hz, 1H), 7.948 (d, J = 7.5 Hz, 1H), 7.858 (d, J = 7.5 Hz, 2H), 7.682 (d, J = 7.5 Hz, 2H), 7.584 (t, J = 7.5 Hz, 1H), 7.525 (t, J = 7.4 Hz, 1H), 7.441 - 7.257 (m, 5H), 7.217 (s, 1H), 7.022 (s, 1H), 6.877 (d, J = 2.9 Hz, 2H), 6.737 (d, J = 7.6 Hz, 1H), 6.564 - 6.373 (m, 4H), 4.231 (m, 5H), 3.261 - 3.165 (m, 2H), 3.089 (m, 2H), 2.886 (s, 12H), 2.383 - 2.353 (m, 1H), 0.528 (s, 3H), 0.487 (s, 3H). HRMS (ESI, pos. mode) m / z calc. for C 53 H 51 F4N6O7S2Si + 1051.2961, found 1051.2952 [M+H] + .

[0167] Probe 9 followed the synthesis steps of Probe 1.

[0168] Probe 9: Blue powder (39 mg, 95% yield). HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 12.6 min. 1 H NMR (400 MHz, DMSO) δ 8.387 (t, J = 5.6 Hz, 1H), 8.089 - 7.989 (m, 2H), 7.946 (d, J = 7.6 Hz, 1H), 7.719 (s, 1H), 7.597 (t, J = 7.4 Hz, 1H), 7.551 - 7.491 (m, 2H), 6.861 (d, J = 2.8 Hz, 2H), 6.752 (d, J = 7.7 Hz, 1H), 6.531 - 6.365 (m, 4H), 3.969 (s, 1H), 3.291 - 3.223 (m, 2H), 3.150 - 3.116 (m, 2H), 2.712 - 2.542 (m, 2H), 0.534 (s, 3H), 0.491 (s, 3H). HRMS (ESI, pos. mode) m / z calc. for C 38 H 41 F4N6O5S2Si + 829.2280, found 829.2283 [M+H] + .

[0169]

[0170] Example 12

[0171] Compound 32: In a mixture of rhodamine 6G dye (1.0 eq, 0.063 mmol, 30 mg), NaOH (10.0 eq, 0.63 mmol, 25.2 mg), add 400 μL acetonitrile solution and 400 μL water, after reaction at room temperature for 3 hours, add glacial acetic acid to quench the reaction. The product was separated and purified by reverse phase high performance liquid chromatography, freeze-dried to obtain compound 31, yield 92%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 17.1 min. MS (ESI, pos. mode) m / z calc. for C 26 H 27 N2O3 + 415.20, found 415.21 [M+H] + .

[0172] Probe 10 refers to the synthesis steps of probe 9.

[0173] Compound 33: Orange yellow powder, yield 82%. HPLC (0.1% TFA water / acetonitrile 50-95%, 35 min). Retention time 22.0 min. MS (ESI, pos. mode) m / z calc. for C 34 H 33 F4N4O4S2 + 801.24, found 801.23 [M+H] + .

[0174] Compound 34: Orange yellow powder, yield 88%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 21.3 min. MS (ESI, pos. mode) m / z calc. for C 34 H 33 F4N4O4S2 + 701.19, found 701.16 [M+H] + .

[0175] Compound 35: Orange yellow powder, yield 90%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 24.4 min. MS (ESI, pos. mode) m / z calc. for C 53 H 49 F4N6O8S2 + 1037.30, found 1037.26 [M+H] + .

[0176] Probe 10: Orange yellow powder, yield 95%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 17.6 min. HRMS (ESI, pos. mode) m / z calc. for C 38 H 39 F4N6O6S2 + 815.2303, found 815.2302 [M+H] + .

[0177]

[0178] Example 13

[0179] Probe 11 follows the synthesis steps of Probe 9.

[0180] Compound 36: Red powder, yield 85%. HPLC (0.1% TFA water / acetonitrile 50-95%, 35 min). Retention time 26.2 min. MS (ESI, pos. mode) m / z calc. for C 45 H 45 F4N4O6S2 + 877.27, found 877.24 [M+H] + .

[0181] Compound 37: Red powder, yield 90%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 22.9 min. MS (ESI, pos. mode) m / z calc. for C 40 H 37 F4N4O4S2 + 777.22, found 777.21 [M+H] + .

[0182] Compound 38: Red powder, yield 88%. HPLC (0.1% TFA water / acetonitrile 30-95%, 35 min). Retention time 24.8 min. MS (ESI, pos. mode) m / z calc. for C 59 H 53 F4N6O8S2 + 1113.33, found 1113.35 [M+H] + .

[0183] Probe 11 : Red powder, yield 92%. HPLC (0.1% TFA water / acetonitrile 30-95%, 35 min). Retention time 17.0 min. HRMS (ESI, pos. mode) m / z calc. for C 44 H 43 F4N6O6S2 + 891.2616, found 891.2617 [M+H] + .

[0184]

[0185] Example 14

[0186] Probe 12 was synthesized following the synthesis steps of Probe 9.

[0187] Compound 39: white powder, yield 83%. HPLC (0.1% TFA water / acetonitrile 30-95%, 35 min). Retention time 26.2 min. MS (ESI, pos. mode) m / z calc. for C 26 H 29 F4N3O7S2 + 635.13,found 635.14[M+H] + .

[0188] Compound 40: white powder, yield 92%. HPLC (0.1% TFA water / acetonitrile 30-95%, 35 min). Retention time 14.2 min. MS (ESI, pos. mode) m / z calc. for C 21 H 19 F4N3O5S2 + 533.07,found 533.09[M+H] + .

[0189] Compound 41: white powder, yield 85%. HPLC (0.1% TFA water / acetonitrile 30-95%, 35 minutes). Retention time 24.7 minutes. MS (ESI, pos. mode) m / z calc. for C 40 H 35 F4N5O9S2 + 869.18,found 869.14[M+H] + .

[0190] Probe 12: White powder, yield 90%. HPLC (0.1% TFA water / acetonitrile 30-95%, 35 minutes). Retention time 14.8 minutes. HRMS (ESI, pos. mode) m / z calc. for C 25 H 25 F4N5O7S2 + 648.1204,found648.1201[M+H] + .

[0191]

[0192] Example 15

[0193] Compound 42: In a mixture of 7-hydroxycoumarin-3-carboxylic acid (1.0 eq, 0.485 mmol, 100 mg), bromo-propene (3.0 eq, 1.455 mmol, 125.6 μί), DIPEA (5.0 eq, 2.43 mmol, 423.3 μί) was added and the reaction was allowed to proceed at room temperature for 6 h. The reaction was quenched by the addition of glacial acetic acid. The product was isolated and purified by reverse phase HPLC and lyophilized to yield Compound 42 as a white powder in 45% yield. HPLC (0.1% TFA water / acetonitrile 20-95% over 35 min). Retention time 28.6 min. MS (ESI, pos. mode) m / z calc. for C 16 H 15 O5 + 287.09, found 287.13 [M+H] + .

[0194] Compound 43: In a mixture of Compound 42 (1.0 eq, 0.245 mmol, 70 mg), LiOH (5.0 eq, 1.225 mmol, 51.5 mg), 1.0 mL of acetonitrile and 1.0 mL of water were added and the reaction was allowed to proceed at room temperature for 3 h. The reaction was quenched by the addition of glacial acetic acid. The product was isolated and purified by reverse phase HPLC and lyophilized to yield Compound 43 as a white powder in 92% yield. HPLC (0.1% TFA water / acetonitrile 20-95% over 35 min). Retention time 24.5 min. MS (ESI, pos. mode) m / z calc. for C 13 H 11 O5 + 247.06, found 247.09 [M+H] + .

[0195] Compounds 44-47 were synthesized following the procedure for Probe 9.

[0196] Compound 44: White powder in 87% yield. HPLC (0.1% TFA water / acetonitrile 40-95% over 35 min). Retention time 28.5 min. MS (ESI, pos. mode) m / z calc. for C 26 H 25 F4N2O8S2 + 633.09, found 633.10 [M+H] + .

[0197] Compound 45: White powder, yield 92%. HPLC (0.1% TFA water / acetonitrile 40-95%, 35 min). Retention time 15.0 min. MS (ESI, pos. mode) m / z calc. for C 21 H 17 F4N2O6S2 + 533.04, found 533.07 [M+H] + .

[0198] Compound 46: White powder, yield 82%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 27.0 min. MS (ESI, pos. mode) m / z calc. for C 40 H 33 F4N4O 10 S2 + 869.15, found 869.19 [M+H] + .

[0199] Compound 47: White powder, yield 87%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 19.3 min. MS (ESI, pos. mode) m / z calc. for C 25 H 23 F4N4O8S2 + 647.08, found 647.11 [M+H] + .

[0200] Probe 13 was synthesized according to the procedure for Compound 7.

[0201] Probe 13: White powder, yield 75%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 16.0 min. HRMS (ESI, pos. mode) m / z calc. for C 22 H 19 F4N4O8S2 + 607.0575, found 607.0573 [M+H] + .

[0202]

[0203] Example 16

[0204] Compound 48 was synthesized according to the procedure for Compound 6.

[0205] Compound 48: white powder, yield 83%. HPLC (0.1% TFA water / acetonitrile 40-95%, 35 min). Retention time 26.7 min. MS (ESI, pos. mode) m / z calc. for C 34 H 38 F4N3O 11 S2 + 804.18, found 804.20 [M+H] + .

[0206] Compound 49: Compound 48 was dissolved in TFA solution, stirred at room temperature for 1 hour, the solvent was removed under vacuum, the product was purified by reverse phase HPLC, freeze-dried to give compound 49, white powder, yield 90%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 19.3 min. MS (ESI, pos. mode) m / z calc. for C 25 H 22 F4N3O9S2 + 648.07, found 648.08 [M+H] + .

[0207] Probe 14 was synthesized according to the method of reference compound 7.

[0208] Probe 14: white powder, yield 90%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 16.2 min. HRMS (ESI, pos. mode) m / z calc. for C 22 H 18 F4N3O9S2 + 608.0415, found 608.0418 [M+H] + .

[0209]

[0210] Example 17

[0211] Compound 50: In a mixture of NBD-Cl (1.0 eq, 0.40 mmol, 80 mg), glycine hydrochloride (1.5 eq, 0.60 mmol, 61.7 mg), DIPEA (5.0 eq, 2.0 mmol, 348.4 μί) was added and the reaction was allowed to proceed at room temperature for 4 h. The reaction was quenched by the addition of glacial acetic acid. The product was isolated and purified by reverse phase HPLC and lyophilized to yield compound 50 as a white powder in 90% yield. HPLC (0.1% TFA water / acetonitrile 10-95% over 35 min). Retention time 20.8 min. MS (ESI, pos. mode) m / z calc. for C8H7N4O5 + 239.04, found 239.04 [M+H] + .

[0212] Compound 51-53 were synthesized following the procedure for compound 29-31.

[0213] Compound 51: white powder, 78% yield. HPLC (0.1% TFA water / acetonitrile 30-95% over 35 min). Retention time 24.3 min. MS (ESI, pos. mode) m / z calc. for C 21 H 21 F4N6O8S2 + 625.07, found 625.08 [M+H] + .

[0214] Compound 52: white powder, 90% yield. HPLC (0.1% TFA water / acetonitrile 30-95% over 35 min). Retention time 12.5 min. MS (ESI, pos. mode) m / z calc. for C 16 H 13 F4N6O6S2 + 525.02, found 525.05 [M+H] + .

[0215] Compound 53: white powder, 92% yield. HPLC (0.1% TFA water / acetonitrile 40-95% over 35 min). Retention time 22.6 min. MS (ESI, pos. mode) m / z calc. for C 29 H 34 F4N7O 11 S2 + 796.16, found 796.15 [M+H] + .

[0216] Probe 15 was synthesized following the procedure for compound 49.

[0217] Probe 15: White powder, yield 82%. HPLC (0.1% TFA water / acetonitrile 10-80%, 35 min). Retention time 18.4 min. HRMS (ESI, pos. mode) m / z calc. for C 20 H 18 F4N7O9S2 + 640.0538, found 640.0533 [M+H] + .

[0218]

[0219] Example 18

[0220] Probe 16 follows the synthesis steps of Probe 6.

[0221] Compound 54: White powder, yield 86%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 20.4 min. MS (ESI, pos. mode) m / z calc. for C 25 H 26 F4N7O 11 S2 + 740.10, found 740.11 [M+H] + .

[0222] Compound 55: White powder, yield 93%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 19.5 min. HRMS (ESI, pos. mode) m / z calc. for C 25 H 27 F4N8O 10 S2 + 739.12, found 739.13 [M+H] + .

[0223] Probe 16: White powder, yield 95%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 14.2 min. HRMS (ESI, pos. mode) m / z calc. for C 20 H 19 F4N8O8S2 + 639.0698, found 639.0696 [M+H] + .

[0224]

[0225] Example 19

[0226] Synthetic steps for probe 17 with reference to probe 9.

[0227] Compound 56: Red powder, yield 75%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 18.1 min. MS (ESI, pos. mode) m / z calc. for C 44 H 53 F4N4O 11 S4 + 1017.25, found 1017.28 [M+H] + .

[0228] Compound 57: Red powder, yield 83%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 13.0 min. MS (ESI, pos. mode) m / z calc. for C 39 H 45 F4N4O9S4 + 917.20, found 917.23 [M+H] + .

[0229] Compound 58: Red powder, yield 78%. HPLC (0.1% TFA water / acetonitrile 10-80%, 35 min). Retention time 27.7 min. MS (ESI, pos. mode) m / z calc. for C 58 H 61 F4N6O 13 S4 + 1253.31, found 1253.30 [M+H] + .

[0230] Probe 17: Red powder, yield 90%. HPLC (0.1% TFA water / acetonitrile 10-80%, 35 min). Retention time 17.1 min. HRMS (ESI, pos. mode) m / z calc. for C 43 H 51 F4N6O 11 S4 + 1031.2429, found 1031.2428 [M+H] + .

[0231]

[0232] Example 20

[0233] Probe 18 reference synthesis steps of probe 9.

[0234] Compound 59: dark blue powder, yield 62%. HPLC (0.1% TFA water / acetonitrile 50-95%, 35 min). Retention time 13.5 min. MS (ESI, pos. mode) m / z calc. for C 33 H 29 F4N4O6S2 + 717.14, found 717.15 [M+H] + .

[0235] Compound 60: dark blue powder, yield 92%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 16.5 min. MS (ESI, pos. mode) m / z calc. for C 28 H 21 F4N4O4S2 + 617.09, found 617.08 [M+H] + .

[0236] Compound 61 : dark blue powder, yield 86%. HPLC (0.1% TFA water / acetonitrile 30-95%, 35 min). Retention time 21.6 min. MS (ESI, pos. mode) m / z calc. for C 47 H 37 F4N6O8S2 + 953.20, found 953.22 [M+H] + .

[0237] Probe 18: dark blue powder, yield 91%. HPLC (0.1% TFA water / acetonitrile 10-80%, 35 min). Retention time 18.4 min. HRMS (ESI, pos. mode) m / z calc. for C 32 H 27 F4N6O6S2 + 731.1364, found 731.1366 [M+H] + .

[0238]

[0239] Example 21

[0240] Probe 19 reference synthesis steps of probe 9.

[0241] Compound 62: dark blue powder, yield 90%. HPLC (0.1% TFA water / acetonitrile 40-95%, 35 min). Retention time 20.9 min. MS (ESI, pos. mode) m / z calc. for C 41 H 45 F4N4O6S2 + 829.27, found 829.27 [M+H] + .

[0242] Compound 63: dark blue powder, yield 92%. HPLC (0.1% TFA water / acetonitrile 20-95%, 35 min). Retention time 22.0 min. MS (ESI, pos. mode) m / z calc. for C 36 H 37 F4N4O4S2 + 729.22, found 729.21 [M+H] + .

[0243] Compound 64: dark blue powder, yield 86%. HPLC (0.1% TFA water / acetonitrile 40-95%, 35 min). Retention time 27.11 min. MS (ESI, pos. mode) m / z calc. for C 55 H 53 F4N6O8S2 + 1065.33, found 1065.31 [M+H] + .

[0244] Probe 19: dark blue powder, yield 91%. HPLC (0.1% TFA water / acetonitrile 30-80%, 35 min). Retention time 17.6 min. HRMS (ESI, pos. mode) m / z calc. for C 40 H 43 F4N6O6S2 + 843.2616, found 843.2620 [M+H] + .

[0245]

[0246] Verification Example

[0247] Because the bacterial cell wall is difficult to purify, the present application uses SDS solution as a reference after activation of the probe. It has been reported that such probes have a significant change in polarity after interaction with SDS, and the fluorescence intensity has a significant activation phenomenon.

[0248] Probes 1-13 and the reference probe were dissolved in dimethyl sulfoxide to prepare a concentration of 1x10 -2 M solution was added to HEPES buffer to prepare a final concentration of 2x10 -6 The fluorescence spectra of the solutions of M were detected under the same conditions using excitation wavelengths matching the probes according to the different probes; 0.4% SDS was added to the reference solution to obtain the reference state after fluorescence activation, as shown in Table 1.

[0249] From the fluorescence intensity of the free probe in Table 1, it can be seen that the fluorescence intensity of the probe of the embodiment is low in HEPES solution; while from the fluorescence intensity of the reference probe in SDS solution, it can be seen that the fluorescence has a significant activation phenomenon and has a high brightness.

[0250] In summary, the fluorescence of the probes of the embodiments can be activated after binding to biomacromolecules, and they have good fluorescent molecular switch properties. Among them, probes 6, 8, and 9 have better fluorescence activation performance than other probes.

[0251] Bacillus subtilis was used as an example to examine the labeling effect of the probe molecule on the bacterial cell wall. The labeling process is simple and easy: the probe molecule is directly dissolved in the agarose gel pad that assists bacterial imaging, and live Bacillus subtilis bacteria are co-incubated for in situ real-time confocal and super-resolution imaging (see Figure 3 The experimental results show that probes 6, 8, 9, 10, 11, 12, 13, and 16 can specifically label bacterial cell walls, and the fluorescence intensity on the cell wall is significantly higher than the background, showing fluorescence activation characteristics (see Figure 4 ).

[0252] Table 1 Spectral properties and bacterial labeling performance of probes 1-19

[0253]

[0254]

[0255] It can be seen from the fluorescence intensity of the free probe in Table 1 that the fluorescence intensity of the probe of the embodiment is low in HEPES solution; while it can be seen from the fluorescence intensity of the reference probe in SDS solution that the fluorescence has obvious activation and high brightness.

[0256] Table 2 Fluorescence signal to background ratio of probe 6 labeling different bacterial cell walls

[0257]

[0258] As shown in Table 2, the probes of the embodiment can achieve live bacterial labeling on a variety of bacteria: the fluorescence intensity of the probes is low in the culture medium, but they are significantly enriched at the bacterial cell wall and have enhanced fluorescence, and the signal-to-background ratio meets the requirements for wash-free labeling.

[0259] Compared with the prior art, the fluorescent emission wavelength range of such compounds is wide (see Figure 1 ); compared with the prior art, the fluorescent intensity of the compound of formula 1 is weak in the unlabeled state (see Table 1), and is significantly enhanced after labeling, with the fluorescent intensity enhancement reaching more than 2.7 times Figure 2 ), wherein the fluorescent intensity of probe 6 is enhanced by 41.9 times, and the fluorescent intensity of probe 8 is enhanced by 5.4 times; the fluorescent signal to background ratio after labeling on various bacteria reaches 1.7-26.3 times (see Table 1). Although the fluorescent enhancement characteristics of the probe molecules 9, 10, 11, 12, 13, 16, 18, and 19 of formula 2 are weak in the in vitro assay, their more rapid labeling rate also enables them to effectively label the cell wall, and the fluorescent signal to background ratio after labeling on bacteria reaches 10.1-35.2 times Figure 2 ). These probe molecules have the characteristic of high fluorescent quantum yield of the probe after fluorescence activation (Table 1), and can be used for no-wash confocal fluorescence imaging and super-resolution STED microscopic imaging (see Figure 4 , Figure 5 ).

[0260] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the scope of protection of the present application.

Claims

1. A compound, characterized in that Its structural formula is shown in Formula 1 or Formula 2; Formula 1, Formula 2; in: A is the residue from which the carboxyl group of the fluorescently labeled signal molecule is removed; L is: a 3- to 6-membered heterocycle or formula 14; R1 is a polar regulating group; R is a D-amino acid or a D-aminoamide residue with the amino group or carboxyl group removed; The structural formula of A is shown in any of the structural formulas of Formula 3 to Formula 12; Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, Formula 8, Formula 9, Formula 10, Formula 11, Formula 12; Formula 14; Wherein: R2 is F; X is S; The polarity regulating group is any one of methyl, allyloxy, allylmercapto and isobutylmercapto; The structural formula of R is shown in Formula 15 or Formula 16; Formula 15, Formula 16.

2. The compound according to claim 1, wherein It has a structural formula as shown in any of Formulas 17 to 35: Formula 17, Formula 18 Formula 19, Formula 20, Formula 21, Formula 22, Formula 23, Formula 24, Formula 25, Formula 26, Formula 27, Formula 28 Formula 29, Formula 30, Formula 31, Formula 32, Formula 33, Formula 34, Formula 35.

3. A probe, characterized in that include: The compound according to claim 1 or 2.

4. A detection reagent, characterized in that include: The compound according to claim 1 or 2 and / or the probe according to claim 3 and an acceptable auxiliary agent.

5. A kit, characterized in that include: The compound according to claim 1 or 2, the probe according to claim 3 and / or the detection reagent according to claim 4, and an acceptable carrier and / or device.

6. Use of the compound according to claim 1 or 2, the probe according to claim 3, the detection reagent according to claim 4 and / or the kit according to claim 5 in any of the following; ( ), fluorescent labeling; ( ), fluorescently labeled bacterial cell wall ( ), fluorescence imaging; ( ), fluorescence detection; ( ), bacterial markers; ( ), detect bacterial growth; ( ), screening of antimicrobial drugs against cell wall synthases; The application does not relate to a method for diagnosing or treating a disease.

Images