A tryptophan c2-position functionalized polypeptide derivative and a method for modifying tryptophan polypeptides or proteins using functionalized oxime esters as labeling sources

Abstract

This invention relates to the field of peptide or protein modification, and more particularly to a tryptophan C2-functionalized peptide derivative and a method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources. Using tryptophan-containing peptides or proteins as substrates and functionalized oxime esters as labeling reagents, the reaction is carried out in a reaction solution in the presence of a photosensitizer, under inert gas protection, and at room temperature light irradiation to obtain tryptophan C2-functionalized peptide derivatives. This method has high labeling efficiency and good site selectivity, and is suitable for the selective derivatization of complex peptides, peptide drugs, and proteins. This invention utilizes photochemical synthesis technology to generate phosphorus-containing free radicals or carbon-center free radicals under mild conditions, and efficiently captures these free radicals through the double bond of the tryptophan indole ring. Subsequently, a re-aromatication process is used to achieve selective modification of tryptophan residues, enabling efficient phosphonylation or acylation modification of tryptophan-containing peptides or proteins.

Description

Technical Field

[0001] This invention relates to the field of peptide or protein modification, and more particularly to a tryptophan C2-functionalized peptide derivative and a method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources. Background Technology

[0002] Late-stage functionalization of peptides and proteins plays a crucial role in expanding their functional diversity and enables numerous biochemical applications, including biochemical imaging, biomaterials, and drug delivery. However, current site-specific modification techniques for peptides and proteins remain highly dependent on specific cell systems or in vitro enzymatic modification methods. These techniques are limited by the inherent genetic regulation of organisms and the catalytic performance of enzymes, resulting in significant limitations in the range of applicable peptide substrates and the types of functional groups that can be modified. Late-stage chemical modification provides a direct route for introducing functional groups into native peptides and proteins, avoiding complex molecular biology operations and cumbersome protein synthesis. It is important to note, however, that the functionalization of peptides and proteins is closely related to their chemical structure, and the subtle structural perturbations caused by late-stage functionalization must be strictly controlled to maintain structural integrity and homogeneity.

[0003] Over the past few decades, there has been increasing focus on transition metal catalysis, photochemical, and electrochemical methods for modifying peptides and proteins. However, due to the stringent requirements of peptides and proteins, including the need for aqueous environments, temperature sensitivity, low substrate concentrations, and regio / chemoselectivity, there is a strong need for a mild and selective late-stage functionalization method for peptides and proteins.

[0004] Tryptophan (Trp, W) is an essential aromatic amino acid for the human body. It is the only naturally occurring amino acid carrying an indole ring and is also the residue with the highest energy consumption and lowest content (approximately 1%) in the biosynthesis of 20 standard natural amino acids. Its molecular structure contains the largest planar conjugated π-electron system, which endows proteins with rich biological activity and unique biophysical properties. This amino acid can participate in stabilizing protein secondary structure, mediating protein-protein interactions, and regulating the recognition and binding of proteins to small molecule ligands through non-covalent interactions such as cation-π interactions and π-π stacking interactions. This characteristic makes it indispensable in bioactive peptides, peptide drugs, and natural products. For example, in ribosome-synthesized and post-translational modified peptides (RiPPs), peptide hormones such as somatostatin, and clinically commonly used peptide drugs such as vapreotide and tirzepatide, tryptophan residues play a key role in ligand-receptor interactions.

[0005] Currently, considerable efforts have been made to achieve late-stage diversification of tryptophan-containing peptides (Org. Biomol. Chem. 2025, 23, 1773). For example, transition metal-catalyzed arylation, amidation, alkylation, alkenylation, and alkynylation reactions of tryptophan residues have been developed. Utilizing the inherent nucleophilicity of tryptophan (Trp) residues, a range of functional groups, including SCF2X, triazolinide, 9-azabicyclo[3.3.1]nonane-3-one-N-oxy(keto-ABNO), oxazoline, etc., can be introduced into the structures of tryptophan-containing peptides and proteins. Furthermore, tryptophan residues can act as excellent free radical scavengers, leading to the formation of peptides and proteins containing trifluoromethylated, alkylated, thiolated, and azidolated tryptophan. Although a series of methods for selective late-stage modification of tryptophan have been established, the need for stoichiometric redox reagents, high-temperature conditions, and the low solubility of peptides pose significant challenges to the late-stage diversification of tryptophan-containing peptides, which is still far from the late-stage diversification of tryptophan-containing proteins.

[0006] Currently, only two reports exist regarding tryptophan (Trp) C2 phosphonylation (Org. Lett. 2024, 26, 4700; Org. Chem. Front. 2024, 11, 6287). However, existing methods are incompatible with peptide sequences containing easily oxidized amino acid residues (e.g., Cys, Tyr, Met, His), thus limiting their further application in complex peptide and protein systems. Therefore, developing a method that operates in a protein-compatible aqueous environment, is compatible with 19 natural amino acids, and achieves selective phosphonylation labeling of tryptophan at the C2 position is of great significance. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of the prior art by providing a tryptophan C2-functionalized polypeptide derivative and a method for modifying tryptophan polypeptides or proteins using functionalized oxime esters as labeling sources.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention is to provide a method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, comprising the steps of: using tryptophan-containing peptides or proteins as substrates, using functionalized oxime esters as labeling reagents, reacting in a reaction solution in the presence of a photosensitizer, under inert gas protection and room temperature light conditions, to obtain tryptophan C2-functionalized peptide derivatives.

[0010] Preferably, the tryptophan-containing polypeptide or protein is as shown in formula (I), including but not limited to: protected-terminal polypeptides, free-terminal polypeptides, random-sequence natural polypeptides, polypeptide drugs, and functional proteins;

[0011] .

[0012] More preferably, the polypeptide drugs include: daptomycin, exenatide, telpoxetine, and lanreotide;

[0013] Preferably, the functionalized oxime ester is as shown in formula (II), wherein -R 1 Selected from -aryl or -alkyl; -R 2 Selected from -aryl or -alkyl; -R 3 Selected from -P(O)R 4 R 4 -P(O)R 4 R 5 or -C(O)R 7 ;

[0014] .

[0015] More preferably, the functionalized oxime esters are as shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5;

[0016]

[0017] In Equation 1, R 4 It consists of a series of C1-C18 alkoxy groups of varying lengths, including: -OMe, -OEt, -OiPr, -OC4H9 to OC. 18 H 37 ;

[0018] In Equation 2, R 4 It consists of a series of C1-C18 alkoxy groups of varying lengths, including: -OMe, -OEt, -OiPr, -OC4H9 to OC. 18 H 37 ;R 5 It can be a series of aryl groups with different substitutions and varied structures, or a series of alkyl groups with different lengths;

[0019] In Equation 3, R 4 It is a series of structurally variable benzyl groups with different substitutions, wherein R 6 These are substituents at different positions, including: -H, -alkoxyl, -alkyl, -halogen, -amino, -ester;

[0020] In Equation 4, R 7These are a series of C1-C18 alkyl groups of varying lengths with diverse structures, including: -Me, -Et, - i Pr, -C4H9 to -C 18 H 37 ;

[0021] In Equation 5, R 7 It consists of a series of differently substituted benzoyl groups with varying structures, where R 8 These are substituents at different positions, including: -H, -alkoxyl, -alkyl, -halogen, -amino, -alkenyl, -alkynyl, -azide, fluorescent molecule substituents, and drug molecule groups.

[0022] Preferably, the photosensitizer includes at least one of fac-Ir(ppy)3, Ir[dF(CF3)ppy]2(dtbbpy)PF6, Eosin Y, Ru(phen)3Cl2, and 4-CzIPN.

[0023] Preferably, the reaction solution comprises: a buffer solution with pH 6-9 and a co-solvent; the volume ratio of the co-solvent is 2-50%;

[0024] The buffers include at least one of the following: PBS buffer, PB buffer, Tris HCl buffer, TES buffer, HEPES buffer, NaPi buffer, and Sorensen buffer.

[0025] The co-solvents include at least one of acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0026] Preferably, the molar ratio of substrate to labeling reagent is 1:1.05-1.25; the molar ratio of substrate to photosensitizer is 1:0.005-0.015.

[0027] Preferably, the concentration of the polypeptide is 2-80 mM, the concentration of the protein is 0.1-0.5 μM, the reaction temperature is 20-30℃, the reaction time is 1-10 h, and the light irradiation is visible light at 390-500 nm.

[0028] The second aspect of the present invention is to provide a tryptophan C2-functionalized polypeptide derivative, which is prepared by the above modification method and has the structural formula shown in formula (Ⅲ);

[0029]

[0030] Among them, -R 3 Selected from -P(O)R 4 R 4 -P(O)R4 R 5 or -C(O)R 7 .

[0031] Preferably, the structural formula is as shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5;

[0032]

[0033] In Equation 1, R 4 It consists of a series of C1-C18 alkoxy groups of varying lengths, including: -OMe, -OEt, -OiPr, -OC4H9 to OC. 18 H 37 ;

[0034] In Equation 2, R 4 It consists of a series of C1-C18 alkoxy groups of varying lengths, including: -OMe, -OEt, -OiPr, -OC4H9 to OC. 18 H 37 ;R 5 It can be a series of aryl groups with different substitutions and varied structures, or a series of alkyl groups with different lengths;

[0035] In Equation 3, R 4 It is a series of structurally variable benzyl groups with different substitutions, wherein R 6 These are substituents at different positions, including: -H, -alkoxyl, -alkyl, -halogen, -amino, -ester;

[0036] In Equation 4, R 7 These are a series of C1-C18 alkyl groups of varying lengths with diverse structures, including: -Me, -Et, - i Pr, -C4H9 to -C 18 H 37 ;

[0037] In Equation 5, R 7 It consists of a series of differently substituted benzoyl groups with varying structures, where R 8 These are substituents at different positions, including: -H, -alkoxyl, -alkyl, -halogen, -amino, -alkenyl, -alkynyl, -azide, fluorescent molecule substituents, and drug molecule groups.

[0038] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0039] (1) This invention provides a method for achieving tryptophan C2 phosphonylation or acylation in an aqueous buffer solution system. This method is green and efficient, requires no additional redox reagents, is mild and easy to operate, has high labeling efficiency and good site selectivity, and is compatible with 19 natural amino acid residues. It is suitable for the selective derivatization of complex peptides, peptide drugs and proteins.

[0040] (2) This invention utilizes photochemical synthesis technology to generate phosphorus-containing free radicals or carbon-centered free radicals under mild conditions, and efficiently captures the above free radicals through the double bond of the tryptophan indole ring. Subsequently, selective modification of tryptophan residues is achieved through a re-aromatication process. This method has good substrate applicability and can efficiently achieve phosphonylation or acylation modification of tryptophan-containing peptides or proteins, with product yields reaching 36%-88%.

[0041] (3) The method of the present invention has mild reaction conditions, low cost and simple operation. At the same time, the substrate of the present invention has wide applicability, good reaction selectivity and high efficiency, and can be applied to large-scale industrial production. Attached Figure Description

[0042] Figure 1 This is a synthetic route diagram of the present invention;

[0043] Figure 2 The product 3a of Example 1 in this invention 1 H nuclear magnetic resonance (H nuclear magnetic resonance) 1 H-NMR spectrum;

[0044] Figure 3 The product 3a of Example 1 in this invention 13 C nuclear magnetic resonance (C 13 C-NMR spectrum;

[0045] Figure 4 The product 3a of Example 1 in this invention 31 P nuclear magnetic resonance (P nuclear magnetic resonance) 31 P-NMR spectrum;

[0046] Figure 5 The HPLC and ESI-MS spectra of product 3u from Example 21 of this invention are shown below.

[0047] Figure 6 The HPLC and ESI-MS spectra of product 3v from Example 22 of this invention are shown below.

[0048] Figure 7 The HPLC and ESI-MS spectra of product 3w from Example 23 of this invention are shown below.

[0049] Figure 8The HPLC and ESI-MS spectra of product 3x from Example 24 of this invention are shown below.

[0050] Figure 9 The HPLC and ESI-MS spectra of product 3y from Example 25 of this invention are shown. Detailed Implementation

[0051] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0053] This invention provides a method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, comprising the following steps:

[0054] S1. Add photosensitizer, tryptophan-containing polypeptide or protein, and functionalized oxime ester to the reaction solvent. After evacuation, stir the reaction for 1-10 hours under light, room temperature, and inert gas protection to obtain the reaction solution.

[0055] S2. After the reaction is complete, the reaction solution is extracted, separated and purified, and then evaporated to dryness under reduced pressure to obtain a tryptophan C2-phosphonylated / acylated polypeptide derivative.

[0056] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0057] Example 1

[0058] This embodiment provides a method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0059] 150.6 mg (0.3 mmol) of tryptophan-containing polypeptide substrate 1a, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3a in 72% yield.

[0060] Large-scale reaction: 1.506 g (3.0 mmol) of tryptophan-containing polypeptide substrate, 0.721 g (2.0 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 13.1 mg (0.02 mmol) of tris(2-phenylpyridine)iridium were added to a dried 50 mL flask. The system was purged three times with argon gas, and then 16.0 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, and the reaction was monitored by TLC. The reaction was complete in 9 h. The crude product was extracted with ethyl acetate (150 mL × 3), and the organic phases were combined, washed with saturated brine (200 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3a in 65% yield.

[0061] The synthesis path is as follows:

[0062]

[0063] Product 3a obtained in Example 1 1 H NMR spectrum, 13 C NMR and 31 The P NMR spectra are attached. Figure 2-4 As shown, the identification data is as follows:

[0064] 1H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.63 (br, 1H), 7.77 (d, J = 7.8Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.18 (t, J = 7.3Hz, 1H), 6.98 (br, 1H), 6.94 (br, 1H), 6.74 (br, 1H), 4.52-4.51 (m, 1H),4.46-4.40 (m, 1H), 4.28-4.06 (m, 5H), 3.71 (s, 3H), 3.58 (d, J = 14.3 Hz,1H), 3.39 (t, J = 12.0 Hz, 1H), 1.67-1.57 (m, 3H), 1.38-1.35 (m, 6H), 1.32(s, 9H), 1.18 (d, J = 6.3 Hz, 3H), 0.91 (t, J = 6.2 Hz, 6H).

[0065] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 173.1, 172.1, 156.7, 137.6 (d,J = 11.8 Hz), 127.6 (d, J = 16.2 Hz), 125.7, 123.3 (d, J = 18.9 Hz), 121.0(d, J = 216.7 Hz), 120.9, 120.7, 111.9, 80.1, 63.1 (d, J = 4.4 Hz), 63.0 (d,J = 4.6 Hz), 56.8, 52.3, 51.0, 49.1, 41.1, 30.3, 29.8, 28.3, 26.7, 24.9,22.9, 22.0, 17.5, 16.5 (d, J = 6.2 Hz), 16.4 (d, J = 5.5 Hz).

[0066] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.2.

[0067] HRMS-ESI: calcd. for C 30 H 48 N4O9P + [M+H]+ 639.3154, found 639.3128.

[0068] Example 2

[0069] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0070] 112.5 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3b in 68% yield.

[0071] The synthesis path is as follows:

[0072]

[0073] The identification data of product 3b obtained in Example 2 are as follows:

[0074] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 9.00 (br, 1H), 7.76 (d, J = 7.9Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.15 (t, J = 7.1Hz, 1H), 7.09 (br, 1H), 6.52 (br, 1H), 4.42-4.37 (m, 1H), 4.28-4.07 (m, 4H), 4.05-3.96 (m, 2H), 3.71 (s, 3H), 3.59 (dd, J = 11.6 Hz, J = 3.6 Hz, 1H), 3.34(t, J = 12.3 Hz, 1H), 1.36-1.32 (m, 6H), 1.30-1.13 (m, 9H).

[0075] 13C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.6, 170.1, 156.1, 137.7 (d,J = 11.9 Hz), 127.5 (d, J = 16.3 Hz), 125.4, 123.3 (d, J = 18.6 Hz), 120.9(d, J = 216.8 Hz), 120.7, 120.5, 111.9, 79.8, 62.9 (d, J = 5.1 Hz), 56.2,52.3, 41.3, 38.8, 28.3, 27.3, 23.8, 23.1, 16.5 (d, J = 4.7 Hz), 16.4 (d, J = 4.7 Hz).

[0076] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.3, 11.0 (dr 85:15).

[0077] HRMS-ESI: calcd. for C 23 H 35 N3O8P + [M+H] + 512.2157, found 512.2182.

[0078] Example 3

[0079] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0080] 137.7 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3c in 88% yield.

[0081] The synthesis path is as follows:

[0082]

[0083] The identification data of product 3c obtained in Example 3 are as follows:

[0084] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 9.46 (br, 1H), 7.73 (d, J = 8.6Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 7.3 Hz, 1H), 7.21 (d, J = 8.2Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 6.59 (d, J = 6.1 Hz, 1H), 4.42-4.35 (m,2H), 4.24-4.06 (m, 4H), 3.55 (d, J = 14.3 Hz, 1H), 3.36 (t, J = 13.3 Hz, 1H), 2.14-2.08 (m, 1H), 1.43 (s, 9H), 1.34-1.30 (m, 6H), 1.27 (s, 9H), 0.89 (t, J= 7.5 Hz, 6H).

[0085] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.3, 170.6, 169.1, 156.1,137.8 (d, J = 12.9 Hz), 133.0, 130.5, 130.1, 128.3, 127.4 (d, J = 15.9 Hz), 125.1, 123.1 (d, J = 19.9 Hz), 120.8 (d, J = 216.2 Hz), 120.4 (d, J = 8.3Hz), 120.0, 112.0, 81.6, 79.5, 62.9 (d, J = 3.3 Hz), 62.8 (d, J = 5.4 Hz),31.5, 28.3, 28.0, 26.9, 18.8, 17.7, 16.5 (d, J = 4.7 Hz), 16.4 (d, J = 4.7Hz).

[0086] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.7, 11.2 (dr 85:15).

[0087] HRMS-ESI: calcd. for C 29 H 47 N3O7P + [M+H] + 596.3696, found 596.3696.

[0088] Example 4

[0089] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0090] 133.8 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the product 3d was obtained, with a yield of 43%.

[0091] The synthesis path is as follows:

[0092]

[0093] The identification data of the product obtained in Example 4 at 3 days are as follows:

[0094] 1H NMR (500 MHz, CDCl3, 300K): δ (ppm) 9.05 (br, 1H), 7.75 (d, J = 7.8Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.15 (t, J = 7.3Hz, 1H), 6.53 (br, 2H), 5.79 (br, 1H), 4.65-4.55 (m, 1H), 4.41-4.34 (m, 1H),4.23-4.13 (m, 4H), 3.70 (s, 2.7H), 3.64 (s, 0.3H), 3.57-3.47 (m, 1H), 3.37(t, J = 11.6 Hz, 1H), 2.28-2.15 (m, 3H), 1.97-1.87 (m, 1H), 1.39-1.27 (m,14.4H), 1.17-1.12 (m, 0.6H).

[0095] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 175.0, 172.9, 172.1, 156.1,137.8 (d, J = 12.4 Hz), 127.5 (d, J = 16.8 Hz), 125.4, 122.9 (d, J = 18.5Hz), 121.0 (d, J = 219.4 Hz), 120.8, 120.4, 112.1, 79.9, 63.0 (d, J = 5.1Hz), 56.4, 52.6, 51.7, 31.7, 28.8, 28.3, 27.3, 16.4 (t, J = 7.0 Hz).

[0096] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.4, 11.3 (dr 90:10).

[0097] HRMS-ESI: calcd. for C 26 H 40 N4O9P + [M+H] + 583.2528, found 583.2522.

[0098] Example 5

[0099] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0100] 129.6 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with blue LED light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3e in 51% yield.

[0101] The synthesis path is as follows:

[0102]

[0103] The identification data of product 3e obtained in Example 5 are as follows:

[0104] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 9.56 (br, 1H), 7.76 (d, J = 8.2Hz, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.28 (t, J = 7.2Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 6.53 (d, J = 4.7 Hz, 1H), 6.08 (br, 1H), 5.96 (br, 1H), 4.78-4.74 (m, 1H), 4.40-4.36 (m, 1H), 4.23-4.08 (m, 4H), 3.68(s, 3H), 3.50 (d, J = 13.1 Hz, 1H), 3.40 (t, J = 10.1 Hz, 1H), 2.78-2.74 (m,1H), 2.53-2.49 (m, 1H), 1.35-1.18 (m, 15H).

[0105] 13C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.5, 172.4, 171.4, 156.0,137.9 (d, J = 12.6 Hz), 127.6 (d, J = 16.5 Hz), 125.2, 122.1 (d, J = 18.4Hz), 121.1 (d, J = 216.9 Hz), 120.6, 120.5, 112.2, 79.8, 63.0 (d, J = 4.9Hz), 56.3, 52.7, 49.1, 37.3, 28.3, 27.1, 16.4 (d, J = 3.1 Hz), 16.3 (d, J =3.1 Hz).

[0106] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.6, 11.2 (dr 85:15).

[0107] HRMS-ESI: calcd. for C 25 H 38 N4O9P + [M+H] + 569.2371, found 569.2390.

[0108] Example 6

[0109] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0110] 134.7 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3f, with a yield of 51%.

[0111] The synthesis path is as follows:

[0112]

[0113] The identification data of product 3f obtained in Example 6 are as follows:

[0114] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.96 (br, 1H), 7.75 (d, J = 8.3Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.24 (d, J = 7.8Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 6.59 (d, J = 5.9 Hz, 1H), 4.67 (dd, J =7.2 Hz, J = 5.5 Hz, 1H), 4.37-4.33 (m, 1H), 4.28-4.09 (m, 4H), 3.70 (s, 3H), 3.54 (d, J = 14.8 Hz, 1H), 3.35 (t, J = 12.0 Hz, 1H), 2.47-2.37 (m, 2H), 2.12-2.08 (m, 1H), 2.05 (s, 3H), 1.98-1.91 (m, 1H), 1.36-1.32 (m, 6H), 1.30(s, 7.65H), 1.12(s, 1.35H).

[0115] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.3, 172.1, 156.1, 137.7 (d,J = 11.7 Hz), 127.5, 127.4, 125.4, 123.2 (d,J = 18.5 Hz), 121.0 (d, J =218.8 Hz), 120.6 (d, J = 27.6 Hz), 111.9, 79.7, 62.9 (d, J = 5.2 Hz), 56.2,52.5, 51.5, 31.9, 29.8, 28.3, 27.1, 16.5 (d, J = 4.7 Hz), 16.4 (d, J = 4.7Hz), 15.4.

[0116] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.4, 10.9 (dr 85:15).

[0117] HRMS-ESI: calcd. for C 26 H 41 N3O8PS + [M+H] + 586.2347, found 586.2348.

[0118] Example 7

[0119] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0120] 1 g (126.3 mg, 0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with blue LED light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, evaporated under reduced pressure, and 3 g of product was obtained, with a yield of 62%.

[0121] The synthesis path is as follows:

[0122]

[0123] The identification data for 3g of the product obtained in Example 7 are as follows:

[0124] 1H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.92 (br, 0.4H), 8.72 (br,0.6H), 7.78 (d, J = 8.8 Hz, 0.6H), 7.62 (d, J = 8.8 Hz, 0.4H), 7.41 (d, J =8.2 Hz, 0.6H), 7.38 (d, J = 8.6 Hz, 0.4H), 7.32 (t, J = 7.7 Hz, 1H), 7.16-7.03 (m, 3H), 5.33 (s, 0.6H), 4.78 (s, 1H), 4.62 (s, 0.4H), 4.42-4.08 (m,4H), 3.69 (s, 1.8H), 3.65 (s, 1.2H), 3.50-2.77 (m, 4H), 1.45 (s, 9H), 1.37-1.25 (m, 7H).

[0125] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.5, 172.0, 170.7, 170.6,156.2, 155.8, 137.7 (d, J = 12.7 Hz), 136.4, 127.9, 127.5 (d, J = 17.6 Hz),125.6, 123.9, 123.2, 122.0, 120.9, 119.7 (d, J = 222.0 Hz), 119.5, 112.0,111.5, 80.2, 80.0, 63.1 (d, J = 4.8 Hz), 56.4, 55.2, 52.7 (d, J = 3.2 Hz),52.1, 51.6, 40.4, 40.1, 28.5, 28.4, 28.1, 27.2, 16.4 (d, J = 6.5 Hz), 16.3(d, J = 6.9 Hz).

[0126] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.3, 11.0 (dr 85:15).

[0127] HRMS-ESI: calcd. for C 24 H 37 N3O8PS + [M+H] +558.2034, found 558.2030.

[0128] Example 8

[0129] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0130] 209.1 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the product was separated after 3 h, with a yield of 64%.

[0131] The synthesis path is as follows:

[0132]

[0133] The identification data of the product obtained in Example 8 after 3 hours are as follows:

[0134] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.71 (br, 1H), 7.81-7.78 (m,1H), 7.38-7.28 (m, 12H), 7.16-7.00 (m, 8H), 6.51 (br, 1H), 6.33 (br, 1H), 4.82-4.74 (m, 1H), 4.43-4.32 (m, 1H), 4.22-4.08 (m, 4H), 3.57 (s, 3H), 3.52(d, J = 15.4 Hz, 1H), 3.32 (t, J = 12.2 Hz, 1H), 3.01-2.97 (m, 1H), 2.87-2.85(m, 1H), 1.37-1.13 (m, 15H).

[0135] 13C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.2, 171.7, 155.7, 142.4,138.6, 137.6 (d, J = 12.4 Hz), 136.6, 129.9, 128.2, 127.7 (d, J = 15.5 Hz),125.3, 123.0, 121.2 (d, J = 226.6 Hz), 120.8, 120.6, 119.7, 111.8, 79.3,75.4, 62.92 (d, J = 4.3 Hz), 62.85 (d, J = 4.5 Hz), 56.3, 52.7, 52.1, 30.3,29.8, 28.4, 27.9, 27.6, 26.4, 16.4 (t, J = 6.4 Hz).

[0136] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm)11.3, 11.0 (dr 80:20).

[0137] HRMS-ESI: calcd. for C 46 H 53 N5O8P + [M+H] + 834.3627, found 834.3612.

[0138] Example 9

[0139] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0140] 188.4 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3i, with a yield of 46%.

[0141] The synthesis path is as follows:

[0142]

[0143] The identification data of product 3i obtained in Example 9 are as follows:

[0144] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 9.80 (br, 1H), 8.18 (br,1H), 7.76 (d, J = 6.7 Hz, 2H), 7.63 (d, J = 7.5 Hz, 1H), 7.54 (br, 1H), 7.30 (t, J= 7.3 Hz, 1H), 7.23 (d, J = 7.1 Hz, 2H), 7.14 (t, J = 6.8 Hz, 1H), 6.33-6.01(m, 3H), 5.60 (d, J = 6.1 Hz, 1H), 4.81-4.59 (m, 1H), 4.35-4.20 (m, 2H),4.18-3.96 (m, 3H), 3.71 (s, 2.7H), 3.66 (s, 0.3H), 3.53 (d, J = 13.9 Hz, 1H), 3.37 (t, J = 13.0 Hz, 1H), 3.22-3.11 (m, 1H), 3.06-2.95 (m, 1H), 2.39 (s,3H), 1.62-1.28 (m, 19H).

[0145] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.5, 172.2, 157.0, 155.5,141.9, 141.2, 137.8 (d, J = 12.3 Hz), 129.3, 127.0 (d, J = 15.9 Hz), 126.0,125.5, 121.3 (d, J = 216.9 Hz), 120.7, 119.4, 112.9, 79.8, 63.5 (d, J = 5.2Hz), 63.3 (d, J = 5.9 Hz), 53.5, 52.6, 40.0, 29.8, 28.4, 26.9, 24.7, 21.5,16.4 (d, J = 6.0 Hz), 16.2 (d, J = 6.5 Hz).

[0146] 31P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.0.

[0147] HRMS-ESI: calcd. for C 34 H 50 N6O 10 PS + [M+H] + 765.3042, found 765.3032.

[0148] Example 10

[0149] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0150] 1 J of 229.5 mg (0.3 mmol) tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3J, with a yield of 40%.

[0151] The synthesis path is as follows:

[0152]

[0153] The identification data of product 3j obtained in Example 10 are as follows:

[0154] 1H NMR (500 MHz, CDCl3, 300K): δ (ppm) 9.08 (br, 1H), 7.72 (d, J = 8.2Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.39 (d, J = 7.7 Hz, 1H), 7.32 (t, J = 8.1Hz, 1H), 7.16 (t, J = 7.2 Hz, 1H), 7.12 (br, 1H), 6.93 (br, 1H), 6.91 (br,1H), 4.59-4.53 (m, 1H), 4.36 (dd, J = 5.6 Hz, J = 4.5 Hz, 1H), 4.27-4.07 (m,6H), 3.63-3.54 (m, 2H), 3.38 (dd, J = 10.1 Hz, J = 3.9 Hz, 1H), 2.35-2.19 (m,2H), 1.48 (s, 9H), 1.40 (d, J = 7.5 Hz, 3H), 1.36 (t, J = 6.8 Hz, 7H), 1.32(s, 9H), 1.26 (d, J = 13.9 Hz, 2H), 0.95 (t, J = 5.7 Hz, 6H).

[0155] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 173.3, 172.2, 170.8, 168.8,157.0, 137.8 (d, J = 11.9 Hz), 127.4 (d, J = 15.8 Hz), 126.3, 125.7, 124.6,122.6 (d, J = 17.8 Hz), 121.2 (d, J = 216.8 Hz), 120.9, 120.4, 112.1, 81.7,80.4, 63.1 (d, J = 5.1 Hz), 63.0 (d, J = 5.6 Hz) 58.1, 57.6, 49.2, 43.4,31.5, 31.2, 30.3, 29.8, 28.4, 28.1, 26.6, 19.1, 18.0, 17.7, 16.4 (d, J = 3.3Hz), 16.4 (d, J = 3.6 Hz).

[0156] 31P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.3.

[0157] HRMS-ESI: calcd. for C 37 H 61 N5O 10 P + [M+H] + 766.4151, found 766.4182.

[0158] Example 11

[0159] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0160] 159.6 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 66.6 mg (0.2 mmol) of (Z)-(((benzoyloxy)imino)(phenyl)methyl)phosphonate dimethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3K, with a yield of 70%.

[0161] The synthesis path is as follows:

[0162]

[0163] The identification data of product 3k obtained in Example 11 are as follows:

[0164] 1H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.72 (br, 1H), 7.68 (d, J = 8.2Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.07 (t, J = 7.1Hz, 2H), 6.64 (br, 1H), 6.59 (br, 1H), 4.40-4.39 (m, 1H), 4.32 (d, J = 7.1Hz, 1H), 4.31-3.23 (m, 1H), 3.74-3.71 (m, 6H), 3.62 (s, 3H), 3.54 (s, 3H),3.46 (d, J = 13.6 Hz, 1H), 3.29 (t, J = 9.4 Hz, 1H), 2.37-2.23 (m, 2H), 2.13-2.07 (m, 1H), 1.99-1.88 (m, 1H), 1.21 (s, 9H), 1.07 (d, J = 5.6 Hz, 3H).

[0165] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 173.4, 172.3, 172.1, 171.9,156.5, 137.8 (d, J = 13.5 Hz), 127.6 (d, J = 16.2 Hz), 125.8, 123.7 (d, J =17.3 Hz), 121.2 (d, J = 216.5 Hz), 120.9, 120.7, 112.0, 80.1, 56.6, 53.28,53.23, 52.6, 51.9, 49.1, 30.1, 29.8, 28.1, 27.0, 17.6.

[0166] 31 P NMR (162 MHz, CDCl3 , 300K): δ (ppm) 14.2.

[0167] HRMS-ESI: calcd. for C 28 H 42 N4O 11 P + [M+H] + 641.2583, found 641.2578.

[0168] Example 12

[0169] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0170] 1 L of 134.1 mg (0.3 mmol) tryptophan-containing polypeptide substrate, 77.8 mg (0.2 mmol) (Z)-(((benzoyloxy)imino)(phenyl)methyl)phosphonate diisopropyl ester, and 1.31 mg (0.002 mmol) tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, evaporated under reduced pressure, and 3 L of product was obtained, with a yield of 75%.

[0171] The synthesis path is as follows:

[0172]

[0173] The identification data of product 3l obtained in Example 12 are as follows:

[0174] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 9.04 (br, 1H), 7.76 (d, J = 8.4Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 7.4 Hz, 1H), 7.30 (t, J = 8.1Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 6.74 (br, 1H), 4.84-4.74 (m, 3H), 4.36-4.32 (m, 1H), 3.71 (s, 3H), 3.64 (s, 3H), 3.57-3.52 (m, 1H), 3.38-3.33 (m, 1H), 2.90 (dd, J = 4.5 Hz, J = 12.3 Hz, 1H), 2.65 (dd, J = 4.8 Hz, J = 11.6Hz, 1H), 1.41 (t, J = 6.0 Hz, 6H), 1.32 (s, 9H), 1.27 (d, J = 6.1 Hz, 3H),1.20 (d, J = 6.1 Hz, 3H).

[0175] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.4, 171.09, 171.04, 156.1,137.6 (d, J = 11.8 Hz), 127.5 (d, J = 16.2 Hz), 125.2, 122.5 (d, J = 218.1Hz), 122.0 (d, J = 17.5 Hz), 120.6, 120.5, 111.9, 79.7, 72.2 (d, J = 5.5 Hz), 72.0 (d, J = 3.8 Hz), 56.3, 52.7, 51.9, 48.6, 34.3, 36.2, 28.3, 28.0, 26.7,24.0 (d, J = 4.5 Hz), 24.1 (dd, J = 4.3 Hz), 24.0 (d, J = 4.6 Hz), 23.8 (d, J= 4.7 Hz).

[0176] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 9.0, 8.6 (dr 85:15).

[0177] HRMS-ESI: calcd. for C 28 H 43 N3O 10 P + [M+H] + 612.2681, found 612.2702.

[0178] Example 13

[0179] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0180] 1 mL of 129.3 mg (0.3 mmol) tryptophan-containing polypeptide substrate, 94.6 mg (0.2 mmol) (Z)-(((benzoyloxy)imino)(phenyl)methyl)phosphonate dihexyl ester, and 1.31 mg (0.002 mmol) tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the product 3 mL was obtained, with a yield of 49%.

[0181] The synthesis path is as follows:

[0182]

[0183] The identification data of product 3m obtained in Example 13 are as follows:

[0184] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 9.03 (br, 0.1H), 8.82 (br,0.9H), 8.16-8.11 (m, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H),7.31 (t, J = 8.2 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 5.21 (d, J = 8.9 Hz, 1H),4.66-4.64 (m, 1H), 4.26-4.22 (m, 1H), 4.14-3.97 (m, 4H), 3.69 (s, 3H), 3.51-3.42 (m, 2H), 1.78-1.71 (m, 3H), 1.69-1.61 (m, 2H), 1.41-1.38 (m,3H), 1.35(s, 9H), 1.32-1.29 (m, 7H), 1.24-1.21 (m, 4H), 0.91-0.82 (m, 12H).

[0185] 13C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.2, 171.9, 155.5, 137.6 (d,J = 11.5 Hz), 130.1, 128.4, 127.3 (d, J = 15.3 Hz), 125.4, 121.4 (d, J =215.5 Hz), 120.8, 119.9, 112.1, 79.1, 67.4 (d, J = 5.1 Hz), 66.9 (d, J = 5.4Hz), 58.7, 54.0, 52.3, 38.7, 31.4, 31.3, 30.5 (d, J = 6.4 Hz), 30.3 (d, J =6.6 Hz), 28.4, 26.7, 25.3, 25.25, 25.18, 24.3, 22.60, 22.56, 15.2, 14.1,14.0, 11.7.

[0186] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.6, 11.2 (dr 90:10).

[0187] HRMS-ESI: calcd. for C 35 H 59 N3O8P + [M+H] + 680.4035, found 680.4021.

[0188] Example 14

[0189] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0190] 129.3 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 78.6 mg (0.2 mmol) of (Z)-(((benzoyloxy)imino)(phenyl)methyl)(phenyl)phosphine ethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3n in 35% yield.

[0191] The synthesis path is as follows:

[0192]

[0193] The identification data of product 3n obtained in Example 14 are as follows:

[0194] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.84 (d, J =4.5 Hz, 1H), 8.63(br, 1H), 7.78-7.74 (m, 2H), 7.71 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 7.4 Hz,1H), 7.44-7.40 (m, 3H), 7.31 (t, J = 8.0 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 5.33 (d, J = 9.4 Hz, 1H), 4.63-4.53 (m, 1H), 4.29-4.13 (m, 3H), 3.76 (s, 3H), 3.44 (d, J = 13.8 Hz, 1H), 3.35 (t, J = 13.8 Hz, 1H), 2.07-2.06 (m, 1H), 1.84(s, 1H), 1.45 (t, J = 6.9 Hz, 3H), 1.36 (s, 9H), 1.13 (d, J = 6.3 Hz, 0.4H), 0.97 (d, J = 6.2 Hz, 2.6H), 0.90 (t, J = 6.7 Hz, 3H).

[0195] 13C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.3, 172.2, 155.5, 137.8 (d,J = 9.5 Hz), 132.8, 131.3, (d, J = 145.7 Hz), 131.1 (d, J = 11.5 Hz), 128.9(d, J = 13.6 Hz), 127.5 (d, J = 14.2 Hz), 125.6, 125.4, 124.1, 122.7 (d, J =16.7 Hz), 121.0, 120.0, 112.1, 178.9, 62.6 (d, J = 5.5 Hz), 58.7, 53.9, 52.4,32.9, 29.8, 28.5, 26.5, 24.4, 16.7 (d, J = 6.4 Hz), 15.3, 11.9.

[0196] 31 P NMR NMR (162 MHz, CDCl3, 300K): δ (ppm) 24.6.

[0197] HRMS-ESI: calcd. for C 31 H 43 N3O7P + [M+H] + 600.2834, found 600.2860.

[0198] Example 15

[0199] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0200] 136.8 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the product 3O was obtained, with a yield of 61%.

[0201] The synthesis path is as follows:

[0202]

[0203] The identification data of product 3o obtained in Example 15 are as follows:

[0204] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.98 (br, 1H), 7.72 (d, J = 8.1Hz, 1H), 7.67 (d, J = 6.4 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H), 7.30 (t, J = 7.7Hz, 1H), 7.17 (t, J = 7.3 Hz, 1H), 4.69-4.65 (m, 1H), 4.23-4.07 (m, 4H), 3.69(s, 3H), 3.49-3.40 (m, 2H), 2.18-2.04 (m, 2H), 1.57-1.47 (m, 2H), 1.37 (t, J= 7.1 Hz, 3H), 1.33 (t, J = 7.1 Hz, 3H), 1.25 (s, 15H), 1.19 (s, 9H), 0.87(t, J = 6.9 Hz, 3H).

[0205] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 173.8, 172.6, 137.6 (d, J =12.0 Hz), 127.3 (d, J = 15.9 Hz), 125.4, 121.9 (d, J = 18.8 Hz), 121.2 (d, J= 216.6 Hz), 120.8, 120.1, 112.1, 62.9 (d, J = 5.6 Hz), 62.8 (d, J = 5.5 Hz), 53.8, 52.3, 36.3, 32.0, 31.5, 30.3, 29.78, 29.76, 29.72, 29.6, 29.5, 29.4,29.4, 29.3, 26.7, 25.6, 22.8, 16.5 (d, J = 5.7 Hz), 16.3 (d, J = 6.5 Hz),14.2.

[0206] 31P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.0.

[0207] HRMS-ESI: calcd. for C 32 H 54 N2O6P + [M+H] + 593.3714, found 593.3723.

[0208] Example 16

[0209] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0210] 171.3 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3p in 44% yield.

[0211] The synthesis path is as follows:

[0212]

[0213] The identification data of product 3p obtained in Example 16 are as follows:

[0214] 1H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.83 (br, 1H), 7.77 (d, J = 8.3Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.33-7.28 (m, 1H), 7.16 (t, J = 8.7 Hz,1H), 7.13 (t, J = 8.2 Hz, 1H), 6.96-6.94 (m, 1H), 6.86(s, 1H), 6.67 (br, 1H),6.54 (br, 1H), 4.36-4.32 (m, 1H), 4.20-4.03 (m, 4H), 3.58-3.55 (m, 1H), 3.41-3.37 (m, 1H), 3.30 (t, J = 11.3 Hz, 1H), 2.93-2.78 (m, 4H), 2.23 (d, J = 12.6Hz, 1H), 2.01-1.96 (m, 1H), 1.89-1.81 (m, 1H), 1.77-1.66 (m, 2H), 1.64-1.59(m, 1H), 1.37-1.27 (m, 9H), 1.21-1.18 (m, 15H), 0.98-0.95 (m, 1H), 0.90 (s,3H), 0.87 (t, J = 7.0 Hz, 1H), 0.83-0.81 (m, 1H).

[0215] 13C NMR (125 MHz, CDCl3, 300K): δ (ppm) 172.4, 156.1, 147.2, 145.6,137.7 (d, J = 12.0 Hz), 134.8, 127.5 (d, J = 16.3 Hz), 126.9, 125.4, 124.4,123.9, 123.7 (d, J = 18.8 Hz), 120.9 (d, J = 215.8 Hz), 120.7, 120.6, 111.9,79.5, 62.9 (d, J = 6.0 Hz), 62.8 (d, J = 5.1 Hz), 55.5, 50.1, 46.1, 40.6,38.4, 37.7, 37.6, 37.5, 35.9, 33.6, 30.4, 28.4, 28.2, 27.4, 25.5, 24.1, 19.0,18.7, 18.4, 16.5 (d, J = 6.1 Hz), 16.3 (d, J = 6.4 Hz).

[0216] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.5, 11.4 (dr 85:15).

[0217] HRMS-ESI: calcd. for C 40 H 59 N3O6P + [M+H] + 708.4136, found 708.4128.

[0218] Example 17

[0219] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0220] 139.2 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 72.1 mg (0.2 mmol) of (Z)-((benzoyloxy)imino)(phenyl)methylphosphonic acid diethyl ester, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3q, with a yield of 61%.

[0221] The synthesis path is as follows:

[0222]

[0223] The identification data of product 3q obtained in Example 17 are as follows:

[0224] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.99 (br, 1H), 7.83 (d, J = 8.1Hz, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.24 (t, J = 7.5Hz, 2H), 7.19 (t, J = 7.5 Hz, 2H), 7.24 (d, J = 6.2 Hz, 1H), 4.78-4.75 (m,1H), 4.28-4.12 (m, 4H), 3.69 (d, J =13.4 Hz, 1H), 3.55 (t, J = 12.2 Hz, 1H),3.13-3.05 (m, 2H), 1.41-1.33 (m, 15H), 1.21 (d, J = 6.6 Hz, 12H).

[0225] 13C NMR (125 MHz, CDCl3, 300K): δ (ppm) 171.9, 155.8, 145.7, 140.8,137.6 (d, J = 12.3 Hz), 127.4 (d, J = 16.0 Hz), 126.7, 124.1, 121.72 (d, J =219.3 Hz), 121.69, 121.5 (d, J = 12.5 Hz), 121.0, 120.8, 120.2, 121.2, 79.5,63.1 (d, J = 5.4 Hz), 63.0 (d, J = 5.3 Hz), 55.3, 29.9, 28.4 (overlapped),28.0, 27.5, 27.3, 26.8, 23.9 (overlapped), 23.1 (overlapped), 16.4 (d, J =6.6 Hz, overlapped).

[0226] 31 P NMR (162 MHz, CDCl3, 300K): δ (ppm) 11.2, 10.9 (dr 90:10).

[0227] HRMS-ESI: calcd. for C 32 H 46 N2O7P + [M+H] + 601.3038, found 601.3041.

[0228] Example 18

[0229] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0230] 116.7 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 43.8 mg (0.2 mmol) of (Z)-2-(acetoxyimino)-1-(p-tolyl)prop-1-one, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with blue LED light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 3r, with a yield of 76%.

[0231] The synthesis path is as follows:

[0232]

[0233] The identification data of product 3r obtained in Example 18 are as follows:

[0234] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.82 (br, 1H), 7.89 (br, 1H), 7.71 (d, J = 7.7 Hz, 2H), 7.67 (d, J = 8.3 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H),7.27 (d, J = 7.1 Hz, 3H), 6.48 (d, J = 7.1 Hz, 1H), 5.25 (br, 1H), 4.46 (t, J= 7.0 Hz, 2H), 3.65 (s, 3H), 3.35-3.28 (m, 1H), 3.24-3.20 (m, 1H), 2.44 (s, 3H), 1.42 (s, 9H), 1.29 (d, J = 7.1 Hz, 3H).

[0235] 13 C NMR (125 MHz, CDCl3, 300K): δ (ppm) 197.2, 173.0, 171.2, 155.6,142.7, 136.0, 135.7, 131.9, 131.0, 130.4, 128.9, 127.2, 122.1, 118.3, 114.5,111.2, 80.3, 55.3, 52.6, 48.3, 28.4, 21.8, 18.4.

[0236] HRMS-ESI: calcd. for C 28 H 34 N3O6 + [M+H] + 508.2443, found 508.2465.

[0237] Example 19

[0238] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0239] 124.5 mg (0.3 mmol) of tryptophan-containing polypeptide substrate, 43.8 mg (0.2 mmol) of (Z)-2-(acetoxyimino)-1-(p-tolyl)prop-1-one, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with blue LED light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the product 3s was obtained, with a yield of 43%.

[0240] The synthesis path is as follows:

[0241]

[0242] The identification data of product 3s obtained in Example 19 are as follows:

[0243] 1H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.78 (br, 0.17H), 8.69 (br,0.83H), 7.75 (s, 1H), 7.58 (d, J = 7.8 Hz, 2H), 7.45 (d, J = 8.3 Hz, 0.83H),7.41 (t, J = 8.5 Hz, 0.17H), 7.13 (d, J = 7.7 Hz, 3H), 5.36 (d, J = 8.6 Hz,0.17H), 5.21 (d, J = 8.4 Hz, 0.83H), 5.02-4.93 (m, 0.17H), 4.65-4.64 (m,0.83H), 4.39-4.34 (m, 1H), 3.55 (s, 2.49H), 3.51 (s, 0.51H), 3.46-3.40 (m,1H), 3.17-3.13 (m, 1H), 3.03-3.01 (m, 2H), 2.30 (s, 3H), 2.01-2.00 (m, 1H),1.77-1.65 (m, 3H), 1.29 (s, 1.53H), 1.25 (s, 7.47H), 1.14-1.09 (m, 1H).

[0244] 13C NMR (125 MHz, CDCl3, 300K): δ (ppm) 197.2, 172.7, 172.0 (minor),171.3 (minor), 171.0, 155.4, 155.0 (minor), 142.7 (minor), 142.5, 136.2,136.0 (minor), 135.4, 132.0 (minor), 131.6, 131.5, 130.8 (minor), 130.38,130.35 (minor), 128.95 (minor), 128.89, 128.0, 127.1 (minor), 122.02 (minor),121.98, 118.4 (minor), 118.2, 114.5, 114.3(minor), 111.4 (minor), 110.5,79.8, 79.7 (minor), 59.0, 53.6, 52.8, 52.5, 52.3, 47.1, 46.3 (minor), 30.5,29.8 (minor), 29.2, 28.6(minor), 28.5, 28.4(minor), 25.0, 22.2(minor),21.7.

[0245] HRMS-ESI: calcd. for C 30 H 36 N3O6 + [M+H] + 534.2599, found 534.2608.

[0246] Example 20

[0247] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0248] 1 ton (147.9 mg, 0.3 mmol) of tryptophan-containing polypeptide substrate, 43.8 mg (0.2 mmol) of (Z)-2-(acetoxyimino)-1-(p-tolyl)prop-1-one, and 1.31 mg (0.002 mmol) of tris(2-phenylpyridine)iridium were added to a dried reaction flask. The system was purged three times with argon gas. Then, 1.6 mL of N,N-dimethylformamide was added under argon conditions. The mixture was stirred at room temperature, irradiated with blue LED light, and the reaction was monitored by TLC. The reaction was completed in 6.5 h. The crude product was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, evaporated under reduced pressure, and 3 tonnes of product were obtained, with a yield of 54%.

[0249] The synthesis path is as follows:

[0250]

[0251] The identification data of product 3t obtained in Example 20 are as follows:

[0252] 1 H NMR (500 MHz, CDCl3, 300K): δ (ppm) 8.53 (d, J = 13.5 Hz, 0.2H), 8.25 (d, J = 14.0 Hz, 0.8H), 7.79 (d, J = 7.9 Hz, 0.4H), 7.74 (s, 0.8H), 7.68(d, J = 7.7 Hz, 1.6H), 7.59-7.51 (m, 6H), 7.34-7.32 (m, 6H), 7.25 (d, J = 8.7Hz, 2H), 7.14 (s, 0.4H), 7.11 (s, 0.2H), 7.09 (s, 0.8H), 6.69 (d, J = 6.7 Hz,0.8H), 5.00-4.91 (m, 0.8H), 4.73-4.68 (m, 0.2H), 3.65 (s, 2.4H), 3.63 (s,0.6H), 3.37-3.20 (m, 2H), 3.16-3.10 (m, 1H), 3.06-2.97 (m, 0.8H), 2.85-2.67(m, 2H), 2.61-2.55 (m, 0.2H), 2.43 (s, 3H).

[0253] 13C NMR (125 MHz, CDCl3, 300K): δ (ppm) 198.6 (minor), 197.1, 172.6(minor), 172.3, 171.6 (minor), 171.1, 162.5 (minor), 162.4, 145.7, 145.4(minor), 144.3 (minor), 142.6, 137.1 (minor), 136.0, 135.8 (minor), 135.3,135.0 (minor), 134.9, 132.72 (minor), 132.66, 131.8, 131.4 (minor), 131.2,130.9, 130.3, 130.2 (minor), 129.2, 129.0 (minor), 128.92, 128.85, 128.81,128.76, 128.71, 128.67, 128.5 (minor), 128.3, 128.19, 128.16 (minor), 128.13,127.1 (minor), 126.5, 125.6, 125.0 (minor), 123.0 (minor), 121.9, 120.5(minor), 118.0, 114.7 (minor), 114.6, 111.5 (minor), 110.3, 54.5 (minor),53.1, 52.6, 52.4 (minor), 32.8, 32.7 (minor), 28.0 (minor), 27.5, 23.8, 23.7 (minor), 21.9 (minor), 21.7.

[0254] HRMS-ESI: calcd. for C 38 H 34 N3O5 + [M+H] + 612.2493, found 612.2505.

[0255] Example 21

[0256] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0257] 1 µL (2 mM, 1 equiv.) of tryptophan-containing polypeptide substrate, ethyl (Z)-(((benzoyloxy)imino)(phenyl)methyl)(phenyl)phosphinate (2.2 mM, 1.1 equiv.), and tris(2-phenylpyridine)iridium (0.002 mM, 1% equiv.) were added to a dried reaction flask. The system was purged three times with argon gas. Then, N,N-dimethylformamide and water were added under argon conditions, and the volume was adjusted to 250 µL. The mixture was stirred at room temperature, irradiated with blue LED light, and the reaction was monitored by TLC. The reaction was complete in 8 h. The product 3 µL was detected by reversed-phase high-performance liquid chromatography (RP-HPLC), with a yield of 76%. The phosphonylated daptomycin 3 µL exhibits stronger antibacterial activity. The method of this invention can be further applied to the selective modification of tryptophan-containing polypeptide drugs such as daptomycin. This invention also includes derivatives obtained by phosphonylation or acylation modification of daptomycin using the above method, their preparation methods, and subsequent applications in biological activity.

[0258] The synthesis path is as follows:

[0259]

[0260] Example 22

[0261] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0262] 1 v (2 mM, 1 equiv.) of tryptophan-containing polypeptide substrate, ethyl (Z)-(((benzoyloxy)imino)(phenyl)methyl)(phenyl)phosphine (4.4 mM, 2.2 equiv.), and tris(2-phenylpyridine)iridium (0.004 mM, 2% equiv.) were added to a dried reaction flask. The system was purged three times with argon gas. Then, N,N-dimethylformamide and water were added under argon atmosphere, and the volume was adjusted to 250 µL. The mixture was stirred at room temperature, irradiated with blue LED light, and the reaction was monitored by TLC. The reaction was complete in 8 h. Product 3 v was detected by reversed-phase high-performance liquid chromatography (RP-HPLC), with a yield of 76%.

[0263] The synthesis path is as follows:

[0264]

[0265] Example 23

[0266] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0267] 1 w (2 mM, 1 equiv.) of tryptophan-containing polypeptide substrate, ethyl (Z)-(((benzoyloxy)imino)(phenyl)methyl)(phenyl)phosphine (2.2 mM, 1.1 equiv.), and tris(2-phenylpyridine)iridium (0.002 mM, 1% equiv.) were added to a dried reaction flask. The system was purged three times with argon gas. Then, N,N-dimethylformamide and water were added under argon atmosphere, and the volume was adjusted to 250 µL. The mixture was stirred at room temperature, irradiated with blue LED light, and the reaction was monitored by TLC. The reaction was completed in 8 h. The product was detected by reversed-phase high-performance liquid chromatography (RP-HPLC), with a yield of 73%.

[0268] The synthesis path is as follows:

[0269]

[0270] Example 24

[0271] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0272] Add 1x (2 mM, 1 equiv.) of tryptophan-containing polypeptide substrate, (Z)-(((benzoyloxy)imino)(phenyl)methyl)(phenyl)phosphine ethyl ester (2.2 mM, 1.1 equiv.), and tris(2-phenylpyridine)iridium (0.002 mM, 1% equiv.) to a dried reaction flask. The system was purged three times with argon gas. Then, N,N-dimethylformamide and water were added under argon conditions, and the volume was adjusted to 250 µL. The mixture was stirred at room temperature, irradiated with LED blue light, and the reaction was monitored by TLC. The reaction was complete in 8 hours. The product 3x was detected by reversed-phase high-performance liquid chromatography (RP-HPLC), with a yield of 43%. The method of this invention can be further applied to the selective modification of tryptophan-containing polypeptide drugs such as lanreotide. This invention also includes derivatives obtained by phosphonylation or acylation modification of lanreotide using the above method, their preparation methods, and their bioactive applications.

[0273] The synthesis path is as follows:

[0274]

[0275] Example 25

[0276] This embodiment provides another method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, the steps of which include:

[0277] 1 y (2 mM, 1 equiv.) of tryptophan-containing polypeptide substrate, (Z)-(((benzoyloxy)imino)(phenyl)methyl)(phenyl)phosphine hexyl ester (2.2 mM, 1.1 equiv.), and tris(2-phenylpyridine)iridium (0.002 mM, 1% equiv.) were added to a dried reaction flask. The system was purged three times with argon gas. Then, N,N-dimethylformamide and water were added under argon conditions, and the volume was adjusted to 250 µL. The mixture was stirred at room temperature, irradiated with blue LED light, and the reaction was monitored by TLC. The reaction was complete in 4 h. The product 3y was detected by reversed-phase high-performance liquid chromatography integration, with a yield of 65%. The method of this invention can be further applied to the selective modification of tryptophan-containing polypeptide drugs such as exenatide. This invention also includes derivatives obtained by phosphonylation or acylation modification of exenatide using the above method, their preparation methods, and their bioactive applications.

[0278] The synthesis path is as follows:

[0279]

[0280] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for modifying tryptophan peptides or proteins using functionalized oxime esters as labeling sources, characterized in that, The steps include: Tryptophan-containing peptides or proteins were prepared by reacting them in a reaction solution in the presence of a photosensitizer, under inert gas protection and room temperature light conditions, using functionalized oxime esters as substrates and functionalized oxime esters as labeling reagents.

2. The method for modifying tryptophan peptides or proteins according to claim 1, characterized in that, Tryptophan-containing polypeptides or proteins are as shown in Formula (I), including but not limited to: protected-terminal polypeptides, free-terminal polypeptides, random-sequence natural polypeptides, polypeptide drugs, and functional proteins; 。 3. The method for modifying tryptophan peptides or proteins according to claim 1, characterized in that, Functionalized oxime esters are shown in formula (II), wherein -R 1 Selected from -aryl or -alkyl; -R 2 Selected from -aryl or -alkyl; -R 3 Selected from -P(O)R 4 R 4 -P(O)R 4 R 5 or -C(O)R 7 ; 。 4. The method for modifying tryptophan peptides or proteins according to claim 3, characterized in that, Functionalized oxime esters are shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5; In Equation 1, R 4 It consists of a series of C1-C18 alkoxy groups of varying lengths, including: -OMe, -OEt, -OiPr, -OC4H9 to OC. 18 H 37 ; In Equation 2, R 4 It consists of a series of C1-C18 alkoxy groups of varying lengths, including: -OMe, -OEt, -OiPr, -OC4H9 to OC. 18 H 37 ;R 5 It can be a series of aryl groups with different substitutions and varied structures, or a series of alkyl groups with different lengths; In Equation 3, R 4 It is a series of structurally variable benzyl groups with different substitutions, wherein R 6 These are substituents at different positions, including: -H, -alkoxyl, -alkyl, -halogen, -amino, -ester; In Equation 4, R 7 These are a series of C1-C18 alkyl groups of varying lengths with diverse structures, including: -Me, -Et, - i Pr, -C4H9 to -C 18 H 37 ; In Equation 5, R 7 It consists of a series of differently substituted benzoyl groups with varying structures, where R 8 These are substituents at different positions, including: -H, -alkoxyl, -alkyl, -halogen, -amino, -alkenyl, -alkynyl, -azide, fluorescent molecule substituents, and drug molecule groups.

5. The method for modifying tryptophan peptides or proteins according to claim 1, characterized in that, Photosensitizers include: At least one of fac-Ir(ppy)3, Ir[dF(CF3)ppy]2(dtbbpy)PF6, Eosin Y, Ru(phen)3Cl2, and 4-CzIPN.

6. The method for modifying tryptophan peptides or proteins according to claim 1, characterized in that, The reaction solution includes: a buffer solution with pH 6-9 and a co-solvent; the volume ratio of the co-solvent is 2-50%. The buffers include at least one of the following: PBS buffer, PB buffer, Tris HCl buffer, TES buffer, HEPES buffer, NaPi buffer, and Sorensen buffer. The co-solvents include at least one of acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

7. The method for modifying tryptophan peptides or proteins according to claim 1, characterized in that, The molar ratio of substrate to labeling reagent is 1:1.05-1.25; the molar ratio of substrate to photosensitizer is 1:0.005-0.

015.

8. The method for modifying tryptophan peptides or proteins according to claim 1, characterized in that, The concentration of polypeptides is 2-80 mM, and the concentration of protein is 0.1-0.5 μM; the reaction temperature is 20-30℃, the reaction time is 1-10 h, and the light conditions are visible light at 390-500 nm.

9. A tryptophan C2-functionalized polypeptide derivative, prepared by the modification method according to any one of claims 1-8, characterized in that, The structural formula is shown in equation (Ⅲ); Among them, -R 3 Selected from -P(O)R 4 R 4 -P(O)R 4 R 5 or -C(O)R 7 .

10. The tryptophan C2-functionalized polypeptide derivative according to claim 9, characterized in that, The structural formulas are shown in Equation 1, Equation 2, Equation 3, Equation 4 or Equation 5; In Equation 1, R 4 It consists of a series of C1-C18 alkoxy groups of varying lengths, including: -OMe, -OEt, -OiPr, -OC4H9 to OC. 18 H 37 ; In Equation 2, R 4 It consists of a series of C1-C18 alkoxy groups of varying lengths, including: -OMe, -OEt, -OiPr, -OC4H9 to OC. 18 H 37 ;R 5 It can be a series of aryl groups with different substitutions and varied structures, or a series of alkyl groups with different lengths; In Equation 3, R 4 It is a series of structurally variable benzyl groups with different substitutions, wherein R 6 These are substituents at different positions, including: -H, -alkoxyl, -alkyl, -halogen, -amino, -ester; In Equation 4, R 7 These are a series of C1-C18 alkyl groups of varying lengths with diverse structures, including: -Me, -Et, - i Pr, -C4H9 to -C 18 H 37 ; In Equation 5, R 7 It consists of a series of differently substituted benzoyl groups with varying structures, where R 8 These are substituents at different positions, including: -H, -alkoxyl, -alkyl, -halogen, -amino, -alkenyl, -alkynyl, -azide, fluorescent molecule substituents, and drug molecule groups.

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