Cysteine specific coupling nucleophilic reagent as well as preparation method and application thereof

Abstract

The invention discloses a cysteine specific coupling nucleophilic reagent as well as a preparation method and application thereof, and the cysteine specific coupling nucleophilic reagent comprises alpha / beta-ADPr-bromoethyl ether, alpha / beta-ADPr-bromopropyl ether and alpha / beta-ADPr-bromobutyl ether. The invention also discloses a preparation method of the coupling nucleophilic reagent and application of the coupling nucleophilic reagent in modification of cysteine and oligopeptide / polypeptide / protein containing cysteine. According to the invention, a substrate containing cysteine is used for carrying out an ADP-ribosylation modification coupling reaction, an ADP-ribosylation substrate is enriched, ADPr-Ub obtained by the ADP-ribosylation reaction can still be recognized by a corresponding antibody, and original ubiquitin characteristics are retained; the invention provides a new thought for obtaining a mono-ADP-ribosylation molecular tool and even a double-mono-ADP-ribosylation modified molecular tool for the continuous exploration of the ADP-ribosylation by researchers.

Description

A cysteine-specific conjugated nucleophilic reagent, its preparation method and application Technical Field

[0001] This invention belongs to the field of cysteine-specific coupling technology, specifically relating to a cysteine-specific coupling nucleophile reagent, its preparation method, and its application. Background Technology

[0002] Current research on the synthesis of mono-ADP-ribosylated peptides / proteins via proteases is limited. A mainstream method for obtaining mono-ADP-ribosylated peptides / proteins using different amino acid modification sites relies on total synthesis. However, this method suffers from lengthy synthetic steps, high operational complexity, and low yields. Current techniques for mono-ADP-ribosylation have significant shortcomings, such as low detection sensitivity, lack of site-specific tools, and difficulty in distinguishing between mono-ADP-ribosylation and poly-ADP-ribosylation, severely hindering the development of this field. Developing and enriching methods for mono-ADP-ribosylation could advance the discovery of new disease biomarkers, the development of highly selective drugs, and precise experimental efficacy assessment. This invention proposes a method for site-specific mono-ADP-ribosylation modification of cysteine ​​using a chemical approach. Cysteine ​​has extremely low abundance in proteins, ensuring site specificity, and its thiol group has high nucleophilicity. By chemically modifying NAD+ into an electrophilic group for coupling with cysteine, the "neighbor group participation" effect can be used to achieve efficient cysteine ​​linkage. However, there are currently no reports on this approach. Summary of the Invention

[0003] The technical problem solved by this invention is to provide a cysteine-specific coupling nucleophile reagent with mild reaction conditions and high reaction yield, and a method for preparing the same. The cysteine-specific coupling nucleophile reagent prepared by this method can be used to modify oligopeptides, polypeptides, or proteins containing cysteine.

[0004] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a cysteine-specific coupling nucleophile, comprising compounds with the following structures:

[0005] , , , , , .

[0006] The preparation method of the cysteine-specific conjugated nucleophilic reagent described in this invention specifically involves: β-nicotinamide adenine dinucleotide β-NAD... + The target product, cysteine, was prepared by reacting cysteine-specific coupling nucleophiles with bromoalkyl alcohols in a system containing the ionic liquid [TMG][BF4] at 80–100 °C with stirring. The corresponding reaction route for the preparation process is as follows:

[0007]

[0008] Where n is 1, 2 or 3.

[0009] Furthermore, the β-nicotinamide adenine dinucleotide β-NAD + The molar ratio of bromoalkyl alcohol compounds to ionic liquids [TMG][BF4] is 1:10~70:30~70.

[0010] Furthermore, the β-nicotinamide adenine dinucleotide β-NAD + The molar ratio of bromoalkyl alcohol compounds to ionic liquid [TMG][BF4] is 1:50:50, the preferred reaction temperature is 90℃, and the preferred reaction time is 6h.

[0011] The present invention relates to the application of the cysteine-specific coupling nucleophile reagent in the modification of cysteine, cysteine-containing oligopeptides, cysteine-containing polypeptides, or cysteine-containing proteins.

[0012] Furthermore, the specific process of modifying cysteine ​​with a cysteine-specific coupling nucleophile is as follows: ADPr-bromoalkyl alcohol, N-acetylcysteine, and reducing agent DTT are placed in a reaction flask, water is added as a solvent, the pH of the reaction system is adjusted to 6-9, and the reaction is stirred at 25-40℃ to achieve the coupling reaction between ADPr-bromoalkyl alcohol and N-acetylcysteine. The molar ratio of ADPr-bromoalkyl alcohol, N-acetylcysteine, and reducing agent DTT is 1:2 to 10:5, and the corresponding reaction route is as follows:

[0013]

[0014] Where n is 1, 2 or 3.

[0015] Furthermore, the specific process of modifying cysteine-containing oligopeptides with cysteine-specific coupling nucleophilic reagent is as follows: ADPr-bromoalkyl alcohol, cysteine-containing oligopeptides and reducing agent DTT are placed in a reaction flask, water is added as a solvent, the pH of the reaction system is adjusted to 6-9, and the reaction is stirred at 25-40℃ to finally achieve the coupling reaction between ADPr-bromoalkyl alcohol and cysteine-containing oligopeptides.

[0016] Furthermore, the specific reaction route for modifying cysteine-containing oligopeptides using cysteine-specific coupling nucleophilic reagents is as follows:

[0017] .

[0018] Furthermore, the specific process for modifying cysteine-containing proteins using the cysteine-specific coupling nucleophilic reagent is as follows: ADPr-bromoalkyl alcohol, the cysteine-containing polypeptide / protein, and the reducing agent DTT are placed in a reaction flask, water is added as a solvent, the pH of the reaction system is adjusted to 6-9, and the reaction is stirred at 25-40℃ to ultimately achieve the coupling reaction between ADPr-bromoalkyl alcohol and the cysteine-containing polypeptide / protein. The corresponding reaction route is as follows:

[0019] .

[0020] Furthermore, the amino acid sequences of the cysteine-containing polypeptides / proteins are shown in Sequence Listings SEQ No. 2 and SEQ No. 3, respectively.

[0021] Furthermore, the specific process for modifying cysteine-containing peptides using cysteine-specific coupling nucleophiles is as follows: ADPr-bromoalkyl alcohol, the cysteine-containing peptide, and the reducing agent DTT are placed in a reaction flask, water is added as a solvent, the pH of the reaction system is adjusted to 6-9, and the reaction is stirred at 25-40℃ to achieve the coupling reaction between ADPr-bromoalkyl alcohol and the cysteine-containing peptide. The corresponding reaction route using ADPr-bromoethanol and the peptide SRIF-14 (somatostatin-14) is as follows:

[0022] .

[0023] This invention offers the following advantages and benefits: By synthesizing cysteine-containing oligopeptides in the liquid phase and polypeptides in the solid phase, and expressing mutant proteins using *E. coli* prokaryotic cells, a series of cysteine-containing substrates were obtained. These substrates were then subjected to ADP-ribosylation modification under optimized conditions, enriching the ADP-ribosylation substrate pool. Subsequent Western blot experiments demonstrated that the ADPr-Ub obtained using this newly developed ADP-ribosylation reaction could still be recognized by corresponding antibodies (anti-Ub antibody and anti-ADPr antibody), retaining the original ubiquitin characteristics. This provides researchers with a new approach to obtaining molecular tools with single-ADP-ribosylation or even double-single-ADP-ribosylation modifications for continued exploration of ADP-ribosylation. In mass spectrometry-based proteomics, synthesizing well-defined dual-modified peptides is crucial for standards; these synthetic peptides serve as a key bridge between biochemical discoveries and the understanding of physiological and pathological functions. Attached Figure Description

[0024] Figure 1 shows the binding of ubiquitin and ADPr-Ub to anti-ubiquitin and anti-ADPr antibodies. Detailed Implementation

[0025] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0026] Example 1

[0027] Synthesis of α / β-ADPr-bromoethyl ether:

[0028] β-Nicotinamide adenine dinucleotide β-NAD + Under the conditions of ionic liquid [TMG][BF4], it reacts more readily with nucleophiles, thus facilitating the synthesis of NAD. + When using analogues, the ionic liquid [TMG][BF4] was directly selected as one of the reaction conditions. Next, the reaction was carried out on β-NAD... + Other conditions for the nucleophilic substitution reaction with bromoethanol were optimized.

[0029]

[0030] β-NAD + Nucleophilic substitution reaction route with bromoethanol

[0031] Under anhydrous and oxygen-free conditions, β-NAD +0.015 mmol (1 equiv) and different equivalences of bromoethanol were added to systems containing different equivalences of ionic liquid [TMG][BF4]. The resulting mixtures were stirred and heated with stirring. After the reaction was completed, the reaction system was extracted three times with water and dichloromethane, and then the aqueous phase was collected. The product was separated and purified using semi-preparative HPLC (C18, CH3CN / 0.1M TEAB=20 / 80, within 40 min, flow rate=3.0 mL / min, I=254 nm). The solvent was then removed using a freeze dryer to obtain a white powder product. The reaction conditions, yields, and α:β ratios (α-configuration:β-configuration) are shown in Table 1.

[0032] Table 1 β-NAD + Screening of reaction conditions and yield of nucleophilic substitution reaction with bromoethanol

[0033] Item bromine ethanol equivalent ionic liquid type and equivalent reaction time (h) reaction temperature (°C) yield (%) / α:β 110 [TMG][BF4] 5069032 / 1:1230 [TMG][BF4] 5069078 / 4:1350 [TMG][BF4] 5069094 / 6:1470 [TMG][BF4] 5069090 / 6:1550 [TMG][BF4] 7069092 / 6:1650 [TMG][BF4] 3069090 / 6:1750 [TMG][BF4] 5059085 / 6:1850 [TMG][BF4] 5068078 / 6:1950 [TMG][BF4] 50610075 / 6:1 surface

[0034]

[0035] Compound (4aa): 11H NMR (600 MHz, D2O) δ 8.53 (s, 1H), 8.28 (s, 1H), 6.15(d, J = 6.0 Hz, 1H), 5.01 (d, J = 2.4 Hz, 1H), 4.71 (t, J = 6.0 Hz, 1H), 4.55– 4.52 (m, 1H), 4.40 (t, J = 3.0 Hz, 1H), 4.24 – 4.21 (m, 3H), 4.14 – 4.12(m, 2H), 4.02 (t, J = 4.2 Hz, 2H), 3.97 – 3.92 (m, 1H), 3.82 – 3.77 (m, 1H),3.54 (t, J = 5.4 Hz, 2H). 31 31P NMR (162 MHz, D2O) δ -11.3 (s). MALDI-TOF-MS: m / z C 17 H 26 BrN5O 14 P2, calculated [M-H] - = 665.01, found 665.00。

[0036]

[0037] Compound (4ab): 1 1H NMR (600 MHz, D2O) δ 8.53 (s, 1H), 8.27 (s, 1H), 6.15(d, J = 5.4 Hz, 1H), 5.00 (s, 1H), 4.56 – 4.53 (m, 1H), 4.40 (s, 1H), 4.29 –4.26 (m, 1H), 4.25 – 4.22 (m, 2H), 4.14 – 4.09 (m, 2H), 4.08 – 4.06 (m, 1H),4.03 – 3.97 (m, 2H), 3.77 – 3.54 (m, 2H), 3.53 – 3.46 (m, 2H). 31 31P NMR (162MHz, D2O) δ -11.2 (s). MALDI-TOF-MS: m / z C 17 H 26 BrN5O 14 P2, calculated [M-H] -=665.01, found 665.01.

[0038] After screening and optimization of reaction conditions, it was found that the highest yield (94%) was achieved when bromoethanol and ionic liquid [TMG][BF4] were used in a 50-equivalent ratio, the reaction time was 6 h, and the reaction temperature was 90 °C, with an α:β ratio of 6:1. To explore the effect of the linker arm length (carbon chain length) on the coupling efficiency when ADPr-bromoethanol compounds are coupled with cysteine, the range of reaction substrates was expanded.

[0039] Example 2

[0040] Synthesis of α / β-ADPr-bromopropyl ether and α / β-ADPr-bromobutyl ether:

[0041]

[0042] Reaction routes of α / β-ADPr-bromopropanol and α / β-ADPr-bromobutanol

[0043] Under anhydrous and oxygen-free conditions, β-NAD + 0.015 mmol (1 equiv) of bromopropanol / bromobutanol and 0.75 mmol (50 equiv) of bromopropanol / bromobutanol were added to a system containing 0.75 mmol (50 equiv) of ionic liquid [TMG][BF4]. The mixture was stirred and reacted at 90 °C for 6 h. After the reaction was completed, the reaction system was extracted three times with water and dichloromethane. The aqueous phase was then collected, and the product was separated and purified by semi-preparative HPLC (C18, CH3CN / 0.1M TEAB=20 / 80, within 40 min, flow rate=3.0 mL / min, I=254 nm). The solvent was then removed by freeze drying to obtain a white powder product.

[0044]

[0045] Compound (4ac): 11H NMR (600 MHz, D2O) δ 8.43 (s, 1H), 8.28 (s, 1H), 6.15 (d, J = 6.0 Hz, 1H), 4.94 (d, J = 4.2 Hz, 1H), 4.77 – 4.76 (m, 1H), 4.55 – 4.52 (m, 1H), 4.40 (s, 1H), 4.25 – 4.21 (m, 2H), 4.17 (s, 1H), 4.14 – 4.10 (m, 2H), 4.04 – 4.01 (m, 2H), 3.54 – 3.51 (m, 2H), 2.09 – 2.05 (m, 2H), 1.26 – 1.23 (m, 2H). 31 31P NMR (162 MHz, D2O) δ -11.2 (d). MALDI-TOF-MS: m / z C 18 H 28 BrN5O 14 P2, calculated [M-H] - = 679.03, found 679.03。

[0046]

[0047] Compound (4ad): 1 1H NMR (600 MHz, D2O) δ 8.53 (s, 1H), 8.27 (s, 1H), 6.14 (d, J = 6.0 Hz, 1H), 4.95 (s, 1H), 4.53 (s, 1H), 4.39 (s, 1H), 4.26 – 4.22 (m, 3H), 4.14 – 4.06 (m, 2H), 4.05 – 3.95 (m, 2H), 3.45 (t, J = 6.6 Hz, 2H), 2.00 (t, J = 6.0 Hz, 2H), 1.26 – 1.22 (m, 2H). 31 31P NMR (162 MHz, D2O) δ -11.2(d). MALDI-TOF-MS: m / z C 18 H 28 BrN5O 14 P2, calculated [M-H] - = 679.03, found 679.05。

[0048]

[0049] Compound (4ae): 1 H NMR (600 MHz, D2O) δ 8.28 (d, J = 23.4 Hz, 1H), 8.06(d, J = 13.8 Hz, 1H), 5.93 (t, J = 8.4 Hz, 1H), 5.60 (s, 1H), 4.63 – 4.57 (m,2H), 4.43 – 4.40 (m, 2H), 4.30 (s, 1H), 4.28 (s, 1H), 4.24 – 4.22 (m, 1H),4.19 – 4.18 (m, 2H), 4.13 (s, 2H), 4.12 – 4.09 (m, 1H), 4.03 – 4.01 (m, 1H),3.97 – 3.94 (m, 1H), 2.90 (s, 2H), 1.19 – 1.18 (m, 2H). 31 P NMR (162 MHz, D2O)δ -11.4 (s). MALDI-TOF-MS: m / z C 19 H 30 BrN5O 14 P2, calculated [M-H] - = 693.04,found 693.02。

[0050]

[0051] Compound (4af): 1 H NMR (600 MHz, D2O) δ 8.45 (s, 1H), 8.19 (s, 1H), 6.07(d, J = 6.0 Hz, 1H), 5.63 (d, J = 5.4 Hz, 1H), 4.81 (s, 1H), 4.71 (t, J = 6.0Hz, 2H), 4.47 (t, J = 4.8 Hz, 2H), 4.32 (d, J = 2.4 Hz, 2H), 4.16 - 4.14 (m,4H), 4.12 – 4.10 (m, 2H), 4.02 – 3.97 (m, 2H), 2.88 (m, 2H), 1.19 – 1.16 (m,2H). 31P NMR (162 MHz, D2O) δ -11.3 (s). MALDI-TOF-MS: m / z C 19 H 30 BrN5O 14 P2, calculated [MH] - = 693.04, found 693.03.

[0052] Example 3

[0053] The reaction of ADPr-bromoethanol with N-acetylcysteine:

[0054] Before modifying proteins with ADP-ribosylation, we first attempt ADP-ribosylation reactions at the amino acid level to screen for optimal conditions and explore the effect of linker arm (carbon chain length) on the coupling reaction.

[0055] ADPr-bromoethanol, different equivalents of N-acetylcysteine, and reducing agent DTT (dithiothreitol, 5 equiv) were placed in a reaction flask. Water was added as a solvent, the pH of the reaction system was adjusted, and the reaction was heated and stirred. After the reaction was completed, the product was separated and purified by semi-preparative HPLC (C18, CH3CN / 0.1M TEAB=20 / 80, within 40 min, flow rate=3.0 mL / min, I=254 nm). The solvent was then removed by freeze drying to obtain a white powder product.

[0056]

[0057] ADP-ribosylation pathway at the amino acid level

[0058] Table 2. Optimization of reaction conditions and yields for ADP-ribosylation at the amino acid level.

[0059] EntrytimeTemperature (℃)pHEquiv of cysYield (%) b 11h409.01084240min409.01096330min409.01079540min259.01084640min379.01094740min37 8.01092840min377.01012940min376.01051040min379.05971140min379.02921240min379.0394 surface

[0060]

[0061] Compound (4ag): 1 H NMR (600 MHz, D2O) δ 8.55 (s, 1H), 8.30 (s, 1H), 6.10 (d, J = 5.4 Hz, 1H), 4.91 (s, 1H), 4.48 (t, J = 4.2 Hz, 1H), 4.33 (s, 1H),4.29 – 4.26 (m, 1H), 4.21 – 4.16 (m, 3H), 4.13 – 4.09 (m, 3H), 4.07 – 4.02(m, 2H), 3.94 – 3.91 (m, 1H), 3.82 – 3.78 (m, 1H), 3.70 (s, 3H), 2.66 (t, J =6.0 Hz, 2H), 1.35 (s, 9H). 31 P NMR (162 MHz, D2O) δ -11.1 (d). MALDI-TOF-MS:m / z C 25 H 40 N6O 18 P2S, calculated [MH] - = 806.16, found 806.15.

[0062] After screening and optimization of the conditions, it was found that the highest yield (94%) was achieved when 5 equivalents of N-acetylcysteine ​​were used, the pH of the reaction system was 9.0, the reaction time was 40 min, and the reaction temperature was 37℃. To further investigate the effect of the linker arm length (carbon chain length) on the coupling efficiency when ADPr-bromoethanol compounds are coupled with cysteine, this study aimed to explore the following steps.

[0063] Example 4

[0064] The reaction of ADPr-bromoethanol / bromopropanol with N-acetylcysteine ​​containing linker arms of varying lengths:

[0065]

[0066] Reaction routes of ADPr-bromoethanol analogs with N-acetylcysteine ​​using linker arms of varying lengths

[0067] 0.01 mmol (1 equiv) of ADPr-bromoethanol, 0.05 mmol (5 equiv) of N-acetylcysteine, and 0.05 mmol (5 equiv) of reducing agent DTT were placed in a reaction flask. Water was added as a solvent, and the pH of the reaction system was adjusted to 9.0. The reaction was then heated and stirred. After the reaction was completed, the product was separated and purified by semi-preparative HPLC (C18, CH3CN / 0.1M TEAB=20 / 80, within 40 min, flow rate=3.0 mL / min, I=254 nm). The solvent was then removed by freeze drying to obtain a white powder product.

[0068] Analysis of the reaction results revealed that when bromoethyl ether was used as the linker, the yield was significantly higher than that when bromopropyl ether and bromobutyl ether were used as the linkers, indicating that ADPr-bromoethanol is a more suitable reactant for subsequent ADP-ribosylation reactions.

[0069] Example 5

[0070] Synthesis of oligopeptides containing cysteine:

[0071]

[0072]

[0073] Compound (4ah): 1 H NMR (600 MHz, CD3OD) δ 7.53 (d, J = 7.8 Hz, 2H), 7.23(t, J = 7.8 Hz, 2H), 7.03 (t, J = 7.8 Hz, 1H), 4.38 (q, J = 4.8 Hz, 1H), 4.03– 3.89 (m, 2H), 3.21 (dd, J = 13.8 Hz, 4.2 Hz, 1H), 2.96 – 2.88 (m, 1H), 1.38(s, 9H). 13 C NMR (151 MHz, CD3OD) δ 173.7, 169.3, 157.9, 139.1, 129.6, 125.2, 121.3, 81.1, 55.5, 44.0, 41.2, 28.5.

[0074]

[0075]

[0076] Compound (4ai): 1 H NMR (600 MHz, CD3OD) δ 7.55 (d, J = 7.8 Hz, 2H), 7.25(t, J = 7.2 Hz, 2H), 7.05 (t, J = 7.2 Hz, 1H), 4.00 (q, J = 3.6 Hz, 1H), 3.98(q, J = 17.4 Hz, 2H), 3.29 – 3.20 (m, 2H), 2.98 – 2.92 (m, 1H), 2.77 (s, 2H), 1.97 (s, 1H), 1.40 (s, 9H), 1.20 (t, J = 7.2 Hz, 1H). 13 C NMR (151 MHz, CD3OD) δ 171.4, 170.1, 155.6, 138.0, 128.9, 124.3, 119.8, 80.3, 64.1, 61.2, 53.5, 47.9, 29.1, 28.4, 24.8.

[0077]

[0078]

[0079] Compound (4aj): 1 H NMR (600 MHz, CD3OD) δ 7.51 (d, J = 7.8 Hz, 2H), 7.26(t, 7.8 Hz, 2H), 7.06 (t, J = 7.8 Hz, 1H), 4.54 – 4.41 (m, 2H), 4.10 – 4.02(m, 1H), 3.55 – 3.50 (m, 1H), 3.27 (s, 2H), 3.23 – 3.16 (m, 1H), 2.90 = 2.80(m, 1H), 2.25 – 2.16 (m, 1H), 2.06 – 1.93 (m, 3H), 1.39 (s, 9H). 13 C NMR (151MHz, CD3OD) δ 172.7, 172.1, 168.8, 157.0, 138.9, 129.2, 129.1, 124.7, 120.9,80.2, 61.7, 54.4, 47.1, 42.1, 41.5, 30.0, 28.0, 25.1.

[0080] Example 6

[0081] Coupling reaction of ADPr-bromoethyl ether with cysteine-containing oligopeptides:

[0082]

[0083] ADPr-bromoethanol reaction route with cysteine-containing oligopeptides

[0084] 0.01 mmol (1 equiv) of ADPr-bromoethanol, 0.05 mmol (5 equiv) of cysteine-containing oligopeptide, and 0.05 mmol (5 equiv) of reducing agent DTT were placed in a reaction flask. 300 μL of water was added as a solvent to adjust the pH of the reaction system to 9.0. Then, 100 μL of DMSO was added to aid dissolution, and the reaction was heated and stirred at 37 °C. After the reaction was completed, the product was separated and purified by semi-preparative HPLC (C18, CH3CN / 0.1M TEAB=20 / 80, within 40 min, flow rate=3.0 mL / min, I=254 nm). The solvent was then removed by freeze drying to obtain a white powder product.

[0085]

[0086] Compound (4ak): 1 H NMR (600 MHz, D2O) δ 8.51 (s, 1H), 8.22 (s, 1H), 7.39– 7.36 (m, 4H), 7.22 (s, 1H), 6.11 (d, J = 6.0 Hz, 1H), 4.95 (s, 1H), 4.73(t, J = 5.4 Hz, 1H), 4.53 (t, J = 4.8 Hz, 1H), 4.39 (s, 1H), 4.30 – 4.22 (m,4H), 4.14 – 4.08 (m, 4H), 4.03 – 3.97 (m, 2H), 3.89 – 3.84 (m, 1H), 3.59 –3.54 (m, 1H), 3.05 – 2.99 (m, 1H), 2.94 – 2.88 (m, 1H), 2.80 – 2.74 (m, 2H), 1.40 (s, 9H). 31 P NMR (162 MHz, D2O) δ -11.3 (d). MALDI-TOF-MS: m / zC 33 H 48N8O 18 P2S, calculated [M-H] - = 938.23, found 938.23。

[0087]

[0088] Compound (4al): 1 H NMR (600 MHz, D2O) δ 8.51 (s, 1H), 8.23 (s, 1H), 7.39– 7.36 (m, 4H), 7.22 (s, 1H), 6.11 (d, J = 6.0 Hz, 1H), 4.95 (s, 1H), 4.73(t, J = 5.4 Hz, 1H), 4.54 – 4.52 (m, 1H), 4.39 (s, 1H), 4.29 – 4.23 (m, 4H),4.14 – 4.08 (m, 4H), 4.04 – 3.98 (m, 2H), 3.89 – 3.84 (m, 1H), 3.59 – 3.54(m, 1H), 3.06 – 3.00 (m, 1H), 2.94 – 2.88 (m, 1H), 2.80 – 2.75 (m, 2H), 1.40(s, 9H). 31 P NMR (162 MHz, D2O) δ -11.3 (d). MALDI-TOF-MS: m / z C 36 H 52 N8O 18 P2S,calculated [M-H] - = 978.26, found 978.26。

[0089]

[0090] Compound (4am): 1H NMR (600 MHz, D2O) δ 8.51 (s, 1H), 8.23 ​​(s, 1H), 7.40– 7.30 (m, 4H), 7.23 – 7.15 (m, 1H), 6.11 (d, J = 6.0 Hz, 1H), 4.86 (s, 1H),4.57 – 4.51 (m, 2H), 4.39 (s, 1H), 4.32 (s, 1H), 4.23 – 4.20 (m, 2H), 4.19 –4.14 (m, 3H), 4.10 – 4.07 (m, 1H), 4.05 – 4.02 (m, 1H), 3.98 – 3.94 (m, 2H), 3.80 – 3.72 (m, 2H), 3.72 – 3.66 (m, 1H), 3.60 – 3.54 (m, 1H), 3.10 – 3.02(m, 2H), 2.91 – 2.82 (m, 1H), 2.78 – 2.72 (m, 2H), 2.40 – 2.32 (m, 1H), 2.14– 2.06 (m, 2H), 1.41 (s, 9H). 31 P NMR (162 MHz, D2O) δ -11.3 (d). MALDI-TOF-MS: m / z C 38 H 55 N9O 19 P2S, calculated [MH] - = 1035.28, found 1035.17.

[0091] Example 7

[0092] The reaction of ADPr-bromoethanol with a polypeptide containing two cysteine ​​residues:

[0093] The core significance of synthesizing a polypeptide containing two single-ADP-ribosylation modifications lies in moving from single-modification research to combinatorial modification research. This allows for a deeper exploration of the complexity and precision of the ADP-ribosylation signaling pathway, studying site specificity and synergy. Proteins may contain multiple ADP-ribosylation sites; synthesizing dual-modified polypeptides helps investigate whether the modification of these two sites is independent, mutually promoting, or mutually inhibiting; and whether a synergistic effect occurs, significantly altering protein function or interactions with other molecules—something that single-site studies cannot reveal. Furthermore, the synthesis of dual-modified single-ADP-ribosylated polypeptides is unprecedented. This example uses cyclic somatostatin (SRIF-14, amino acid sequence AGCKNFFWKTFTSC) as a substrate to attempt the synthesis of dual-modified single-ADP-ribosylated polypeptides.

[0094] 0.03 mmol (3 equiv) of ADPr-bromoethanol, 0.01 mmol (1 equiv) of cysteine-containing oligopeptide, and 0.02 mmol (2 equiv) of reducing agent DTT were placed in a reaction flask, and 300 μL of water was added as a solvent. The pH of the reaction system was adjusted to 9.0, and the reaction was heated and stirred at 37 °C. After the reaction was completed, the product was separated and purified by semi-preparative HPLC (C18, CH3CN / 0.1M TEAB=20 / 80, within 40 min, flow rate=3.0 mL / min, I=254 nm). The solvent was then removed by freeze drying to obtain a white powder product.

[0095]

[0096] Reaction route of dual-modified mono-ADPr-SRIF-14

[0097]

[0098] Compound (4an): 1 H NMR (600 MHz, D2O) δ 8.63 (s, 2H), 8.39 (s, 2H), 7.78– 7.70 (m, 10H), 7.60 – 7.46 (m, 9H), 6.42 (s, 2H), 6.18 – 6.10 (m, 2H).MALDI-TOF-MS: m / z C 38 H 55 N9O 19 P2S, calculated [MH] -= 2809.91, found 2809.91.

[0099] Example 8

[0100] The reaction of ADPr-bromoethanol with proteins containing cysteine:

[0101] 0.03 mmol (3 equiv) of ADPr-bromoethanol, 0.01 mmol (1 equiv) of cysteine-containing protein and 0.02 mmol (2 equiv) of reducing agent DTT were placed in a reaction flask, 300 μL of water was added as a solvent, the pH of the reaction system was adjusted to 9.0 and then heated and stirred at 37 °C.

[0102] This example uses two proteins for the reaction. zEGFR was purified by semi-preparative HPLC after ADP-ribosylation, and the product was identified by LC-MS. Western blot verification was performed after the reaction. The reaction process is shown below, and the Western blot results for ADPr-Ub (R42C) are shown in Figure 1.

[0103]

[0104] ADP-ribosylation pathway of proteins

[0105] Pro-Cys(α-ADPr-bromoethanol)-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Ala-Val-Thr-Lys compound (4ao): 1 H NMR (600 MHz, D2O) δ 8.54 (s,1H), 8.29 (s, 1H), 6.16 (s, 1H). 31 P NMR (162 MHz, D2O) δ -11.30 (s).

[0106] MALDI-TOF-MS: m / z C 105 H 181 N 31 O 38 P2S calculated [MH] - = 2579.33, found2579.33.

[0107] The amino acid sequence of zEGFR is: Ac-C-VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK

[0108] ADPr-zEGFR LC-MS: m / z C 313 H 484 N 85 O 105 P2S3, calculated [M+H] + = 7275.95,found [M+3H] 3+ =2424.21, [M+5H] 5+ =1212.63.

[0109] The amino acid sequence of Mutation-Ub (R42C) is: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQ-C-LIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGHHHHHH

[0110] Figure 1 shows the binding of ubiquitin and ADPr-Ub to anti-ubiquitin and anti-ADPr antibodies. The results indicate that the anti-ubiquitin antibody can recognize ubiquitin before and after the reaction, while the anti-ADP-ribosyl antibody specifically recognizes the ADP-ribosyl group attached to ubiquitin.

[0111] Through experimentation, a protein modified with mono-ADP-ribosylation was successfully synthesized. This demonstrates the feasibility of a one-step strategy to modify proteins with ADPr groups via nucleophilic substitution reactions, and also proves the high substrate tolerance of this reaction type, allowing modification of substrates containing cysteine ​​at varying levels of complexity.

[0112] This invention uses β-NAD + A series of nucleophilic reagents specifically coupled to cysteine ​​were synthesized for the reaction substrates, and a scheme for single-ADP-ribosylation modification of cysteine-containing peptides / proteins using the principle of nucleophilic substitution was developed. This invention avoids the introduction of photoinitiators, eliminates the need for light exposure, protects peptides / proteins from light damage, results in milder reaction conditions, and fewer reaction byproducts. NAD+ is linked through NAD+ with connecting arms of different lengths. + Analogs (ADPr-bromoethanol, ADPr-bromopropanol, and ADPr-bromobutanol) were reacted with N-acetylcysteine ​​under the same reaction conditions. The analysis results showed that the efficiency of ADP-ribosylation modification of the substrate gradually decreased with the elongation of the linker arm, and ADPr-bromoethanol was the optimal nucleophile.

[0113] A series of cysteine-containing substrates were obtained through liquid-phase synthesis of cysteine-containing oligopeptides, solid-phase synthesis of cysteine-containing peptides, and expression of mutant proteins in *E. coli* prokaryotic cells. These substrates were then subjected to ADP-ribosylation modification under optimized conditions, enriching the ADP-ribosylation substrate pool. Western blot experiments demonstrated that ADPr-Ub obtained using this newly developed ADP-ribosylation reaction could still be recognized by corresponding antibodies (anti-Ub antibody and anti-ADPr antibody), retaining the original ubiquitin characteristics. This provides researchers with a new approach to obtaining molecular tools with single-ADP-ribosylation or even double-single-ADP-ribosylation modifications for continued exploration of ADP-ribosylation. In mass spectrometry-based proteomics, the synthesis of well-defined dual-modified peptides is crucial for standards; these synthetic peptides serve as a key bridge between biochemical discoveries and the understanding of physiological and pathological functions.

[0114] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

[0115] SEQUENCE LISTING

[0116] <110> Henan Normal University

[0117] <120> A cysteine-specific conjugated nucleophilic reagent, its preparation method and application

[0118] <130> 2026

[0119] <160> 3

[0120] <170> Patentin version 3.3

[0121] <210> 1

[0122] <211> 14

[0123] <212> RNA

[0124] <400> 1

[0125] AGCKNFFWKTFTSC 14

[0126] <210> 2

[0127] <211> 59

[0128] <212> RNA

[0129] <400> 2

[0130] CVDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK

[0131] <210> 3

[0132] <211> 82

[0133] <212> RNA

[0134] <400> 3

[0135] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQCLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGHHHHHH 82。

Claims

1. A cysteine-specific nucleophilic conjugate, characterized in that... Compounds including those with the following structures: 、 、 、 、 、 。 2. A method for preparing the cysteine-specific conjugated nucleophilic reagent according to claim 1, characterized in that... The specific preparation process is as follows: β-nicotinamide adenine dinucleotide β-NAD... + The target product, cysteine, was prepared by reacting cysteine-specific coupling nucleophiles with bromoalkyl alcohols in a system containing the ionic liquid [TMG][BF4] at 80–100 °C with stirring. The corresponding reaction route for the preparation process is as follows: Where n is 1, 2 or 3.

3. The method for preparing the cysteine-specific conjugated nucleophilic reagent according to claim 2, characterized in that: The β-nicotinamide adenine dinucleotide β-NAD + The molar ratio of bromoalkyl alcohol compounds to ionic liquids [TMG][BF4] is 1:10~70:30~70.

4. The use of the cysteine-specific coupling nucleophile reagent according to claim 1 in the modification of cysteine, cysteine-containing oligopeptides, cysteine-containing polypeptides, or cysteine-containing proteins.

5. The application according to claim 4, characterized in that... The specific process of modifying cysteine ​​with a cysteine-specific coupling nucleophile is as follows: ADPr-bromoalkyl alcohol, N-acetylcysteine, and reducing agent DTT are placed in a reaction flask, water is added as a solvent, the pH of the reaction system is adjusted to 6-9, and the reaction is stirred at 25-40℃ to achieve the coupling reaction between ADPr-bromoalkyl alcohol and N-acetylcysteine. The molar ratio of ADPr-bromoalkyl alcohol, N-acetylcysteine, and reducing agent DTT is 1:2 to 10:5, and the corresponding reaction route is as follows: Where n is 1, 2 or 3.

6. The application according to claim 4, characterized in that... The specific process of modifying cysteine-containing oligopeptides with cysteine-specific coupling nucleophilic reagent is as follows: ADPr-bromoalkyl alcohol, cysteine-containing oligopeptides and reducing agent DTT are placed in a reaction flask, water is added as a solvent, the pH of the reaction system is adjusted to 6-9 and then stirred at 25-40℃ to achieve the coupling reaction between ADPr-bromoalkyl alcohol and cysteine-containing oligopeptides.

7. The application according to claim 6, characterized in that... The specific reaction route for modifying cysteine-containing oligopeptides using cysteine-specific coupling nucleophilic reagents is as follows: 。 8. The application according to claim 4, characterized in that... The specific process for modifying cysteine-containing proteins using a cysteine-specific coupling nucleophilic reagent is as follows: ADPr-bromoalkyl alcohol, a cysteine-containing polypeptide / protein, and the reducing agent DTT are placed in a reaction flask. Water is added as a solvent, and the pH of the reaction system is adjusted to 6-9. The reaction is then stirred at 25-40°C to achieve the coupling reaction between ADPr-bromoalkyl alcohol and the cysteine-containing polypeptide / protein. The corresponding reaction route is as follows: 。 9. The application according to claim 8, characterized in that: The amino acid sequences of the polypeptides / proteins containing cysteine ​​are shown in SEQ No. 2 and SEQ No. 3, respectively.

10. The application according to claim 4, characterized in that... The specific process for modifying cysteine-containing peptides using a cysteine-specific coupling nucleophilic reagent is as follows: ADPr-bromoalkyl alcohol, the cysteine-containing peptide, and the reducing agent DTT are placed in a reaction flask. Water is added as a solvent, and the pH of the reaction system is adjusted to 6-9. The reaction is then stirred at 25-40°C to achieve the coupling reaction between ADPr-bromoalkyl alcohol and the cysteine-containing peptide. The corresponding reaction route, represented by ADPr-bromoethanol and the peptide SRIF-14, is as follows: 。

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