Use of selenium-containing polypeptide compounds in the preparation of antibacterial drugs

Abstract

The application prepares L-selenomethionine, and applies the L-selenomethionine to synthesis of tripeptide and tetrapeptide, and prepares new selenium-containing polypeptide compounds; the selenium-containing polypeptide compounds have antibacterial activity, and have good inhibition effect on gram-positive bacteria; the results obtained by the application show that the selenium-containing polypeptide compounds have wide application prospect in a drug development system, and provide a new and wider idea for synthesis and screening of selenium-containing polypeptide drug.

Description

(I) Technical Field

[0001] This invention relates to the application of a selenium-containing polypeptide compound in the preparation of antibacterial drugs. (II) Background Technology

[0002] Selenium is an essential trace element for the human body. Selenocysteine, found in nature, is considered the 21st amino acid, and bioactive peptides are an important source of organic selenium. Selenopeptides are novel peptides with significant research value in the field of bioactive peptides. Compared to ordinary bioactive peptides and single selenium compounds, selenopeptides exhibit higher levels of antioxidant, anticancer, and immunomodulatory activities. However, research on selenopeptides is currently in its early stages, and further exploration in this area is needed.

[0003] Multiple studies have shown that short peptides have a superior absorption mechanism compared to amino acids, and their nutritional effects are stronger than those of free amino acids. Peptides possess diverse physiological activities and are widely used in medicine, hygiene, healthcare, food, cosmetics, and other fields. They hold significant theoretical research value and practical application value, playing a crucial role in advancing fields such as biochemistry, medicine, immunology, chemistry, and microbiology. Synthetic methods are increasingly becoming highly useful for studying the structure-function relationship of peptides. Currently, peptide synthesis falls into two main categories: chemical synthesis and biosynthesis. In chemical synthesis, the yield is affected by various factors, such as reaction time, temperature, and the molar ratio of reactants. Furthermore, amino acids do not spontaneously condense under mild reaction conditions; special reagents are required to enhance the reactivity of the amino or carboxyl groups of amino acids to form peptides.

[0004] This invention employs a liquid-phase organic synthesis method. By studying the reactant ratio, reaction time, reaction temperature, and the type and ratio of condensing agents during the reaction process, the yield of peptide synthesis is improved. Using L-selenomethionine with high optical purity as a substrate, a series of selenium-containing peptide compounds are obtained through multi-step condensation reactions with amino acids. These compounds have been tested and found to have antibacterial activity, and can be developed into corresponding drugs. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a selenium polypeptide compound and its preparation method, as well as the application of this type of compound in antibacterial processes. This type of compound has high yield and simple synthesis route, and has a significant inhibitory effect on Gram-positive bacteria, showing good application prospects.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides the application of a selenium-containing polypeptide compound with the structure shown in formula (I) in the preparation of antibacterial drugs.

[0008]

[0009] R1 is one of the following structures:

[0010]

[0011] R2 is a hydroxyl group or one of the following structures:

[0012]

[0013] Where X is H and Y is C 1-10 Alkyl (preferably C) 1-5 Alkyl group or benzyl group; or X, Y and N between them are linked to form a ring, forming a tetrahydropyrrole ring.

[0014] Preferably, R2 is a hydroxyl group or one of the following structures:

[0015]

[0016] Furthermore, the selenium-containing polypeptide compound of the present invention is preferably one of the following:

[0017]

[0018] Preferably, the selenium-containing polypeptide compound is compound I-4, I-6, I-9, I-10 or I-12; most preferably, compound I-4.

[0019] More preferably, the bacteria are Gram-positive bacteria, especially Staphylococcus aureus or Bacillus subtilis.

[0020] This invention provides a method for preparing short peptides. Taking tripeptide synthesis as an example, the method is as follows:

[0021] Weigh out a certain amount of II-1 and II-2, add condensing agents (diisopropylcarbodiimide DIC, 1-hydroxybenzotriazole HOBt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI, N,N-diisopropylethylamine DIPEA, 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate HATU, 1H-benzotriazole-1-yloxytripyrrolidinylphosphonium hexafluorophosphate PyBOP), use DCM (dichloromethane) as solvent, stir and react at 0-45℃ for 2-8 h. After the reaction is complete as monitored by thin-layer chromatography (TLC), add DCM and stir evenly. Then wash with 5% phosphoric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence. Dry the organic phase with anhydrous sodium sulfate, filter and concentrate the filtrate under vacuum to obtain II-3.

[0022] The preferred combination of condensing agents is EDCI / HOBt / DIPEA, and the preferred ratio of II-1, II-2 to EDCI / HOBt / DIPEA is 1:1.3:1.2:1.2:2.4.

[0023] The preferred reaction temperature is 0-25℃, and the preferred reaction time is 6h.

[0024] Dissolve II-3 in methanol and add 1 M·L under ice bath conditions. -1 The lithium hydroxide aqueous solution was stirred thoroughly, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was diluted with 0.5 M·L⁻¹ water. -1 The pH was adjusted to 3 with hydrochloric acid, then extracted with ethyl acetate. The organic phases were combined, dried with anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain III-4.

[0025] Weigh out a certain amount of II-4 and II-5, add condensing agents (DIC, HOBt, EDCI, DIPEA, HATU, PyBOP), use DCM as solvent, and stir the reaction at 0-45℃ for 2-8 hours. After the reaction is complete as monitored by TLC, add DCM and stir until homogeneous. Then wash successively with 5% phosphoric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum to obtain III-6.

[0026] The preferred combination of condensing agents is EDCI / HOBt / DIPEA, and the preferred ratio of II-4, II-5 to EDCI / HOBt / DIPEA is 1:1.3:1.2:1.2:2.4.

[0027] The preferred reaction temperature is 0-25℃, and the preferred reaction time is 6h.

[0028] Dissolve II-3 in methanol and add 1 M·L under ice bath conditions. -1 The lithium hydroxide aqueous solution was stirred thoroughly, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was diluted with 0.5 M·L⁻¹ water. -1 The pH was adjusted to 3 with hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain crude product II-7. The crude product was purified by column chromatography with an eluent ratio of V(petroleum ether, Pet:V(ethyl acetate, EtoAc)) of 4:1 to obtain product II-7.

[0029] Dissolve III-7 in 4 M·L under ice bath conditions. -1 HCl (1,4-dioxane) was added and stirred at room temperature; the reaction was monitored by TLC. After the substrate had reacted completely, diethyl ether was added, and the mixture was stirred thoroughly for 2 hours. The filtered solid was washed with diethyl ether to give II-8.

[0030]

[0031]

[0032] Figure: Peptide synthesis preparation process

[0033] The selenium-containing polypeptide compounds described in this invention can be used to prepare antibacterial drugs, which have a good inhibitory effect on Gram-positive bacteria, especially Staphylococcus aureus and Bacillus subtilis.

[0034] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: (1) L-selenomethionine was prepared by the present invention and applied to the synthesis of tripeptides and tetrapeptides to prepare new selenium-containing polypeptide compounds; (2) This type of selenium-containing polypeptide compound has antibacterial activity and has a good inhibitory effect on Gram-positive bacteria; (3) The results obtained by the present invention show that selenium-containing polypeptide compounds have broad application prospects in the drug development system, and provide new and broader ideas for the synthesis and screening of selenium-containing polypeptide compound drugs. (IV) Detailed Implementation

[0035] Example 1: Preparation of compound Leu-Gly-Se-Met-OH (Ⅰ-1)

[0036] The reaction formula is as follows:

[0037]

[0038] a. Synthesis of N-tert-butoxycarbonylleucylglycine methyl ester Boc-Leu-Gly-OMe

[0039] Take a round-bottom flask, weigh 1-1 (1 mmol, 0.231 g) and dissolve it in DCM (5 mL). Add DIPEA (2.4 mmol, 0.310 g) at 0 °C, followed by EDCI (1.2 mmol, 0.230 g) and HOBt (1.2 mmol, 0.162 g). Stir and activate for 5 min, then add glycine methyl ester hydrochloride (1.3 mmol, 0.163 g) and react for 5 h. After the reaction is complete as monitored by TLC, add DCM (60 mL) and stir well. Then wash successively with 5% phosphoric acid (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum. 0.286 g of product is recovered, yield 94.8%.

[0040] b. Synthesis of N-tert-butoxycarbonylleucylglycine Boc-Leu-Gly-OH

[0041] Dissolve product 1-2 from the previous step in 5 mL of methanol, and add 1 M·L⁻¹ under ice bath conditions. -1 Add 5 mL of lithium hydroxide aqueous solution and stir thoroughly. Monitor the reaction progress by TLC. After the reaction is complete, dilute the reaction solution with 0.5 M·L⁻¹ water. -1The pH was adjusted to 3 with hydrochloric acid, and then extracted with ethyl acetate. The organic phases were combined, dried with anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the pure product –1-3. 0.260 g of product was recovered, with a yield of 95.2%.

[0042] c. Synthesis of N-tert-butoxycarbonylleucylglycylselenomethionine methyl ester Boc-Leu-Gly–Se-Met-OMe

[0043] The 1-3 (0.9 mmol, 0.259 g) obtained in the previous step were dissolved in DCM (5 mL). DIPEA (2.16 mmol, 0.279 g) was added at 0 °C, followed by EDCI (1.08 mmol, 0.207 g) and HOBt (1.08 mmol, 0.146 g). The mixture was stirred and activated for 5 min, and then selenomethionine methyl ester hydrochloride (1.17 mmol, 0.289 g) was added and reacted for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.403 g of the product, with a yield of 96.3%.

[0044] d. Synthesis of N-tert-butoxycarbonylleucylglycylselenomethionine (Boc-Leu-Gly–Se-Met-OH)

[0045] Dissolve products 1-4 from the previous step in 5 mL of methanol, and add 1 M·L⁻¹ under ice bath conditions. -1 Add 5 mL of lithium hydroxide aqueous solution and stir thoroughly. Monitor the reaction progress by TLC. After the reaction is complete, dilute the reaction solution with 0.5 M·L⁻¹ water. -1 The pH was adjusted to 3 with hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product –1-5. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.382 g of product was recovered, with a yield of 94.5%.

[0046] e. Synthesis of leucine, glycine, selenomethionine (NH2-Leu-Gly–Se-Met-OH)

[0047] The above-obtained –1-5 was dissolved in 4 M·L under ice bath conditions. -1 The reaction was monitored by TLC using HCl 1,4-dioxane solution. Once the substrate had reacted completely, 50 mL of diethyl ether was added, and the mixture was stirred thoroughly for 2 h. The mixture was filtered to obtain –Ⅰ-1, which was washed three times with 150 mL of diethyl ether. The solvent was removed by vacuum evaporation, yielding 0.289 g of the product (96.5% yield). Its structure is characterized as follows: 1H NMR(500MHz,D2O)δ4.27-4.29(dd,J=9.0,4.4Hz,1H),3.96(d,J=2.1Hz,2H),3.67(d,J=7.1Hz,1H),2.59(ddd,J=12.2,9.0,5.1Hz ,1H),2.54(s,1H),2.14(dddd,J=13.7,9.0,7.5,4.3Hz,1H),2.05-1.99(m,4H),1.70–1.50(m,3H),0.92(dd,J=10.7,6.5Hz,6H); 13 C NMR (125MHz, D2O) δ173.45,169.76,168.77,66.82,52.30,51.37,42.34,32.09,24.01,22.93,22.78,21.28,3.98.

[0048] Example 2: Preparation of compound Pro-Ile-Se-Met-OH(Ⅰ-2)

[0049] The reaction formula is as follows:

[0050]

[0051] a. Synthesis of N-tert-butoxycarbonylprolyl isoleucine methyl ester Boc-Pro-Ile-OMe

[0052] The procedure was the same as step a in Example 1, except that 1-1 (1 mmol, 0.231 g) was replaced with 2-1 (1 mmol, 0.215 g) and glycine methyl ester hydrochloride (1.3 mmol, 0.163 g) was replaced with isoleucine methyl ester hydrochloride (1.3 mmol, 0.236 g), resulting in a final product of 0.313 g and a yield of 95.7%.

[0053] b. Synthesis of N-tert-butoxycarbonylprolyl isoleucine Boc-Pro-Ile-OH

[0054] The procedure is the same as step b in Example 1, except that 1-2 is replaced with 2-2, and the final product is 0.302g, with a yield of 96.2%.

[0055] c. Synthesis of N-tert-butoxycarbonylprolyl isoleucyl selenomethionine methyl ester Boc-Pro-Ile-Se-Met-OMe

[0056] The procedure was the same as step c in Example 1, except that 1-3 (0.9 mmol, 0.259 g) was replaced with –2-3 (0.9 mmol, 0.296 g), resulting in a final product of 0.452 g and a yield of 96.4%.

[0057] d. Synthesis of N-tert-butoxycarbonylprolyl isoleucyl selenomethionine Boc-Pro-Ile-Se-Met-OH

[0058] The operation was the same as step d in Example 1, except that 1-4 was replaced with 2-4, and the final product was 0.414 g, with a yield of 94.3%.

[0059] e. Synthesis of prolyl isoleucyl selenomethionine NH2-Pro-Ile-Se-Met-OH

[0060] The procedure was the same as step e in Example 1, except that 1-5 was replaced with 2-5, ultimately yielding 0.342 g of product, with a yield of 97.1%. Its structural characterization is as follows: 1 H NMR(500MHz, (CD3)2SO) δ12.88(s,1H),10.50(dd,J=11.7,6.1Hz,1H),8.96(d,J=8.6Hz,1H),8.69(p,J=6.2,5. 7Hz, 1H), 8.61 (d, J = 7.7Hz, 1H), 4.54 (ddd, J = 9.5, 7.7, 4.5Hz, 1H), 4.42 (t, J = 8.2Hz, 2H), 3.39 (ddp, J = 27.0, 17. 3,5.7Hz,2H),2.78(ddd,J=12.4,8.6,5.1Hz,1H),2.72(d,J=6.9Hz,1H),2.58–2.43(m,1H),2.14(s,4H),2.11–1 .91(m,4H),1.69(dtd,J=15.0,7.5,3.3Hz,1H),1.41–1.25(m,1H),1.10(d,J=6.7Hz,3H),1.04(t,J=7.5Hz,3H). 13 C NMR (125MHz, (CD3)2SO) δ173.43,171.17,168.54,66.82,58.91,57.97,52.15,46.05,36.87,30.37,24.80,23.90,21.23,15.71,11.37,3.99.

[0061] Example 3: Preparation of compound Phe-Val-Se-Met-OH (Ⅰ-3)

[0062] The reaction formula is as follows:

[0063]

[0064] a. Synthesis of N-tert-butoxycarbonylphenylalanylvaline methyl ester Boc-Phe-Val-OMe

[0065] The procedure was the same as step a in Example 1, except that 1-1 (1 mmol, 0.231 g) was replaced with 3-1 (1 mmol, 0.265 g) and glycine methyl ester hydrochloride (1.3 mmol, 0.163 g) was replaced with valine methyl ester hydrochloride (1.3 mmol, 0.218 g), resulting in a final product of 0.362 g and a yield of 95.7%.

[0066] b. Synthesis of N-tert-butoxycarbonylphenylalanylvaline Boc-Phe-Val-OH

[0067] The procedure was the same as step b in Example 1, except that 1-2 was replaced with 3-2, ultimately yielding 0.356 g of product, with a yield of 96.3%. c. Synthesis of N-tert-butoxycarbonylphenylalanylvaline selenomethionine methyl ester Boc-Phe-Val-Se-Met-OMe

[0068] The procedure was the same as step c in Example 1, except that 1-3 (0.9 mmol, 0.259 g) was replaced with 2-3 (0.9 mmol, 0.328 g), and the final product was 0.488 g, with a yield of 97.5%.

[0069] The synthesis of N-tert-butoxycarbonylphenylalanylvaline selenomethionine Boc-Phe-Val-Se-Met-OH was performed in the same manner as step d in Example 1, except that 1-4 was replaced with 2-4. The final product was 0.447 g, with a yield of 93.8%.

[0070] e. Synthesis of phenylalanylvaline selenomethionine NH2-Phe-Val-Se-Met-OH

[0071] The procedure was the same as step e in Example 1, except that 1-5 was replaced with 2-5, ultimately yielding 0.352 g of product, with a yield of 96.6%. Its structural characterization is as follows:

[0072] 1 H NMR(500MHz, (CD3)2SO) δ12.65(s,1H),8.78(d,J=8.8Hz,1H),8.44(d,J=7.6Hz,1H),8.42–8.25(m,2H),7.31–7.17(m,5H),4.32(ddd,J=9.6,7.6, 4.4Hz, 1H), 4.27–4.10 (m, 2H), 3.16 (dd, J=14.0, 5.6Hz, 1H), 3.01 (dd, J= 14.1, 6.8Hz, 1H), 2.56 (s, 2H), 2.11–1.92 (m, 6H), 0.91 (t, J = 6.9Hz, 6H). 13C NMR(125MHz,(CD3)2SO)δ173.38,171.01,168.14,135.36,130.14,128.80,127. 42,66.82,58.38,53.47,52.34,37.17,32.08,31.36,21.24,19.58,18.89,3.95.

[0073] Example 4: Preparation of Lys-SeMet-Ile-OH(I-4)

[0074] The reaction formula is as follows:

[0075]

[0076] a. Synthesis of 2,6-di-tert-butoxycarbonylaminohexanoic acid N-tert-butoxycarbonylalanylselenomethionine methyl ester Boc-Lys-SeMet-OMe

[0077] The procedure was the same as step a in Example 1, except that 1-1 (1 mmol, 0.231 g) was replaced with 4-1 (1 mmol, 0.346 g) and glycine methyl ester hydrochloride (1.3 mmol, 0.163 g) was replaced with selenomethionine methyl ester hydrochloride (1.3 mmol, 0.321 g), resulting in a final product of 0.517 g and a yield of 95.9%.

[0078] b. Synthesis of 2,6-di-tert-butoxycarbonylaminohexanoic acid N-tert-butoxycarbonylalanylselenomethionine Boc-Lys-Se-Met-OH

[0079] The procedure is the same as step b in Example 1, except that 1-2 is replaced with 4-2, and the final product is 0.484 g, with a yield of 96.2%.

[0080] c. Synthesis of 2,6-di-tert-butoxycarbonylaminohexanoic acid N-tert-butoxycarbonylalanylselenomethionylisoleucine methyl ester Boc-Lys-SeMet-Ile-OMe

[0081] The procedure was the same as step c in Example 1, except that 1-3 (0.9 mmol, 0.259 g) was replaced with 4-3 (0.9 mmol, 0.472 g), and selenomethionine methyl ester hydrochloride (1.17 mmol, 0.289 g) was replaced with isoleucine methyl ester hydrochloride (1.17 mmol, 0.229 g). The final product was 0.574 g, with a yield of 95.8%.

[0082] Synthesis of d. 2,6-Di-tert-butoxycarbonylaminohexanoic acid, N-tert-butoxycarbonylalanylselenomethionylisoleucine, Boc-Lys-SeMet-Ile-OH

[0083] The operation was the same as step d in Example 1, except that 1-4 was replaced with –4-4, and the final product was 0.529g, with a yield of 94.1%.

[0084] e. Synthesis of lysylselenomethionine isoleucine NH2-Lys-SeMet-Ile-OH

[0085] The procedure was the same as step e in Example 1, except that 1-5 was replaced with 4-5, ultimately yielding 0.459 g of product, with a yield of 97.8%. Its structural characterization is as follows: 1 H NMR(500MHz, (CD3)2SO) δ12.62(s,1H),8.95(d,J=7.8Hz,1H),8.40(d,J=5.2Hz,3H),8.24–8.09( m,4H),4.43(td,J=7.9,5.2Hz,1H),4.14(d,J=2.3Hz,1H),3.86(h,J=6.3,5.8Hz,1H),2.70(d,J=6 .6Hz,2H),2.53(dddd,J=42.8,12.1,10.3,5.8Hz,2H),1.93(s,5H),1.82–1.70(m,3H),1.58(tt, J=13.4,7.0Hz,2H),1.42–1.32(m,3H),1.20(ddd,J=13.7,9.0,7.0Hz,1H),0.85–0.78(m,6H).13C NMR(126MHz,(CD3)2SO)δ173.15,171.26,168.88,56.87,53.55,52.10,38 .64,36.57,33.56,30.67,26.59,25.06,21.37,20.72,16.01,11.78,3.93.

[0086] Example 5: Preparation of Leu-Gly-Se-Met-Ile-OH (Ⅰ-5)

[0087] The reaction formula is as follows:

[0088]

[0089] a. Synthesis of N-tert-butoxycarbonylleucylglycylselenomethionylisoleucine methyl ester Boc-Leu-Gly-Se-Met-Ile-OMe

[0090] The procedure was the same as step a in Example 1, except that 1-1 (1 mmol, 0.231 g) was replaced with 1-5 (1 mmol, 0.466 g) and glycine methyl ester hydrochloride (1.3 mmol, 0.163 g) was replaced with isoleucine methyl ester hydrochloride (1.3 mmol, 0.236 g), resulting in a final product of 0.573 g and a yield of 96.5%.

[0091] b. Synthesis of N-tert-butoxycarbonylleucylglycylselenomethionylisoleucine Boc-Leu-Gly-Se-Met-Ile-OH

[0092] The procedure was the same as step d in Example 1, except that 1-4 was replaced with –5-1, ultimately yielding 0.534 g of product, with a yield of 95.2%. c. Synthesis of leucylglycylselenomethionine isoleucine NH2-Leu-Gly-Se-Met-Ile-OH

[0093] The procedure was the same as step e in Example 1, except that 1-6 was replaced with –5-2, ultimately yielding 0.429 g of product, with a yield of 97.4%. Its structural characterization is as follows: 1 H NMR(500MHz, (CD3)2SO) δ12.56(s,1H),8.95(s,1H),8.41(d,J=5.6Hz,3H),8.22(d,J=8.2Hz,1H),8.09(d,J =8.1Hz,1H),4.45(td,J=8.0,5.9Hz,1H),4.13(d,J=2.3Hz,1H),3.93–3.67(m,3H),2.48(dddt,J=19.0,12. 1,9.6,6.5Hz,2H),2.00–1.86(m,4H),1.86–1.75(m,1H),1.70(dq,J=13.2,6.6Hz,1H),1.58(d,J=7.2Hz,2H ),1.40(dqd,J=15.1,7.6,4.2Hz,1H),1.20(ddddd,J=17.7,13.8,10.7,7.1,2.8Hz,1H),0.92–0.78(m,12H). 13 C NMR(125MHz,(CD3)2SO)δ173.17,171.56,169.76,168.48,66.82,65.37,56.91,53.19 ,51.36,42.45,36.54,33.76,25.18,24.01,22.93,22.77,20.73,16.02,11.79,3.94.

[0094] Example 6: Preparation of compound Leu-Gly-Se-Met-Phe-OH(Ⅰ-6)

[0095] The reaction formula is as follows:

[0096]

[0097]

[0098] a. Synthesis of N-tert-butoxycarbonylleucylglycylselenomethionylphenylalanine methyl ester Boc-Leu-Gly-Se-Met-Phe-OMe

[0099] The procedure was the same as step a in Example 5, except that isoleucine methyl ester hydrochloride (1.3 mmol, 0.236 g) was replaced with phenylalanine methyl ester hydrochloride (1.3 mmol, 0.280 g), and the final product was 0.595 g, with a yield of 94.8%.

[0100] b. Synthesis of N-tert-butoxycarbonylleucylglycylselenomethionylphenylalanine (Boc-Leu-Gly-Se-Met-Phe-OH)

[0101] The procedure was the same as step b in Example 5, except that 5-1 was replaced with 6-1, and the final product was 0.554 g, with a yield of 95.3%.

[0102] c. Synthesis of leucylglycylselenomethionine NH2-Leu-Gly-Se-Met-Phe-OH

[0103] The procedure was the same as step c in Example 5, except that 5-2 was replaced with –6-2, ultimately yielding 0.453 g of product, with a yield of 97.7%. Its structural characterization is as follows: 1 H NMR(500MHz, (CD3)2SO) δ8.94(s,1H),8.41(d,J=4.9Hz,3H),8.35(d,J=7.8Hz,1H),8.19(d,J= 8.2Hz,1H),7.26(m,4H),7.19(d,J=4.4Hz,1H),4.39(dd,J=18.1,5.3Hz,2H),3.85–3.78(m,2H) ,3.75(s,1H),3.05(d,J=5.0Hz,1H),2.93(s,1H),2.42(dddt,J=18.9,12.3,10.0,6.2Hz,2H), 1.91(s,4H),1.70(dq,J=13.1,6.6Hz,1H),1.58(t,J=7.3Hz,2H),0.89(dd,J=11.9,6.5Hz,7H). 13C NMR (126MHz, (CD3)2SO) δ173.13,171.39,169.77,168.45,138.04,129.62,128.61,126. 87,54.05,53.16,51.37,42.48,40.62,36.89,33.69,24.01,22.95,22.77,20.70,3.93.

[0104] Example 7: Preparation of compound Pro-Ile-Se-Met-Ala-OH (Ⅰ-7)

[0105] The reaction formula is as follows:

[0106]

[0107] a. Synthesis of N-tert-butoxycarbonylleucylglycylselenomethionylalanine methyl ester Boc-Pro-Ile-Se-Met-Ala-OMe

[0108] The procedure was the same as step a in Example 1, except that 1-1 (1 mmol, 0.231 g) was replaced with 2-5 (1 mmol, 0.507 g) and glycine methyl ester hydrochloride (1.3 mmol, 0.163 g) was replaced with alanine methyl ester hydrochloride (1.3 mmol, 0.182 g), resulting in a final product of 0.560 g and a yield of 94.6%.

[0109] b. The synthesis of N-tert-butoxycarbonylleucylglycylselenomethionylalanine Boc-Pro-Ile-Se-Met-Ala-OH was performed in the same manner as step d in Example 1, except that 1-4 was replaced with 7-1. The final product was 0.518 g, with a yield of 94.9%.

[0110] c. Synthesis of leucylglycylselenomethionylalanine NH2-Pro-Ile-Se-Met-Ala-OH

[0111] The procedure was the same as step e in Example 1, except that 1-5 was replaced with –7-2, ultimately yielding 0.421 g of product, with a yield of 98.2%. Its structural characterization is as follows: 1H NMR (500MHz, (CD3)2SO) δ12.56 (s, 1H), 10.27 (d, J = 10.5Hz, 1H), 8.74 (d, J = 8.6Hz, 1H), 8.46 (dt, J = 14. 1,8.0Hz,1H),8.24(d,J=7.0Hz,1H),8.18(d,J=7.8Hz,1H),4.35(td,J=8.1,5.1Hz,1H),4.27(p,J=5.2 Hz,1H),4.24–4.15(m,2H),3.18(dq,J=25.3,10.3,8.1Hz,2H),2.49(d,J=10.2Hz,2H),2.37–2.22(m,1 H),1.92(s,4H),1.81(m,4H),1.44(m,1H),1.26(d,J=7.3Hz,3H),1.18–1.09(m,1H),0.87–0.78(m,6H). 13 C NMR(125MHz,(CD3)2SO)δ174.32,170.97,170.80,168.61,58.94,58.06,52.92,47 .99,46.06,36.76,33.51,30.36,24.84,23.92,20.63,17.45,15.78,11.42,3.92.

[0112] Example 8: Preparation of compound Pro-Ile-Se-Met-Phe-OH (Ⅰ-8)

[0113] The reaction formula is as follows:

[0114]

[0115] a. Synthesis of N-tert-butoxycarbonylprolyl isoleucyl selenomethionyl phenylalanine methyl ester Boc-Pro-Ile-Se-Met-Phe-OMe

[0116] The procedure was the same as step a in Example 7, except that alanine methyl ester hydrochloride (1.3 mmol, 0.182 g) was replaced with phenylalanine methyl ester hydrochloride (1.3 mmol, 0.280 g), and the final product was 0.645 g, with a yield of 96.6%.

[0117] b. Synthesis of N-tert-butoxycarbonylprolyl isoleucyl selenomethionylphenylalanine Boc-Pro-Ile-Se-Met-Phe-OH

[0118] The procedure was the same as step b in Example 7, except that 7-1 was replaced with –8-1, and the final product was 0.593g, with a yield of 93.9%.

[0119] c. Synthesis of prolyl-isoleucyl-selenomethionine phenylalanine NH2-Pro-Ile-Se-Met-Phe-OH

[0120] The procedure was the same as step c in Example 7, except that –7-2 was replaced with –8-2, ultimately yielding 0.483 g of product, with a yield of 96.2%. Its structural characterization is as follows: 1 H NMR(500MHz,(CD3)2SO)δ12.77(s,1H),10.28(s,1H),8.90–8.63(m,1H),8.44( d,J=31.9Hz,1H),8.36–8.02(m,2H),7.21(d,J=27.2Hz,5H),4.40(d,J=39.1Hz ,2H),4.33–4.06(m,2H),3.32–2.98(m,3H),2.93(d,J=10.6Hz,1H),2.39(dd,J =47.3,33.2Hz,3H),2.12–1.57(m,8H),1.43(s,1H),1.09(s,1H),0.80(s,6H). 13 C NMR (125MHz, (CD3)2SO) δ173.08,171.18,170.72,168.59,137.88,129.54,128.58,126.85,66.83,58. 93,58.06,53.83,53.03,46.05,36.98,36.77,33.71,30.38,24.84,23.93,20.62,15.79,11.41,3.91.

[0121] Example 9: Preparation of compound Pro-Ile-Se-Met-Val-OH (Ⅰ-9)

[0122] The reaction formula is as follows:

[0123]

[0124] a. Synthesis of N-tert-butoxycarbonylprolyl isoleucyl selenomethionylvaline methyl ester Boc-Pro-Ile-Se-Met-Val-OMe

[0125] The procedure was the same as step a in Example 1, except that alanine methyl ester hydrochloride (1.3 mmol, 0.182 g) was replaced with valine methyl ester hydrochloride (1.3 mmol, 0.218 g), and the final product was 0.603 g, with a yield of 97.3%.

[0126] b. Synthesis of N-tert-butoxycarbonylprolyl isoleucyl selenomethionylvaline Boc-Pro-Ile-Se-Met-Val-OH

[0127] The procedure was the same as step b in Example 7, except that 7-1 was replaced with –9-1, and the final product was 0.558g, with a yield of 94.6%.

[0128] c. Synthesis of prolyl isoleucyl selenomethionine valine NH2-Pro-Ile-Se-Met-Val-OH

[0129] The procedure was the same as step c in Example 7, except that –7-2 was replaced with –9-2, resulting in 0.434 g of product with a yield of 95.9%. Its structural characterization is as follows: 1 H NMR (500MHz, (CD3)2SO) δ12.65(s,1H),10.29(s,1H),8.75(d,J=8.6Hz,1H),8.48(t,J=9.9Hz,1H),8.28(d,J=8 .0Hz,1H),7.94(d,J=8.3Hz,1H),4.44(td,J=8.1,5.1Hz,1H),4.23(t,J=8.1Hz,2H),4.13(dd,J=8.4,5.6Hz,1H) ,3.53–3.31(m,1H),3.17(tq,J=20.7,6.3Hz,2H),2.51(d,J=3.0Hz,2H),2.38–2.22(m,1H),2.07(tq,J=13.4,6 .8Hz,1H),1.92(m,4H),1.89–1.72(m,3H),1.45(dqd,J=15.1,7.5,3.3Hz,1H),1.08(s,1H),0.91–0.76(m,12H). 13 C NMR(125MHz,(CD3)2SO)δ172.60,170.83,170.13,166.36,66.82,58.92,58.14,52.54 ,52.00,46.06,41.11,36.69,33.49,30.37,24.85,23.93,20.71,15.81,11.41,3.93.

[0130] Example 10: Preparation of compound Phe-Val-Se-Met-Pro-OH (Ⅰ-10)

[0131] The reaction formula is as follows:

[0132]

[0133] a. Synthesis of N-tert-butoxycarbonylphenylalanylvaline selenomethionylproline methyl ester Boc-Phe-Val-Se-Met-Pro-OMe

[0134] The procedure was the same as step a in Example 9, except that valine methyl ester hydrochloride (1.3 mmol, 0.218 g) was replaced with proline methyl ester hydrochloride (1.3 mmol, 0.215 g), ultimately yielding 0.616 g of product, with a yield of 94.3%.

[0135] b. The synthesis of N-tert-butoxycarbonylphenylalanylvaline selenomethionine proline Boc-Phe-Val-Se-Met-Pro-OH was performed in the same manner as in step b of Example 9, except that –9-1 was replaced with –10-1, and the final product was 0.622 g, with a yield of 95.1%.

[0136] c. Synthesis of phenylalanylvaline selenomethionine proline NH2-Phe-Val-Se-Met-Pro-OH

[0137] The procedure was the same as step c in Example 9, except that –9-2 was replaced with 10-2, ultimately yielding 0.466 g of product, with a yield of 96.2%. Its structural characterization is as follows: 1 H NMR(500MHz, (CD3)2SO) δ8.75(d,J=8.7Hz,1H),8.35(d,J=6.7Hz,4H),7.26(dt,J=1 7.5,4.3Hz,5H),4.60(q,J=7.1Hz,1H),4.21(ddt,J=11.8,8.5,4.5Hz,4H),3.76(dt, J=10.0,6.7Hz,1H),3.61(dq,J=10.0,6.7Hz,1H),3.17(dp,J=14.5,4.6Hz,1H),3.02 (s,1H),2.65–2.40(m,2H),2.21–2.07(m,1H),2.06–1.77(m,8H),1.00–0.77(m,6H). 13 C NMR (126MHz, (CD3)2SO) δ173.60,170.77,169.82,168.15,135.37,130.18,128.78,127.40,66. 83,58.95,53.47,50.90,47.09,37.14,32.35,31.26,29.11,25.09,20.81,19.61,18.90,4.06.

[0138] Example 11: Preparation of compound Phe-Val-Se-Met-Gly-OH (Ⅰ-11)

[0139] The reaction formula is as follows:

[0140]

[0141] a. Synthesis of N-tert-butoxycarbonylphenylalanylvaline selenomethionylglycine methyl ester Boc-Phe-Val-Se-Met-Gly-OMe

[0142] The procedure was the same as step a in Example 9, except that valine methyl ester hydrochloride (1.3 mmol, 0.218 g) was replaced with glycine methyl ester hydrochloride (1.3 mmol, 0.163 g), and the final product was 0.587 g, with a yield of 95.7%.

[0143] b. Synthesis of N-tert-butoxycarbonylphenylalanylvaline selenomethionylglycine Boc-Phe-Val-Se-Met-Gly-OH

[0144] The procedure was the same as step b in Example 9, except that –9-1 was replaced with 11-1, and the final product was 0.549 g, with a yield of 95.6%.

[0145] c. Synthesis of phenylalanylvaline selenomethionine NH2-Phe-Val-Se-Met-Gly-OH

[0146] The procedure was the same as step c in Example 9, except that –9-2 was replaced with 11-2, ultimately yielding 0.445 g of product, with a yield of 97.3%. Its structural characterization is as follows: White soild... 1 H NMR(500MHz,(CD3)2SO)δ12.55(s,1H),8.74(d,J=8.7Hz,1H),8.53–8.17(m ,4H),7.35–7.17(m,5H),4.40(s,1H),4.22(dd,J=10.1,3.5Hz,2H),3.80(d, J=6.0Hz,1H),3.70(d,J=5.7Hz,1H),3.17(dd,J=14.1,5.5Hz,1H),3.09–2.9 3(m,1H),2.62–2.42(m,2H),2.08–1.84(m,6H),0.88(dd,J=6.9,2.4Hz,6H). 13C NMR (125MHz, (CD3)2SO) δ171.65,171.44,170.77,168.22,135.37,130.16,128.80,127. 43,66.89,58.50,53.46,53.16,41.13,37.17,33.61,31.16,20.80,19.69,18.86,3.93.

[0147] Example 12: Preparation of compound Phe-Val-Gly-Se-Met-OH (Ⅰ-12)

[0148] The reaction formula is as follows:

[0149]

[0150] a. Synthesis of N-tert-butoxycarbonylphenylalanylvaline glycine methyl ester Boc-Phe-Val-Gly-OMe

[0151] The procedure was the same as step a in Example 1, except that 1-1 (1 mmol, 0.231 g) was replaced with 4-3 (1 mmol, 0.364 g), and the final product was 0.417 g, with a yield of 95.8%.

[0152] b. Synthesis of N-tert-butoxycarbonylphenylalanylvaline glycine Boc-Phe-Val-Gly-OH

[0153] The procedure is the same as step b in Example 1, except that 1-2 is replaced with 12-1, and the final product is 0.385g, with a yield of 95.3%.

[0154] c. Synthesis of N-tert-butoxycarbonylphenylalanylvaline-glycyl selenomethionine methyl ester Boc-Phe-Val-Gly-Se-Met-OMe

[0155] The procedure was the same as step c in Example 1, except that 1-3 (0.9 mmol, 0.259 g) was replaced with –12-2 (0.9 mmol, 0.379 g), resulting in a final product of 0.527 g and a yield of 95.4%.

[0156] d. Synthesis of N-tert-butoxycarbonylphenylalanylvalineglycylselenomethionine Boc-Phe-Val-Gly-Se-Met-OH

[0157] The operation was the same as step d in Example 1, except that 1-4 was replaced with 12-3, and the final product was 0.484 g, with a yield of 94.1%.

[0158] e. Synthesis of phenylalanylvaline-glycylselenomethionine NH2-Phe-Val-Gly-Se-Met-OH

[0159] The procedure was the same as step e in Example 1, except that 1-5 was replaced with –12-4, ultimately yielding 0.389 g of product, with a yield of 96.3%. Its structural characterization is as follows: 1 H NMR(500MHz,(CD3)2SO)δ8.58(s,1H),8.50–8.29(m,1H),7.65–7.50(m,1H),7.32–7.16(m,5 H),4.26–4.10(m,1H),4.04(q,J=7.0,6.6Hz,1H),3.71(dd,J=12.0,5.9Hz,2H),3.59(ddq,J= 12.9,8.2,4.5,4.0Hz,1H),3.01(dt,J=13.6,4.7Hz,1H),2.67(dd,J=13.6,8.4Hz,1H),2.41 (ddq,J=17.4,12.3,5.6Hz,1H),2.08–1.93(m,2H),1.87(d,J=3.6Hz,7H),0.91–0.74(m,6H). 13 C NMR (125MHz, (CD3)2SO) δ 178.93, 178.16, 176.51, 173.07, 143.48, 134.62, 133.37, 131.42, 63.08, 60.89, 47.57, 38.87, 35.81, 35.71, 27.24, 26.07, 24.50, 23.40, 23.28, 8.50. Example 13: Preparation of sample Phe-Pro-Se-Met-OH (Ⅰ-13)

[0160] The reaction formula is as follows:

[0161]

[0162] a. Synthesis of N-tert-butoxycarbonylphenylalanylproline methyl ester Boc-Phe-Pro-OMe

[0163] The procedure was the same as step a in Example 1, except that 1-1 (1 mmol, 0.231 g) was replaced with 13-1 (1 mmol, 0.265 g) and glycine methyl ester hydrochloride (1.3 mmol, 0.163 g) was replaced with proline methyl ester hydrochloride (1.3 mmol, 0.215 g), resulting in a final product of 0.361 g and a yield of 95.9%.

[0164] b. Synthesis of N-tert-butoxycarbonylphenylalanylproline Boc-Phe-Pro-OH

[0165] The procedure was the same as step b in Example 1, except that 1-2 was replaced with –13-2, ultimately yielding 0.336 g of product, with a yield of 96.8%. c. Synthesis of N-tert-butoxycarbonylphenylalanylprolyl selenomethionine methyl ester Boc-Phe-Pro-Se-Met-OMe

[0166] The procedure was the same as step c in Example 1, except that 1-3 (0.9 mmol, 0.259 g) was replaced with 13-3 (0.9 mmol, 0.326 g), and the final product was 0.477 g, with a yield of 95.5%.

[0167] The synthesis of N-tert-butoxycarbonylphenylalanylprolyl selenomethionine Boc-Phe-Val-Se-Met-OH was performed in the same manner as step d in Example 1, except that 1-4 was replaced with 13-4. The final product was 0.437 g, with a yield of 94.1%.

[0168] e. Synthesis of phenylalanylprolyl selenomethionine NH2-Phe-Pro-Se-Met-OH

[0169] The procedure was the same as step e in Example 1, except that 1-5 was replaced with 13-5, ultimately yielding 0.348 g of product, with a yield of 97.6%. Its structural characterization is as follows: 1 H NMR(500MHz,(CD3)2SO)δ12.48(s,1H),8.74–8.59(m,2H),8.39(d,J=7.8Hz,1H),7.35–7.27(m,3H),7 .21(d,J=6.8Hz,2H),4.28–4.21(m,1H),4.19–4.07(m,2H),3.55(s,2H),3.21(s,1H),2.92(s,1H),2. 63–2.51(m,1H),2.49–2.43(m,1H),2.10(dtd,J=14.3,9.4,4.9Hz,1H),2.04–1.96(m,1H),1.91(d,J= 8.9Hz,3H),1.74(dhept,J=14.1,5.1,4.5Hz,1H),1.63(d,J=7.5Hz,2H),1.37(dp,J=14.4,5.3Hz,1H). 13C NMR(125MHz,(CD3)2SO)δ173.20,171.32,167.40,135.16,129.91,128.98,127.81, 66.82,60.14,52.56,48.51,47.08,37.09,31.53,29.67,27.36,23.89,21.61,3.95.

[0170] Example 14: Synthesis of sample Lys-Se-Met-Gly-OH (Ⅰ-14)

[0171] The reaction formula is as follows:

[0172]

[0173] Synthesis of 2,6-di-tert-butoxycarbonylaminohexanoic acid N-tert-butoxycarbonylalanylselenomethionylglycine methyl ester Boc-Lys-SeMet-Gly-OMe

[0174] The procedure was the same as step a in Example 1, except that 1-1 (1 mmol, 0.231 g) was replaced with 4-3 (1 mmol, 0.525 g), and the final product was 0.574 g, with a yield of 96.3%.

[0175] b. Synthesis of 2,6-di-tert-butoxycarbonylaminohexanoic acid, N-tert-butoxycarbonylalanylselenomethionylglycine, Boc-Lys-SeMet-Gly-OH

[0176] The operation was the same as step d in Example 1, except that 1-4 was replaced with –14-1, and the final product was 0.530g, with a yield of 94.7%.

[0177] c. Synthesis of lysylselenomethionine isoleucine NH2-Lys-SeMet-Gly-OH

[0178] The procedure was the same as step e in Example 1, except that 1-5 was replaced with –14-2, resulting in 0.416 g of product with a yield of 97.5%. Its structural characterization is as follows: White solid. 1H NMR(500MHz, (CD3)2SO) δ12.58(s,1H),8.88(d,J=7.9Hz,1H),8.58–8.30(m,3H),8.17(t,J=5.7Hz,3H),4.36(td,J=8.1,5.0Hz,1H),3.78(s,2 H),3.68(s,1H),2.71(d,J=7.1Hz,2H),2.64–2.46(m,2H),1.93(s,4H),1.75(d,J=6.6Hz,2H),1.57(dd,J=10.8,4.3Hz,2H),1.45–1.30(m,2H). 13 C NMR (125MHz, (CD3)2SO) δ171.46,171.37,168.93,66.80,53.53,52.23,38.69,33.37,30.62,26.59,21.46,20.76,3.90.

[0179] Example 15: Synthesis of sample Leu-Gly-Se-Met-Val-OH (Ⅰ-15)

[0180] The reaction formula is as follows:

[0181]

[0182] a. Synthesis of N-tert-butoxycarbonylleucylglycylselenomethionylvaline methyl ester Boc-Leu-Gly-Se-Met-Val-OMe

[0183] The procedure was the same as step a in Example 1, except that 1-1 (1 mmol, 0.231 g) was replaced with 1-5 (1 mmol, 0.466 g), and glycine methyl ester hydrochloride (1.3 mmol, 0.163 g) was replaced with valine methyl ester hydrochloride (1.3 mmol, 0.218 g). The final product was 0.561 g, with a yield of 96.8%.

[0184] b. The synthesis of N-tert-butoxycarbonylleucylglycylselenomethionylvaline Boc-Leu-Gly-Se-Met-Val-OH was performed in the same manner as step d in Example 1, except that 1-4 was replaced with –15-1, and the final product was 0.519 g with a yield of 94.7%.

[0185] c. Synthesis of leucylglycylselenomethionine NH2-Leu-Gly-Se-Met-Val-OH

[0186] The procedure was the same as step e in Example 1, except that 1-5 was replaced with –15-2, resulting in 0.415 g of product with a yield of 97.2%. Its structural characterization is as follows: White solid. 1 H NMR(500MHz,D2O)δ4.41(d,J=2.3Hz,1H),4.15(d,J=6.0Hz,1H),3.99–3.83(m,3H),2.49(s,2H),2. 10(dq,J=13.3,7.0Hz,1H),2.06–1.93(m,2H),1.88(s,3H),1.72–1.54(m,3H),0.92–0.77(m,12H). 13 C NMR(126MHz,D2O)δ174.97,173.40,170.94,170.58,66.63,58.53,53.63,5 1.98,42.30,31.73,29.91,23.90,21.74,21.26,20.07,18.44,17.40,3.65.

[0187] Example 16: Preparation of L-selenomethionine (8)

[0188] The reaction formula is as follows:

[0189]

[0190] a. Preparation of L-aminobutyrolactone hydrochloride 2

[0191] Weigh 0.595 g (5 mmol) of L-homoserine into a 50 mL thick-bottom flask and add 6 M·L -1 7.5 mL of HCl was added, and the mixture was heated under reflux for 4 h. When the substrate was completely reacted, the mixture was cooled and filtered to obtain a white solid. After washing, product II-2 was obtained, with a yield of 94.3%. The measured melting point of the product was 218 °C.

[0192] b. Preparation of L-bromohomoserine hydrobromide 3

[0193] Weigh 0.548 g (4 mmol) of the above product 2 into a 50 mL pressure-resistant bottle, add acetic acid solution (HBr mass fraction of 33%), stir and heat at 110 °C for 5.5 h. After the substrate reaction is complete, cool to room temperature, a white precipitate is produced, and filter to obtain product 3 with a yield of 94.5%. 1 H NMR (500MHz, CD3OD) δ4.17(s,1H),3.66(td,J=6.9,2.4Hz,2H),2.63–2.47(m,1H),2.39(d,J=6.9Hz,1H). 13C NMR (126MHz, CD3OD) δ174.33,55.91,38.18,31.80.

[0194] c. Preparation of L-bromohomoserine methyl ester hydrochloride 4

[0195] Weigh 0.973 g (3.5 mmol) of the above product 3 into a 25 mL round-bottom flask, add 3 mL of SOCl2, reflux under nitrogen for 1 h, evaporate the solvent, cool to 0 °C, add 3 mL of cold methanol, and then slowly heat to 50 °C. After the reaction is complete, evaporate the solvent to obtain a pale yellow syrupy substance 4, with a yield of 93.2%.

[0196] d. Preparation of L-Boc-bromohomoserine methyl ester 5

[0197] Weigh 0.693 g (3 mmol) of the dried product 4 into a 25 mL round-bottom flask, add dichloromethane and stir to dissolve it, then add 0.334 g Et3N (3.3 mmol) and 0.719 g (Boc)2O (3.3 mmol), and react overnight at room temperature. After the substrate has reacted completely, evaporate the solvent to obtain a solid substance, which is purified by column chromatography to obtain the target product II-5 in 93.7%. 1 H NMR (500MHz, CDCl3) δ5.19(s,1H),4.41(s,1H),3.73(s,3H),3.42(s,2H),2.37(s,1H),2.19(s,1H),1.41(s,9H).

[0198] e. Preparation of L-Boc-selenomethionine methyl ester 6

[0199] First, 0.564 g (3 mmol) of dimethyl diselenide was weighed into a 25 mL flask, and then 3 mL of anhydrous methanol was added to dissolve it. Subsequently, 0.158 g (6.6 mmol) of dry NaH was rapidly added. This process was carried out under strictly anhydrous and oxygen-free conditions. A vigorous initial reaction was observed, and the reaction proceeded at room temperature for 40 min. Then, 0.590 g (2 mmol) of the completely dried product II-5 was weighed and dissolved in 3 mL of anhydrous methanol, and added to the above reaction solution. The substrate consumption and product formation during the reaction were monitored by GC-MS. After 5 h of reaction, the substrate reaction was complete, and the crude product was a pale yellow syrupy substance. The solvent was evaporated, and the product was purified by column chromatography to obtain the target product 6.

[0200] f. Preparation of L-selenomethionine methyl ester 7

[0201] Weigh 0.467 g (1.5 mmol) of the above substance into a 25 mL flask and add 5 mL of 4 M·L⁻¹ solution. -1Dissolve 1,4-dioxane in HCl solution and stir at room temperature for 2 hours. A white precipitate forms. Filter and wash with diethyl ether to obtain II-6, with a yield of 93.1%.

[0202] Preparation of g, L-selenomethionine 8

[0203] Weigh 72.11 g (10 mmol) of the above substance into a 100 mL flask, add 10 mL of methanol and 5 mL of water. Stir to dissolve, then add 48 mg (20 mmol) of LiOH. Monitor the reaction by TLC. After the reaction is complete and cooled, a white precipitate forms. After filtration, washing, and drying, L-selenomethionine is obtained. ESI-MS, m / z: M + 197. 1 H NMR (500MHz, D2O) δ3.87–3.77(m,1H),2.62(td,J=7.9,1.4Hz,2H),2.30–2.12(m,2H),2.02(d,J=1.5Hz,3H). 13 CNMR(125MHz,D2O)δ174.32,55.91,38.18,31.80.[α] D +21.7°(C=0.5, 2N HCl), mp=275℃.

[0204] Example 17: Detection of the antibacterial ability of selenium-containing peptides

[0205] (1) Sample solution preparation

[0206] Weigh 40 mg of the sample and dissolve it in 1 mL of LB broth (containing 1% sodium chloride, 1% peptone, and 0.5% yeast extract). Then dilute with LB broth to concentrations of 20 mg / mL, 10 mg / mL, and 5 mg / mL. Label the four concentrations of 5 mg / mL, 10 mg / mL, 20 mg / mL, and 40 mg / mL as A, B, C, and D. Similarly, label the prepared concentrations of 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL as A. 1 B 1 C 1 D 1 .

[0207] (2) Preparation of bacterial culture

[0208] Take eight test tubes containing 3 mL of LB medium and divide them into two groups. In one group, each tube is inoculated with 100 μL of Staphylococcus aureus (without resistance) culture, and in the other group, each tube is inoculated with 100 μL of Bacillus subtilis (without resistance) culture. Incubate at 37°C in a shaker for 12 h.

[0209] (3) Preparation of petri dishes

[0210] Add 1% agar to LB medium, autoclave, cool to about 60°C, and slowly pour into petri dishes, about 15 mL per dish, and place horizontally until completely solidified.

[0211] (4) Oxford cup method for detecting antibacterial activity of samples

[0212] Divide the plate into four equal areas and mark the bottom of each plate. Add 100 μL of bacterial suspension to each petri dish and spread evenly until no visible liquid remains on the surface. Place an Oxford cup in the center of each area and at the center of the plate. Add 100 μL of LB medium to the center Oxford cup as a control. Add 100 μL of sample solution of different concentrations to the other four Oxford cups. Cover the plates, seal with sealing film, and incubate at 37°C for 12 hours. Measure the size of the inhibition zone using calipers. If the diameter of the inhibition zone is less than 8 mm, the substance has no antibacterial activity (marked as "-"); between 8-10 mm, the substance has antibacterial activity (marked as "+"); 11-15 mm indicates moderate sensitivity (marked as "++"); and a diameter of 16 mm or more indicates high sensitivity (marked as "+++").

[0213] Table: Results of Antibacterial Experiment

[0214]

[0215]

[0216] Note: A=5mg / mL, B=10mg / mL, C=20mg / mL, D=40mg / mL

[0217] A 1 =0.05mg / mL, B 1 =0.5mg / mL, C 1 =1 mg / mL, D 1 =2mg / mL

[0218] As shown in the table above, when the dosage concentration is from 10 mg / mL to 40 mg / mL, compounds I-4 to I-12 have moderate to high levels of antibacterial activity against Staphylococcus aureus and Bacillus subtilis, with compound I-4 exhibiting the best sensitivity.

[0219] When the dosage is 0.05-2 mg / mL, the antibacterial activity of compound I-4 against Staphylococcus aureus is close to that of ampicillin and significantly superior to clotrimazole.

[0220] When administered at a concentration of 1-2 mg / mL, compound I-4 exhibits antibacterial activity against Bacillus subtilis.

Claims

1. The application of a selenium-containing polypeptide compound with the structure shown in formula (I) in the preparation of antibacterial drugs, wherein the bacteria is Staphylococcus aureus or Bacillus subtilis, and each of the formulas (I) is independently one of the following formulas: 。 2. Use according to claim 1, wherein The selenium-containing polypeptide compounds are compounds I-4, I-6, I-9, I-10, or I-12.

3. Use according to claim 2, wherein The selenium-containing polypeptide compound is compound I-4.

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