CN105461742B - It is a kind of to be used to verify compound of amino acid inducing polypeptide formation β sheet secondary structure abilities and preparation method thereof - Google Patents

Abstract

The invention discloses a compound for verifying the ability of amino acids to induce polypeptides to form a β-sheet secondary structure, which is characterized in that the structure is shown in the following formula. The present invention determines whether compound 1 can induce the connected polypeptide to form a stable β-sheet secondary structure by testing whether the amino hydrogen and carbonyl oxygen in compound 2 can form intramolecular hydrogen bonds, and whether this can be applied to synthetic fluorescent amino acids It has important guiding significance in the fields of fluorescence kit development and drug research and development.

Description

A method for verifying the ability of amino acids to induce polypeptides to form β-sheet secondary structures Compounds and methods for their preparation

technical field

The invention relates to the field of medical bioengineering, in particular to a compound for verifying the ability of amino acids to induce polypeptides to form a β-sheet secondary structure and a preparation method thereof.

Background technique

As one of the three major material foundations of the life system, protein is involved in almost every link of life activities and plays a decisive role in the birth, growth and reproduction of life. The occurrence of many diseases is also closely related to the variation of polypeptides and proteins in the body. Therefore, it is of great significance to select proteins, peptides and amino acid residues closely related to major diseases as targets, and develop highly selective and sensitive detection methods to reveal the mysteries of life, early diagnosis of diseases and screening of drugs.

Fluorescence detection is one of the important detection methods for diagnosing peptides and proteins at present. It has been widely used in protein detection because of its high sensitivity, good selectivity, wide dynamic response range, and in vivo detection. The fluorescence detection method needs to design polypeptide fluorescent probes according to the structure and properties of the target protein. One of the methods is to first synthesize fluorescent amino acids (by introducing fluorescent groups on the side chains), and then directly use them as a structural unit for peptide synthesis .

There are three kinds of secondary structures of proteins: β-turn, β-sheet and α-helix, which are mainly realized by the formation of hydrogen bonds between the hydrogen atoms on the -NH- in the peptide chain and the oxygen atoms on the carbonyl. By synthesizing β-turn mimics and introducing them into a polypeptide sequence to induce the formation of β-sheet structure, this kind of work has been reported a lot. When the β-turn mimic is combined with the fluorescent amino acid structure and the fluorescent amino acid of the specific β-turn mimic is synthesized, whether it can successfully induce the connected polypeptide to form a β-sheet secondary structure, the verification of this ability is currently available There is no report yet in the technology.

Contents of the invention

The purpose of the present invention is to solve the shortcomings of the above-mentioned background technology, to provide a compound and its preparation method for verifying the ability of amino acids to induce polypeptides to form β-sheet secondary structures, and to determine whether the verified specific amino acids can induce the formation of linked polypeptides Whether the stable β-sheet secondary structure can be applied to the development of fluorescent kits, drug development and other fields.

Technical scheme of the present invention is:

A compound for verifying the ability of amino acids to induce polypeptides to form a β-sheet secondary structure, characterized in that the structure is shown in the following formula.

The present invention also provides the synthetic method of above-mentioned compound, it is characterized in that, synthetic step is:

Preferably, the steps are:

(1) Add compound I-3, N-acetylcysteamine, and triethylamine to acetonitrile for reaction, stir at room temperature for 1-3 hours, and obtain solid intermediate I-8 after post-processing;

(2) Add intermediate I-8 and N-methyl-β-alaninamide to acetonitrile for reaction, stir at room temperature for 12-16 hours, and obtain solid compound 2 after post-processing;

Feeding ratio is following mol ratio in above-mentioned each step:

(1) Compound I-3: N-acetyl cysteamine: triethylamine=1:4.8-6:1-1.5;

(2) Intermediate I-8: N-methyl-β-alaninamide=1:1.5-2.

Further, the steps are:

(1) Add triethylamine to the acetonitrile solution in which compound I-3 is dissolved, then slowly add the acetonitrile solution in which N-acetylcysteamine is dissolved, stir at room temperature for 1-3 hours, extract, dry, filter and concentrate , separation and purification by silica gel column chromatography to obtain solid intermediate I-8;

(2) Add N-methyl-β-alaninamide to the acetonitrile solution in which intermediate I-8 is dissolved, stir at room temperature for 12-16h, add water, dry, filter, concentrate, and separate and purify by silica gel column chromatography to obtain a solid Compound 2.

Further, in each step, the feed ratio is the following molar ratio:

(1) Compound I-3: N-acetyl cysteamine: triethylamine=1:4.8:1;

(2) Intermediate I-8: N-methyl-β-alaninamide=1:1.5.

Compound 2 in the present invention is to verify the ability of compound 1 to induce the formation of β-sheet secondary structure of the polypeptide as shown below.

Compound 1 is a fluorescent amino acid for the specific synthesis of β-turn mimics. The synthetic route is as follows:

The specific synthetic steps of compound 1 are:

a. Preparation of Intermediate I-1:

Under a nitrogen atmosphere, pyrrole, benzaldehyde, and trifluoroacetic acid TFA were mixed and stirred at room temperature for 1-2 hours, and the solid intermediate I-1 was obtained after post-treatment;

b. Preparation of Intermediate I-2:

Under nitrogen atmosphere, add the tetrahydrofuran THF solution dissolved in chlorosuccinimide NCS to the tetrahydrofuran THF solution dissolved in intermediate I-1 at -78°C, keep stirring at -78°C for 2-3 hours, and continue stirring at room temperature for 3 hours -4h, the product was obtained after post-treatment; the obtained product was dissolved in dichloromethane, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DDQ was added, stirred at room temperature for 1-2h, after Process to obtain solid intermediate I-2;

c. Preparation of Intermediate I-3:

Under nitrogen atmosphere, dissolve intermediate I-2 and triethylamine in anhydrous dichloromethane, add boron trifluoride ether solution, stir at room temperature for 12-16 hours, and obtain solid intermediate I-3 after post-treatment;

d. Preparation of Intermediate I-4:

Intermediate I-3, Fmoc-protected aminoethanethiol, and triethylamine were reacted in acetonitrile at room temperature for 1-3 hours, and the solid intermediate I-4 was obtained after post-treatment;

e. Preparation of intermediate I-5:

Intermediate I-4, β-alanine tert-butanol ester was reacted in acetonitrile at room temperature for 12-16 hours, and the solid intermediate I-5 was obtained after post-treatment;

Preparation of f intermediate 1-6:

Intermediate I-5, Fmoc-Osu, and triethylamine were reacted in dioxane at room temperature for 3-5 hours, and the solid intermediate I-6 was obtained after post-treatment;

g. Preparation of compound 1:

Dissolve intermediate I-6 in dichloromethane, add trifluoroacetic acid under ice bath, react at room temperature for 2-4 hours, and obtain compound 1 after post-treatment;

Feeding ratio is following mol ratio in above-mentioned each step:

a. Pyrrole: benzaldehyde: trifluoroacetic acid TFA=20-25:1:0.1-0.2;

b. Intermediate I-1: chlorosuccinimide NCS: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DDQ=1:2.2-3:1.2-2;

c. Intermediate I-2: triethylamine: boron trifluoride ether = 1:2.2-3:4.2-5;

d. Intermediate I-3: Fmoc-protected aminoethanethiol: triethylamine=1:1.2-2:1.2-2.4;

e. Intermediate I-4: β-alanine tert-butanol ester=1:5-7;

f. Intermediate I-5: Fmoc-Osu: triethylamine=1:1.2-2:7.2-8;

g. Intermediate I-6: trifluoroacetic acid = 1:90-100.

The intermediate I-3 in the synthesis of compound 1 is the raw material in step a of the synthesis of compound 2, and I-3 is a known compound in the prior art.

The beneficial effects of the present invention are as follows: for the fluorescent amino acid compound 1 of a specific β-turn mimic, synthesize compound 2, and determine whether compound 1 can Induced linked peptides form a stable β-sheet secondary structure, which has important guiding significance for the application of synthetic fluorescent amino acids in the development of fluorescent kits, drug development and other fields.

Description of drawings

Fig. 1 is the infrared spectrogram of the dichloromethane solution of compound 2

Figure 2 is a graph of the NMR shift values of the amino hydrogen of compound 2 at different temperatures

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Preparation of compound 1

a. Preparation of Intermediate I-1:

Take a dry 500ml two-neck flask, replace the bottle with nitrogen, add pyrrole (104ml, 1.5mol), benzaldehyde (6mL, 60mmol) and trifluoroacetic acid (0.67mL, 6mmol), stir at room temperature for 1h, add 1M concentration of Quenched with NaOH solution (100 mL), extracted with ethyl acetate (200 mL×3), dried over anhydrous magnesium sulfate, filtered, concentrated, and recrystallized from ethanol to obtain 10.5 g of a brown solid with a yield of 80.7%.

b. Preparation of Intermediate I-2:

Intermediate I-1 (5g, 22mmol) was dissolved in 200mL of anhydrous tetrahydrofuran, replaced with a nitrogen system, and a tetrahydrofuran solution (60mL) of chlorosuccinimide (6.5g, 48.4mmol) was added at -78°C, Keep stirring at -78°C for 2 hours, then stir at room temperature for 3 hours. After the reaction, add water (300 mL), extract with dichloromethane (200 mL×3), dry over anhydrous magnesium sulfate, filter, concentrate, and dissolve the obtained product in dichloromethane (250mL), add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (6g, 26.4mmol), stir at room temperature for 1h, after the reaction, wash with water (100mL×3), Dry over anhydrous magnesium sulfate, filter, concentrate, and separate and purify by silica gel column chromatography (volume ratio of petroleum ether: ethyl acetate = 50:1) to obtain 5 g of yellow solid with a yield of 79%.

c. Preparation of Intermediate I-3:

Intermediate I-2 (5g, 17mmol) was dissolved in anhydrous dichloromethane (150mL), replaced by nitrogen system, triethylamine (5.2mL, 37.4mmol) was added, and then boron trifluoride ether solution (8.9 mL, 71.4mmol), stirred at room temperature for 12h, washed with water (100mL×3), extracted with dichloromethane (200mL×3), dried over anhydrous magnesium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography (volume ratio petroleum ether: acetic acid Ethyl ester=10:1) to obtain 5.1 g of dark red solid with a yield of 89%.

d. Preparation of Intermediate I-4:

Intermediate I-3 (2g, 6mmol) was dissolved in 100mL acetonitrile, catalyst triethylamine (1mL, 7.2mmol) was added, Fmoc-protected aminoethanethiol Fmoc-NHCH ₂ CH ₂ SH (2.2g, 7.2mmol ) was dissolved in 80mL of acetonitrile, and this solution was slowly added dropwise to the acetonitrile solution of intermediate I-3 and triethylamine, reacted at room temperature for 1h, added water (200mL), extracted with dichloromethane (200mL×3), anhydrous magnesium sulfate Dry, filter, concentrate, and separate and purify by silica gel column chromatography (volume ratio of petroleum ether: ethyl acetate: dichloromethane = 4:1:0.5) to obtain 2.5 g of a deep red solid with a yield of 70%. mp103.8-104.2°C.

¹ H NMR (400MHz, CDCl ₃ ) δ7.75 (d, J=7.5Hz, 2H), 7.54-7.51 (m, 4H), 7.46-7.36 (m, 7H), 7.29-7.27 (m, 2H), 6.87(d, J=4.0Hz, 1H), 6.72(d, J=4.0Hz, 1H), 6.67(d, J=4.0Hz, 1H), 6.35(d, J=4.0Hz, 1H), 5.40( ^13C NMR (100MHz, CDCl ₃ )δ156.4, 143.7, 141.3, 140.4, 136.6, 133.2, 133.0, 132.4, 130.4, 130.4, 130.3, 128.4, 128.3, 127.7, 127.1, 125.1, 119.9, 118.9, 1629, 4.9 32.8. MS(ESI) calcd for C ₂₃ H ₂₅ BClF ₂ N ₃ NaO ₂ S ⁺ (M+Na) ⁺ 622 found 622.

e. Preparation of intermediate I-5:

Intermediate I-4 (1.0g, 1.7mmol) was dissolved in 80mL of acetonitrile, then β-alanine tert-butanol ester (1.2mL, 8.5mmol) was added, reacted at room temperature for 12h, added water (100mL), dichloromethane Extract (100mL×3), dry over anhydrous magnesium sulfate, filter, concentrate, and separate and purify by silica gel column chromatography (dichloromethane:methanol=50:1 by volume) to obtain 0.6g of red solid with a yield of 73%. m.p.101.5-103.2°C.

¹ H NMR (400MHz, CDCl ₃ ) δ7.48–7.43(m, 5H), 6.88(d, J=8.0Hz, 1H), 6.40(d, J=4.0Hz, 1H), 6.35(d, J= 4.0Hz,1H),6.24(d,J＝8.0Hz,1H),3.69-3.66(m,2H),3.09-3.06(m,2H),2.99-2.94(m,2H),2.63-2.60(m ,2H),1.47(s,9H). ¹³ C NMR(100MHz,CDCl ₃ )δ169.9,161.4,137.6,135.4,134.4,134.2,133.3,131.6,130.3,129.1,128.1,120.8,117.9,110.2,82.0, 40.7, 40.6, 39.4, 35.9, 28.0; MS(ESI) calcd for C ₂₄ H ₃₀ BF ₂ N ₄ O ₂ S ⁺ (M+H) ⁺ 487found 487.

f. Preparation of Intermediate I-6:

Intermediate I-5 (0.60g, 1.2mmol) was dissolved in 50mL dioxane, Fmoc-OSu 9-fluorenylmethylsuccinimidyl carbonate (0.50g, 1.4mmol), triethylamine (1.2 mL, 8.6mmol), reacted at room temperature for 3h, added 50mL of dilute hydrochloric acid with a concentration of 1M, extracted with ethyl acetate (50mL×3), dried over anhydrous magnesium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography (volume ratio of petroleum ether : ethyl acetate=4:1) to obtain 0.52 g of red solid, the yield was 96%; m.p.86.1-87.1 °C.

¹ H NMR (400MHz, CDCl ₃ )δ7.76-7.74(m,2H),7.61-7.60(m,3H),7.47-7.30(m,8H),7.28-7.27(m,2H),6.87(d ,J=4.0Hz,1H),6.63(s,1H),6.41(m,2H),6.25(d,J=4.0Hz,1H),5.76(s,1H),4.31-4.29(m,2H) ,4.21-4.18(m,1H),3.68-3.61(m,2H),3.45-3.44(m,2H),3.09(m,2H),2.60-2.57(m,2H),1.46(s,9H) . ¹³ C NMR (100MHz, CDCl ₃ ) δ169.8, 161.5, 156.4, 144.0, 141.2, 136.9, 135.6, 134.3, 134.1, 133.5, 131.7, 130.2, 129.1, 128.1, 127.60, 1267.0, 125.9, 189 110.4,82.0,67.0,47.2,40.6,40.1,36.5,35.8,28.0; MS(ESI) calcd for C ₃₉ H ₃₉ BF ₂ N ₄ NaO ₄ S ⁺ (M+Na) ⁺ 731 found 731.

g. Preparation of compound 1:

Dissolve intermediate I-6 (0.50 g, 1 mmol) in 10 mL of dichloromethane, add (10 mL, 90 mmol) trifluoroacetic acid under ice-cooling, react at room temperature for 2 h, add water (30 mL), extract with dichloromethane (20 mL×3 ), dried over anhydrous magnesium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography (volume ratio dichloromethane:methanol:acetic acid=70:1:0.1) to obtain 0.32g of red solid, yield 77%; m.p.82.1-82.9 ℃.

¹ H NMR (400MHz, CDCl ₃ ) δ7.75(d, J=8.0Hz, 2H), 7.58(d, J=8.0Hz, 2H), 7.53-7.33(m, 9H), 7.31-7.27(m, 2H),6.97-6.96(m,1H),6.82(m,1H),6.43(m,1H),6.27-6.26(m,1H),4.35(d,J＝8.0Hz,2H),4.19(t ,J=8.0Hz,1H),3.75-3.71(m,2H),3.35-3.34(m,2H),2.77-2.74(m,4H); ¹³ C NMR(100MHz,CDCl ₃ )δ176.11,161.91,143.98 , _{141.30,136.85,134.07,133.10,130.25,129.39,128.36,127.66,127.07,125.14,123.51,119.93,110.87,66.82,47.23,40.16,36.858,34.21c} F for MS _B1,20 . ₂ N ₄ NaO ₄ S ⁺ (M+Na) ⁺ 675 found675.

Each step charge ratio is following mol ratio:

a. pyrrole:benzaldehyde:trifluoroacetic acid TFA=25:1:0.1;

b. Intermediate I-1: chlorosuccinimide NCS: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DDQ=1:2.2:1.2;

c. Intermediate I-2: triethylamine: boron trifluoride ether = 1:2.2:4.2;

d. Intermediate I-3: Fmoc-protected aminoethanethiol: triethylamine=1:1.2:1.2;

e. Intermediate I-4: β-alanine tert-butanol ester=1:5;

f. Intermediate I-5: Fmoc-Osu: triethylamine=1:1.2:7.2;

g. Intermediate I-6: trifluoroacetic acid = 1:90.

The synthesis of compound 1 is carried out at room temperature 18-30°C (preferably 25°C).

Example 1

The steps for preparing compound 2 are:

(1) Dissolve intermediate I-3 (100 mg, 0.30 mmol) in the process of preparing compound 1 in 30 mL of acetonitrile, add triethylamine (0.2 mL, 1.44 mmol), N-acetyl cysteamine (32 μL, 0.30 mmol) was dissolved in 10 mL of acetonitrile, this solution was slowly added to the reaction system, stirred at room temperature for 1 h, added water (50 mL), extracted with dichloromethane (50 mL×3 times), dried over anhydrous magnesium sulfate, filtered, concentrated, and silica gel column layer Analysis, separation and purification (volume ratio dichloromethane:methanol=30:1) gave 105 mg of red solid with a yield of 84%. m.p.208.8-209.6°C.

¹ H NMR (400MHz, CDCl ₃ ) δ7.56-7.45 (m, 5H), 6.91 (d, J = 4.5Hz, 1H), 6.73 (d, J = 4.5Hz, 1H), 6.70 (d, J = 4.0Hz, 1H), 6.36(d, J=4.0Hz, 1H), 6.19(s, 1H), 3.58(q, J=6.3Hz, 2H), 3.27(t, J=6.4Hz, 2H), 1.95 (s,3H) ^.13 C NMR(100MHz,CDCl ₃ )δ170.7,159.8,140.6,136.5,133.2,132.9,132.4,130.4,130.3,128.6,128.5,119.4,117.1,39.27,32.96,23.10; MS(ESI )calcd for C ₁₉ H ₁₇ BClF ₂ N ₃ NaOS ⁺ (M+Na) ⁺ 422 found 422.

(2) Intermediate I-8 (50 mg, 0.12 mmol) was dissolved in 20 mL of acetonitrile, N-methyl-β-alaninamide (25 mg, 0.18 mmol) was added, and N-methyl-β-alaninamide was also It can be called N-methyl-3-aminopropanamide, react at room temperature for 12h, add water (20mL), extract with dichloromethane (20mL×3 times), dry over anhydrous magnesium sulfate, filter, concentrate, and separate and purify by silica gel column chromatography (Volume ratio of dichloromethane:methanol=20:1) 45 mg of red solid was obtained with a yield of 78%. m.p.104.8-105.4°C.

1H NMR (400MHz, CDCl3) δ7.53-7.39(m, 5H), 6.89(d, J=5.0Hz, 1H), 6.75(s, 1H), 6.58(s, 1H), 6.45-6.38(m, 2H), 6.31(d, J=5.0Hz, 1H), 5.78(s, 1H), 3.80-3.75(m, 2H), 3.47-3.43(m, 2H), 3.06-3.03(m, 2H), 2.80 (d,J=4.8Hz,3H),2.54(t,J=6.4Hz,2H),1.92(s,3H).13C NMR(100MHz,CDCl3)δ170.4,170.2,161.7,136.6,135.7,134.2,134.0 , 133.5, 131.5, 130.2, 129.2, 128.2, 120.9, 118.9, 110.8, 41.1, 38.3, 36.8, 36.5, 26.4, 23.1; MS (ESI) calcd for C23H26BF2N5NaO2S+(M+Na)+508 found 508.

Each step charge ratio is following mol ratio:

Compound I-3: N-acetyl cysteamine: triethylamine = 1:4.8:1;

Intermediate I-8: N-methyl-β-alaninamide=1:1.5.

In the present invention, the room temperature is 18-30° C. (preferably 25° C. in this embodiment).

Example 2

The steps for preparing compound 2 are:

(1) Dissolve intermediate I-3 (100 mg, 0.30 mmol) in the process of preparing compound 1 in 40 mL of acetonitrile, add triethylamine (0.25 mL, 1.8 mmol), N-acetyl cysteamine (48 μL, 0.45 mmol) was dissolved in 20 mL of acetonitrile, this solution was slowly added to the reaction system, stirred at room temperature for 3 h, added water (50 mL), extracted with dichloromethane (50 mL×3 times), dried over anhydrous magnesium sulfate, filtered, concentrated, and silica gel column layer Analysis, separation and purification (volume ratio dichloromethane:methanol=30:1) gave red solid intermediate I-8;

(2) Dissolve intermediate I-8 (50 mg, 0.12 mmol) in 30 mL of acetonitrile, add N-methyl-β-alaninamide (33.3 mg, 0.24 mmol), react at room temperature for 16 h, add water (20 mL), and Extracted with methyl chloride (20 mL×3 times), dried over anhydrous magnesium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography (volume ratio of dichloromethane:methanol=20:1) to obtain compound 2 as a red solid.

Each step charge ratio is following mol ratio:

Compound I-3: N-acetyl cysteamine: triethylamine = 1:6:1.5;

Intermediate I-8: N-methyl-β-alaninamide=1:2.

In the present invention, the room temperature is 18-30° C. (preferably 18° C. in this embodiment).

Example 3

The steps for preparing compound 2 are:

(1) Dissolve intermediate I-3 (100 mg, 0.30 mmol) in the process of preparing compound 1 in 50 mL of acetonitrile, add triethylamine (0.21 mL, 1.5 mmol), N-acetyl cysteamine (40 μL, 0.375 mmol) was dissolved in 15 mL of acetonitrile, this solution was slowly added to the reaction system, stirred at room temperature for 2 h, added water (50 mL), extracted with dichloromethane (50 mL×3 times), dried over anhydrous magnesium sulfate, filtered, concentrated, and silica gel column layer Analysis, separation and purification (volume ratio dichloromethane:methanol=30:1) gave red solid intermediate I-8;

(2) Dissolve intermediate I-8 (50 mg, 0.12 mmol) in 25 mL of acetonitrile, add N-methyl-β-alaninamide (29.2 mg, 0.21 mmol), react at room temperature for 14 h, add water (20 mL), and Extracted with methyl chloride (20 mL×3 times), dried over anhydrous magnesium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography (volume ratio of dichloromethane:methanol=20:1) to obtain compound 2 as a red solid.

Each step charge ratio is following mol ratio:

Compound I-3: N-acetyl cysteamine: triethylamine = 1:5:1.25;

Intermediate I-8: N-methyl-β-alaninamide=1:1.75.

In the present invention, the room temperature is 18-30° C. (preferably 30° C. in this embodiment).

Intramolecular Hydrogen Bonding Test of Compound 2

There are mainly two types of intramolecular hydrogen bonds in compound 2, as shown in the following formulas 2-A and 2-B, according to literature reports (Nesloney, CL; Kelly, JWSynthesis and hydrogen bonding capabilities of biphenyl-based amino acids designed to nucleate β -sheetstructure.J.Org.Chem.1996,61,3127-3137.) The nitrogen-hydrogen stretching vibration peak of the amide bond is in the range of 3200-3500cm ^-1 , usually a narrower peak in the range of 3400-3500cm ^-1 It is the stretching vibration peak of free nitrogen-hydrogen bond, and the broad peak in the range of 3200-3400cm ^-1 is the stretching vibration peak of nitrogen-hydrogen bond with hydrogen bond.

We dissolve the compound 2 obtained by synthesis in anhydrous dichloromethane, and measure the infrared spectrum at room temperature, as shown in Figure 1, there is a broad peak at the highest peak at 3310cm ^-1 , mainly 2-A and 2- The nitrogen-hydrogen stretching vibration of intramolecular hydrogen bonds exists in the two mixed states of B, because the infrared detection speed is much faster than the speed at which the two conformations reach equilibrium. The narrow peak at the highest peak at 3410cm ^-1 is the sum of nitrogen-hydrogen stretching vibrations of amino groups in 2-A and 2-B and amide bonds without forming intramolecular hydrogen bonds.

While conducting infrared spectrum research on compound 2, the NMR shift values of the amino hydrogen of the amide bond at different temperatures were also studied. As shown in Figure 2, under normal circumstances, 25 ° C, CD ₂ Cl ₂ as the solvent for hydrogen spectrum scanning, the displacement value of the amino hydrogen with hydrogen bonds is generally in the low field (~ 7.0-9.0ppm), while the free amino hydrogen The displacement value is generally (~5.5-6.0ppm). And there is a great relationship between the nuclear magnetic shift value of the amino hydrogen with intramolecular hydrogen bonds and the temperature (Δδ/ΔT～-10 to -13ppb/K), while the hydrogen of the free amino group changes relatively little with the temperature (Δδ/ΔT～-13ppb/K). ΔT～-3ppb/K). The H NMR spectrum of compound 2 was studied at different temperatures. The amino hydrogen in compound 2 moves like a downfield as the temperature gradually decreases. There is an equilibrium between 2-A and 2-B, and their Δδ/ΔT is about -12ppb/ K.

Based on the research on the infrared spectrum and the variable temperature NMR spectrum of compound 2, it can be proved that there are intramolecular hydrogen bonds in this kind of compound, and compound 1 has the ability to induce its linked polypeptide to form a β-sheet structure.

Claims (5)

1. A compound for verifying the ability of amino acids to induce polypeptides to form a β-sheet secondary structure, characterized in that the structure is shown in the following formula 2. a synthetic method of compound as claimed in claim 1, is characterized in that, synthetic step is: 3. synthetic method as claimed in claim 2, is characterized in that, step is: (1) Add compound I-3, N-acetylcysteamine, and triethylamine to acetonitrile for reaction, stir at room temperature for 1-3 hours, and obtain solid intermediate I-8 after post-processing; (2) Add intermediate I-8 and N-methyl-β-alaninamide to acetonitrile for reaction, stir at room temperature for 12-16 hours, and obtain solid compound 2 after post-processing; Feeding ratio is following mol ratio in above-mentioned each step: (1) Compound I-3: N-acetyl cysteamine: triethylamine=1:4.8-6:1-1.5; (2) Intermediate I-8: N-methyl-β-alaninamide=1:1.5-2. 4. synthetic method as claimed in claim 3, is characterized in that, step is: (1) Add triethylamine to the acetonitrile solution in which compound I-3 is dissolved, then slowly add the acetonitrile solution in which N-acetylcysteamine is dissolved, stir at room temperature for 1-3 hours, extract, dry, filter and concentrate , separation and purification by silica gel column chromatography to obtain solid intermediate I-8; (2) Add N-methyl-β-alaninamide to the acetonitrile solution in which intermediate I-8 is dissolved, stir at room temperature for 12-16h, add water, dry, filter, concentrate, and separate and purify by silica gel column chromatography to obtain a solid Compound 2. 5. synthetic method as claimed in claim 3 or 4, is characterized in that, in each step, feed ratio is following mol ratio: (1) Compound I-3: N-acetyl cysteamine: triethylamine=1:4.8:1; (2) Intermediate I-8: N-methyl-β-alaninamide=1:1.5.