Disulfide substitute of somatostatin and preparation method thereof
阅读说明：本技术 一种生长抑素的二硫替代物及其制备方法 (Disulfide substitute of somatostatin and preparation method thereof ) 是由 孟祥明 朱良静 方葛敏 朱满洲 于 2019-12-16 设计创作，主要内容包括：本发明公开了一种生长抑素的二硫替代物及其制备方法,通过使用硫醚键替换生长抑素二硫键后得到一种生长抑素二硫替代物,目标多肽是通过Fmoc固相合成方法实现,粗品经纯化,冻干得到生长抑素二硫替代物。本发明的生长抑素二硫替代物相比于生长抑素在二硫苏糖醇还原条件下具有良好的还原稳定性。(The invention discloses a dithio substitute of somatostatin and a preparation method thereof. Compared with somatostatin, the somatostatin disulfide substitute has good reduction stability under the condition of dithiothreitol reduction.)
1. A disulfide substitute for somatostatin, characterized by:
the disulfide substitute of somatostatin is obtained after the replacement of the disulfide bond of somatostatin is realized by using diamino diacid(ii) a The diamino diacid is Fmoc-A2(all/Alloy) -OH, having the formula:
2. the dithio substitute of somatostatin according to claim 1, characterized by the following structure:
3. a method for preparing the disulfide substitute of somatostatin according to claim 1 or 2, characterized by comprising the steps of:
step 1: synthesis of Fmoc-A2(Alloy/Alloy) resin
Swelling 2-CI-Trt resin with DMF, adding Fmoc-A2Reacting the mixture of (all/Alloy) -OH, DIPEA and DCM, and adding the mixture of DCM, MeOH and DIPEA to react to obtain Fmoc-A2(Alloy/Alloy) resin;
step 2: fmoc method for solid-phase synthesis of somatostatin disulfide substitute
Fmoc-A obtained in step 12(all/Alloy) resin is transferred into a solid phase synthesis tube, DMF is swelled and then condensed and connected with amino acid by adopting Fmoc solid phase synthesis method according to peptide sequence, after the condensation of 11 th amino acid Fmoc-Lys (Boc) -OH at the C end of the dithio substitute of somatostatin is finished, resin peptide Alloy and an all protective group are removed, the molecules are subjected to intramolecular cyclization after the Fmoc protective group, and amino acid is continuously condensed by adopting Fmoc solid phase synthesis method according to peptide sequence; after the solid phase synthesis is finished, a cutting reagent is added for reaction for 2 hours, and the somatostatin disulfide substitute H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Ala-OH (Cys3-Ala14, thioether bond) is obtained after the glacial ethyl ether precipitation and purification.
Technical Field
The invention relates to a dithio substitute of somatostatin and a preparation method thereof.
Background
Somatostatin is currently used as a disulfide cyclic peptide drug, has a half-life of 2 to 3 minutes, and has certain clinical value in the aspect of treating esophageal variceal bleeding. Studies have shown that disulfide bonds are closely related to the spatial structure and biological activity of polypeptides, however, disulfide bonds are easily reduced in vivo by thiol and disulfide isomerase, resulting in structural rearrangement and loss of biological activity of polypeptide drugs. Therefore, it is important to find a stable covalent bond to replace disulfide bond to constrain the spatial structure of polypeptide.
Disclosure of Invention
Aiming at the defect that the disulfide bond of somatostatin is easy to break under the reduction condition, the invention aims to provide a disulfide substitute of somatostatin and a preparation method thereof. The invention utilizes Fmoc-A2The thioether bond of (all/Alloy) -OH replaces a pair of disulfide bonds of somatostatin to form a more structurally stable disulfide substitute. Compared with the existing somatostatin, the somatostatin disulfide substitute has good reduction stability under the condition of dithiothreitol reduction.
The disulfide substitute of the somatostatin is prepared by using diamino diacid Fmoc-A2(aly/Alloy) -OH thioether bond to replace the disulfide bond of somatostatin.
The special amino acid Fmoc-A2The structural formula of (Alloy/Alloy) -OH is:
the disulfide substitute of somatostatin has the following structure:
the preparation method of the disulfide substitute of somatostatin comprises the following steps:
step 1: synthesis of Fmoc-A2(Alloy/Alloy) resin
Swelling 2-CI-Trt resin with DMF, adding Fmoc-A2Reacting the mixture of (all/Alloy) -OH, DIPEA and DCM, and adding the mixture of DCM, MeOH and DIPEA to react to obtain Fmoc-A2(Alloy/Alloy) resin;
step 2: fmoc method for solid-phase synthesis of somatostatin disulfide substitute
Fmoc-A obtained in step 12(Ally/Alloy) resin is transferred into a solid phase synthesis tube, DMF is swelled and then condensed and connected with amino acid by adopting Fmoc solid phase synthesis method according to peptide sequence, after the condensation of 11 th amino acid Fmoc-Lys (Boc) -OH at the C end of the somatostatin disulfide substitute is finished, resin peptide Alloy and Alloy protective group are removed, the molecules are subjected to intramolecular cyclization after the Fmoc protective group, and amino acid is continuously condensed by adopting Fmoc solid phase synthesis method according to peptide sequence; after the solid phase synthesis is finished, a cutting reagent is added for reaction for 2 hours, and the somatostatin disulfide substitute H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Ala-OH (Cys3-Ala14, thioether bond) is obtained after the glacial ethyl ether precipitation and purification.
The preparation process comprises the following steps:
step 1: synthesis of Fmoc-A2(Alloy/Alloy) resin
12.5mg of 2-CI-Trt resin (loading 0.40mmol/g,5umol) was weighed into a solid phase synthesis tube, DMF was added to swell for 15 minutes, 5.68mg of Fmoc-A was added2(all/Alloy) -OH, 7uL DIPEA, 150uL DCM, normal temperature reaction for 2 hours, adding 100uL DCM/MeOH/DIPEA (17:2:1), normal temperature end capping reaction for 20 minutes; the resin was washed 3 times with DCM and DMF.
Step 2: fmoc method for solid-phase synthesis of somatostatin disulfide substitute
2a, 20% piperidine in DMF was added to Fmoc-A obtained in step 12(all/Alloy) resin was soaked for 8 min and 6 min, respectively, and washed with DMF; adding a DMF mixture of the 2 nd amino acid from the C-terminus of the currently prepared DIC, Oxyma, somatostatin disulfide substitute (4.5-fold equivalent DIC: 4.5-fold equivalent Oxyma: 4.5-fold equivalent Fmoc-Ser (tBu) -OH, 200 uLDMF), reacting at 55 ℃ for 40 minutes, washing with DMF, and then condensing the amino acids by peptide sequence to repeat the above procedure, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc C-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Asn (trt) -OH, Fmoc-Lys (Boc) -OH; after completion of Fmoc-Lys (Boc) -OH condensation of the 11 th amino acid from the C-terminus of the somatostatin disulfide substitute, 6mg Pd (Ph P) was added3)4A mixed solution of (5.2umol), 15uL of N-methylaniline (138.6umol) and 250uL of THF, and reacting for 2 hours at normal temperature in a dark place; after the reaction was completed, 0.5% NaS was prepared from DMF (1 mL. times.3) and DMF in this order2CN(C2H5)2The resin was washed with solution (1 mL. times.6), DCM (1 mL. times.2), DMF (1 mL. times.2); adding 20% piperidine DMF solution to soak the resin for 8 min and 6 min respectively, and washing with DMF; adding a mixture of 13.0mg PyAOP (25umol), 3.5mg HOAT (25umol), 5.7uL NMM (50umol) and 250uL DMF, reacting at normal temperature for 4 hours, repeatedly condensing once, and washing with DMF; and (4) carrying out solid phase condensation on amino acids Fmoc-Gly-OH and Fmoc-Ala-OH sequentially.
2b, after the solid phase synthesis of the somatostatin disulfide substitute is finished, the resin is washed by a large amount of DMF and DCM, and after natural drying, a cleavage reagent (TFA: phenol: water: TIPS ═ 88:5:5:2, V%) is added for treatment of 0.3mL-0.5mL for 2 hours. Collecting a cutting reagent, adding 15 times of glacial ethyl ether with volume equivalent for precipitation, centrifuging powdery crude peptide, and semi-preparing HPLC purified polypeptide; vacuum freeze-drying to obtain purified somatostatin disulfide substitute H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Ala-OH (Cys3-Ala14, thioether bond), HPLC spectrum is shown in figure 3, mass spectrum is shown in figure 4, [ M + H ] +:1619.72, [ M + Na ] +:1642.65, theoretical molecular weight of somatostatin disulfide substitute: 1618.76, the mass spectrum result of the sample is consistent with the theoretical molecular weight, and the structure is correct.
Compared with the existing somatostatin, the somatostatin disulfide substitute has reduction stability, has only one atom difference in structure, and avoids larger structural change.
Drawings
Figure 1 is a schematic representation of a structural comparison of somatostatin and a somatostatin disulfide substitute of the invention.
FIG. 2 is a schematic diagram of the synthetic route of the present invention.
Figure 3 is an HPLC profile of a somatostatin disulfide substitute.
Figure 4 is a mass spectrum of a somatostatin disulfide surrogate.
FIG. 5 is a HPLC trace for somatostatin reduction stability test (a) and for somatostatin sulphur analogue reduction stability test (b).
Detailed description of the invention
The following is further illustrated with reference to the examples:
the meanings of the abbreviations used in the present invention are listed in the following table:
