阅读说明：本技术 一种rgd环化五肽的制备工艺 (Preparation process of RGD cyclized pentapeptide ) 是由 胡峻 计炜 徐春夏 徐佩 于 2019-11-13 设计创作，主要内容包括：本发明提供一种RGD环化五肽的制备工艺,针对该环肽的特殊结构,将天冬氨酸侧链的羧基固定在树脂上,在偶联氨基酸结束后,分别脱去天冬氨酸主链羧基的保护基团(OMe或者Oall)和D-苯丙氨酸上的Fmoc基团,然后进行固相成环,成环结束,切割树脂即可得到纯度较高的环形RGD五肽,且收率较高。该工艺价格操作简单,易于工业化生产。(The invention provides a preparation process of RGD cyclized pentapeptide, which is characterized in that aiming at the special structure of the cyclic peptide, carboxyl of an aspartic acid side chain is fixed on resin, after coupling amino acid is finished, a protecting group (OMe or Oall) of aspartic acid main chain carboxyl and an Fmoc group on D-phenylalanine are respectively removed, then solid phase cyclization is carried out, cyclization is finished, and the resin is cut to obtain the ring-shaped RGD pentapeptide with higher purity and higher yield. The process is simple in price operation and easy for industrial production.)

1. A preparation process of RGD cyclized pentapeptide is characterized in that the reaction equation is as follows:

the method comprises the following specific steps:

(1) preparation of intermediate 5: coupling the resin with the carboxyl group on the Fmoc-aspartic acid (X) side chain to obtain an intermediate 5, wherein the resin is CTC resin; x-is a protecting group of aspartic acid carboxyl, and is OMe and Oall;

(2) preparation of intermediate 4: sequentially coupling R-Asp (X) resin with Fmoc-glycine, Fmoc-arginine (R1), Fmoc-lysine (R2) and Fmoc-D-phenylalanine by using a solid-phase synthesis method to obtain an intermediate 4, wherein R1-is a protective group of arginine amino and is Pbf; r2-is a protecting group of lysine amino and is Boc and Dde;

(3) preparation of intermediate 3: removing OMe groups on aspartic acid by using NaOH/DMF solution, or removing Oall groups on aspartic acid by using palladium tetrakis (triphenylphosphine); the Fmoc group on D-phenylalanine was removed using DBLK solution (20%) to give intermediate 3;

(4) preparation of intermediate 2: cyclizing the linear peptide on the resin by using condensing agent HOBT/DIC, HBTU/DIEA to obtain an intermediate 2;

(5) preparation of the target compound: the RGD cyclized pentapeptide is cleaved from the resin under weak acid conditions.

Technical Field

The invention relates to the field of solid-phase polypeptide synthesis, in particular to a preparation process of RGD cyclized pentapeptide.

Background

With the change of environmental factors, the morbidity and mortality of various cancers are increased. Certain integrin receptors are often highly expressed specifically on some tumor cells or tumor neovascular endothelial cells: such as α v β 3, but is present in very small amounts in normal tissue vessels. RGD peptides are short peptides containing arginine, glycine, and aspartic acid (Arg-Gly-Asp), which are used as recognition sites for the interaction between integrin and its ligand, and can bind to some integrins specifically expressed by tumor cells or new blood vessels, such as α v β 3. Therefore, the receptor can be used as a target for tumor targeted therapy, and the tumor can be targeted and marked by using the competition of the exogenous RGD peptide and the integrin receptor, so that the aims of more effective, accurate and safe therapy are fulfilled. Most endogenous linear RGD peptides are in the bloodThe half-life period in the pulp is extremely short, the affinity to the alpha v beta 3 receptor is low, and the positioning effect and the biological activity are not ideal enough. Therefore, people modify and transform the RGD peptide to improve the stability, affinity, specificity and targeting property of the RGD peptide and prolong the action time in vivo. The RGD peptide primary sequence was found to have maximum affinity immediately following the glutamic acid residue and confirmed that the cyclized peptide has a more stable structure and stronger affinity. A plurality of medicines related to RGD peptide are developed, wherein CN101428148, CN101485891 and CN101474415 all relate to an RGD polypeptide radiopharmaceutical, and the structure of the radiopharmaceutical is^99mTc-HYNIC-PKM-E[L-c(RGDxK)]₂Wherein c (RGDxK) is a cyclic pentapeptide containing arginine, glycine and aspartic acid, and is an important starting material. CN106518966A discloses a synthesis method of RGD cyclopeptide, which is to perform liquid phase cyclization after solid phase synthesis of linear full-protection pentapeptide, wherein the yield is about 75%. Since the linear peptide inevitably undergoes side reactions such as polymerization during the liquid phase cyclization, the yield is lowered, and the condensation agent used in the cyclization is mixed with the final product, which makes the separation difficult. CN103588863B discloses a synthesis method of RGD cyclopeptide, which comprises the steps of fixing carboxyl of an Asp main chain on resin, after solid-phase synthesis of linear full-protection pentapeptide, respectively removing a protecting group Oall of an Asp side chain and an Fmoc group of phenylalanine, and carrying out solid-phase cyclization. The Asp used in the process protects amino acid, the protecting group is Oall, the price is high, in addition, Oall is removed, expensive tetrakis (triphenylphosphine) palladium is needed, and in addition, the use of the tetrakis (triphenylphosphine) palladium brings hidden danger of residual heavy metal palladium to the medicine.

Disclosure of Invention

The invention aims to provide a preparation process of RGD cyclized pentapeptide with simple synthetic route, low production cost, high yield and high purity aiming at the technical problems.

The technical scheme of the invention is as follows:

a preparation method of RGD cyclized pentapeptide has the following reaction equation:

the abbreviations used in the present invention and their meanings are listed in the following table:

the method comprises the following specific steps:

(1) preparation of intermediate 5: the resin was coupled with the carboxyl group on the side chain of Fmoc-aspartic acid (X) to give intermediate 5. The resin is a CTC resin; x-is a protecting group of aspartic acid carboxyl, and is OMe and Oall;

(2) preparation of intermediate 4: the intermediate 4 was obtained by coupling R-Asp (X) resin with Fmoc-glycine, Fmoc-arginine (R1), Fmoc-lysine (R2) and Fmoc-D-phenylalanine in this order by solid phase synthesis. R1-is a protecting group for arginine amino and is Pbf; r2-is a protecting group of lysine amino and is Boc and Dde;

(3) preparation of intermediate 3: removing OMe groups on aspartic acid by using NaOH/DMF solution, or removing Oall groups on aspartic acid by using palladium tetrakis (triphenylphosphine); the Fmoc group on D-phenylalanine was removed using DBLK solution (20%) to give intermediate 3;

(4) preparation of intermediate 2: cyclizing the linear peptide on the resin by using condensing agent HOBT/DIC, HBTU/DIEA to obtain an intermediate 2;

(5) preparation of the target compound: the RGD cyclized pentapeptide is cleaved from the resin under weak acid conditions.

The invention has the beneficial effects that: aiming at the special structure of the cyclopeptide, the carboxyl of the side chain of aspartic acid is fixed on resin, after the coupling of amino acid is finished, the protecting group (OMe or Oall) of the carboxyl of the main chain of aspartic acid and the Fmoc group on D-phenylalanine are respectively removed, then solid phase cyclization is carried out, the cyclization is finished, and the resin is cut to obtain the annular RGD pentapeptide with higher purity and higher yield. The process is simple in price and operation and easy for industrial production, compared with Fmoc-aspartic acid (Oall), the price of the used Fmoc-aspartic acid (OMe) is only one third of that of the Fmoc-aspartic acid (Oall), the reaction condition for removing the OMe group is relatively simple, the process only needs to be carried out under the alkaline condition of NaOH, and expensive tetrakis (triphenylphosphine) palladium is not needed.

Detailed Description