Coupled peptide chain for dissolving poorly soluble polypeptides and application thereof for separation and purification in liquid chromatography

Abstract

The invention discloses a coupled peptide chain for dissolving poorly soluble polypeptides and an application thereof for separation and purification in liquid chromatography, belonging to the field of biochemistry. A special linker arm is used to link a hydrophilic polypeptide chain with a poorly soluble polypeptide chain to solve the problem that the poorly soluble polypeptide chains cannot be operated in the liquid chromatography, and optimize the combination of hydrophilic amino acids, and then the poorly soluble polypeptide chain and hydrophilic polypeptide chain are broken by hydrolyzing an ester bond, so that the target peptide chain is directly precipitated, the method has the characteristics of simplicity and high efficiency, and the poorly soluble polypeptide product obtained by the method fully meets the standards required by customers.

Claims

1. A coupled peptide chain for dissolving poorly soluble polypeptides, wherein the peptide chain is as shown in formula (I):
X-Ph-Y(B).sub.n(I), wherein Ph represents a phenyl ring substituted at 1 and 4 positions; X represents a hydroxy group or a C1, C3 or C4 monohydroxy alkyl group; Y represents a carbonyl group or a C2-C4 carbonylalkyl group formed after the dehydration and condensation reaction with (B).sub.n; (B).sub.n represents a hydrophilic polypeptide chain formed by the condensation of n identical or different hydrophilic amino acids, and n is 1.2 to 2 times the number of amino acids of the poorly soluble polypeptide, wherein the hydrophilic amino acid is any one selected from a group consisting of arginine, lysine, asparagine, aspartic acid, glutamine, glutamic acid, histidine and proline, wherein the total number of glutamic acid or aspartic acid accounts for 30% to 80% of the total number of hydrophilic amino acids, and wherein the hydrophilic polypeptide chain includes DDDDDEEKEEEE (SEQ ID NO: 2).

2. A coupled peptide chain for dissolving poorly soluble polypeptides according to claim 1, wherein X is any one selected from a group consisting of a linear or branched CH.sub.2OH, CH.sub.2CH.sub.2CH.sub.2OH and CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH; and Y is any one selected from a group consisting of a linear or branched CH.sub.2CO, CH.sub.2CH.sub.2CO and CH.sub.2CH.sub.2CH.sub.2CO.

3. A coupled peptide chain for dissolving poorly soluble polypeptides according to claim 2, wherein the X-Ph-Y is any one selected from a group consisting of 4-hydroxybenzoic acid, p-hydroxymethylbenzoic acid, 4-(3-hydroxypropyl)benzoic acid, 4-(4-hydroxybutyl)benzoic acid, p-hydroxyphenylacetic acid, 4-(hydroxymethyl)phenylacetic acid, p-hydroxyphenylpropionic acid, 3-[4-(hydroxymethyl)phenyl]propionic acid, 4-(4-hydroxyphenyl)butyric acid, 2-(4-hydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)butyric acid, and 2-[(4-hydroxyphenyl)methyl]aminobutanoic acid.

4. A method for using the coupled peptide chain according to claim 1, comprising performing liquid chromatographic separation and purification of the poorly soluble polypeptide coupled with the peptide chain.

5. A coupled peptide chain for dissolving poorly soluble polypeptides according to claim 1, wherein the hydrophilic polypeptide chain is DDDDDEEKEEEE (SEQ ID NO: 2).

6. A coupled peptide chain for dissolving poorly soluble polypeptides, wherein the peptide chain is as shown in formula (I):
X-Ph-Y(B).sub.n(I), wherein Ph represents a phenyl ring substituted at 1 and 4 positions; X is CH.sub.2CH.sub.2OH; Y is any one selected from a group consisting of a linear or branched CH.sub.2CO, CH.sub.2CH.sub.2CO and CH.sub.2CH.sub.2CH.sub.2CO; and (B).sub.n represents a hydrophilic polypeptide chain formed by the condensation of n identical or different hydrophilic amino acids, and n is 1.2 to 2 times the number of amino acids of the poorly soluble polypeptide, wherein the hydrophilic amino acid is any one selected from a group consisting of arginine, lysine, asparagine, aspartic acid, glutamine, glutamic acid, histidine and proline, and wherein the hydrophilic polypeptide chain includes RRRRREDKKKKK (SEQ ID NO: 3).

7. A coupled peptide chain for dissolving poorly soluble polypeptides according to claim 6, wherein the X-Ph-Y is one selected from a group consisting of 4-(2-hydroxyethyl)benzoic acid and 4-(1-hydroxyethyl)-benzoic acid.

8. A coupled peptide chain for dissolving poorly soluble polypeptides according to claim 6, wherein the hydrophilic polypeptide chain is RRRRREDKKKKK (SEQ ID NO: 3).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic flow diagram of the polypeptide synthesis steps of embodiment 1 of the present invention; and

(2) FIG. 2 is a schematic flow diagram of the polypeptide synthesis steps of embodiment 2 of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

(3) The present invention is further described below in conjunction with particular embodiments.

Embodiment 1

(4) This embodiment is directed to separation and purification of a poorly soluble polypeptide ILVLLIII (SEQ ID NO: 1), 4-hydroxymethylbenzoic acid was selected as a linker arm, and a hydrophilic polypeptide chain DDDDDEEKEEEE (SEQ ID NO: 2) was linked via the linker arm. D represents aspartic acid, having a hydrophobic parameter of 3.5, protecting the functional group with OtBu; E represents glutamic acid, with a hydrophobic parameter of 3.5, protecting the functional group with OtBu; K represents lysine, having a hydrophobic parameter of 3.9, protecting the functional group with Boc; and all of these amino acids were subjected to solid-phase synthesis by protecting -amino group with Fmoc. As shown in FIG. 1, the specific synthesis steps are as follows:

(5) 1 g Fmoc-Glu(OtBu)-Wang resin was weighed, and [Operation A] was performed, i.e.: piperidine:DMF=1:4 (volume ratio) were added to remove the Fmoc protecting group at the N-terminal, the temperature was controlled at 30 C., the reaction was performed for 20 minutes, after the reaction, the resin was washed for three times with DMF/methanol respectively, a ninhydrin detection reagent was used for detection, and a color of blue indicated that the reaction was complete.

(6) Then [Operation B] was performed, i.e.: DIC and HOBT which were two times the moles of the initial resin, and amino acids having protecting groups in an amount of two times of the initial resin were added for reacting for 1 hour, the temperature was controlled at 30 C., after the reaction was finished, the resin was washed for three times with DMF, and color detection by ninhydrin showing colorless indicated that the reaction was complete.

(7) Subsequently, [Operation A] and [Operation B] were alternately performed, except that the hydrophilic amino acids added in [Operation B] were changed as the proceeding of the synthesis order. Glutamic acid, aspartic acid and lysine with protecting groups are respectively Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Lys(Boc)-OH. The reaction was thus performed until the linking of HMBA was complete, and color detection by a ninhydrin solution showing colorless indicated that the reaction was complete. A hydrophilic coupled peptide chain of X-Ph-Y(B).sub.n was obtained from the above reaction.

(8) Based on the peptide chain obtained above, [Operation B] was directly performed, and was alternated with [Operation A] in the subsequent steps until Wang resin-poorly soluble polypeptide chain-linker arm-hydrophilic polypeptide chain was obtained.

(9) Then the resin was lysed with 10 ml TFA:TIS:EDT=95%: 3%: 2% for 2 hours, and the protecting groups were removed from the coupled peptide chain. Then the resin was filtered to leave the mother liquor, and 100 ml diethyl ether was added thereto to precipitate polypeptide. A crude polypeptide was obtained after centrifuging at 3000 rpm for 2 minutes and precipitating, and after repeated washing and centrifugation, and then drying in a vacuum drier, a crude product of poorly soluble polypeptide chain-linker arm-hydrophilic polypeptide chain was obtained.

(10) The above crude product was dissolved in a mixed liquid of water and acetonitrile, and loaded for separation and purification by high performance liquid chromatography, and after separation and purification by a gradient elution chromatographic system with H.sub.2O/0.1 TFA % as the aqueous phase and ACN/0.1% TFA as the organic phase, target peaks were collected. The purity of the collected target peaks was detected by analytical high performance liquid chromatography. Qualified samples were concentrated by using a rotary evaporator and placed in a freezer to be pre-frozen into solids. Finally, the solids were placed into a vacuum freeze drier for lyophilization to obtain a pure product of ILVLLIII (SEQ ID NO: 1)-HMBA-DDDDDEEKEEEE (SEQ ID NO: 2).

(11) The above pure product was subjected with ester bond hydrolysis by using an LiOH saturated solution, after reacting at room temperature for 3 hours, the target polypeptide chain ILVLLIII (SEQ ID NO: 1) was precipitated, while the added sequence HMBA-DDDDDEEKEEEE (SEQ ID NO: 2) was dissolved in the LiOH solution due to a strong hydrophilicity and the whole acidic peptide chains, and then the precipitated target polypeptide was filtrated and lyophilized to obtain the finished product.

(12) In the above reactions, Fmoc amino acids were purchased from GL Biochem Co., Ltd., with the production batch number of GLS141015-4071, DIC Hobt DMAP was purchased from Suzhou Highfine Co., Ltd., HMBA was purchased from GL Biochem Ltd., TFA, TIS, EDT, ethyl ether, piperidine and DMF were all purchased from Nanjing Wanqing Co., Ltd.

Embodiment 2

(13) The present embodiment is directed to separation and purification of a poorly soluble polypeptide ILVLLIII (SEQ ID NO: 1), 4-(2-hydroxyethyl)benzoicacid (HPA) was selected as a linker arm, and a hydrophilic polypeptide chain RRRRREDKKKKK (SEQ ID NO: 3) was linked via the linker arm. D represents aspartic acid, having a hydrophobic parameter of 3.5, protecting the functional group with OtBu; E represents glutamic acid, having a hydrophobic parameter of 3.5, protecting the functional group with OtBu; K represents lysine, having a hydrophobic parameter of 3.9, protecting the functional group with Boc; R represents arginine, having a hydrophobic parameter of 4.5, protecting the functional group with Pbf; and all of these amino acids were subjected to solid-phase synthesis by protecting -amino group with Fmoc. As shown in FIG. 2, the specific synthesis steps are as follows:

(14) 1 g Fmoc-Lys(boc)-Wang resin was weighed, and [Operation A] was performed, i.e.: piperidine:DMF=1:4 (volume ratio) were added to remove the Fmoc protecting group at the N-terminal, the temperature was controlled at 30 C., the reaction was performed for 20 minutes, after the reaction, the resin was washed for three times with DMF/methanol respectively, a ninhydrin detection reagent was used for detection, and a color of blue indicated that the reaction was complete.

(15) Then [Operation B] was performed, i.e.: DIC and HOBT which were two times the moles of the initial resin, and amino acids having protecting groups in an amount of two times of the initial resin were added for reacting for 1 hour, the temperature was controlled at 30 C., after the reaction was finished, the resin was washed for three times with DMF, and color detection by ninhydrin showing colorless indicated that the reaction was complete.

(16) Subsequently, [Operation A] and [Operation B] were alternately performed, except that the hydrophilic amino acids added in [Operation B] were changed as the proceeding of the synthesis order. Lysine, aspartic acid, glutamic acid and arginine with protecting groups are respectively Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Arg(Pbf)-OH. The reaction was thus performed until the linking of HPA was complete, and color detection by a ninhydrin solution showing colorless indicated that the reaction was complete. A hydrophilic coupled peptide chain of X-Ph-Y(B).sub.n was obtained from the above reaction.

(17) Based on the peptide chain obtained above, [Operation B] was directly performed, and was alternated with [Operation A] in the subsequent steps until Wang resin-poorly soluble polypeptide chain-linker arm-hydrophilic polypeptide chain was obtained.

(18) Then the resin was lysed with 10 ml TFA:TIS:EDT=95%: 3%: 2% for 2 hours, and the protecting groups were removed from the coupled peptide chain. Then the resin was filtered to leave the mother liquor, and 100 ml diethyl ether was added thereto to precipitate polypeptide. A crude polypeptide was obtained after centrifuging at 3000 rpm for 2 minutes and precipitating, and after repeated washing and centrifugation, and then drying in a vacuum drier, a crude product of poorly soluble polypeptide chain-linker arm-hydrophilic polypeptide chain was obtained.

(19) The above crude product was dissolved in a mixed liquid of water and acetonitrile, and loaded for separation and purification by high performance liquid chromatography, and after separation and purification by a gradient elution chromatographic system with H.sub.2O/0.1 TFA % as the aqueous phase and ACN/0.1% TFA as the organic phase, target peaks were collected. The purity of the collected target peaks was detected by analytical high performance liquid chromatography. Qualified samples were concentrated by using a rotary evaporator and placed in a freezer to be pre-frozen into solids. Finally, the solids were placed into a vacuum freeze drier for lyophilization to obtain a pure product of ILVLLIII (SEQ ID NO: 1)-HPA-RRRRREDKKKKK (SEQ ID NO: 3).

(20) The above pure product was subjected with ester bond hydrolysis by using an LiOH saturated solution, after reacting at room temperature for 3 hours, the target polypeptide chain ILVLLIII (SEQ ID NO: 1) was precipitated, while the added sequence HPA-RRRRREDKKKKK (SEQ ID NO: 3) was dissolved in the LiOH solution due to a strong hydrophilicity and the whole acidic peptide chains, and then the precipitated target polypeptide was filtrated and lyophilized to obtain the finished product.

Test Embodiment

(21) This test evaluated the dissolving capacity of each group of peptide chain at 2 mg/mL in a DMF solution, a DMSO solution, an ACN:H.sub.2O (1:3) solution in HPLC, and an LiOH saturated solution, respectively. The poorly soluble polypeptide chain-linker arm-hydrophilic polypeptide chain in embodiments 1 and 2 were taken as Groups 1 and 2, respectively, and a separate poorly soluble polypeptide chain ILVLLIII (SEQ ID NO: 1) was taken as a control group, as shown in Table 3:

(22) TABLE-US-00003 TABLE 3 Dissolving capacity evaluation after the treatment of the poorly soluble polypeptide chain LiOH ACN:H.sub.2O saturated Group Peptide chain DMF DMSO (1:3) solution 1 ILVLLIII (SEQ ID completely completely completely completely NO: 1)-HMBA- dissolved, dissolved, dissolved, dissolved, DDDDDEEKEEEE no solid no solid no solid no solid (SEQ ID NO: 2) precipitating precipitating precipitating precipitating 2 ILVLLIII (SEQ ID partially partially completely partially NO: 1)-HPA- dissolved, dissolved, dissolved, dissolved, RRRRREDKKKKK solids solids no solid solids (SEQ ID NO: 3) precipitating precipitating precipitating precipitating 3 ILVLLIII (SEQ ID very cloudy, very cloudy, very cloudy, very cloudy, NO: 1) solids solids solids solids' precipitating precipitating precipitating precipitating

(23) The target peptide chain sequence was: ILVLLIII (SEQ ID NO: 1), which was very cloudy with solids precipitating after dissolving in DMF, DMSO and common HPLC purification condition of ACN: H.sub.2O=1: 3. ILVLLIII (SEQ ID NO: 1) 4-HMBA-DDDDDEEKEEEE (SEQ ID NO: 2) was clear with no solids precipitating after dissolving in DMF, DMSO and common HPLC purification condition of ACN: H.sub.2O (1: 3). However, ILVLLIII (SEQ ID NO: 1)-HPA-RRRRREDKKKKK (SEQ ID NO: 3) was completely dissolved in ACN:H.sub.2O (1:3), but solids still precipitated in DMF and DMSO.

(24) For the dissolution of the first group and second group of peptide chains in the LiOH saturated solution, it was best to require complete dissolving when adding, and then after waiting for 3 hours of reaction, the polypeptide chain hydrolysis was hydrolyzed to precipitate solids. Furthermore, the dissolution effect of the second group was obviously not better than that of the first group, which resulted in decreased product yield. It can be seen that the amino acid structure of the first group of hydrophilic polypeptide chain was more beneficial for the final precipitation of the poorly soluble polypeptide chain.

(25) The above are merely preferred embodiments of the present invention, and it should be pointed out that for a person skilled in the art, some improvements and modifications can also be made under the premise of not departing from the principle of the present invention, and these improvements and modifications should also be considered to be within the protection scope of the present invention.