Use of a tetraphenylborate (TPB) salt for the separation of biomolecules

Abstract

Process for the separation of a biomolecule containing at least one cationic group from a liquid medium containing said biomolecule, which comprises the use of a tetraphenylborate (TPB) salt.

Claims

1. A process for the separation of a biomolecule containing at least one cationic group from a liquid medium containing said biomolecule, comprising adding a tetraphenylborate (TPB) salt and an alkaline agent to the liquid medium containing said biomolecule to form a biomolecule-TPB salt, and separating the said biomolecule-TPB salt from said liquid medium, wherein said liquid medium is an aqueous solution of the biomolecule wherein the pH of the aqueous solution of the biomolecule is controlled to be higher than or equal to 5 and less than or equal to 10 wherein the pH of the aqueous solution of the biomolecule is controlled by the addition of a mineral salt selected from the group consisting of alkali or earth alkali sulphates, and alkali or earth alkali hydrogen carbonates.

2. The process according to claim 1, wherein the biomolecule-TPB salt is separated from said aqueous solution by precipitation.

3. The process according to claim 1, wherein the aqueous solution is washed, before addition of the TPB salt, with an organic washing solvent which is not miscible with water and is selected from the group consisting of halocarbons, esters, and ethers.

4. The process according to claim 1, wherein the tetraphenylborate (TPB) salt includes an organic or inorganic cation.

5. A process for the manufacture of a biomolecule which comprises the separation process according to claim 1.

6. The process according to claim 1, wherein the biomolecule is a peptide which comprises at least one amino acid selected from the group consisting of arginine, homoarginine, and lysine.

7. The process according to claim 5, being for the manufacture of a peptide or a derivative thereof comprising one or more amino acid units containing a cationic group, comprising the steps: (a) coupling in an organic solution a first amino acid or a first peptide with a second amino acid or second peptide, wherein the first and/or the second amino acid or peptide comprises a cationic group to produce a peptide comprising a cationic group; (b) adding a tetraphenylborate (TPB) salt and an alkaline agent to form a peptide-TPB salt after the completion of the coupling step; and (c) separating the peptide containing the cationic group or a derivative from the organic solution.

8. The process according to claim 1, wherein the biomolecule-TPB salt is separated from said aqueous solution by crystallization.

9. The process according to claim 1, wherein the biomolecule-TPB salt is separated from said aqueous solution by extraction into an organic solvent, said extraction providing a first organic solution of the TPB salt of the biomolecule.

10. The process according to claim 1, wherein the alkaline agent is an inorganic base.

11. The process according to claim 1, wherein the cationic group of said biomolecule is selected from the group consisting of a guanidine group; a protonated primary, secondary or tertiary amino group; and a quaternary ammonium group.

12. The process according to claim 1, wherein the biomolecule is selected from the group consisting of a peptide, a peptide derivative, an oligonucleotide, an oligonucleotide derivative, and a cationic polysaccharide.

13. The process according to claim 1, wherein the tetraphenylborate (TPB) salt is added in a quantity from 1 to 10 equivalents per cationic group present in said biomolecule.

14. The process according to claim 4, wherein the organic cation is selected from the group consisting of tetraethylammonium, diisopropylethylammonium, N-ethylpiperidinium, N-methylmorpholinium and N-ethylmorpholinium.

15. The process according to claim 4, wherein the inorganic cation is Na.sup.+ or Li.sup.+.

16. The process according to claim 1, wherein the TPB anion of the tetraphenyl (TPB) salt is substituted on its benzene ring.

17. The process according to claim 10, wherein the TPB anion is tetrakis(3,5-bistrifluoromethylphenyl)borate.

18. The process according to claim 7, wherein the tetraphenylborate (TPB) salt includes an organic or inorganic cation.

19. The process according to claim 18, wherein the organic cation is selected from the group consisting of tetraethylammonium, diisopropylethylammonium, N-ethylpiperidinium, N-methylmorpholinium and N-ethylmorpholinium.

20. The process according to claim 18, wherein the inorganic cation is Na.sup.+ or Li.sup.+.

21. The process according to claim 7, wherein the TPB anion of the tetraphenyl (TPB) salt is substituted on its benzene ring.

22. The process according to claim 21, wherein the TPB anion is tetrakis(3,5-bistrifluoromethylphenyl)borate.

23. The process according to claim 7, wherein the tetraphenylborate (TPB) salt is added in a quantity from 1 to 10 equivalents per cationic group present in said peptide or derivative thereof.

Description

Example 1

Synthesis of HCl.H-Arg-Lys(Boc)-NH2

(1) 1.02 equivalents of Z-Arg-OH.HCl (Mw=344.8) were added at room temperature to a mixture of DMA and CH.sub.2Cl.sub.2 (6/4). Thereafter, 1.03 equivalents of HOBt (N-Hydroxybenzotriazole, Mw=135,12) and 1.00 equivalent of H-Lys(Boc)-NH.sub.2 (Mw=245.3; purity: 99%) were added. After cooling the solution to 05 C., 1.03 equivalents of EDC.HCl (Mw 191.7) were added.

(2) Stirring was continued for further 30 min at 05 C. and then for at least 2 hours at room temperature. After checking for the completion of the reaction by HPLC, the reaction mixture was diluted with a CH.sub.2Cl.sub.2/iso-BuOH (6/4) mixture and extracted with a solution of 0.5 eq. of HCl. The acidic aqueous phase was extracted a second time with a mixture of CH.sub.2Cl.sub.2/iso-BuOH (6/4). The combined organic phases were first washed with a 5% (weight) aqueous solution of Na.sub.2CO.sub.3 containing 1.05 equivalents of sodium tetraphenylborate (TPBNa) (Mw=342 g), and then 4 times with a 5% (weight) aqueous NaCl solution.

(3) After the organic phase had been concentrated in vacuo, methoxyethanol was added in several portions to the concentrate to eliminate traces of iso-butanol. It was then further evaporated. The concentrate was then finally diluted with methoxyethanol and slowly added to a cold (0 to 5 C.) 5% (weight) aqueous NaCl solution. The peptide precipitated and was kept for at least 30 min at low temperature and then filtered.

(4) The solid was washed 3 times with cold (05 C.) demineralised water. Thereafter, the solid was redissolved in MeOH and stirred until a slightly cloudy solution was obtained. The solution was partially concentrated and the methanolic solution was then added slowly to a cooled aqueous NaCl 5% (weight) solution. The precipitate was kept for at least 30 min at low temperature before it was filtered off. Finally the solid was washed 3 times with cold demineralised water (0 C.5 C.) and dried under vacuum (45 C.). An off white solid was finally obtained. The yield based on the NMR measurement of the content was 89%.

Synthesis of HCl.H-Arg-Lys(Boc)-NH2

(5) A methanol solution of 1.00 equivalent of TPB.Z-Arg-Lys(Boc)-NH.sub.2 (Mw=535.6; purity=62.0%) was passed several times through a column containing a methanol washed resin IRA 958 (Mw=1000; 3.00 equivalents). After checking the exchange by HPLC, the resin was filtered and washed three times with methanol. The combined organic phases were partially concentrated in vacuo. The concentrated solution was diluted with water.

(6) Pd catalyst (Mw=106.4; 2% weight) were added and the suspension then hydrogenated for at least 5 hours at 355 C. The catalyst was filtered off, washed twice with a mixture methanol/water. The filtrate was evaporated in vacuo, the residue suspended in DMA and partially evaporated in vacuo in order to eliminate traces of water. After checking the water content, the final solution was titrated by HCl (0.1N) and further used without any purification.

(7) Yield (based on the titration): 90%.

Example 2

Synthesis of 2HCl.H-Trp-Arg-Arg-Lys(Boc)-NH2(SEQ ID NO: 2)

Synthesis of Z-Trp-Arg-Arg-Lys(Boc)-NH2.2TPB (SEQ ID NO: 2)

(8) 1.00 equivalent Z-Trp-Arg-OH (Mw=494.5; purity=85.0%) and 1.10 equivalents HOOBt (Mw=163.13) were added to the DMA solution of 1.15 equivalents HCl.H-Arg-Lys(Boc)-NH.sub.2 (Mw=437.5; purity=20.0%) which had been previously diluted with CH.sub.2Cl.sub.2. After cooling the solution to 55 C., 1.00 equivalent HO/dioxane 4N was slowly poured in and then 1.10 equivalents EDC (Mw=191.7) were added. The reaction mixture was stirred at 55 C. for at least 3 hours and then at least for 8 hours at 55 C. After checking the completion of the reaction by HPLC, the reaction mixture was diluted with a CH.sub.2Cl.sub.2/sec-butanol mixture (6/4), washed first with a 5% (weight) aqueous solution of NaCl containing HCl (0.5 eq.), then with 1900 ml of a 5% (weight) aqueous solution of Na.sub.2CO.sub.3 containing 2.2 equivalents NaTPB (Mw=342), and finally five times with a 5% (weight) aqueous solution of NaCl. After the concentration of the organic layer, the residue was dissolved in methanol and then concentrated in vacuo in order to eliminate most of the remaining CH.sub.2Cl.sub.2. This final solution was titrated by NMR and further used without any purification.

(9) Yield (based on titration content)=83%.

Synthesis of 2HCl.H-Trp-Arg-Arg-Lys(Boc)-NH2(SEQ ID NO: 2)

(10) The methanol solution of 1.00 equivalent of 2TPB.Z-Trp-Arg-Arg-Lys(Boc)-NH.sub.2 (Mw=878.11) were passed several times through a column containing 6.00 equivalents of a methanol washed resin IRA 958 (Mw=1000). After checking the exchange by HPLC, the resin was filtered and washed three times with methanol. The combined organic phases were partially concentrated in vacuo. The concentrated solution was diluted with water and Pd catalyst were added. The suspension was then hydrogenated for at least 3 hours at 355 C. The catalyst was filtered off and washed twice with methanol. The filtrate was evaporated in vacuo, the residue dissolved in DMA and further concentrated in order to eliminate the remaining water. After the water content was checked, the precipitate was dissolved in DMA and partially evaporated in vacuo in order to adapt the weight of the solution. The final solution was titrated by 0.1N HCl and further used without any purification.

(11) Yield (based on the titration): 95%.

Example 3

Synthesis of 2HCl.H-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH2 (SEQ ID NO: 3)

Synthesis of Z-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH2.2TPB (SEQ ID NO: 3)

(12) 1.00 equivalent of Z-Trp-Trp-Pro-OH (Mw=621.7; purity=94.0%) was added to the DMA solution of 1.05 equivalents 2HCl.HTrp-Arg-Arg-Lys(Boc)-NH.sub.2 (Mw=816.9; purity=15.0%) previously diluted with CH.sub.2Cl.sub.2. Then, 1.20 equivalents N,N-Diisopropylethylamine (DIPEA) (Mw=129.2) and 1.05 equivalents of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) (HBTU) (Mw=379.24) were added. The reaction mixture was stirred at room temperature for at least 1 hour. After checking the completion of the reaction by HPLC, the reaction mixture was diluted with a CH.sub.2Cl.sub.2/iso-butanol mixture (8/2), washed first with a 5% (weight) aqueous solution of NaCl and HCl (1.5 eq.), then with a 5% (weight) aqueous solution of Na.sub.2CO.sub.3 and 2.2 equivalents NaTPB (Mw=342 g/mol), and finally three times with a 5% (weight) aqueous solution of NaCl. After concentration of the organic layer, the residual oil was several times dissolved in methoxyethanol, then concentrated in vacuo in order to eliminate most of the remaining iso-butanol. After GC control, the concentrate was precipitated by slowly pouring it into cold (05 C.) 5% (weight) aqueous solution of NaCl. After stirring for at least 1 hour, the suspension was filtered and washed twice with cold water. The precipitate was dried in vacuo at 45 C. A white solid was finally obtained.

(13) Yield (based on NMR content)=98%.

Synthesis of 2HCl.H-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH2(SEQ ID NO: 3)

(14) A methanol solution of 1.00 equivalent of 2TPB.Z-TrpTrp-Pro-Trp-Arg-Arg-Lys(Boc)-NH.sub.2 (SEQ ID NO: 3) (Mw=1347.5; purity=64.0%) was passed several times through a column containing a methanol washed resin IRA 958 (or Amberjet Cl 1000; 6.00 equivalents). After checking the exchange by HPLC, the resin was filtered, washed three times. The combined organic phases were partially concentrated in vacuo and then diluted with water. Finally, 2% (weight) of Pd catalyst were added and the suspension hydrogenated for at least 3 hours at 40 C. The catalyst was filtered off, washed three times with a mixture of methanol/water. The combined filtrates were evaporated in vacuo, the residue suspended in DMA and further concentrated in order to eliminate the remaining water. After checking the water content, the solution was titrated by HCl (0.1 N), AgNO.sub.3 (0.1N) or NMR and further used without any purification.

(15) Yield (based on titration)=82%.

Example 4

Synthesis of Z-Arg-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH2.3TPB (SEQ ID NO: 4)

(16) 1.00 equivalent of Z-Arg-Trp-Pro-OH (Mw=591.65; purity=96.0%), 1.00 equivalent of HO/dioxane (4N) and 1.05 equivalents HOBt (Mw=135.12; purity=98.0%) were added to 1.00 equivalent 2HCl.H-Trp-Trp-Pro-Trp-Arg-Arg-Lys(Boc)-NH.sub.2 (SEQ ID NO: 3) (Mw=1213.4; purity=85.0%) in solution in DMA diluted with CH.sub.2Cl.sub.2. After the solution had been cooled to 105 C., 1.02 equivalents EDC (Mw=191.7) were added. The reaction mixture was stirred at 105 C. for 30 min and then at least 4 hours at room temperature. After the completion of the reaction was confirmed by HPLC, the reaction mixture was diluted with a CH.sub.2Cl.sub.2/iso-butanol mixture (8/3), washed first with a 5% (weight) aqueous NaCl solution with HCl (0.5 eq.), then with a 5% (weight) aqueous solution of Na.sub.2CO.sub.3, containing 3 eq NaTPB (Mw=342), and finally twice with a 5% (weight) aqueous solution of NaCl. After concentration of the organic layer, the residual oil was several times dissolved in methoxyethanol, then concentrated in vacuo in order to eliminate most of the iso-butanol. After GC control, the concentrate was precipitated by slowly pouring it into cold 5% (weight) aqueous solution of NaCl. After stirring for at least 1 hour, the suspension was filtered, washed twice with cold water. The precipitate was dried at 405 C. An off white solid was finally obtained.

(17) Yield (based on NMR content)=87%.

(18) A methanol solution of 1.00 equivalent of 3TPB.Z-Arg-Trp-Pro-TrpTrp-Pro-Trp-Arg-Arg-Lys(Boc)-NH.sub.2 (Mw=1787.1; purity=57.0%) was passed several times through a column containing a methanol washed resin IRA 958 (Mw=1000, 9.00 equivalents). After the exchange had been checked by HPLC, the resin was filtered and washed three times with methanol. The combined organic phases were partially concentrated in vacuo. The concentrated solution was then diluted with water and Pd catalyst was added. The suspension was then hydrogenated for at least 6 hours at 355 C. The catalyst was filtered off, washed three times with a mixture methanol/water. The combined filtrates were evaporated in vacuo, the residue suspended in DMA and further concentrated in order to eliminate the remaining water. After checking the water content, the solution was titrated by HCl (0.1N) or NMR and further used without any purification.

(19) Yield (based on NMR content measurement): 93%.

Example 5

Synthesis of Z-(D)Arg-Gly-Arg-pNA.2HCl

Synthesis of Z-(D)Arg-Gly-Arg-pNA.2HCl

(20) 7.7 g of Z-(D)ArgOH (25 mmoles) and 13.8 g of 2HCl.H-Gly-ArgpNA were dispersed in about 100 ml of DMA at room temperature till complete dissolution. The mixture was then cooled to 0 C. and DIPEA (N,N-diisopropyl ethyl amine) was added to neutralize the excess of HCl, followed by 3.6 g of HOBt (26.13 mmoles) and 5.7 g of DCC (27.5 mmoles). The solution was left to stir at least 1 hour at 0 C. before being conditioned to 255 C. When the reaction was considered as completed (followed by HPLC), the crude was concentrated in vacuo, and the concentrate was then diluted with water to precipitate the DCU which was then removed by filtration and washed with water. The aqueous solution was washed several times with DCM to remove DMA (dimethyl acetamide) and HOBt. Two equivalents of TPBNa and 500 ml of DCM were poured into the aqueous solution while the pH was adjusted between 6.5 and 7.5 by the controlled addition of an aqueous NaHCO3 solution. After 1 hour of mixing, the aqueous phase was discarded and the organic phase was washed several times with an aqueous solution of NaCl and finally with water. The solvent was removed under vacuum and replaced with MeOH.

Isolation of Z-(D)Arg-Gly-Arg-pNA.2TPB

(21) The methanolic solution of Z-(D)Arg-Gly-Arg-pNA.2TPB was then poured into an aqueous 5% solution of NaCl at 05 C. to precipitate Z-(D)Arg-Gly-Arg-pNA.2TPB. After filtration, washing by water and drying under vacuum, Z-(D)Arg-Gly-Arg-pNA.2TPB was obtained as a white solid.

Isolation of the bischlorhydrate salt of Z-(D)Arg-Gly-Arg-pNA

(22) The TPB salt was dissolved in methanol and the solution was passed several times through a column containing a methanol washed resin IRA 958. After checking the exchange by HPLC, the resin was washed several times with methanol. The combined concentrated filtrates were concentrated under vacuum and the solid obtained was purified by HPLC and finally lyophilized to yield Z-(D)Arg-Gly-Arg-pNA as its bischlorhydrate salt.

Example 6

Synthesis of Ac-(D)Arg-Gly-Arg-pNA.2HCl

Isolation of purified Ac-(D)Arg-Gly-Arg-pNA.2TPB

(23) The pH of a Ac-(D)Arg-Gly-Arp-pNA.2TFA solution in water/acetonitrile (31 l) was adjusted to 70.5 by adding an aqueous solution of NaHCO.sub.3. The acetonitrile fraction was evaporated in vacuo (the volume of the solution was maintained by adding water if necessary). 1 kg TPBNa and 1.1 kg NaHCO.sub.3 were dissolved in about 20 l water. The concentrated peptide solution was then gradually poured into the aqueous solution TPBNa/NaHCO.sub.3. The Ac-(D)Arg-Gly-Arg-pNA.2TPB precipitated. After filtration, washing by water (40 l) and drying under vacuum (45 C.), 1.6 kg of Ac-(D)Arg-Gly-Arg-pNA.2TPB were obtained.

Isolation of the Purified Ac-(D)Arg-Gly-Arg-pNA.2HCl

(24) The TPB salt was dissolved in 20 l methanol which was passed several times through a column containing a methanol washed resin IRA 958 (7.6 kg resin). After checking the exchange by HPLC, the resin was washed several times with methanol (11 l each time). The combined concentrated filtrates were precipitated in 44 l cooled (55 C.) acetonitrile. After washing with acetonitrile (12 l), drying under vacuum, the precipitate gave 0.8 kg of off white solid. If necessary, the Ac-(D)Arg-Gly-Arg-pNA.2HCl can be precipitated a second time.

Example 7

Synthesis of Mpr(*)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(*)-NH2 (SEQ ID NO: 5)

(25) (*) indicates a disulfide bridge between the mercaptopropionic acid and the cysteinamid.

(26) 550 ml of a methanol solution of 46.6 g of Mpr(TrO-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(TrO-NH.sub.2 (SEQ ID NO: 5) (Mw=1374.8 g/mol, 20 mmol=1.00 equivalent) was treated with 130 g of a washed resin IRA 958 (Mw=1000 g/mol; 6.72 equivalents). After checking the exchange by HPLC, the resin was filtered and washed several times with 340 ml methanol. The combined organic phases were diluted by adding 1500 ml methanol, 5400 ml dichloromethane and finally 180 ml water. 24 g of iodine was added to the diluted solution. After checking the cyclisation by HPLC, the remaining iodine is quenched by adding 700 ml of Na.sub.2S.sub.2O.sub.3 (3.6% weight) in aqueous solution. The reaction mixture was then neutralized by 190 ml of resin acetate (Mw=720 g/mol; 57 equivalents). After filtration and washing of the resin by 210 ml methanol and 1800 ml water, the organic layer was separated from the aqueous one. The peptide (SEQ ID NO: 5) was extracted with 21 g of NaTPB (Mw=342.2 g/mol; 3 equivalents) from the neutralized aqueous layer by adding 10000 ml dichloromethane. After separation and evaporation of the organic layer, the residue was dissolved in methanol (900 ml) and then concentrated in vacuo in order to eliminate most of the remaining dichloromethane. The methanol solution was suspended into 2400 g of a washed resin IRA 958 (Mw=1000 g/mol; 12.1 equivalents). After checking the exchange by HPLC and washing the resin, the combined organic phases were diluted by adding 240 ml of acetic acid. This solution was further purified by preparative HPLC.

Example 8

Storage of Mpr(TrO-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(TrO-NH2 TPB Salt (SEQ ID NO: 5)

(27) Solid Mpr(Trt)-Har-Gly-Asp(OtBu)-Trp-Pro-Cys(Trt)-NH.sub.2 TPB salt (SEQ ID NO: 5) was obtained by precipitation in accordance with the process according to the invention. The product was stored for more than 1 year at a temperature of 20-25 C. The solid TPB salt had remained substantially stable after the storage.

SEQUENCE LISTING

(28) <110> SOLVAY SA <120> Use of a TPB salt for the separation of biomolecules <130> S200918 <160> 5 <170> PatentIn version 3.3 <210> 1 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 1 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 <210> 2 <211> 4 <212> PRT <213> Artificial <220> <223> Synthetic Peptide <400> 2 Trp Arg Arg Lys 1 <210> 3 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic Peptide <400> 3 Trp Trp Pro Trp Arg Arg Lys 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic Peptide <400> 4 Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (1) . . . (1) <223> Mercaptopropionic acid <220> <221> MISC_FEATURE <222> (2) . . . (2) <223> Homoarginine <400> 5 Xaa Xaa Gly Asp Trp Pro Cys 1 5