COMPOSITION OF A LIPOPHILIC AGENT FOR SOLUTION PHASE SYNTHESIS OF BIOMOLECULES

Abstract

Disclosed is a lipophilic agent comprising a triazine core having lipophilic groups for organic synthesis. The lipophilic agent soluble in one system of solvent(s) wherein the lipophilic agent is participated in a chemical reaction but insoluble by adding a miscible poor solvent to change solution composition after the reaction completes. The lipophilic agent facilitates process improvement wherein practical operation only involves mixing with reactants in solution followed by precipitating with change of solution composition followed by filtering to obtain the precipitated solid, simplifying the purification by isolating the desired solid. The operation is reproducible along the progress in a multi-step synthesis, allowing pure intermediates and pure product as a solid to be rapidly obtained with ease and certainty. This invention can thus accelerate research and development of pharmaceutical biomolecules, representing a tremendous step forward for boosting productivity and greening chemical industry.

Claims

1. A composition of a lipophilic agent including a lipophilic anchor which has a triazine core shown in the following Chemical Formula (1) in an organic synthesis reaction: ##STR00024## wherein R.sup.1 to R.sup.2 may be same or different, and are independently selected from the group comprising alkyl group with a carbon number of 12 to 48 which may have a substituent group, alkoxyl group with a carbon number of 12 to 48 which may have a substituent group, aryl group with a carbon number of 12 to 48 which may have a substituent group, thioalkyl group with a carbon number of 12 to 48 which may have a substituent group, alkylamino group with a carbon number of 12 to 48 which may have a substituent group, and dialkylamino group with a carbon number of 12 to 48 which may have a substituent group, and X represents an active site of the lipophilic agent having one or more atoms including carbon atom, halogen atom, nitrogen atom, oxygen atom, silicon atom or sulfur atom.

2. The composition of claim 1, wherein X represents an active site including the following formulas 1P, 1C, 1H, 1A, 1B, 1W, 1T, 1R: ##STR00025## ##STR00026##

3. The composition of claim 1, wherein R.sup.1 and R.sup.2 are independently selected from the groups including alkylamino group with a carbon number of 12 to 48 which may have a substituent group and alkoxyl group with a carbon number of 12 to 48 which may have a substituent group shown in the following Chemical Formulas (2, 2′ and 2″): ##STR00027## wherein R.sup.3, R.sup.4 and R.sup.5 may be same or different, and are independently selected from the group comprising alkyl group with a carbon number of 12 to 48 which may have a substituent group, and R.sup.6 may include a hydrogen atom, methyl, ethyl, propyl, isopropyl, or butyl and X represents an active site of the lipophilic agent having one or more atoms selected from the group comprising carbon atom, halogen atom, nitrogen atom, oxygen atom, silicon atom or sulfur atom.

4. The composition of claim 3, wherein in the Chemical Formulas (2, 2′ and 2″), R.sup.3, R.sup.4 and R.sup.5 are n-octadecyl group and R.sup.6 is a methyl group shown by the following Chemical Formulas (3M, 3G and 3Q) and X represents an active site of the lipophilic agent having one or more atoms selected from the group comprising carbon atom, halogen atom, nitrogen atom, oxygen atom, silicon atom or sulfur atom. ##STR00028##

5. The composition of claim 4, wherein X represents an active site including the following formulas 3QP, 3C, 3H, 3A, 3B, 3W, 3T, 3R: ##STR00029## ##STR00030##

6. The composition of claim 1, wherein the lipophilic agent is useful for a multi-step synthesis of a biomolecule.

7. The composition of claim 6, wherein the biomolecule is a peptide.

8. A method for synthesizing a composition of a lipophilic agent including a lipophilic anchor which has a triazine core shown in the following Chemical Formula (2″): ##STR00031## wherein R.sup.3 and R.sup.4 may be same or different, and are independently selected from the group comprising alkyl group with a carbon number of 12 to 48 which may have a substituent group, X represents an active site of the lipophilic agent having one or more atoms selected from the group comprising carbon atom, halogen atom, nitrogen atom, oxygen atom, silicon atom or sulfur atom, comprising: i) using two equivalents of a first amine including R.sup.3 and R.sup.4 for nucleophilic substitution to displace two chlorides of cyanuric chloride in presence of a base at elevated temperature; ii) using a second amine for nucleophilic substitution to displace a third chloride of cyanuric chloride at high temperature to install an active site X.

9. The method of claim 8, wherein the elevated temperature is between 20° C. to 50° C.

10. The method of claim 8, wherein the high temperature is between 50° C. to 100° C.

11. The method of claim 8, wherein the base is N,N-diisopropylethylamine.

12. The method of claim 8, wherein R.sup.3 and R.sup.4 are n-octadecyl group shown in the following chemical formula for the lipophilic agent: ##STR00032##

13. The method of claim 12, wherein the active site X includes piperazine shown in the following chemical formula for the lipophilic agent: ##STR00033##

14. The method of claim 12, wherein the active site X comprises functional linkers.

15. The method of claim 14, wherein the functional linkers include the following formulas 3C, 3H: ##STR00034##

16. The method of claim 13, wherein the lipophilic agent is useful for installing functional linkers.

17. The method of claim 16, wherein the functional linkers include the following formulas 3A, 3B, 3W, 3T, 3R: ##STR00035##

18. The method of claim 17, wherein the lipophilic linkers are useful for a multi-step synthesis of a biomolecule.

19. The method of claim 18, wherein the biomolecule is a peptide.

Description

EXAMPLES

[0079] The present invention is explained below with reference to the following examples, but the present invention is not in any way limited by these examples. The practice of the present invention will employ, unless otherwise indicated, techniques of synthetic organic chemistry, biochemistry and the like, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Kirk-Othmer's Encyclopedia of Chemical Technology; and House's Modern Synthetic Reactions.

[0080] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compositions/compound/methods of the invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some experimental error and deviations should, of course, be allowed. All components were obtained commercially unless otherwise indicated.

Example 1

Synthesis of 2-(4′-piperazino)-4-(N-methyl-N-n-octadecyl)amino-6-[N,N-di(n-octadecyl)amino]-1,3,5-triazine (3MP)

[0081] ##STR00020##

[0082] Cyanuric chloride (5 g, 27.1 mmol) was dissolved in toluene (200 mL), and mixed with N,N-diisopropylethylamine (DIPEA) (18.5 ml, 108.8 mmol). N-Methyl-N-n-octadecylamine (7.68 g, 27.1 mmol) was added to the mixture cooled in an ice bath. This was stirred for 4 hours, and then the mixture was allowed to warm to room temperature. Di(n-octadecyl)amine (14.15 g, 27.1 mmol) was added to the mixture. This was stirred for 16 hours at room temperature. After the completion of the reaction, ethanol (400 mL) was added, and after the precipitation, suction filtration was carried out and solid product, 2-chloro-4-(N-methyl-N-n-octadecyl)amino-6-[N,N-di(n-octadecyl)amino]-1,3,5-triazine (23.6 g, 95%), was obtained.

[0083] 2-Chloro-4-(N-methyl-N-n-octadecyl)amino-6-[N,N-di(n-octadecyl)amino]-1,3,5-triazine (20 g, 21.8 mmol) was dissolved in toluene (300 mL). Piperazine (10 g, 116 mmol) dissolved in isopropanol (100 mL) was added to the mixture. This was stirred for 16 hours at 80° C. After completion of the reaction, most solvents were removed by evaporation. Ethanol was added to the residue and after the precipitation, suction filtration was carried out and solid product, 2-(4′-piperazino)-4-(N-methyl-N-n-octadecyl)amino-6-[N,N-di(n-octadecyl)amino]-1,3,5-triazine (20.4 g, 97%), was obtained.

Example 2

Synthesis of 2-(4′-piperazino)-4-n-octadecyloxy-6-[N,N-di(n-octadecyl)amino]-1,3,5-triazine (3GP)

[0084] ##STR00021##

[0085] Cyanuric chloride (25 g, 135.6 mmol) was dissolved in toluene (1 L), and mixed with DIPEA (92.23 ml, 542.32 mmol). n-Octadecyl alcohol (37.4 g, 138.26 mmol) was added to the mixture cooled in an ice bath. This was stirred for 4 hours, and then the mixture was allowed to warm to room temperature and continued stirred for 12 hours. Di(n-octadecyl)amine (78.63 g, 150.64 mmol) was added to the mixture. This was stirred for 16 hours at room temperature. After the completion of the reaction, ethanol (1 L) was added, and after the precipitation, suction filtration was carried out and solid product, 2-chloro-4-n-octadecyloxy-6-[N,N-di(n-octadecyl)amino]-1,3,5-triazine (121.6 g, 99%), was obtained.

[0086] 2-Chloro-4-n-octadecyloxy-6-[N,N-di(n-octadecyl)amino]-1,3,5-triazine (40 g, 44 mmol) was dissolved in toluene (500 ml). Piperazine (20 g, 232 mmol) dissolved in isopropanol (200 mL) was added to the mixture. This was stirred for 16 hours at 50° C. After completion of the reaction, most solvents were removed by evaporation. Ethanol was added to the residue and after the precipitation, suction filtration was carried out and solid product, 2-(4′-piperazino)-4-n-octadecyloxy-6-[N,N-di(n-octadecyl)amino]-1,3,5-triazine (29.6 g, 71%), was obtained.

Example 3

Synthesis of 2-(4′-piperazino)-4,6-di[N,N-di(n-octadecyl)amino]-1,3,5-triazine (3QP)

[0087] ##STR00022##

[0088] Cyanuric chloride (10 g, 54.2 mmol) was dissolved in toluene (400 mL), and mixed with DIPEA (37 ml, 217.6 mmol). Di(n-octadecyl)amine (56.6 g, 108.4 mmol) was added to the mixture cooled in an ice bath. After addition, the ice bath was removed. This was stirred for 16 hours at room temperature. After the completion of the reaction, ethanol (800 mL) was added, and after the precipitation, suction filtration was carried out and solid product, 2-chloro-4,6-di[N,N-di(n-octadecyl)amino]-1,3,5-triazine (55.7 g, 89%), was obtained.

[0089] 2-Chloro-4,6-di[N,N-di(n-octadecyl)amino]-1,3,5-triazine (25 g, 21.6 mmol) was dissolved in toluene (375 mL). Piperazine (10 g, 116 mmol) dissolved in isopropanol (100 mL) was added to the mixture. This was stirred for 16 hours at 80° C. After completion of the reaction, ethanol was added and after the precipitation, suction filtration was carried out and solid product, 2-(4′-piperazino)-4,6-di[N,N-di(n-octadecyl)amino]-1,3,5-triazine (25.1 g, 96%), was obtained.

Example 4

Synthesis of Lipophilic Linker Containing p-oxybenzyl Alcohol (3W)

[0090] ##STR00023##

[0091] 2-(4′-piperazino)-4,6-di[N,N-di(n-octadecyl)amino]-1,3,5-triazine (3QP) (2.41 g, 2.00 mmol) was dissolved in toluene (20 ml). 4-(Hydroxymethyl)phenoxyacetic acid (373 mg, 2.05 mmol), and N,N′-diisopropylcarbodiimide (DIC) (265 mg, 2.10 mmol) were then added to the solution for the condensation reaction. The reaction mixture was stirred at room temperature until the reaction was completed (30 min). After completion, acetonitrile was added to the reaction mixture to give the solid product, the Lipophilic linker (3W) (2.73 g) quantitatively as a precipitate. The precipitate was filtered and washed with acetonitrile.

Example 5

Synthesis of Peptide Using Lipophilic Linker-3W

[0092] Acetyl tetrapeptide-2 was synthesized using Lipophilic linker-3W. This four amino acid peptide could mimic the youth hormone called thymopoietin. By compensating the loss of thymic factors that come with age, the peptide can stimulate the skin immune defenses and help the skin to regenerate. The peptide has the following structure: N2-acetyl-L-lysyl-L-alpha-aspartyl-L-valyl-L-tyrosine. The synthesis was carried out by the following general procedures using Fmoc-O-tert-butyl-L-tyrosine, Fmoc-L-valine, Fmoc-L-aspartic acid 4-tert-butyl ester, Na-Fmoc-Ne-Boc-L-lysine and acetic acid sequentially.

General Procedure for Adding the First Fmoc-Amino Acid

[0093] Lipophilic linker-3W was dissolved in toluene (1 mmole/10 ml). Fmoc-amino acid (1.5 equivalents), N,N′-diisopropylcarbodiimide (DIC) (1.5 equivalents), and 4-(N,N-dimethylamino)pyridine (DMAP) (0.2 equivalents) were then added to the solution. The reaction mixture was stirred at room temperature until the reaction was completed (30 min). After completion, acetonitrile was added to the reaction mixture to give the product quantitatively as a precipitate. The precipitate was filtered and washed with acetonitrile.

General Procedure for Coupling of Fmoc-Amino Acid

[0094] To a solution of Lipophilic linker-3W tagged peptide in THF (0.10 M), respective Fmoc-amino acid (1.2 mol equiv.), O-(1H-benzotriaol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) (1.2 mol equiv.), 1-Hydroxybenzotriazole (HOBt) (1.2 mol equiv.), and DIPEA (2.4 mol equiv.) were added. The resulting reaction mixture was stirred at room temperature until completion of the reaction (determined by TLC), followed by dilution with acetonitrile to give a product as a precipitate.

General Procedure for Basic Deprotection of Lipophilic Linker-3W Tagged Peptide

[0095] Lipophilic linker-3W tagged peptide was dissolved in 1% piperidine and 1% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in THF (0.10 M). The resulting reaction mixture was stirred at room temperature until completion of the reaction (determined by TLC), followed by adding acetonitrile to give a product as a precipitate.

General Procedure for Final Cleavage of Peptide from Lipophilic Linker-3W

[0096] Lipophilic linker-3W tagged peptide was dissolved in 2.5% triisopropylsilane (TIS) and 2.5% water in trifluoroacetic acid (TFA). The reaction mixture was stirred at room temperature until the reaction was completed. After completion, the solution was diluted with ethanol and the precipitate was removed by filtration, and t-butyl methyl ether was added to the filtrate to give the peptide quantitatively as a precipitate.