Granted Patent B2

US 11,542,299 · App. 16/619,388 · Granted Jan 3, 2023

Inventors: Kenichi Nomura (Shizuoka, JP); Terushige Muraoka (Shizuoka, JP); Mikimasa Tanada (Shizuoka, JP); Takashi Emura (Shizuoka, JP)

Assignee: Chugai Seiyaku Kabushiki Kaisha

C07K1/061C07K11/02

Abstract

Claims (114)

1. A method of producing a peptide comprising at least one N-alkylated amino acid or proline, wherein the N-alkyl of the N-alkylated amino acid and the proline are optionally substituted with a substituent independently selected from the group consisting of a halogen group, an ether group, and a hydroxyl group;

wherein the method comprises the steps of:

1) preparing an Fmoc-protected amino acid comprising at least one each of following functional groups i) and ii), or an Fmoc-protected peptide comprising the Fmoc-protected amino acid:

i) a main chain amino group protected by at least one protecting group having an Fmoc skeleton; and

ii) at least one free carboxylic acid group or active esterified carboxylic acid group;

2) making the Fmoc-protected amino acid, or the Fmoc-protected peptide prepared in step 1) to be supported onto a solid phase;

3) deprotecting the protecting group having the Fmoc skeleton of the Fmoc-protected amino acid, or the Fmoc-protected peptide, which is supported onto the solid phase, by using a base to expose its amino group;

4) forming an amide bond by adding a new Fmoc-protected amino acid, or a new Fmoc-protected peptide; and

5) cleaving the peptide obtained in step 4) off from the solid phase under a condition of weaker acidity than TFA;

wherein the condition of weaker acidity than TFA in step 5) is a condition that uses a weakly acidic solution containing a fluoroalcohol.

2. The production method of claim 1 , wherein at least one side chain of the amino acid constituting the peptide obtained in step 4) is protected by a protecting group that is not deprotected under a basic condition but is deprotected under a condition having weaker acidity than TFA, and wherein the method further comprises before or after step 5), a step of deprotecting the protecting group under a condition having weaker acidity than TFA; and wherein the condition having weaker acidity than TFA in the deprotection step is a condition that uses a weakly acidic solution comprising a weak acid having an aqueous pKa value of 1 to 5 in a solvent having an aqueous pKa value of 5 to 14 and whose ionization ability value Y OTs is positive.

3. A method of producing a peptide comprising at least one N-alkylated amino acid or proline, wherein the N-alkyl of the N-alkylated amino acid and the proline are optionally substituted with a substituent independently selected from the group consisting of a halogen group, an ether group, and a hydroxyl group;

wherein the method comprises the steps of:

1) preparing an Fmoc-protected amino acid comprising at least one each of following functional groups i) and ii), or an Fmoc-protected peptide comprising the Fmoc-protected amino acid:

i) a main chain amino group protected by at least one protecting group having an Fmoc skeleton; and

ii) at least one free carboxylic acid group or active esterified carboxylic acid group;

2) deprotecting the protecting group having the Fmoc-skeleton of the Fmoc-protected amino acid, or the Fmoc-protected peptide, by using a base to expose its amino group;

3) forming an amide bond by adding a new Fmoc-protected amino acid, or a new Fmoc-protected peptide, wherein at least one side chain of the amino acid constituting a peptide obtained in this step has a protecting group that is not deprotected under a basic condition and is deprotected under a condition having weaker acidity than TFA; and

4) deprotecting the protecting group of the side chain under the condition having weaker acidity than TFA;

wherein the condition having weaker acidity than TFA is a condition that uses a weakly acidic solution comprising a weak acid having an aqueous pKa value of 1 to 5 in a solvent having an aqueous pKa value of 5 to 14 and whose ionization ability value Y OTs is positive.

4. The production method of claim 3 , wherein peptide production is carried out by a solid phase method.

5. The production method of claim 4 , which further comprises before or after step 4), a step of cleaving the peptide obtained in step 3) off from the solid phase under a condition that uses a weakly acidic solution containing a fluoroalcohol.

6. The production method of claim 3 , wherein peptide production is carried out by a liquid phase method.

7. The production method of claim 1 , wherein step 4) further comprises the steps of:

deprotecting the protecting group having the Fmoc skeleton on the newly added Fmoc-protected amino acid, or the newly added Fmoc-protected peptide, by using a base to expose its amino group; and

forming an amide bond by further adding a new Fmoc-protected amino acid, or a new Fmoc-protected peptide,

and wherein these steps are repeated once or multiple times.

8. The production method of claim 1 , wherein the produced peptide comprises on its C-terminal side an amino acid residue comprising one reactive site, and comprises on its N-terminal side an amino acid residue comprising the other reactive site.

9. The production method of claim 8 , which further comprises the step of bonding said reactive site and said other reactive site to cyclize the peptide.

10. The production method of claim 9 , wherein the amino acid residue having said other reactive site is at the N terminus and the bonding is an amide bonding or a carbon-carbon bonding.

11. The production method of claim 1 , wherein the fluoroalcohol is TFE or HFIP.

12. The production method of claim 2 , wherein the side chain protecting group is a protecting group which is capable of being deprotected in the range of pH 1 to pH 7, or a protecting group which is capable of being deprotected in 10% or lower concentration of TFA.

13. The production method of claim 2 , wherein the side chain protecting group is selected from following a) to d):

a) when the side chain protecting group is a protecting group for the side chain hydroxyl group of Ser, Thr, Hyp, and derivatives thereof, any one protecting group selected from a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt skeleton, a silyl skeleton, and a Boc skeleton represented by the general formulae below;

b) when the side chain protecting group is a protecting group for the side chain hydroxyl group of Tyr and derivatives thereof, any one protecting group selected from a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt skeleton, a silyl skeleton, a Boc skeleton, and a tBu skeleton represented by the general formulae below;

c) when the side chain protecting group is a protecting group for the side chain imidazole ring of His and derivatives thereof, any one protecting group selected from a MOM skeleton, a Bn skeleton, and a Trt skeleton represented by the general formulae below; and

d) when the side chain protecting group is a protecting group for the side chain carboxylic acid group of Asp, Glu, and derivatives thereof, any one protecting group selected from a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt skeleton, a tBu skeleton, a phenyl-EDOTn skeleton, which are represented by the following general formulae, and an orthoester skeleton in which a carbon atom of the carboxylic acid group to be protected is substituted with three alkoxy groups:

wherein the protecting group having a MOM skeleton has the following formula:

wherein

R1 is H, R2 is H, and X is methyl, benzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, or 2-trimethylsilylethyl;

R1 is methyl, R2 is H, and X is ethyl;

R1, R2, and R3 are all methyl; or

R1 and X together form —CH 2 —CH 2 —CH 2 — or —CH 2 —CH 2 —CH 2 —CH 2 —, and R2 is H, wherein when any one of R1, R2, and X is methyl or ethyl, these groups may further be substituted with alkyl, benzyl, or aryl;

wherein the protecting group having a Bn skeleton has the following formula:

wherein

R1 to R5 are each independently H, alkyl, aryl, or halogen, and R6 and R7 are alkyl;

R1, R2, R4, and R5 are each independently H, alkyl, aryl, or halogen, R3 is methoxy, and R6 and R7 are H;

R1 and R3 are methoxy, R2, R4, and R5 are each independently H, alkyl, aryl, or halogen, and R6 and R7 are H; or

R1, R4, and R5 are each independently H, alkyl, aryl, or halogen, and R2 and R3 together form —O—CH2-O—;

wherein the protecting group having a Dpm skeleton has the following formula:

wherein

R1 to R10 are each independently H, alkyl, aryl, alkoxy, or halogen; or

R1 to R4 and R7 to R10 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form —O— or —CH2-CH2-;

wherein the protecting group having a Trt skeleton has the following formula:

wherein

R1 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen;

R1, R2, and R4 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R3 is methyl or methoxy;

R1 is Cl, and R2 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen; or

R1 to R4 and R7 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form —O—;

wherein the protecting group having a silyl skeleton has the following formula:

wherein R1 to R3 are each independently alkyl or aryl;

wherein the protecting group having a Boc skeleton has the following formula:

wherein R1 to R9 are each independently H, alkyl, or aryl;

wherein the protecting group having a tBu skeleton has the following formula:

wherein R1 to R9 are each independently H, alkyl, or aryl; and

wherein the protecting group having a phenyl-EDOTn skeleton has the following formula:

wherein R1 to R3 are each independently H or methoxy.

14. The production method of claim 3 , wherein step 3) further comprises the steps of:

deprotecting the protecting group having the Fmoc skeleton on the newly added Fmoc-protected amino acid, or the newly added Fmoc-protected peptide, by using a base to expose its amino group; and

forming an amide bond by further adding a new Fmoc-protected amino acid, or a new Fmoc-protected peptide,

and wherein these steps are repeated once or multiple times.

15. The production method of claim 3 , wherein the produced peptide comprises on its C-terminal side an amino acid residue comprising one reactive site, and comprises on its N-terminal side an amino acid residue comprising the other reactive site.

16. The production method of claim 15 , which further comprises the step of bonding said reactive site and said other reactive site to cyclize the peptide.

17. The production method of claim 16 , wherein the amino acid residue having said other reactive site is at the N terminus and the bonding is an amide bonding or a carbon-carbon bonding.

18. The production method of claim 3 , wherein the solvent having an aqueous pKa value of 5 to 14 and whose ionization ability value Y OTs is positive is fluoroalcohol.

19. The production method of claim 18 , wherein the fluoroalcohol is TFE or HFIP.

20. The production method of claim 3 , wherein the side chain protecting group is a protecting group which is capable of being deprotected in the range of pH 1 to pH 7, or a protecting group which is capable of being deprotected in 10% or lower concentration of TFA.

21. The production method of claim 3 , wherein the side chain protecting group is selected from following a) to d):

a) when the side chain protecting group is a protecting group for the side chain hydroxyl group of Ser, Thr, Hyp, and derivatives thereof, any one protecting group selected from a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt skeleton, a silyl skeleton, and a Boc skeleton represented by the general formulae below;

b) when the side chain protecting group is a protecting group for the side chain hydroxyl group of Tyr and derivatives thereof, any one protecting group selected from a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt skeleton, a silyl skeleton, a Boc skeleton, and a tBu skeleton represented by the general formulae below;

c) when the side chain protecting group is a protecting group for the side chain imidazole ring of His and derivatives thereof, any one protecting group selected from a MOM skeleton, a Bn skeleton, and a Trt skeleton represented by the general formulae below; and

d) when the side chain protecting group is a protecting group for the side chain carboxylic acid group of Asp, Glu, and derivatives thereof, any one protecting group selected from a MOM skeleton, a Bn skeleton, a Dpm skeleton, a Trt skeleton, a tBu skeleton, a phenyl-EDOTn skeleton, which are represented by the following general formulae, and an orthoester skeleton in which a carbon atom of the carboxylic acid group to be protected is substituted with three alkoxy groups:

wherein the protecting group having a MOM skeleton has the following formula:

wherein

R1 is H, R2 is H, and X is methyl, benzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, or 2-trimethylsilylethyl;

R1 is methyl, R2 is H, and X is ethyl;

R1, R2, and R3 are all methyl; or

R1 and X together form —CH 2 —CH 2 —CH 2 — or —CH 2 —CH 2 —CH 2 —CH 2 —, and R2 is H, wherein when any one of R1, R2, and X is methyl or ethyl, these groups may further be substituted with alkyl, benzyl, or aryl;

wherein the protecting group having a Bn skeleton has the following formula:

wherein

R1 to R5 are each independently H, alkyl, aryl, or halogen, and R6 and R7 are alkyl;

R1, R2, R4, and R5 are each independently H, alkyl, aryl, or halogen, R3 is methoxy, and R6 and R7 are H;

R1 and R3 are methoxy, R2, R4, and R5 are each independently H, alkyl, aryl, or halogen, and R6 and R7 are H; or

R1, R4, and R5 are each independently H, alkyl, aryl, or halogen, and R2 and R3 together form —O—CH2-O—;

wherein the protecting group having a Dpm skeleton has the following formula:

wherein

R1 to R10 are each independently H, alkyl, aryl, alkoxy, or halogen; or

R1 to R4 and R7 to R10 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form —O— or —CH2-CH2-;

wherein the protecting group having a Trt skeleton has the following formula:

wherein

R1 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen;

R1, R2, and R4 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R3 is methyl or methoxy;

R1 is Cl, and R2 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen; or

R1 to R4 and R7 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form —O—;

wherein the protecting group having a silyl skeleton has the following formula:

wherein R1 to R3 are each independently alkyl or aryl;

wherein the protecting group having a Boc skeleton has the following formula:

wherein R1 to R9 are each independently H, alkyl, or aryl;

wherein the protecting group having a tBu skeleton has the following formula:

wherein R1 to R9 are each independently H, alkyl, or aryl; and

wherein the protecting group having a phenyl-EDOTn skeleton has the following formula:

wherein R1 to R3 are each independently H or methoxy.

22. The production method of claim 1 , wherein a side chain of the amino acid constituting the peptide obtained in step 4) is or is not protected by a protecting group which is deprotected under a condition having weaker acidity than TFA, and when the side chain is protected, the method further comprises deprotecting the protecting group under the condition having weaker acidity than TFA; and wherein the condition having weaker acidity than TFA in the deprotection step is a condition that uses a weakly acidic solution comprising a weak acid having an aqueous pKa value of 1 to 5 in a solvent having an aqueous pKa value of 5 to 14 and whose ionization ability value Y OTs is positive.

23. The production method of claim 1 , wherein the solid phase comprises a resin selected from the group consisting of a tritylchloride resin (Trt resin), a 2-chlorotritylchloride resin (Clt resin), a 4-methyltritylchloride resin (Mtt resin), and 4-methoxytritylchloride resin (Mmt).

Assignments (1)

Priority Claims (1)

Continuity (1)

Cited By (9)

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