Process for preparing peptide derivatized oligomeric compounds

• US 6,559,279 B1
• Filed: 09/08/2000
• Issued: 05/06/2003
• Est. Priority Date: 09/08/2000
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Guzaev, Andrei P.
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US09/658,517
• Time in Patent Office: 970 Days
• Field of Search: 514/2.8, 525/54.2, 530/322, 530/333, 530/338, 530/345, 536/25.3, 536/25.31, 536/25.33, 536/25.34
• US Class Current: 530/322
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)

Abstract

Methods of preparing peptide linked oligomeric compounds are provided. The method is useful for preparing larger scale amounts of peptide linked oligomeric compounds. More particularly, the synthesis of peptide linked oligomeric compounds is performed without the problems of aggregation associated with electrostatic interactions. The present method describes using equimolar amounts of oligomeric compounds and peptide reagents providing for an increase in overall efficiency.

Claims

1. A method for preparing a peptide linked oligomeric compound comprising the steps of:

(a) providing a support medium derivatized with a compound wherein said compound comprises a protected hydroxyl group;

(b) treating said protected hydroxyl group with a deprotecting reagent effective to deprotect said hydroxyl group;

(c) reacting said deprotected hydroxyl group with a nucleoside having a protected hydroxyl group and an activated phosphorus containing substituent group thereby forming an extended compound;

(d) optionally treating said extended compound with a capping agent to form a capped compound;

(e) optionally repeating steps (b), (c) and (d) to form a further extended compound;

(f) treating said capped compound or said further extended compound with an oxidizing reagent thereby forming an oxidized compound comprising one or more nucleosides;

(g) repeating steps (b), (c), (d), (e) and (f) for oxidized compounds comprising one nucleoside or optionally repeating steps (b), (c), (d), (e) and (f) for oxidized compounds comprising more than one nucleoside to give a further oxidized compound;

(h) cleaving said oxidized compound or said further oxidized compound from the support medium to give said oligomeric compound comprising a linking moiety attached through a phosphorous substituent group to a 2′

- or 3′

-position of a terminal nucleoside;

(i) treating said linking moiety attached to said oligomeric compound with reagents effective to form a reactive sulfur moiety on said linking moiety; and

(j) reacting said reactive sulfur moiety with a peptide wherein said peptide is functionalized with a functional group reactive with said sulfur moiety thereby forming said peptide linked oligomeric compound.

2. The method of claim 1 wherein said nucleoside is a 2′

-, 3′

-, or 5′

-phosphoramidite.

3. The method of claim 1 wherein said nucleoside is a 2′

-, 3′

-, or 5′

-H-phosphonate.

4. The method of claim 1 wherein said activated phosphorus containing substituent group is a phosphoramidite, H-phosphonate, phosphate triester or a chiral auxiliary.

5. The method of claim 1 wherein one of said reactive sulfur moiety and said functional group is —

SH and the other of said reactive sulfur moiety and said functional group is a disulfide group.

6. The method of claim 1 wherein said support medium derivatized with a compound is 3′

-thiol-modifier C3 S—

S CPG (DMT-O—

(CH₂)₃—

S—

S—

(CH₂)₃—

O-succinyl-LCAA-CPG).

7. The method of claim 1 wherein said hydroxyl protecting groups are acid labile.

8. The method of claim 7 wherein each hydroxyl protecting group is, independently, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) or 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

9. The method of claim 8 wherein said deprotecting reagent is a weak acid.

10. The method of claim 9 wherein said deprotecting reagent is dichloroacetic acid or trichloroacetic acid.

11. The method of claim 1 wherein said capping agent comprises 20% acetic anhydride in acetonitrile mixed with about an equal volume of a solution having 20% N-methylimidazole, 30% pyridine and 50% acetonitrile.

12. The method of claim 1 wherein said cleaving is performed using aqueous ammonium hydroxide.

13. The method of claim 1 wherein said cleaving is performed using a bifunctional compound having an internal disulfide group.

14. The method of claim 13 where said bifunctional compound has the formula H₂N—

(CH₂)₂—

S—

S—

(CH₂)₂—

NH₂.

15. The method of claim 1 wherein said oligomeric compound comprises from about 5 to about 50 nucleosides.

16. The method of claim 1 wherein said oligomeric compound comprises from about 8 to about 30 nucleosides.

17. The method of claim 1 wherein said oligomeric compound comprises from about 15 to about 25 nucleosides.

18. The method of claim 1 wherein said peptide linked oligomeric compound has one of the formulas:

wherein

T₁ is hydrogen or a hydroxyl protecting group;

J is C₁-C₁₂alkyl or —

(CH₂)_m—

G—

(CH₂)_m—

;

G is O, S, —

NH—

C(O)—

, —

NH—

C(O)—

NH—

, —

NH—

O—

, or —

NH—

C(O)—

O—

;

m is from 2 to about 12;

each X₂ is, independently, O or S;

each X₁ is, independently, Pg—

O—

, Pg—

S—

, C₁-C₁₀ straight or branched chain alkyl, CH₃(CH₂)_g—

O—

, R₂R₃N—

or a group remaining from coupling a chiral auxiliary;

g is from 0 to 10;

Pg is CH₃, —

CH₂CH₂CN, —

C(CH₃)(CH₃)—

CCl₃, —

CH₂—

CCl₃, —

CH₂CH═

CH₂, CH₂CH₂SiCH₃, 2-yl-ethyl phenylsulfonate, -cyanobutenyl, cyanop-xylyl, diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl, or a blocking group;

each R₂ and R₃ is, independently, hydrogen, C₁-C₁₀alkyl, cycloalkyl or aryl;

or optionally, R₂ and R₃, together with the nitrogen atom to which they are attached form a cyclic moiety;

each Bx is, independently, a heterocyclic base moiety; and

each R₁ is, independently, H, a blocked hydroxyl group, or a sugar substituent group; and

n is from 2 to about 50.

19. The method of claim 18 wherein one of said reactive sulfur moiety and said functional group is —

SH and the other of said reactive sulfur moiety and said functional group is a disulfide group.

20. The method of claim 18 wherein said support medium derivatized with a compound is 3′

-thiol-modifier C3 S—

S CPG (DMT-O—

(CH₂)₃—

S—

S—

(CH₂)₃—

O-succinyl-LCAA-CPG).

21. The method of claim 18 wherein said cleaving is performed using a bifunctional compound having an internal disulfide group.

22. The method of claim 1 wherein said reactive sulfur moiety is reacted with an equimolar amount of said functionalized peptide.

23. A method for preparing a peptide linked oligomeric compound having one of the formulas:

wherein

T₁ is hydrogen or a hydroxyl protecting group;

each X₂ is, independently, O or S;

each X₁ is, independently, O⁻

—

, Pg—

O—

, S⁻

, Pg—

S—

, C₁-C₁₀ straight or branched chain alkyl, CH₃(CH₂)_g—

O—

, R₂R₃N—

or a group remaining from coupling a chiral auxiliary;

g is from 0 to 10;

Pg is CH₃, —

CH₂CH₂CN, —

C(CH₃)(CH₃)—

CCl₃, —

CH₂—

CCl₃, —

CH₂CH═

CH₂, CH₂CH₂SiCH₃, 2-yl-ethyl phenylsulfonate, -cyanobutenyl, cyano p-xylyl, diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl, or a blocking group;

each R₂ and R₃ is, independently, hydrogen, C₁-C₁₀ alkyl, cycloalkyl or aryl;

or optionally, R₂ and R₃, together with the nitrogen atom to which they are attached form a cyclic moiety;

each Bx is, independently, a heterocyclic base moiety;

each R₁ is, independently, H, a blocked hydroxyl group, or a sugar substituent group;

n is from 2 to about 50; and

JJ has one of the formulas;

wherein * denotes the point of attachment to the peptide;

comprising the steps of;

providing an oligomeric compound of the formula;

 

wherein;

L has one of the formulas;

reacting said oligomeric compound with a functionalized peptide having a —

SH functional group thereby forming said peptide linked oligomeric compound.

24. The method of claim 23 wherein said oligomeric compound is reacted with an equimolar amount of said functionalized peptide.

25. The method of claim 23 wherein said nucleoside is a 2′

-, 3′

-, or 5′

-phosphoramidite.

26. The method of claim 23 wherein said nucleoside is a 2′

-, 3′

-, or 5′

-H-phosponate.

27. A method for the large-scale synthesis of a peptide linked oligomeric compound comprising the steps of:

(a) providing a support medium derivatized with a compound wherein said compound comprises a protected hydroxyl group;

(b) treating said protected hydroxyl group with a deprotecting reagent effective to deprotect said hydroxyl group;

(c) reacting said deprotected hydroxyl group with a nucleoside having a protected hydroxyl group and an activated phosphorus containing substituent group thereby forming an extended compound;

(d) optionally treating said extended compound with a capping agent to form a capped compound;

(e) optionally repeating steps (b), (c) and (d) to form a further extended compound;

(f) treating said capped compound or said further extended compound with an oxidizing reagent thereby forming an oxidized compound comprising one or more nucleosides;

(g) repeating steps (b), (c), (d), (e) and (f) for oxidized compounds comprising one nucleoside or optionally repeating steps (b), (c), (d), (e) and (f) for oxidized compounds comprising more than one nucleoside to give a further oxidized compound;

(h) cleaving said oxidized compound or said further oxidized compound from the support medium to give said oligomeric compound comprising a linking moiety;

(i) treating said linking moiety attached to said oligomeric compound with reagents effective to form a reactive sulfur moiety on said linking moiety; and

(j) reacting said reactive sulfur moiety with an equimolar amount of a peptide wherein said peptide is functionalized with a functional group reactive with said sulfur moiety thereby forming said peptide linked oligomeric compound.

28. A method for preparing a peptide linked oligomeric compound comprising the steps of:

(a) providing a support medium derivatized with a compound wherein said compound comprises a protected hydroxyl group;

(b) treating said protected hydroxyl group with a deprotecting reagent effective to deprotect said hydroxyl group;

(c) reacting said deprotected hydroxyl group with a nucleoside having a protected hydroxyl group and an activated phosphorus containing substituent group thereby forming an extended compound;

(d) optionally treating said extended compound with a capping agent to form a capped compound;

(e) optionally repeating steps (b), (c) and (d) to form a further extended compound;

(f) treating said capped compound or said further extended compound with an oxidizing reagent thereby forming an oxidized compound comprising one or more nucleosides;

(g) repeating steps (b), (c), (d), (e) and (f) for oxidized compounds comprising one nucleoside or optionally repeating steps (b), (c), (d), (e) and (f) for oxidized compounds comprising more than one nucleoside to give a further oxidized compound;

(h) cleaving said oxidized compound or said further oxidized compound from the support medium with a bifunctional compound having an internal disulfide group to give said oligomeric compound comprising a linking moiety;

(i) treating said linking moiety attached to said oligomeric compound with reagents effective to form a reactive sulfur moiety on said linking moiety; and

(j) reacting said reactive sulfur moiety with a peptide wherein said peptide is functionalized with a functional group reactive with said sulfur moiety thereby forming said peptide linked oligomeric compound, wherein said peptide linked oligomeric compound has the formula;

 

wherein

T₁ is hydrogen or a hydroxyl protecting group;

J is C₁-C₁₂alkyl or —

(CH₂)_m—

G—

(CH₂)_m—

;

G is O, S, —

NH—

C(O)—

, —

NH—

C(O)—

NH—

, —

NH—

O—

, or —

NH—

C(O)—

O—

;

m is from 2 to about 12;

each X₂ is, independently, O or S;

each X₁ is, independently, Pg—

O—

, Pg—

S—

, C₁-C₁₀ straight or branched chain alkyl, CH₃(CH₂)_g—

O—

, R₂R₃N—

or a group remaining from coupling a chiral auxiliary;

g is from 0 to 10;

Pg is CH₃, —

CH₂CH₂CN, —

C(CH₃)(CH₃)—

CCl₃, —

CH₂—

CCl₃, —

CH₂CH═

CH₂, CH₂CH₂SiCH₃, 2-yl-ethyl phenylsulfonate, -cyanobutenyl, cyano p-xylyl, diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl, or a blocking group;

each R₂ and R₃ is, independently, hydrogen, C₁-C₁₀alkyl, cycloalkyl or aryl;

or optionally, R₂ and R₃, together with the nitrogen atom to which they are attached form a cyclic moiety;

each Bx is, independently, a heterocyclic base moiety; and

each R₁ is, independently, H, a blocked hydroxyl group, or a sugar substituent group; and

n is from 2 to about 50.