Process for preparing peptide derivatized oligomeric compounds

• US 6,762,281 B2
• Filed: 09/07/2001
• Issued: 07/13/2004
• Est. Priority Date: 09/08/2000
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Guzaev, Andrei P., Manoharan, Muthiah
• Primary Examiner(s): RUSSEL, JEFFREY E
• Application Number:
  US09/949,474
• Publication Number:
  US 20020156235A1
• Time in Patent Office: 1,040 Days
• Field of Search: 530/322, 530/323, 530/338, 530/345, 514/2, 514/8, 525/54.2, 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 2′

-, 3′

-, or 5′

-H-phosphonate 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 a peptide wherein said peptide is functionalized with a functional group reactive with said sulfur moiety thereby forming said peptide linked oligomeric compound.

2.K 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.

2. The method of claim 1 wherein said support medium derivatized with a compound comprising a protected hydroxyl group is 3′

-thiol-modifier C3 S—

S CPG (DMT-O—

(CH₂)₃—

S—

S—

(CH₂)₃—

O-succinyl-LCAA-CPG).

3. The method of claim 1 wherein each hydroxyl protecting group is, independently, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) or 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

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

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

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

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

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

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

wherein

T, is hydrogen or a hydroxyl protecting group;

J is C₁-C₁₂ alkyl or -Q₁-G-Q₂;

Q₁ is C₁-C₁₂ alkyl, alkaryl or [(CH₂)_mm—

O—

(CH₂)_mm,];

each mm and mm′

are, independently, from 1 to about 6;

p is from 1 to about 6;

Q₂ is C₁-C₁₂ alkyl;

G₂ is —

NH—

C(O)—

, —

NH—

C(O)—

NH—

, —

NH—

C(S)—

NH—

, —

NH—

O—

, or —

NH—

C(O)—

O—

;

each X₂ is, independently, O or S;

each X₁ is, independently, R₅—

O—

, Pg-S—

, C₁-C₁₀ straight or branched chain alkyl, CH₃(CH₂)₅—

O—

, R₂R₃N—

or a group remaining from coupling a chiral auxiliary;

g is from 0 to 10;

R₅ is CH₃, —

GH₂CH₂CN, —

C(CH₃)(CH₃)—

CCl₃, —

CH₂—

CCl₃, —

CH₂CH═

GH₂, CH₂CH₂SiCH₃, 2-yl-ethyl phenylsulfonate, δ

-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; and

n is from 2 to about 50.

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

5. The method of claim 5 wherein said deprotecting reagent is a weak acid.

6. The method of claim 6 wherein said deprotecting reagent is dichloroacetic acid or trichloroacetic acid.

13. 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, 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, δ

-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.

14. The method of claim 14 wherein said oligomeric compound is reacted with an equimolar amount of said functionalized peptide.

15. A peptide linked oligomeric compound having the formula:

wherein;

R₉ is an oligomeric compound;

Q₁ is C₁-C₁₂ alkyl, —

[(CH₂)_mm—

O—

CH₂)_mm,], aryl or alkaryl;

p is from 1 to about 6;

each mm and mm′

are, independently, from 1 to about 6; and

Q₂ is C₂-C₁₂ alkyl.

16. The peptide linked oligomeric compound of claim 16, wherein Q₁ is propyl and is ethyl.

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

(a) providing a support medium derivatized with a compound having the formula;

wherein R9 comprises a hydroxy] protecting group;

Q₁ is C₁-C₁₂ alkyl, alkaryl or —

[(CH₂)_mm—

I—

(CH₂)_mm,]_p-;

Q₃ is C₁-C₁₂ alkyl, aryl or alkaryl;

Sm is a support medium;

p is from 1 to about 6; and

each mm and mm′

are, independently, from 1 to about 6;

(b) treating said hydroxyl protecting 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 a peptide wherein said peptide is functionalized with a functional group reactive with said sulfur moiety thereby forming said peptide linked oligomeric compound.

18. The method of claim 18 wherein

is a support medium having long chain alkyl amine groups.

19. The method of claim 18 wherein Q₁ is propyl and Q₃ is methyl.

20. 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) treating said extended compound with a capping agent to form a capped compound, 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;

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

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

(a) providing a support medium derivatized with a compound that 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, wherein said cleaving is performed using a bifunctional compound having an internal disulfide group;

(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.

22. The method of claim 22 where said bifunctional compound has the formula H₂N-(CH₂)_q—

S—

S—

(CH₂)_q—

NH₂ wherein each q is from 2 to about 6.

23. A compound having the formula:

wherein;

R₈ is hydrogen, a hydroxyl protecting group, a nucleoside, a nucleotide or an oligomeric compound;

Q₁ propyl and Q is methyl; and

Sm is a support medium.