Pictet-Spengler ligation for protein chemical modification

• US 9,605,078 B2
• Filed: 11/15/2013
• Issued: 03/28/2017
• Est. Priority Date: 11/16/2012
• Status: Active Grant

• Current Assignee: Gerawan Farming, LLC, Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Bertozzi, Carolyn, Agarwal, Paresh, Sletten, Ellen M.
• Primary Examiner(s): REYNOLDS, FRED H
• Application Number:
  US14/443,149
• Publication Number:
  US 20150291699A1
• Time in Patent Office: 1,229 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07D 311/92 Naphthopyrans; Hydrogenated naphthopyrans
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C07D 498/04 Ortho-condensed systems
• CPC Class Codes: C07K 16/32 against translation products of oncogenes

Abstract

Aldehyde- and ketone-functionalized proteins are promising new substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with a-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C═N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits their utility in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet-Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C—C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. The reaction was utilized for site-specific chemical modification of glyoxal- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a new means to generate stable bioconjugates for medical and materials applications.

Claims

1. A compound having the formula:

2. The compound according to claim 1, having the formula:

3. The compound according to claim 1, having a formula selected from:

4. The compound according to claim 1, wherein said compound has the formula:

5. The compound according to claim 1, wherein L is substituted or unsubstituted alkyl having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

6. The compound according to claim 1 wherein X is a member selected from a fluorophore and biotin.

7. The compound according to claim 1, wherein LX has the formula:

8. The compound according to claim 7, wherein R⁷ comprises:

9. A method of modifying a biomolecule functionalized with a member selected from an aldehyde and a ketone, said method comprising:

(a) contacting said biomolecule with a compound according to claim 1 under reaction conditions appropriate to form a cyclized Pictet-Spengler adduct by reaction of said compound with a moiety selected from said aldehyde and said ketone, thereby modifying said biomolecule.

10. The method according to claim 9, wherein said adduct has a formula selected from:

11. The method according to claim 9, wherein said adduct has a formula selected from:

12. The method according to claim 9, wherein, prior to step (a), a precursor biomolecule is modified with a reactive moiety comprising a member selected from said aldehyde and said ketone, thereby forming said biomolecule functionalized with a member selected from an aldehyde and a ketone.

13. The method of claim 9 wherein said biomolecule is a member selected from a protein, a glycan, a nucleic acid, a metabolite, an inhibitor, a lipid, and a cofactor.

14. The method of claim 9, wherein said biomolecule is an antibody.