Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method
Inventors
Culbertson, Sean M. • Shen, Xiaoming • Kozlowski, Antoni • McManus, Samuel P.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11634540-B2
Publication Date
2023-04-25
Expiration Date
Abstract
Provided herein are polymeric α-hydroxy aldehyde or α-hydroxy ketone reagents which can be conjugated to amine-containing compounds to form stable conjugates in a single-step reaction. In selected embodiments, the polymeric reagent itself incorporates an internal proton-abstracting (basic) functional group, to promote more efficient reaction. The substituent is appropriately situated, via a linker if necessary, to position the group for proton abstraction, preferably providing a 4- or 5-bond spacing between the abstracting atom and the hydrogen atom on the α-carbon. Also provided are methods of using the reagents and stable, solubilized conjugates of the reagents with biologically active compounds. In preferred embodiments, the polymeric component of the reagent or conjugate is a polyethylene glycol.
Core Innovation
The invention relates to polymeric conjugates having a defined structure in which a secondary or tertiary amino group is linked to a water soluble polymer via an optional spacer group. The polymer conjugate further includes a linkage through “—NH—B,” where B represents the residue of an amine-containing biologically active compound.
The invention also relates to polymeric α-hydroxy aldehyde and polymeric α-hydroxy ketone reagents for single-step conjugation to amine-containing biologically active compounds. The described conjugation forms stable polymer–amine conjugates without requiring a separate reductive stabilization step, and the conjugates include linking to amine-containing biologically active compound residues to give an aminoketone-containing conjugation product moiety.
The polymeric conjugation relies on polymeric α-hydroxy aldehyde/ketone end group reagents having defined reagent structures and includes internal proton-abstracting groups positioned by 3–4 bonds, preferably 4-bond spacing, enabling self-catalyzed rearrangement of an imine intermediate to an aminoketone. The documented material also describes mPEG-functional aldehyde reagents, including mPEG-HP-ALD, mPEG-MAHP-ALD, and mPEG-Pip-HP-ALD, and corresponding conjugation experiments with lysozyme.
Claims Coverage
The provided independent claim coverage centers on a defined polymeric conjugate structure IV that ties an amino-biologically active residue to a secondary or tertiary amino group connected to a water soluble polymer through an optional spacer. The combined claim coverage identifies five inventive features.
Polymeric conjugate with structure IV and amino-biologically active residue connection
A polymeric conjugate having structure IV where R1 is selected from H, lower alkyl, and alkoxyalkyl; R4 is a two- or three-carbon chain with specified substituent and ring-forming constraints; NR5 is a secondary or tertiary amino group linked to water soluble polymer POLY via optional spacer Z; and —NH—B represents the residue of an amine-containing biologically active compound.
Secondary or tertiary amino group linked to a water-soluble polymer via optional spacer
NR5 is a secondary or tertiary amino group linked to water soluble polymer POLY via an optional spacer group Z, where R5 is hydrogen or an alkyl group which may form a ring with spacer group Z when present.
R4 chain constraints excluding hydroxy adjacent to carbonyl and enabling ring formation
R4 is a two- or three-carbon chain which may be substituted with one or more groups selected from alkyl, alkenyl, aryl, alkoxy, halo, cyano, and a water soluble polymer, wherein the carbon adjacent to the carbonyl carbon is not substituted with hydroxy, and wherein two substituents on R4 may together form an aliphatic or aromatic ring.
R1 substituent selection
R1 is selected from H, lower alkyl, and alkoxyalkyl.
Water-soluble polymer linked to NR5 through optional spacer Z
The polymeric conjugate includes a water soluble polymer POLY linked to the amino group NR5 via optional spacer Z.
The claim coverage centers on a defined polymeric conjugate structure IV that links an amino-biologically active residue to a secondary or tertiary amino group connected to a water soluble polymer through an optional spacer, with defined R1 and R4 constraints including ring-forming capabilities and an exclusion of hydroxy on the carbon adjacent to the carbonyl carbon.
Stated Advantages
Stable polymer–amine conjugates are formed without requiring a separate reductive stabilization step.
Self-catalyzed rearrangement of an imine intermediate to an aminoketone is enabled by internal proton-abstracting groups positioned by 3–4 bonds, preferably 4-bond spacing.
Saccharide-derived 3-deoxy analogs are described as improving conjugate stability.
Documented Applications
Conjugation experiments with lysozyme are described to form PEGylated conjugates (mono-, di-, and tri-PEGylated products) using mPEG-functional aldehyde reagents, with analytical outcomes reported by HPLC and SDS-PAGE.
An example is described for preparation of ruPEG2-piperazine aldehyde reagents for keto-amine conjugate formation.
Single-step conjugation to amine-containing biologically active compounds, including proteins and polypeptides.
Use of the polymeric α-hydroxy aldehyde/ketone reagent approach for forming polymer–amine conjugates.
Interested in licensing this patent?