Boronate-mediated delivery of molecules into cells

摘要

Methods for enhancing cellular uptake of cargo molecules by boronating the cargo molecule, particularly with one or more phenylboronic acid groups. Cellular uptake includes at least partial uptake into the cytosol. Boronation includes ligating, crosslinking or otherwise bonding one or more phenylboronic acids substituted to contain a reactive group to a cargo molecule. Boronation also includes ligating, crosslinking or otherwise bonding a phenylboronated oligopeptide to a cargo molecule. The phenylboronate groups are optionally conjugated to the cargo molecule via linking moieties that can be selectively cleaved, such cleavable linkers can allow the phenylboronate groups to be removed from the cargo molecule after the boronated cargo molecule is introduced into the cell. The invention includes certain phenylboronates which are boronation reagents, certain boronated oligopeptides and certain boronated peptides and proteins. The invention also includes kits for enhancing cellular uptake of cargo molecules by boronation with one or more phenylboronates or boronated oligopeptides.

主权项

1. A method for increasing cellular uptake of a cargo molecule by boronating the cargo molecule by binding one or more phenylboronate compounds to the cargo molecule, wherein the one or more phenylboronate compounds are selected from those of formulas: or salts thereof, where: x is 1 or 2; R1 and R5 are independently selected from hydrogen an optionally substituted straight-chain or branched aliphatic group having 1-8 carbon atoms a —CO2R10 group, a —OCOR10 group, a —CON(R12)2 group, a —N(R12)2 group, a —OR10 group, a —(CH2)2—OR10 group, a —(CH2)2—N(R12)2 group, a halogen, a nitro group, or a cyano group; R2, R3, R4 and R6 are independently selected from hydrogen a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, a heteroaryl group, a —CO2R10 group, a —OCO—R10group, a —CON(R12)2 hoop, a —N(R12)2 group, a —OR10 group, a —(CH2)mOH group, a halogen, a nitro group, a cyano group, -M, or two adjacent R2-R6, together with the ring carbons to which they are attached, optionally form a 5-8-member alicyclic, heterocyclic, aryl or heteroaryl ring moiety, each of which groups or moieties is optionally substituted,
wherein:each R10 is independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, or a heteroaryl group, each of which groups is optionally substituted;each R12 is independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, a heteroaryl group, or where two R12 together with the nitrogen to which they are attached can form a 5-8 member heterocyclic or heteroaryl ring moiety, each of which groups or moieties is optionally substituted;m is an integer from 1-8; andM is a reactive group or a linker carrying a reactive group, wherein the reactive group is functional for covalent attachment of the phenylboronate to the cargo molecule and which:(1) reacts with one or more of: an amine group, a carboxylic acid group, a sulfhydryl group or a hydroxyl group, particularly where that group is a group of a natural or unnatural amino acid of such an amino acid of a peptide or a protein;(2) reacts with an aldehyde or ketone group, an azide group, an activated ester group, a thioester group, phosphinothioester, or other group which is introduced into or generated in the amino acid, peptide or protein; or(3) reacts with one reactive group of a homobifunctional or a heterobifunctional crosslinking reagent,wherein at least one of R1-R6 is M andwherein optional substitution is substitution by one or more substituents selected from halogen; a nitro group; a cyano group; a C1-C6 alkyl group; a C1-C6 alkoxy group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a 3-7 member alicyclic ring, wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds; an aryl group having 6-14 carbon ring atoms; a phenyl group; a benzyl group; a 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms; a —CO2R13 group; —OCO—R13 group; —CON(R14)2 group; —N(R14)2 group; or —OR13 group, where each R13 or R14 is independently hydrogen; an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having 6-14 carbon atoms; an unsubstituted phenyl group; an unsubstituted benzyl group; an unsubstituted 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which optionally contains one or two double bonds; or a heteroaryl group having 1-3 heteroatoms and in addition two R14 to ether with the nitro en to which the are attached optionally forms a heterocyclic or heteroaryl ring moiety, each of which groups or moieties is optionally substituted; each of which R13 and R14 groups is in turn optionally substituted with one or more unsubstituted C1-C3 alkyl groups, halogens, nitro groups, cyano groups, —CO2R15 groups, —OCO—R15 groups, —CON(R16)2 groups, —N(R16)2 groups, or —OR15 groups, where each of R15 and R16 independently are hydrogen an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having 6-14 carbon ring atoms; an unsubstituted phenyl group; an unsubstituted benzyl group, an unsubstituted 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms and a total of 5-14 ring atoms; and in addition two R16 to ether with the nitro en to which the are attached optionally form an unsubstituted heterocyclic or heteroaryl ring moiety.