Bioreversible boronates for delivery of molecules into cells

1. A boronation reagent of formula:

where:

PB is a phenylboronate group;

Y 4 , Y 5 and Y 6 are independently selected from —O—, —S—, —NRc-, —CO—, —O—CO—, —CO—O—, —CO—NRc-, —NRc-CO—, —NRc-CO—NRc-, —OCO—NRc-, —NRc-CO—O—, —N═N—, —N═N—NRc-, —CO—S—, —S—CO—, —S—S—, —SO 2 —, and —CRc(OH)—CRc(OH)—, where Rc is hydrogen or C1-C3 alkyl;

L 5 and L 6 are divalent spacer moieties;

X 6 is a leaving group and —Y 6 —X 6 together is a reactive group;

R 33 and each R 32 are independently selected from a C1-C8 alkyl group or a C3-C8 cycloalkyl group;

R 34 -R 36 are selected from the group consisting of hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, a C3-C8 cycloalkyl group, an alicyclic group, an aryl group, a heterocyclic group, a heteroaryl group, a —CO 2 R 10 group, a —O—CO—R 10 group, a —CON(R 12 ) 2 group, a —O—CON(R 12 ) 2 group; a —N(R 12 ) 2 group, a —OR 10 group, a —(CH 2 ) m —OH group, a —(CH 2 ) m —N(R 12 ) 2 group, a halogen, a nitro group, a cyano group, a —SO 2 —OR 10 group, or R 36 together with R 35 or R 35 together with R 34 and the ring carbons to which R 34 -R 36 are attached, form a 5-8-member alicyclic, heterocyclic, aryl or heteroaryl ring moiety, each of which groups or moieties is optionally substituted;

m is an integer from 1-8;

each R 10 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 R 12 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 two R 12 together with the nitrogen to which they are attached form a 5-8 member heterocyclic or heteroaryl ring moiety, each of which groups or moieties is optionally substituted; and

wherein optional substitution is substitution by one or more substituents selected from halogen, an oxo group, 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 of the alicyclic ring are optionally replaced with —CO— and which alicyclic ring optionally contains one or two double bonds, an aryl group having 6-14 carbon ring atoms, a 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein one or two ring carbons of the heterocyclic group are optionally replaced with —CO— and which heterocyclic group optionally contains one or two double bonds, a heteroaryl group having 1-3 heteroatoms, a —CO 2 R 13 group, a —OCO—R 13 group, a —CON(R 14 ) 2 group, a —OCON(R 14 ) 2 group, a —N(R 14 ) 2 group, a —SO 2 —OR 13 group, a —OR 13 group, a —(CH 2 ) m —OR 13 group, a —(CH 2 ) m —N(R 14 ) 2 group, where m is 1-8 and each R 13 or R 14 is independently hydrogen, an unsubstituted C1-C6 alkyl group, an unsubstituted aryl group having 6-14 carbon atoms, an unsubstituted 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein one or two ring carbons of the heterocyclic group are optionally replaced with —CO— and which heterocyclic group optionally contains one or two double bonds, and a heteroaryl group having 1-3 heteroatoms and wherein two R 14 together with the nitrogen to which they are attached optionally form a heterocyclic or heteroaryl ring moiety.

2. The reagent of claim 1 wherein Y 6 —X 6 together is a reactive group that reacts with one or more of an amino group, a carboxylic acid group or ester thereof, a sulfhydryl group, a hydroxyl group, an azide group, a thioester group, a phosphinothioester group, an aldehyde group or a ketone group of an amino acid, peptide or protein.

3. The reagent of claim 1 wherein Y 6 is a —CO— and X 6 is a leaving group of an activated ester.

4. The reagent of claim 3 wherein X 6 is selected from the group consisting of a nitro-substituted phenolate group, a halogen-substituted phenolate group, an N-oxido-succinimidyl or an N-oxido-sulfosuccinimidyl group.

5. The reagent of claim 4 wherein X 6 is pentafluorophenolate, tetrafluorophenolate, p-nitrophenolate or p-sulfo-tetrafluorophenolate.

6. The reagent of claim 1 wherein L 5 and L 6 are selected from:

alkylene linkers (—(CH 2 ) y —) wherein y is 1-12, and alkoxyalkyl linkers —[(CH 2 ) q —O—(CH 2 ) r ] a — wherein q and r are zero or integers from 1-4, one of q and r is not zero, and a is 1-6.

7. The reagent of claim 1 wherein L 5 is —(CH 2 ) 2 — and L 6 is —CH 2 —.

8. The reagent of claim 1 wherein R 33 and each R 32 are C1-C3 alkyl groups.

9. The reagent of claim 1 wherein R 33 and each R 32 are methyl groups.

10. The reagent of claim 1 wherein R 34 -R 36 are selected from hydrogen, a C1-C8 alkyl group, a C3-C8 cycloalkyl group, a halogen, a nitro group, or a cyano group.

11. The reagent of claim 1 wherein R 34 -R 36 are selected from hydrogen, a C1-C3 alkyl group, or a halogen.

12. The reagent of claim 1 wherein Y 4 is —NH—CO—NH—.

13. The reagent of claim 1 wherein Y 5 is —CO—O—.

14. The reagent of claim 1 wherein PB is selected from:

15. A method for reversibly boronating a cargo molecule having one or more amino groups which comprises reacting the cargo molecule with a reagent of claim 1 wherein Y 6 —X 6 together is a reactive group that reacts with an amino group.

16. A method for delivering a cargo molecule into a cell which comprises boronating the cargo molecule by reacting the cargo molecule with a reagent of claim 1 and contacting the cell with the boronated cargo molecule.

17. The reagent of claim 1 wherein:

Y 4 is —NH—CO—NH—, Y 5 is —CO—O—, each R 32 , R 33 and R 35 are methyl groups, and R 34 and R 36 are hydrogens.

18. The reagent of claim 17 wherein PB is selected from:

where R 2 -R 5 are all hydrogens.

19. The reagent of claim 17 wherein X 6 is selected from an N-oxido-succinimidyl or an N-oxido-sulfosuccinimidyl group.

20. The reagent of claim 17 which is:

21. The reagent of claim 1 wherein optional substitution is substitution by one or more substituents selected from halogen, an oxo group; 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 phenyl group, and a benzyl group.

22. The reagent of claim 21 wherein Y 6 —X 6 together is a reactive group that reacts with an amine group.

23. The reagent of claim 1 wherein Y 4 and Y 5 are independently selected from —O—, —S—, —NRc-, —CO—, —O—CO—, —CO—O—, —CO—NRc-, —NRc-CO—, and —NRc-CO—NRc, where Rc is hydrogen or C1-C3 alkyl.

24. The reagent of claim 14 wherein:

each R 32 , R 33 and R 35 are methyl groups, and R 34 and R 36 are hydrogens.

25. The reagent of claim 24 wherein Y 6 —X 6 together is a reactive group that reacts with one or more of an amine group, a carboxylic acid group or ester thereof, a sulfhydryl group, a hydroxyl group, an azide group, a thioester group, a phosphinothioester group, an aldehyde group or a ketone group of an amino acid, peptide or protein.

26. The reagent of claim 25 wherein L 5 and L 6 are selected from:

alkylene linkers (—(CH 2 ) y —) wherein y is 1-12.

27. The reagent of claim 26 wherein Y 4 and Y 5 are independently selected from —O—, —S—, —NRc-, —CO—, —O—CO—, —CO—O—, —CO—NRc-, —NRc-CO—, and —NRc-CO—NRc, where Rc is hydrogen or C1-C3 alkyl.