Triorthogonal reagents for dual protein conjugation
The present invention relates to triorthogonal reagents useful for site-specifically modifying a protein with two orthogonal groups that can be subsequently functionalized in a single one-pot procedure. This approach relies on the selective tagging of proteins containing an appended farnesyltransferase or geranylgeranyltransferase I substrate sequence. The incorporation of a bifunctional ethynyl-hydroxybenzaldehyde into the farnesyl or geranylgeranyl group facilitates the facile labeling of proteins with two different moieties.
1. A compound of formula I or a salt thereof:
wherein n is an integer from 1 to 2.
2. A compound of formula II or a salt thereof:
wherein
n is an integer from 1 to 2; and
R 1 is a protein containing a CaaX motif which is attached the cysteine residue of said CaaX motif.
3. The compound of claim 2 , wherein the CaaX motif is the amino acid sequence CVIA (SEQ ID NO: 1).
4. The compound of claim 2 , wherein R 1 is Ciliary Neurotrophic Factor.
5. A compound of formula III or a salt thereof:
wherein:
n is an integer from 1 to 2;
R 1 is a protein containing a CaaX motif which is attached the cysteine residue of said CaaX motif;
X is O or NH;
F 1 ′ is a first functional group; and
F 2 ′ is a second functional group.
6. The compound of claim 5 , wherein the CaaX motif is CVIA (SEQ ID NO: 1).
7. The compound of claim 5 , wherein X is O.
8. The compound of claim 5 , wherein X is NH.
9. The compound of claim 5 , wherein F 1 ′ is a first functional group formed from reacting a compound of formula II with a compound F 1 containing an azide group that reacts with the ethynyl group of the compound of formula II to form a triazole linkage thereto.
10. The compound of claim 5 , wherein F 2 ′ is a second functional group formed from reacting a compound of formula II with a compound F2 containing an aminooxy group that reacts with the formyl group of the compound of formula II to form an oxime linkage thereto.
11. The compound of claim 5 , wherein F 2 ′ is a second functional group formed from reacting a compound of formula II with a compound F2 containing an hydrazinyl group that reacts with the formyl group of the compound of formula II to form a hydrazone linkage thereto.
12. A method of functionalizing a protein having a CaaX motif, comprising:
(a) reacting said protein with a compound of formula I or a salt thereof in the presence of a protein farnesyltransferase or a geranyl-geranyltransferase I to produce a compound of formula II or a salt thereof:
wherein
n is an integer from 1 to 2; and R 1 is said protein which is attached to the remainder of the compound at the cysteine residue of the CaaX motif; and
(b) reacting the compound of formula II or a salt thereof with a first functional compound F 1 containing an azide group that reacts with the ethynyl group of the compound of formula II to form a triazole linkage thereto, and a second functional compound F 2 containing a reactive aminooxy or hydrazine group that reacts with the formyl group of the compound of formula II to form an oxime or hydrazone linkage thereto, thereby forming a compound of formula III or a salt thereof:
wherein
X is O or NH;
F 1 ′ is a first functional group; and
F 2 ′ is a second functional group.
13. The method of claim 12 , wherein n is 1.
14. The method of claim 13 , wherein the compound of formula I is reacted with said protein in the presence of protein farnesyltransferase.
15. The method of claim 12 , wherein n is 2.
16. The method of claim 15 , wherein the compound of formula I or a salt thereof is reacted with said protein in the presence of geranylgeranyltransferase I.
17. The method of claim 12 , wherein the CaaX is CVIA (SEQ ID NO: 1).
18. The method of claim 12 , wherein R 1 is Ciliary Neurotrophic Factor.
19. The method of claim 12 , wherein the first functional compound F 1 is azido-TAMRA fluorophore.
20. The method of claim 12 , wherein the first functional compound F 1 is azido-bis-methotrexate.
21. The method of claim 12 , wherein the second functional compound F 2 is aminooxy-PEG.
22. The method of claim 12 , wherein the second functional compound F 2 is aminooxy-TAMRA.
23. The method of claim 12 , wherein the first and second functional compounds F 1 and F 2 are reacted with the compound of formula II simultaneously.
24. A pharmaceutical composition comprising a compound of formula III or a salt thereof as described in claim 12 and a pharmaceutically acceptable carrier.
25. A method of functionalizing a protein having a CaaX motif, comprising:
a) reacting said protein with a compound of formula I or a salt thereof:
wherein X and Y are bioorthogonal groups that are identical or different which are capable of conjugating to a functional compound; Z is H, OH, halogen or haloalkyl; and n is an integer from 1 to 2; in the presence of a protein farnesyltransferase or a geranyl-geranyltransferase I to produce a compound of formula II or a salt thereof:
wherein
X and Y are bioorthogonal groups that are identical or different which are capable of conjugating to a functional compound; Z is H, OH, halogen or haloalkyl; and n is an integer from 1 to 2; and R 1 is said protein which is attached to the remainder of the compound at the cysteine residue of the CaaX motif; and
(b) reacting the compound of formula II or a salt thereof with a first functional compound containing a reactive group that reacts with X to form a linkage to said first functional compound, and a second functional compound containing a reactive group that reacts with Y to form a linkage to said second functional compound, thereby forming a compound of formula III or a salt thereof:
wherein
F 1 ′ is a first functional group; and F 2 ′ is a second functional group.