Lipo-Conjugation of Peptides

1 . A peptide conjugate comprising:

i) a peptide; and

ii) a modified lipid,

wherein

said modified lipid comprises at least one lipid linking group and at least one modifying group; and

said modifying group is covalently attached to said peptide at a preselected glycosyl and/or amino acid residue of said peptide via the lipid linking group.

2 . The peptide conjugate of claim 1 , wherein the modified lipid is a member selected from:

wherein

n is an integer selected from 1 to 20;

R 1 is the modifying group; and

R X is a member selected from substituted or unsubstituted, saturated or unsaturated C 1 -C 40 alkyl;

R T comprises at least one moiety which has a structure according to the formula:

wherein

R z is a member selected from H and substituted or unsubstituted methyl;

and describe the points of attachment between said moiety and the remainder of the main chain of the modified lipid;

describes the point of attachment between the modified lipid and either the glycosyl residue or the amino acid residue of the peptide.

3 . The peptide conjugate of claim 2 , wherein the modified lipid is Formula II, and Formula II has a structure according to:

wherein

n is an integer from 1 to 6.

4 . The peptide conjugate of claim 3 , wherein at least one of said modified lipids is conjugated to said peptide through a sulfur atom on one or more cysteine residues near the C-terminal end of the protein.

5 . The peptide conjugate of claim 4 , wherein at least one of said modified lipids is conjugated to a single C-terminal cysteine residue that is embedded within a C-terminal amino acid sequence of CAAX,

wherein

A is any aliphatic amino acid, and

X is a member selected from methionine, glutamine, serine and leucine.

6 . The peptide conjugate of claim 5 , wherein n is 3.

7 . The peptide conjugate of claim 6 , wherein X is a member selected from methionine, glutamine and serine.

8 . The peptide conjugate of claim 5 , wherein n is 4.

9 . The peptide conjugate of claim 6 , wherein X is leucine.

10 . The peptide conjugate of claim 4 , wherein at least two modified lipids are conjugated separately to two cysteine residues, and said cysteine residues are embedded within a C-terminal amino acid sequence which is a member selected from: cysteine-cysteine and cysteine-X 2 -cysteine,

wherein

X 2 is any amino acid.

11 . The peptide conjugate of claim 2 , wherein said modified lipid is

wherein

R X is a member selected from substituted or unsubstituted, saturated or unsaturated C 1 -C 40 alkyl.

12 . The peptide conjugate of claim 11 , wherein said R X is a member selected from C 14 to C 18 alkyl.

13 . The peptide conjugate of claim 11 , wherein said R X is C 14 .

14 . The peptide conjugate of claim 13 , wherein at least one of said modified lipids is conjugated to the peptide through a thioester linkage with one or more cysteine residues of the peptide.

15 . The peptide conjugate of claim 14 , wherein said peptide comprises a first cysteine residue, and said first cysteine residue is near one of the peptide termini.

16 . The peptide conjugate of claim 15 , wherein said first cysteine residue is near the C-terminus of the peptide, and said peptide further comprises a second cysteine residue, and said second cysteine residue is conjugated through a sulfur atom on one or more cysteine residues to a modified lipid having a structure according to the following formula:

wherein

said first cysteine residue is internal relative to said second cysteine residue.

17 . The peptide conjugate of claim 15 , wherein said first cysteine residue is near the N-terminus of the peptide, and the peptide is further conjugated through a nitrogen atom at its N-terminus to a modified lipid having a structure according to the following formula:

wherein

said first cysteine residue near the N-terminus of the peptide is internal relative to the N-terminus.

18 . The peptide conjugate of claim 16 , wherein said modified lipid is attached to said peptide through a nitrogen atom.

19 . The peptide conjugate of claim 18 , wherein R X is a member selected from C 14 to C 18 alkyl.

20 . The peptide conjugate of claim 19 , wherein said modified lipid is conjugated to the peptide through a nitrogen atom on an N-terminal glycine residue.

21 . The peptide conjugate of claim 20 , wherein the N-terminal glycine residue is located at position in the peptide sequence which is a member selected from one and two.

22 . The peptide conjugate of claim 1 , wherein said modifying group is a water soluble polymer which is a member selected from poly(ether), poly(saccharide) and poly(peptide).

23 . The peptide conjugate of claim 22 , wherein said poly(peptide) is an enzyme.

24 . The peptide conjugate of claim 22 , wherein said poly(saccharide) is poly(sialic acid).

25 . The peptide conjugate of claim 22 , wherein said poly(ether) is a poly(ethylene glycol) which is a member selected from linear PEG and branched PEG.

26 . The peptide conjugate of claim 2 , wherein said modified lipid has a structure which is a member selected from the following formulas:

in which

R 2 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroalkyl, e.g., acetal, OHC—, H 2 N—CH 2 CH 2 —, HS—CH 2 CH 2 —, and —(CH 2 ) q C(Y 1 )Z 2 ; -sugar-nucleotide, or protein;

R X is a member selected from substituted or unsubstituted, saturated or unsaturated C 1 -C 40 alkyl;

q is an integer selected from 1 to 2500;

e is an integer selected from 0 and 1;

m and o are integers independently selected from 0 to 20;

Z 1 is a member selected from O, S, N—R 4 , —(CH 2 ) p C(Y 2 )V, —(CH 2 ) p U(CH 2 ) n C(Y 2 ) v ;

X, Y 1 , Y 2 , W and U are independently selected from O, S, N—R 4 ;

V is a member selected from OH, NH 2 , halogen, S—R 5 , the alcohol component of activated esters, the amine component of activated amides, sugar-nucleotides, and proteins;

p, s and v are integers independently selected from 0 to 20; and

R 3 , R 4 and R 5 are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl and substituted or unsubstituted heteroaryl.

27 . The peptide conjugate of claim 2 , wherein said PEG moiety is branched PEG and said modified lipid has a structure which is a member selected from the following formulas:

wherein

L a is a linker selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

n is an integer selected from 1 to 20;

R X is a member selected from substituted or unsubstituted, saturated or unsaturated C 1 -C 40 alkyl;

R z is a member selected from H and substituted or unsubstituted methyl;

R 16 and R 17 are independently selected polymeric arms;

X 2 and X 4 are independently selected linkage fragments joining polymeric moieties R 16 and R 17 to C; and

X 5 is a non-reactive group.

28 . The peptide conjugate of claim 3 , wherein said modified lipid has a structure according to the following formula:

wherein

A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , A 10 and A 11 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —NA 12 A 13 , —OA 12 and —SiA 12 A 13

wherein

A 12 and A 13 are members independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

29 . The peptide conjugate of claim 1 , wherein said modifying group is a water insoluble polymer which is a member selected from poly(vinyl alcohols), polyamides, polyalkylenes, polyacrylamides, polyalkylene glycols and polyalkylene oxides.

30 . A method of preparing the peptide conjugate of claim 1 , said method comprising:

(a) contacting a peptide with

(i) a modified lipid precursor having a formula selected from

wherein

P O is a member selected from monophosphate and diphosphate;

C A is a member selected from N-hydroxysuccinimidyl, N-hydroxybenztriazolyl, halogen, substituted or unsubstituted imidazolyl, thioethers, p-nitrophenyl ethers, alkyl, alkenyl, alkynyl and aromatic ethers, Coenzyme A and derivatives thereof;

R X is a member selected from substituted or unsubstituted, saturated or unsaturated C 1 -C 40 alkyl;

R z is a member selected from H and substituted or unsubstituted methyl;

R 1 is a modifying group which is a member selected from a water soluble polymer, a water insoluble polymer, a therapeutic moiety, and a diagnostic moiety; and

(ii) an enzyme for which said modified lipid precursor is a substrate, under conditions appropriate to link said modified lipid precursor to said peptide, thereby preparing said conjugate.

31 . A pharmaceutical formulation comprising an effective amount of a peptide conjugate according to claim 1 , and a pharmaceutically acceptable excipient.

32 . The peptide conjugate of claim 2 , wherein said modified lipid is Formula II, and wherein said Formula II is a member selected from: