Methods for synthesizing amatoxin building block and amatoxins

1. A method for the synthesis of γ, δ-dihydroxyisoleucine 1, or of a synthon for compound 1, comprising the step of methylating compound 3 or 3*, wherein the 3* has a second benzyl group at the nitrogen atom instead of the phenyl fluorenyl group in compound 3, with methyl iodide in the presence of lithium bis(trimethylsilyl)amide (LHMDS):

2. The method of claim 1 , wherein the methylation is performed at a temperature between about −10° C. and about −80° C. in an ether for between about 12 and about 20 hours.

3. The method of claim 1 , further comprising at least one of (a), (b), and (c):

(a) reaction of L-aspartic acid, monomethyl ester A with 2-methyl propene to create compound B:

(b) reaction of B with benzaldehyde to create compound C:

(c) reaction of C with phenyl fluorenyl bromide to create compound 3, or with benzyl bromide to create compound 3*:

4. The method of claim 1 , further comprising at least one of (a), (b), (c), (d), (e), and (f):

(a) reduction of compound 2 or compound 2*, wherein the 2* has a second benzyl group at the nitrogen atom instead of the phenyl fluorenyl group in compound 2, to create compound D or D*, wherein the D* has a second benzyl group at the nitrogen atom instead of the phenyl fluorenyl group in compound D, respectively:

(b) oxidation of hydroxy compound D or D* to create compound E or E*, wherein the E* has a second benzyl group at the nitrogen atom instead of the phenyl fluorenyl group in compound E, respectively:

(c) conversion of E or E* to create compound 4 or 4*, wherein the 4* has a second benzyl group at the nitrogen atom instead of the phenyl fluorenyl group in compound 4, respectively:

(d) conversion of 4 or 4* to create compound F or F*, wherein the F* has a second benzyl group at the nitrogen atom instead of the phenyl fluorenyl group in compound F, respectively:

(e) conversion of F or F* to create compound 5 or 5*, wherein the 5* has a second benzyl group at the nitrogen atom instead of the phenyl fluorenyl group in compound 5, respectively:

(f) N-deprotection of 5 or 5* to create compound 6:

5. The method of claim 1 , further comprising the step of isolating and purifying compound 6.

6. A compound of structure 6:

7. The compound of claim 6 , wherein the compound has a purity greater than 90%.

8. The compound of claim 6 , wherein the compound has a diastereomeric purity greater than 70:30.

9. A kit comprising the compound of claim 6 , and one or more additional reagents for the synthesis of amatoxins or precursors thereof.

10. The kit of claim 9 , wherein said one or more additional reagents are selected from the list of:

(i) a resin;

(ii) a protected hydroxyproline;

(iii) a protected asparagine;

(iv) a protected Cys-Trp dipeptide;

(v) a protected glycine;

(vi) a protected isoleucine;

(vii) a peptide coupling reagent;

(viii) a tertiary amine;

and combinations thereof.

11. A method for synthesizing an amatoxin, or precursor molecule therefore, comprising the step of (a) coupling compound 6 to hydroxyproline L to create compound G:

12. The method of claim 11 , further comprising at least one of (b), (c), (d), and (e):

(b) iterative Fmoc-N-deprotection and coupling of G with Fmoc-(N-Tri)Asn OH; FmocCys(S-2-((o-NO 2 Ph)SO 2 Trp-O-Allyl))]OH, Fmoc-Gly OH, Fmoc-lle OH, and Fmoc-Gly OH to create compound H:

(c) O-allyl- and Fmoc-N-deprotection of H followed by cyclisation to create compound I:

(d) 2-nitro aryl sulfonamide-N-deprotection and secession of I from resin to create compound J:

(e) solution phase cyclisation of J creating Amanitin derivative K:

13. The method of claim 11 , wherein the amatoxin comprises an amatoxin with a dihydroxyisoleucine moiety as its third amino acid.

14. The method of claim 4 , comprising at least one of:

(i) the reduction of compound 2 or compound 2* in (a) with diisobutylaluminium hydride;

(ii) the oxidation of hydroxy compound D or D* in (b) using a Swern oxidation;

(iii) the conversion of E or E* in (c) under conditions of a Wittig reaction;

(iv) the conversion of 4 or 4* in (d) under conditions of a Sharpless oxidation;

(v) the conversion of F or F* in (e) under catalytic esterification conditions; and

(vi) the N-deprotection of 5 or 5* in (f) using Palladium-catalyzed hydrogenation.

15. The kit of claim 10 , wherein the resin is a Merrifield resin, a Rink-Amid resin, a THP-resin, or a combination thereof.

16. The kit of claim 10 , comprising at least one of: a fluorenylmethyloxycarbonyl-(Fmoc-)-protected O-allyl hydroxyproline (FmocHypOAll); an Fmoc-protected N-trityl asparagine (Fmoc(N-Tri)AsnOH); an Fmoc-protected Cys-Trp dipeptide with -SH and -OH protection groups (FmocCys(S-2-((o-NO 2 Ph)SO 2 Trp-O-Allyl))]OH); an Fmoc-protected glycine (FmocGly); an Fmoc-protected isoleucine (Fmoclle); a peptide coupling reagent selected from the group consisting of: O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), and o-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU); and N,N-diisopropylethylamine (DiPEA).

17. The method of claim 11 , wherein compound 6 is coupled to hydroxyproline by reacting compound 6 with a hydroxyproline-preloaded resin.

18. The method of claim 17 , wherein compound 6 is coupled to the free C-terminus of FmocHypOH immobilized on a resin L.

19. The method of claim 18 , wherein the resin L is a tetrahydropyranyl (THP) resin.