Synthesis of (2S,3R,4R)-4,5-dihydroxyisoleucine and derivatives

Abstract

The invention relates to a method for the preparation of a 4,5-dihydroxyisoleucine derivative comprising the steps of asymmetric Claisen rearrangement of a Z-aminocrotyl-glycin ester and subsequent kinetic resolution of the product diastereomer mix by acylase, and subsequent Sharpless dihydroxylation of the resulting 2-amino-3-methylpent-4-enoic acid derivative.

Claims

1. A method for the preparation of a (2S,3R, 4R)-4,5-dihydroxyisoleucine derivative (400) ##STR00069## wherein: R.sup.1 is H or a carboxylic acid moiety protecting group, R.sup.2 is H or an amino moiety protecting group and R.sup.3 and R.sup.4 are independently from another selected from H and a hydroxyl moiety protecting group, or both R.sup.3 and R.sup.4 are one single vicinal diol protecting group moiety, from a crotyl glycinester derivative (200), wherein said method comprises the method for the preparation of ##STR00070## is converted to yield (2S,3S)-2-amino-3-methylpent-4-enoic acid (30) (2S,3S)-2-amino-3-methylpent-4-enoic acid (30), ##STR00071## wherein said method comprises a) the Claisen rearrangement step of reacting a Z-crotyl-glycin ester derivative 200 ##STR00072## wherein R.sup.2A is amino protecting group stable under conditions favourable to Claisen rearrangement, in the presence of a chiral ligand to yield 2-amino-3-methylpent-4-enoic acid derivative (300) ##STR00073## and b) an enzymatic resolution step, wherein 2-amino-3-methylpent-4-enoic acid derivative (310) and further comprises a) a Sharpless dihydroxylation step, wherein 2-amino-3-methylpent-4-enoic acid derivative (330) ##STR00074## is reacted in the presence of Os(VIII) complexed by a chiral ligand, particularly by hydroquinidine 1,4-phthalazinediyl diether, to the 2-amino-3-methyl-4,5-dihydroxypentanoic acid derivative (430) ##STR00075## or b) a Sharpless epoxidation step, wherein 2-amino-3-methylpent-4-enoic acid derivative (330) ##STR00076## is reacted in the presence of a chiral ligand and a catalyst comprising titanium isopropoxide to a (2S,3S, 4R)-2-amino-3-methyl-4,5-epoxy-pentanoic acid derivative (360), ##STR00077## and and subsequent enzymatic conversion catalysed by an epoxide hydrolase into 2-amino-3-methyl-4,5-dihydroxypentanoic acid derivative (430), ##STR00078## and wherein optionally, subsequent to step a) or b), any amino group protecting moiety R.sup.2C and/or carboxylic acid group protecting moiety R.sup.1C is selectively removed from (430) to yield the free (2S,3R, 4R)-4,5-dihydroxyisoleucine, or (430) is reacted with a vicinal diol protecting group, particularly to yield a compound selected from ##STR00079## wherein R.sup.1C and R.sup.2C have the same meaning as defined above and R.sup.5 and R.sup.5′ are independently selected from H and CH.sub.3.

2. The method according to claim 1, wherein a. R.sup.1 is H or tert-butyl, and/or b. R.sup.2 is fluorenylmethyloxycarbonyl (Fmoc) or H, and/or c. R.sup.3 and/or R.sup.4 is selected from p-methoxy benzylidene, acetonide, acetate and trialkylsilyl, and/or d. R.sup.2 is Fmoc and R.sup.3 and R.sup.4 together are a benzyl or a substituted phenylmethyl, a dialkyl-substituted silyl group or a tetrasubstituted siloxane moiety.

3. The method according to claim 1, which comprises step (c).

4. The method according to claim 3, wherein the chiral ligand in step (c) is hydroquinidine 1,4-phthalazinediyl diether.

5. The method according to claim 3, wherein, subsequent to step (c), any amino group protecting moiety R.sup.2C and/or carboxylic acid group protecting moiety R.sup.1C is selectively removed from 2-amino-3-methyl-4,5-dihydroxypentanoic acid derivative (430) to yield the free (2S, 3R, 4R)-4,5-dihydroxyisoleucine.

6. The method according to claim 3, wherein, subsequent to step (c), 2-amino-3-methyl-4,5-dihydroxypentanoic acid derivative (430) is reacted with a vicinal diol protecting group.

7. The method according to claim 6, wherein 2-amino-3-methyl-4,5-dihydroxypentanoic acid derivative (430) is reacted with a vicinal diol protecting group to yield a compound selected from ##STR00080## wherein R.sup.1C and R.sup.2C have the same meaning as defined above and R.sup.5 and R.sup.5′ are independently selected from H and CH.sub.3.

8. The method according to claim 1, which comprises step (d).

9. The method according to claim 8, wherein, subsequent to step (d), any amino group protecting moiety R.sup.2C and/or carboxylic acid group protecting moiety R.sup.1C is selectively removed from 2-amino-3-methyl-4,5-dihydroxypentanoic acid derivative (430) to yield the free (2S, 3R, 4R)-4,5-dihydroxyisoleucine.

10. The method according to claim 8, wherein, subsequent to step (d), 2-amino-3-methyl-4,5-dihydroxypentanoic acid derivative (430) is reacted with a vicinal diol protecting group.

11. The method according to claim 10, wherein 2-amino-3-methyl-4,5-dihydroxypentanoic acid derivative (430) is reacted with a vicinal diol protecting group to yield a compound selected from ##STR00081## wherein R.sup.1C and R.sup.2C have the same meaning as defined above and R.sup.5 and R.sup.5′ are independently selected from H and CH.sub.3.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 a chiral GC-MS chromatogram of the methylated and TFA-protected 4,5-didehydroisoleucine after the asymmetric Claisen rearrangement with quinine as ligand.

(2) FIG. 2 shows a chiral GC-MS chromatogram of the methylated and TFA-protected 4,5-didehydroisoleucine after the asymmetric Claisen rearrangement with quinidine as ligand.

(3) FIG. 3 shows a chiral GC-MS chromatogram of the trifluoroacetylated and methylated 4,5-didehydroisoleucine after enzymatic kinetic resolution of 31a-d.

(4) FIG. 4 shows a Chiral GC-MS chromatogram of the methylated and TFA-protected 4,5-didehydroisoleucine

EXAMPLES

(5) General

(6) Chemicals and Solvents

(7) Chemicals and solvents were purchased from ABCR (Karlsruhe, Germany), ACROS ORGANICS (Geel, Belgium), ALFA AESAR (Karlsruhe, Germany), FLUKA (Buchs, Switzerland), FLUOROCHEM LIMITED (Derbyshire, UK), IRIS BIOTECH (Marktredwitz, Germany), CARL ROTH (Karlsruhe, Germany), MERCK (Darmstadt, Germany), NOVABIOCHEM (Darmstadt, Germany), BACHEM (Bubendorf, Switzerland) or SIGMA-ALDRICH (Taufkirchen, Germany) and used without further purification.

(8) DMSO-d.sub.6 (99.9%), CDCl.sub.3 (99.9%) and D.sub.2O (98.8%) for NMR spectroscopy were purchased from DEUTERO (Kastellaun, Germany) and SIGMA-ALDRICH (Taufkirchen, Germany).

(9) Inert Gas

(10) Reactions sensitive to air and moisture were carried out under argon atmosphere in oven-dried flasks. Liquid reactants were added with PE-syringes, which were flushed with argon several times, via septum. Solids were either dissolved and added as a solution or introduced into the reaction vessel as powder under a flow of argon.

(11) Thin Layer Chromatography (TLC)

(12) TLC was performed on silica plates purchased from MERCK (Silica gel 60 F.sub.254). The compounds were detected by UV-irradiation (λ=254 nm) and the use of permanganate-, ninhydrin- and molybdate staining reagents.

(13) Column Chromatography

(14) Column chromatography was carried out on silica gel 40-63 μm purchased from GRACE DAVISON (Deerfield, USA) using pressurized air.

(15) NMR Spectra

(16) .sup.1H NMR and. .sup.13C NMR spectra were measured on Bruker DRX 500 and AM 400 instruments. Chemical shifts δ are reported in parts per million (ppm) and relative to the remaining proton signals of CDCl.sub.3 (.sup.1H: δ=7.26 ppm; .sup.13C: δ=77.16 ppm) and DMSO-d.sub.6 (.sup.1H: δ=2.5 ppm; .sup.13C: δ=39.5 ppm). Coupling constants J are given in Hertz (Hz) and refer to H—H-coupling. Integrals are in accordance with assignments. Multiplicities: s=singulet, d=duplet, t=triplet, q=quartet. When the multiplicity could not be identified, the chemical shift range of the signal was given

(17) (m=multiplet). 13C NMR spectra are proton-decoupled.

(18) Mass Spectra

(19) LC-HRMS (ESI) was measured with Orbitrap XL-mass spectrometer from THERMO SCIENTIFIC (Waltham, Mass., USA) coupled with 1200-HPLC from AGILENT TECHNOLOGIES using a hypersil 100-C.sub.18-column from THERMO SCIENTIFIC (solvent A: water/0.025% HCOOH, solvent B: AcN/0.025% HCOOH; flow rate: 1.3 ml/min). Xcalibur (THERMO SCIENTIFIC) was used for the evaluation of the spectra.

(20) Chiral GC-MS

(21) Chiral GC-MS was measured with 5975C from AGILENT TECHNOLOGIES) using a CS EnantioSELECT GC column (30 m, 250 μm×0.3 μm; CS Chromatographie Service GmbH, Langerwehe, Germany).

(22) The temperature gradient (44 min) started with an initial hold for 2 min at 70° C. followed by an increase of 4° C./min up to 240° C. Scans were performed in electron impact (EI) mode (MS source: 300° C., MS Quad: 150° C., Emission: 108.8 μA, Energy: 50 eV) with a flow of 1.2 mL/min using helium as carrier gas.

(23) Preparative HPLC

(24) Purification by Preparative HPLC was performed with 1260 Infinity from AGILENT TECHNOLOGIEs using a PLRP-S column (partial size 10 μm×100 Å) from AGILENT TECHNOLOGIES. Used solvents were water and AcN, each with addition of 0.1% TFA.

(Z)-but-2-En-1-ol (10)

(25) ##STR00050##

(26) A solution of 12 ml but-2-yn-1-ol (10) (11.28 g, 160.94 mmol, 1.0 eq) in MeOH was degassed with nitrogen for 30 min. Then 1.2 g of Lindlar's catalyst (7.5 g/mmol) was added after which the reaction mixture was degassed with hydrogen for 1 h. After vigorous stirring at room temperature under 1.0 atm of hydrogen overnight the catalyst was filtered through a pad of Celite. Afterwards the mixture was concentrated and purified by distillation to obtain the product (10) (7.1 g, 98.6 mmol, 61%) as a transparent liquid.

(27) HRMS (APCI): m/z calc for C.sub.4H.sub.8O [M+H].sup.+ 73.0648 found 73.0647.

(28) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=5.57-5.67 (m, 2H), 4.17-4.21 (m, 2H), 1.85 (s, 1H), 1.64-1.67 (m, 3H)

(29) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=129.35, 127.36, 58.40, 13.22

Trifluoroacetyl Glycine (11)

(30) ##STR00051##

(31) To a solution of glycine (2.5 g, 33.3 mmol, 1.0 eq) in MeOH triethylamine (4.6 ml, 33.3 mmol, 1.0 eq) was added dropwise. After stirring for 5 min ethyl trifluoroacetate (5.2 ml, 43.3 mmol, 1.3 eq) was added and the mixture was stirred for 16 h at room temperature during which time a clear solution formed. Then the reaction mixture was concentrated under reduced pressure and the resulting residue acidified with 2 N HCl before being extracted with EtOAc (3×75 ml). The organic layers were combined then washed with brine and dried over MgSO.sub.4. The solvent was removed in vacuo to give the product (11) as a white crystalline solid (5.4 g, 31.6 mmol, 95%).

(32) HRMS (ESI): m/z calc for C.sub.4H.sub.4F.sub.3NO.sub.3[M−H].sup.− 170.0060 found 170.0073.

(33) DC (n-Hexan/EtOAc/n-Bu/H.sub.2O/AcOH, 2:1:1:1): R.sub.f=0.6.

(34) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=12.97 (br, 1H), 9.85 (t, J=6.0 Hz, 1H), 3.92 (d, J=6.0 Hz, 2H)

(35) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=169.96, 156.87, 117.61, 41.08

(Z)-but-2-Enyl Trifluoroacetyl Glycinate (21)

(36) ##STR00052##

(37) DCC (14.9 g, 72.1 mmol, 1.3 eq) and DMAP (0.7 g, 5.6 mmol, 0.1 eq) were added to a solution of the allylic alcohol (10) (4.0 g, 55.5 mmol, 1.0 eq) in DCM at 0° C. The solution was cooled to −20° C. and the TFA-protected amino acid (11) (11.4 g, 66.6 mmol, 1.2 eq) was added after 5 min. The reaction mixture was allowed to warm to room temperature overnight. After filtration of the DCU and washing of the precipitate with cold DCM the organic phase was washed with 1N HCl (2×200 ml), saturated NaHCO.sub.3 (1×200 ml) and brine (1×200 ml). The organic layer was dried with MgSO.sub.4 and evaporated in vacuo. After purification by flash column chromatography on silica gel (hexane/ethyl acetate, 4:1) the product (21) was obtained as light yellow crystals (11.2 g, 49.9 mmol, 90%).

(38) HRMS (ESI): m/z calc for C.sub.8H.sub.10F.sub.3NO.sub.3[M+H].sup.+ 226.0686 found 226.0681.

(39) DC (n-Hexan/EtOAc, 4:1): R.sub.f=0.4.

(40) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=6.94 (br, 1H), 5.74-5.84 (m, 1H), 5.51-5.60 (m, 1H), 4.76 (d, J=7.05 Hz, 2H), 4.13 (d, J=5.1 Hz, 2H), 1.72 (dd, J=7.1, 1.6 Hz, 3H)

(41) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=176.98, 160.81, 83.29, 66.67, 61.57, 56.92, 33.45, 32.01

Boc-Glycine (18)

(42) ##STR00053##

(43) Glycine (10 g, 133.2 mmol, 1.0 eq) and NaHCO.sub.3 (33.6 g, 399.6 mmol, 3.0 eq) were dissolved in a 1:2 mixture of water/dioxane. Then Boc.sub.2O was added dropwise and the reaction mixture of stirred at room temperature for 16 h. After concentrating the mixture under reduced pressure the resultant residue was diluted with water until the precipitate was dissolved and then acidified with 1N HCl to pH 3. Afterwards the aqueous solution was extracted with EtOAc (3×250 ml). The organic layers were combined then washed with brine and dried over MgSO.sub.4. The solvent was removed in vacuo to give the product (18) as a white crystalline solid (20.7 g, 118.0 mmol, 87%).

(44) HRMS (ESI): m/z calc for C.sub.7H.sub.13NO.sub.4[M+H].sup.+ 176.0917 found 176.0919 [M+Na].sup.+ 198.0737 found 198.0738.

(45) TLC (n-Hexan/EtOAc/n-Bu/H.sub.2O/AcOH, 2:1:1:1): R.sub.f=0.9

(46) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=3.84-3.98 (m, 2H), 1.44 (s, 9H)

(47) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=174.21, 156.34, 80.63, 42.44, 28.65

(Z)-but-2-Enyl Boc-Glycinate (29)

(48) ##STR00054##

(49) DCC (3.7 g, 18.0 mmol, 1.3 eq) and DMAP (0.2 g, 1.4 mmol, 0.1 eq) were added to a solution of the allylic alcohol (10) (1.0 g, 13.9 mmol, 1.0 eq) in DCM at 0° C. The solution was cooled to −20° C. and the boc-protected amino acid (18) (2.7 g, 15.3 mmol, 1.2 eq) was added after 5 min. The reaction mixture was allowed to warm to room temperature overnight. After filtration of the DCU and washing of the precipitate with cold DCM the organic phase was washed with 1N HCl (2×150 ml), saturated NaHCO.sub.3 (1×150 ml) and brine (1×150 ml). The organic layer was dried with MgSO.sub.4 and evaporated in vacuo. After purification by column chromatography (hexane/ethyl acetate, 4:1) the product (29) was obtained as a colourless oil (2.6 g, 11.1 mmol, 80%)

(50) HRMS (ESI): m/z calc for C.sub.11H.sub.19NO.sub.4[M+H].sup.+ 231.1420 found 231.1422 [M+Na].sup.+ 252.1206 found 252.1207.

(51) TLC (n-Hexan/EtOAc, 5:1): R.sub.f=0.3

(52) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=5.67-5.77 (m, 1H), 5.47-5.57 (m, 1H), 5.06 (br, 1H), 4.69 (d, J=7.2 Hz, 2H), 3.89 (s, 2H), 1.69 (d, J=7.0 Hz, 3H), 1.43 (s, 9H)

(53) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=170.75, 156.04, 130.46, 123.90, 79.94, 61.07, 42.67, 28.46, 13.27

(Z)-but-2-Enyl Cbz-Glycinate (29x)

(54) ##STR00055##

(55) DCC (3.7 g, 72.1 mmol, 1.3 eq) and DMAP (0.2 g, 1.4 mmol, 0.1 eq) were added to a solution of the allylic alcohol (10) (1.0 g, 13.9 mmol, 1.0 eq) in DCM at 0° C. The solution was cooled to −20° C. and the cbz-protected amino acid (3.3 g, 15.3 mmol, 1.2 eq) was added after 5 min. The reaction mixture was allowed to warm to room temperature overnight. After filtration of the DCU and washing of the precipitate with cold DCM the organic phase was washed with 1N HCl (2×150 ml), saturated NaHCO.sub.3 (1×150 ml) and brine (1×150 ml). The organic layer was dried with MgSO.sub.4 and evaporated in vacuo. After purification by column chromatography (hexane/ethyl acetate, 4:1) the product was obtained as a colourless oil (2.7 g, 10.4 mmol, 75%).

(56) HRMS (ESI): m/z calc for C.sub.14H.sub.17NO.sub.4[M+H].sup.+ 264.1230 found 264.1223 [M+Na].sup.+ 286.1050 found 286.1043.

(57) TLC (n-Hexan/EtOAc, 5:1): R.sub.f=0.3

(58) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=7.20-7.29 (m, 5H), 5.61-5.71 (m, 1H), 5.40-5.50 (m, 1H), 5.24 (br, 1H), 5.04 (s, 2H), 4.43 (d, J=7.2 Hz, 2H), 3.89 (d, J=5.6 Hz, 2H), 1.61 (d, J=7.0 Hz, 3H)

(59) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=170.26, 156.65, 136.39, 130.67, 128.69, 128.35, 128.25, 123.87, 67.42, 61.08, 42.99, 13.45

Trifluoroacetyl Didehydroisoleucine (31)

(60) ##STR00056##

(61) A LHMDS solution was freshly prepared by adding 2.5 M nBuLi in hexane (8.9 ml, 22.2 mmol, 1.0 eq) to hexamethyldisilazane (4.3 g, 26.7 mmol, 1.2 eq) in 7.76 ml dry THF at −20° C. under argon atmosphere. The trifluoroacetyl protected glycine crotyl ester (21) (1.0 g, 4.4 mmol, 1.0 eq) and dry zinc chloride (1.2 g, 8.9 mmol, 2.0 eq) were dissolved in 8 ml dry THF and cooled to −78° C. Then the previously prepared LHMDS solution was added slowly. The reaction mixture was allowed to warm to room temperature overnight and stirred for 3 d. After diluting with EtO.sub.2 (200 ml) the reaction mixture was hydrolyzed by addition of 1 N KHSO.sub.4 until the precipitate was fully dissolved in the organic layer. Then the reaction mixture was extracted with saturated NaHCO.sub.3 (3×100 ml). Afterwards the basic aqueous solution was acidified by careful addition of solid KHSO.sub.4 to pH 1 and extracted with EtO.sub.2 (3×50 ml). The combined organic layers were dried over MgSO.sub.4 and the solvent was evaporated in vacuo. The product (31) was obtained as a yellow oil (700.1 mg, 3.1 mmol, 70%). For the determination of the enantiomeric and diastereomeric ratios of the product, the residue was treated with trimethylsilyl diazomethane in ether solution.

(62) HRMS (ESI): m/z calc for C.sub.8H.sub.9F.sub.3NO.sub.3[M−H].sup.− 224.0529 found 224.0536.

(63) TLC (n-Hexan/EtOAc/n-Bu/H.sub.2O/AcOH, 2:1:1:1): R.sub.f=0.95

(64) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=6.63 (br, 1H), 5.66-5.77 (m, 1H) 5.19-5.27 (m, 2H), 4.66 (q, J=4.2 Hz, 1H), 2.90-2.99 (m, 1H), 1.15 (d, J=7.04 Hz, 3H)

(65) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=175.37, 157.67, 136.37, 118.37, 117.00, 56.43, 39.56, 16.32

Trifluoroacetyl Didehydroisoleucine (31)

(66) ##STR00057##

(67) The trifluoroacetyl protected glycine crotyl ester (21) (1.0 eq), dry aluminium isopropoxide (1.25 eq) and quinidine (2.5 eq) were dissolved in dry THF and cooled to −78° C. Then, LHMDS (1 M in THF, 5.5 eq) was added slowly. The reaction mixture was allowed to warm to room temperature overnight and stirred for 2 d. After diluting with EtO.sub.2 the reaction mixture was hydrolyzed by addition of 1 M KHSO.sub.4 until the precipitate was fully dissolved in the organic layer. Then the reaction mixture was extracted with saturated NaHCO.sub.3. Afterwards the basic aqueous solution was acidified by careful addition of solid KHSO.sub.4 to pH 1 and extracted with EtO.sub.2. The combined organic layers were dried over MgSO.sub.4 and the solvent was evaporated in vacuo. The rearrangement product (31) was obtained as a yellow oil (72%). For the determination of the enantiomeric and diastereomeric ratios of the product, the residue was treated with trimethylsilyl diazomethane in ether solution.

Boc-4,5-Didehydroisoleucine (39)

(68) ##STR00058##

(69) A LHMDS solution was freshly prepared by adding 2.5 M nBuLi in hexane (7.0 ml, 17.5 mmol, 1.0 eq) to hexamethyldisilazane (3.4 g, 20.9 mmol, 1.2 eq) in 6.1 ml dry THF at −20° C. under argon atmosphere. The Boc-protected glycine crotyl ester (83) (800.0 mg, 3.5 mmol, 1.0 eq) and dry zinc chloride (951.0 g, 8.9 mmol, 2.0 eq) were dissolved in 7 ml dry THF and cooled to −78° C. Then the previously prepared LHMDS solution was added slowly. The reaction mixture was allowed to warm to room temperature overnight and stirred for 3 d. After diluting with EtO.sub.2 (200 ml) the reaction mixture was hydrolyzed by addition of 1N KHSO.sub.4 until the precipitate was fully dissolved in the organic layer. Then the reaction mixture was extracted with saturated NaHCO.sub.3 (3×100 ml). Afterwards the basic aqueous solution was acidified by careful addition of solid KHSO.sub.4 to pH 1 and extracted with EtO.sub.2 (3×50 ml). The combined organic layers were dried over MgSO.sub.4 and the solvent was evaporated in vacuo. After purification by flash column chromatography (dichloromethane/2% methanol) the product (39) was obtained as pale yellow oil (267.2 mg, 1.2 mmol, 34%).

(70) HRMS (ESI): m/z calc for C.sub.8H.sub.9F.sub.3NO.sub.3[M−H].sup.− 224.0529 found 224.0536.

(71) TLC (n-Hexan/EtOAc/n-Bu/H.sub.2O/AcOH, 2:1:1:1): R.sub.f=0.95

(72) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=6.63 (br, 1H), 5.66-5.77 (m, 1H) 5.19-5.27 (m, 2H), 4.66 (q, J=4.2 Hz, 1H), 2.90-2.99 (m, 1H), 1.15 (d, J=7.04 Hz, 3H)

(73) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=175.37, 157.67, 136.37, 118.37, 117.00, 56.43, 39.56, 16.32

Trifluoroacetyl 4,5-Didehydroisoleucine Methyl Ester

(74) ##STR00059##

(75) Trifluoroacetyl 4,5-didehydroisoleucine (67) (30.0 mg, 133.2 μmol, 1.0 eq) was dissolved in a 1:1 mixture of MeOH/toluol. Then trimethylsilyl diazomethane (15.2 mg, 0.1 mmol, 1.0 eq) was added dropwise and the reaction mixture of stirred at room temperature for 30 min. After evaporation of the solvent under reduced pressure the resultant the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate, 5:1) to give the product as a colourless oil (29.5 mg, 123.3 μmol, 93%).

(76) TLC (n-Hexan/EtOAc, 4:1): R.sub.f=0.6

(77) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=6.68 (br, 1H), 5.62-5.72 (m, 1H) 5.11-5.21 (m, 2H), 4.61 (q, J=4.2 Hz, 1H), 3.80 (s, 3H), 2.81-2.90 (m, 1H), 1.11 (d, J=7.2 Hz, 3H)

(78) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=170.67, 157.39, 136.92, 118.11, 114.24, 56.98, 53.11, 40.38, 16.04

N-Acetyl 4,5-Didehydroisoleucine (32)

(79) ##STR00060##

(80) The trifluoroacetylated amino acid (31) (375.0 mg, 1.7 mmol, 1.0 eq) was dissolved in 8 ml MeOH and K.sub.2CO.sub.3 (1153.9 mg, 8.4 mmol, 5 eq) was added. Then the reaction mixture was refluxed for 2 hours until the deprotection was complete which was monitored by TLC. Afterwards the mixture was filtered to remove the K.sub.2CO.sub.3. The solvent was removed under reduced pressure and the resulting residue was dissolved in 8 ml THF. Then acetic anhydride (787.8 μl, 8.4 mmol, 3.0 eq) was added dropwise. After the reaction mixture was refluxed for 6 hours the solvent was removed in vacuo. After purification by flash column chromatography (dichloromethane/5% methanol) the product (91) was obtained as a brown oil (260.0 mg, 1.5 mmol, 90%).

(81) HRMS (ESI): m/z calc for C8H13NO3 [MH].sup.− 170.0812 found 170.0819.

(82) TLC (n-Hexan/EtOAc/n-Bu/H.sub.2O/AcOH, 2:1:1:1): R.sub.f=0.98

(83) .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ (ppm)=7.98 (t, J=9.96 Hz, 1H), 5.68-5.83 (m, 1H) 4.96-5.08 (m, 2H), 4.30-4.22 (m, 1H), 2.56-2.66 (m, 1H), 1.83-1.92 (m, 3H), 0.97 (dd, J=4.4, 2.7 Hz 3H)

(84) .sup.13C-NMR (100 MHz, DMSO-d.sub.6): δ (ppm)=173.01, 169.63, 139.92, 115.34, 56.38, 22.49, 16.87, 15.26

4,5-Didehydroisoleucine (30)

(85) ##STR00061##

(86) To a solution of racemic N-acetylated 2-amino-3-methylpentanoic acid (1412.4 mg, 8.3 mmol, 1.0 eq) in 0.1 M phosphate buffer (30 ml, pH 7.5) and 4 M KOH (2.1 ml, 8.3 mmol, 1.0 eq) acylase I from Aspergillus melleus (700 mg) was added. After 6 h at 35° C. the reaction mixture was acidified to pH 3 and filtered through a PTFE membrane filter (pore size 0.2 μm). Afterwards the filtrate was extracted with diethylether (3×20 ml) and concentrated in vacuo. The crude product was submitted to the next reaction step without further purification.

(87) HRMS (ESI): m/z calc for C.sub.6H.sub.11NO.sub.2[M+H].sup.+ 130.0863 found 130.0858. [M+Na].sup.+ 152.0682 found 152.0680.

(88) TLC (n-Hexan/EtOAc/n-Bu/H.sub.2O/AcOH, 2:1:1:1): R.sub.f=0.45

Fmoc-4,5-Didehydroisoleucine (35)

(89) ##STR00062##

(90) To a stirring solution of 4,5-didehydroisoleucine 30 (300.0 mg, 2.32 mmol, 1.0 eq) in 10 ml of a 1:1 mixture of water/acetonitrile, NaHCO.sub.3 (585.4 mg, 6.9 mmol, 3.0 eq) and Fmoc-OSu (1020.0 mg, 3.02 mmol, 1.3 eq) were added. After stirring for 16 h the reaction mixture was diluted with 50 ml water, acidified to pH 2 with 1 N HCl and extracted with ethyl acetate (3×30 ml). The combined organic layers were washed with brine, dried over MgSO.sub.4 and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (dichloromethane/5% methanol) to obtain the Fmoc-protected amino acid 35 as a white crystalline solid (700.0 mg, 2.0 mmol, 86%).

(91) HRMS (ESI): m/z calc for C.sub.21H.sub.21NO.sub.4[M+H].sup.+ 374.1363 found 374.1360.

(92) TLC (n-Hexan/EtOAc/n-Bu/H.sub.2O/AcOH, 2:1:1:1): R.sub.f=0.9

(93) .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ (ppm)=8.49 (br, 1H), 7.68 (d, J=7.5 Hz, 2H), 7.44-7.54 (m, 2H), 7.32 (t, J=7.02 Hz, 2H), 7.23 (t, J=7.02 Hz, 2H), 5.45-5.73 (m, 1H), 5.18-5.32 (m, 1H), 5.01-5.17 (m, 1H), 4.30-4.37 (m, 2H), 4.12-4.19 (m, 1H), 2.39-2.54 (m, 1H), 2.44-2.52 (m, 1H), 1.60 (d, J=6.0 Hz, 2H), 1.05 (d, J=7.0 Hz, 1H)

(94) .sup.13C-NMR (100 MHz, DMSO-d.sub.6): δ (ppm)=177.01, 156.04, 143.58, 141.45, 137.20, 130.67, 127.93, 127.02, 125.20, 124.13, 120.18, 117.60, 67.46, 58.04, 53.33, 47.25, 39.65, 35.09

Fmoc-4,5-Didehydroisoleucine Tert-Butyl Ester (36)

(95) ##STR00063##

(96) p-Toluenesulfonyl chloride (244.1 mg, 1.3 mmol, 1.5 eq) was added to a stirring solution of Fmoc-protected 4,5-didehydroisoleucine (35) (300.0 mg, 0.8 mmol, 1.0 eq) in 3 ml pyridine. The reaction mixture was cooled to 0° C. and tert-butanol (160.0 μl, 1.7 mmol, 2.0 eq) was added and allowed to warm to room temperature overnight. After stirring for 16 h dichloromethane (100 ml) was added. The organic solution was washed with 1 N HCl (3×50 ml) and evaporated in vacuo. The crude product was purified by column chromatography on silica gel (hexane/ethyl acetate, 10:1) to obtain the fully protected amino acid 36 as a colourless oil (200.5 mg, 0.5 mmol, 57%).

(97) HRMS (ESI): m/z calc for C.sub.25H.sub.29NO.sub.4[M+H].sup.+ 442.2224 found 442.2230.

(98) TLC (n-Hexan/EtOAc, 10:1): R.sub.f=0.3.

(99) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ (ppm)=7.68 (d, J=7.5 Hz, 2H), 7.52 (d, J=7.5 Hz, 2H), 7.31 (t, J=7.2 Hz, 2H), 7.22 (t, J=7.2 Hz, 2H), 5.59-5.69 (m, 1H), 5.12-5.19 (m, 1H), 4.99-5.09 (m, 2H), 4.25-4.34 (m, 2H), 4.12-4.25 (m, 2H), 2.64-2.76 (m, 1H), 1.39 (s, 9H), 1.01 (d, J=6.9 Hz, 3H),

(100) .sup.13C-NMR (100 MHz, CDCl.sub.3): δ (ppm)=170.63, 156.50, 144.04, 141.76, 137.96, 127.93, 127.02, 125.20, 125.20, 120.18, 116.69, 82.35, 67.15 58.34, 47.10, 40.56, 16.02

Fmoc-4,5-Dihydroxyisoleucine Tert-Butyl Ester (46)

(101) ##STR00064##

(102) Terminal alkene 36 (177.0 mg, 0.4 mmol, 1.0 eq) was dissolved in 2.7 ml of a 1:1 mixture of water/t-BuOH, then AD-mix-ß (1120.0 mg, 2800 mg/mmol; Aldrich catalogue no. 392766) was added. After stirring vigorously for 16 h, the reaction was terminated by adding Na.sub.2SO.sub.3 (600.0 mg, 1500 mg/mmol). The mixture was then diluted with 30 ml of water and extracted with ethyl acetate (3×20 ml). The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (dichloromethane/5% methanol) to obtain the dihydroxylated amino acid 46 as a white powder (7.9 mg, 17.9 μmol, 4%).

(103) HRMS (ESI): m/z calc for C.sub.25H.sub.31NO.sub.6[M+H].sup.+ 1104.5468 found 1104.5454 [M+Na].sup.+ 464.2044 found 464.2046.

Fmoc-4,5-Didehydroisoleucine Dicyclopropylmethyl Ester (47)

(104) ##STR00065##

(105) Compound 30a (860 mg, 2.4 mmol, 1.0 eq) was dissolved in 10 ml dichloromethane and treated with DMAP (30 mg, 0.24 mmol, 0.1 eq), EDC×HCl (910 mg, 4.7 mmol, 1.9 eq) and with dicyclopropylmethyl alcohol (420 mg, 3.7 mmol, 1.5 eq). The solution was stirred for 24 hours at 22° C. The solvent was removed under reduced pressure and the residue was taken up in 20 ml methyl tert-butyl ether (MTBE) and 20 ml water. The organic layer was washed with water (3×20 ml), dried over MgSO.sub.4 and the solvent was evaporated in vacuum. The crude was purified via column chromatography on silica gel (iso-hexane/MTBE 4:1 (v/v)) to obtain the desired product 47 as a colorless oil (600 mg, 1.3 mmol, 56%).

(106) MS (ESI): m/z calc for C.sub.28H.sub.31NO.sub.4[m+H].sup.+ 446.23 found 446.21 [m+H].sup.+.

Fmoc-4,5-Dihydroxyisoleucine Dicyclopropylmethyl Ester (48)

(107) ##STR00066##

(108) The full protected Fmoc-4,5-didehydroisoleucine derivative 47 (255 mg, 0.57 mmol, 1 eq) was dissolved in 12 ml of a 1:1 mixture (v/v) CHCl.sub.3/H.sub.2O, treated with AD-mix-ß (1596.0 mg, 2800 mg/mmol; Aldrich catalogue no. 392766) and stirred for 72 hours at 22° C. After no starting material was detectable (IPC via HPLC-MS) the solution was filtered and the two layers were separated. The organic layer was dried over MgSO.sub.4 and the solvent was evaporated in vacuum. Finally, 260 mg crude of 48 (0.54 mmol, crude yield 95%) were obtained and the residue was used without further purification in subsequent reactions.

(109) MS (ESI): m/z calc for C.sub.28H.sub.34NO.sub.6[m+H].sup.+ 480.29 found 480.18 [M+H].sup.+ and 502.18 [M+Na].sup.+.

Fmoc-4,5-dioxy(tetra-isopropyl-disiloxane)isoleucine dicyclopropylmethyl ester (49)

(110) ##STR00067##

(111) Substance 48 (260 mg, 0.54 mmol, 1 eq) was dissolved in 5 ml dry DMF and treated with imidazole (155 mg, 2.3 mmol, 4.2 eq) and 1,3-dichloro-tetra-isopropyl-disiloxane (341 mg, 1.08 mmol, 2 eq). The solution was stirred for 16 hours at 22° C. After the IPC (HPLC-MS) showed a full conversion 20 ml EtOAc and 20 ml water were added to the reaction solution. The layers were separated and the water layer was extracted with EtOAc (3×20 ml) again. The combined organic layers were dried over MgSO.sub.4 and the solvent was removed under vacuum. The obtained residue of 49 was used without further purification for the subsequent de-protection reaction.

(112) MS (ESI): m/z calc for C.sub.40H.sub.60NO.sub.7Si.sub.2[M+H].sup.+ 722.39 found 722.31 [M+H].sup.+ and 744.31 [M+Na].sup.+.

Fmoc-4,5-dioxy(tetra-isopropyl-disiloxane)isoleucine (50)

(113) ##STR00068##

(114) The crude of 49 (0.54 mmol), from the diol protection reaction, was dissolved in 10 ml 0.03 N HCl/dioxane and stirred for 48 hours at 22° C. After the mentioned duration, the IPC (HPLC-MS) showed full conversion to the desired target molecule. Therefore, the solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (CH.sub.2Cl.sub.2/MeOH/AcOH 190:10:1 (v/v/v)) to obtain the desired amino acid derivative 50 (218 mg, 0.3 mmol, 53%) as a colourless to slightly yellow oil.

(115) MS (ESI): m/z calc for C.sub.33H.sub.50NO.sub.7Si.sub.2[M+H].sup.+ 628.31 found 628.23 [M+H].sup.+.

(116) TLC (CH.sub.2Cl.sub.2/MeOH/AcOH 190:10:1): R.sub.f=0.24