Synthesis of (2S,4S)- and (2S,4R)-5,5prime;-dihydroxy5,5-2H2leucine by two independent routes

摘要

Synthesis of (2S,4S)- and (2S,4R)-5,5'-dihydroxy 5,5-2H2leucine by two independent routes Xavier Durand, Piiitrick Hudhomme, Jeffrey A. Khan and Douglas W. Young* W mIIx-nz School oj Chemistry and Molecular Sciences, University of Sussex, Falmer, Brighton BNI 9QJ, UK In studies directed at discovering the absolute stereochemistry of the reaction catalysed by the enzyme glutamate y-carboxylase, two independent stereoselective syntheses of (2S,4S)-5,5'-dihydroxy 53- 2H2-leucine 4a and its (2S,4R)-diastereoisomer 4b have been developed. The first synthesis uses (29- pyroglutamic acid as starting material and provides (2S,4S)-5,5'-dihydroxy 5,5-2H2leucine 4a, while the second starts with (2S,4R)-4-hydroxyproline and provides (2S,4R)-5,5'-dihydroxy 5,5-2H2leucine 4b.The unusual proteinogenic amino acid residue y-carboxy- glutamic acid 1 was not discovered until 1974 because of the lability of the terminal malonate moiety to decarboxylation. Two research groups found this amino acid to be present as residues in normal prothrombin 2.3 whereas the corresponding residues in abnormal prothrombin, obtained from patients treated with anticoagulants, were glutamic acid residues. y-Carboxyglutamic acid was later discovered to be present in other proteins of the blood clotting cascade. This amino acid occurs most abundantly at the amino terminal end of these proteins and it arises through post-translational modification of glutamate residues 2 in precursor proteins as shown in Scheme 1.The enzyme responsible for the y-carboxylation of glutamate residues is unusual, being linked to the oxidation of the hydroquinone 5 of vitamin K to yield vitamin K epoxide 6.4 Inhibitors of this enzyme are potential anti-thrombotic drugs. The mechanism of the reaction has excited much speculation and elucidation of the stereochemistry of the process is important in understanding the mechanism of action of the enzyme and in designing enzyme inhibitors. Marquet, Azerad and co-workers have shown6-' that the hydrogen, 4-H,, is abstracted in the carboxylation process and they have implied that the 4-pro-Rcarboxy group in the non-fluorinated product is derived from CO,, using a pentapeptide containing (49-4- fluoroglutamate. lo This suggests inversion of stereochemistry in the enzyme catalysed reaction.In our own studies in this area, we noted that reduction of peptide bound y-carboxyglutamate with diborane followed by hydrolysis had been reported to yield 5,5'-dihydroxyleucine 4 so that, if 13C-labelled carbon dioxide were used in the enzyme catalysed reaction, we might obtain a sample of dihydroxyleu-cine 4 labelled in one of the two diastereotopic CH,OH groups. Thus synthesis of a sample of this compound labelled in an unambiguously defined manner and comparison of its 13C NMR spectrum with that of an enzymically derived sample would define the stereochemistry of the enzymic reaction. Since deuterium labelling will allow the resonances in the 13C NMR spectrum to be defined, we opted to synthesise samples of 5,5'-dihydroxyleucine 4 labelled with deuterium specifically in one of the diastereotopic CH20H groups so that the synthesis would define the chirality of the labelled compound.For our first synthesis of stereospecifically labelled dihydroxyleucine 4, we chose (2S)-pyroglutamic acid 7 as a chiral template. We had already prepared the enaminone 9 l2 from this commercially available amido acid and had used it in our synthesis of stereospecifically labelled leucine, l2 and in the synthesis of various non-proteinogenic amino acids l3-l5 and glutamate antagonists. l6 We expected that hydrolysis of the H,N$CO,H 1 5 6 *co, 2 3 11 4 Scheme 1 enaminone 9 to the aldehyde 10 might involve stereochemical control from the bulky ester group at C-2, and that reduction of the aldehyde 10 would lead to the required hydroxymethyl group as in compound 11 (Scheme 2). Hydrolytic ring opening would then afford the acid 12, R = Bu', and subsequent reduction of the carboxyl group in this compound with deuteriated reagents would lead to the second, labelled hydroxymethyl group of the target compound. Hydrolysis of the ester at C-2 of the pyroglutamate derivative 11 during the ring opening process might lead to problems of regioselectivity in reactions of the diacid 12, R = H, but we expected to avoid this problem by using the tert-butyl ester in the synthesis.We therefore hydrolysed the enaminone 912 at pH 4.5 in methanolic HCl until the UV spectrum indicated that the chromophore due to the enaminone at A,,, 313 nm had been J.Chem. Soc., Perkin Trans. 1, 1996 1131 8 9 I 11 10 H CH2OH bu0,C NHpcozH ROamp; 12 Scheme 2 replaced by that due to the enol of aldehyde 10 at Amax 257 nm. We then added NaB(CN)H, to the solution in situ, keeping the pH constant at 4.5 to avoid deprotection. When the chromophore due to the aldehyde 10 was no longer present in the UV spectrum, the product was isolated. The product was evidently a mixture of the trans and cis alcohols 11 and 13 respectivcly in a ratio of 5 :2.rdquo; The isomers were separated and their stereochemistry was deduced by H NMR spectroscopic experiments. Irradiation of the absorbance at 6 2.47 corresponding to H-3S of the cis isomer 13 showed NOE at both 6 4.41 for H-2 and 6 2.76 for H-4.The trans isomer 11 showed separate enhancements in the lsquo;H NMR spectrum between H-4 at 6 2.85 and H-3R at 6 2.10, and between H-2 at 6 4.46 and H-3S at 6 2.24. These results are in keeping with the thermodynamically more stable trans diastereoisomer of the aldehyde 10 being formed in the hydrolysis step. A small amount of the unsubstituted compound 8 was also obtained from this reaction, presumably resulting from retro-aldol reaction of the alcohols 11 and 13. 13 Although a good yield of the mixture was obtained, separation of the isomers in useful amounts proved an obstacle to progress and so we sought an alternative route. We were encouraged by an early report 18,19 that hydroboronation of 4-methylideneproline derivatives with disiamylborane followed by oxidation with aqueous hydrogen peroxide gave cis-4-hydroxymethylproline derivatives in good yield.(2s)-N-tert-Butoxycarbonyl-4-methylideneproline14 was therefore prepared by the method of Herdewijn et d2rsquo; from (2S,4R)-4-hydroxyproline via the protected 4-ketone and Wittig reaction. This compound was crystalline rather than an oil as reported.20 It was converted into the tert-butyl ester 15 in ca. 82 yield using either di-tert-butyl dicarbonate, DMAP and triethylamine or tert-butyl alcohol, DMAP and dicyclohexylcar- bodiimidc. Reaction with disiamylborane followed by oxidation using aqueous hydrogen peroxide gave the protected alcohol 16 (Scheme 3).The lsquo;H NMR spectrum was complicated by the 1132 J. Chem. SOC.,Perkin Trans. I, 1996 14 15 16 H, CH20TBDPS H, CH20TBDPS A H +-----kH 0 y c02rsquo;Bu y C0,tsu C0,rsquo;BU C0,lsquo;Bu 18 17 Scheme 3 well known 22 rotational isomerism of proline amides and urethanes and so an estimate of the stereoselectivity of the reaction could not be made by NMR spectroscopic methods. Fractional crystallisation from light petroleum gave a pure sample of the cis isomer, tert-butyl (2S,4S)-N-tert-butoxycarbonyl-4-hydroxymethylprolinate16 which showed a coalescence temperature of 333 K on variable temperature lsquo;H NMR spectroscopy in C2H6DMSO. To proceed to ring opened compounds, it was necessary to convert the proline derivative into a pyroglutamic acid derivative.This had been achieved for simpler compounds by oxidation with ruthenium tetraoxide 23 and we decided to protect the alcohol as the TBDPS derivative before attempting this step. Protection was achieved and the TBDPS ether 17 was obtained in 79 yield using tert-butyldiphenylsilyl chloride and imidazole in dimethylformamide. The protected compound was reacted with Ru0,-NaIO, to yield the corresponding pyroglutamic acid derivative in 39 yield together with a 20 yield of two products which were not fully characterised but the lsquo;H NMR spectra of which suggested that, although oxidation to a pyroglutamate had proceded normally, the protecting group had been modified. The pyroglutamic acid derivatives had simpler NMR spectra than the corresponding proline derivatives and so analysis of the spectra of the oxidised products allowed us to show that the hydroboronation step had given a mixture containing ca.SO of the cis isomer 16 and ca. 20 of the corresponding trans isomer. The stereochemistry of the products was confirmed by NOE studies in the lsquo;H NMR spectra of the products 18 and 19. Interestingly, when the hydroboronation step was accomplished using BH,-Me,amp; the ratio of the isomers 18:19 in the final product was ca. 3:2. Most usefully, we now found that the tert-butyldiphenylsilyl ethers, unlike the corresponding alcohols, could be separated chromatographically in excellent yield. We therefore converted the alcohols 11 and 13 obtained by reduction of the aldehydes 10 into the tert-butyldiphenylsilyl ethers which separated chromatographically giving a 34 yield of the trans isomer 19 and a 12 yield of the cis isomer 18.The route from pyroglutamic acid therefore gave predominantly the trans isomer 19 whilst the route from 4-hydroxyproline gave predominantly the cis isomer 18. The remainder of the synthesis now involved ring opening and further elaboration of the molecule to obtain separate samples of the target compounds (2S,4S)-and (2S,4R)-5,5lsquo;- dihydro~y5,5-~H,leucineso that the rsquo;3C NMR spectrum could be assigned for studies on the enzyme glutamate y-carboxylase. Hydrolytic ring opening of the 4-methylpyroglut- amate 20 using aqueous LiOH in tetrahydrofuran had proved successful in other work in our laboratory l2 but this reaction could not be successfully applied to the TBDPS ether 19.We therefore investigated ring opening of the silyl ether 19 using methanol containing a catalytic quantity of triethylamine. The TBDPSOCH2 H FH2OTBDPS H zCHzOTBDPS H02Cc0zbu -H02HzCQHN C02rsquo;Bu C0,LBU kO2tsU COzbU - 19 20 24 26 21 22 H FHZOTBDPS bu02CN H g c S HOZCQ HN C0;Bu tsu0,c c0,bu 23 24 25 product was evidently a mixture of the diastereoisomers 21 and 22 in a ratio of 4 : I and so epimerisation had occurred either at C-4 or at C-2 under the conditions used for ring opening. In order to define the centre at which epimerisation had occurred, the reaction was repeated using methan2H,ol. The 2HNMR spectrum of the crude product showed a single resonance at 6 2.73 which corresponded to incorporation of the label at C-4 and there was no labelling at C-2.Epimerisation had therefore occurred solely at C-4. When the unprotected alcohol 11 was reacted with aqueous lithium hydroxide in tetrahydrofuran, then ring opening proceeded normally without epimerisation to yield the substituted glutamic acid 23. When the silyl ether 19 was used and the solvent was changed to tetrahydrofuran or acetonitrile, then ring opening using either aqueous LiOH or KOH gave a partially purified compound from the alkali soluble fraction. The lsquo;H NMR spectrum of this crude product indicated the presence not only of the desired product 24 in less than 20 yield but also of the olefin 25 resulting from elimination of the tert-butyldiphenylsilyl ether group, with olefinic singlets at 6 5.81 and 6.44. We evidently needed to alter the ratio of basicity to nucleophilicity in our reagent for the ring opening reaction to occur, and so lithium hydroperoxide in aqueous tetrahydrofuran was chosen to effect this reaction.With the trans lactam 19, this gave a clean product 24 in 57 yield. To prepare our target (2S,4S)-5,5rsquo;-dihydro~y5,5-~H,lleu-cine 4a, we now proceeded as outlined in Scheme 4. The acid 24 was first converted into the mixed anhydride with isobutyl chloroformate and this was reduced in situ with NaB2H, in 2H20 to give the labelled alcohol 26 in 68 yield after purification.Deprotection was now effected in two stages, first reacting the compound 26 with ammonium fluoride in methanol to obtain the diol27 in 72 yield. The final hydrolysis of the diol 27 using trifluoroacetic acid was complicated by cyclisation of the product to diastereoisomeric lactones 28 and 29 but these could be hydrolysed to the sodium salt of the acid 4a with sodium hydroxide. Use of NaBH, in the synthesis gave unlabelled 5,Srsquo;-dihydroxyleucine 4, the lsquo;H and 13C NMR 4a 27 Scheme 4 spectra of which were in keeping with those reported24 for a sample obtained by an alternative synthesis. The diastereoiso- merically labelled alcohol 4b was prepared by the same route, as shown in Scheme 5, using the lactam 18 as starting material.28 29 TBDPSOCHZ 0 18 30 32 31 HOCHZ H H02H2C3c02H 4b Scheme 5 The I3C NMR spectra of the products in Na02H-2H20 are shown in Fig. 1. The proximity of the 13C shifts of the hydroxymethylene groups, and the deuterium isotope shift, caused overlap of the C2H20H with the CH20H resonance in the spectrum of the (2S,4R)-isomer 4b as shown in Fig. l(a) but the spectrum could be assigned by addition of unlabelled diol4 to the sample, as in Fig. l(c). The spectrum of the (2S,4S)-isomer 4a, shown in Fig. l(b), was unambiguous. From the J. Chem. SOC.,Perkin Trans. 1,1996 1133 I I I I60 50 40 8, /PPm Fig. 1 Broad band 'H-decoupled ''C NMR spectra in 10 Na02H-2H,0 of: (a) (2S,4R)-5,5'-dihydro~y5,5-~H~leucine4b, (b) (2S,4S)-5,5'-dihydro~y5,5-~H~Ieucine4a and (c) 4b mixed with unlabelled 5,5'-dihydroxyleucine4 spectra, it is evident that the higher field absorption can be assigned to the 4-pr0-S hydroxymethyl group and the lower field absorption to the 4-pro-R hydroxymethyl group.Experimenta1 Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. Optical rotations (given in units of 10 deg cm g ') were measured on a Perkin Elmer PE241 polarimeter, using a 1 dm path length micro cell. IR spectra were recorded on a Perkin Elmer 1720 Fourier transform instrument, and UV spectra on a Phillips PUS720 UVjVIS scanning spectrophotometer. 'H NMR spectra were recorded on Bruker WM 360 (360 MHz) and AMX 500 (500 MHz) Fourier transform instruments.J Values are given in Hz. I3C NMR spectra (broad band 'H decoupled) were recorded on Bruker WM 360 (90.6 MHz), AMX 500 (125.8 MHz) and 1134 J. Chem. Soc., Perkin Trans. I, 1996 AC-P 250 (62.9 MHz) Fourier transform instruments. INEPT experiments were used to help assign 13C NMR resonances where necessary. ,H NMR spectra were recorded on a Bruker AC-P 250 (38.4 MHz) Fourier transform instrument. Unless otherwise stated, residual solvent peaks were used as an internal reference in the NMR spectra. Mass spectra were recorded on Kratos MSSORF, MS50 and MS25 and Fisons/VG Autospec spectrometers and the accurate mass measurements were recorded on a Fisons VG Autospec by Dr A. Abdul-Sada. Microanalyses were performed by Mrs P.Firmin (Wellcome Research Laboratories), and Miss K. Plowman and Miss M. Patel (Sussex). Thin layer chromatography was performed using Merck Kieselgel 60 F254 pre-coated silica gel plates of thickness 0.2 mm (ART 5554) and column chromatography was performed using Merck Kieselgel 60 (230400 mesh, ART 9385). tevt-Butyl(2S,4RS)-N-tevt-butoxycarbonyl-4-hydroxymethyl-pyroglutamate, 11 and 13 tert-Butyl (2S)-N-tert-butoxycarbonyl-4-(N,N-dimethylamino-methylidene)pyroglutamate, 9 (6.0 g, 18 mmol) was dissolved in methanol (140 cm3) and aq. hydrochloric acid (0.2 mol dm 3; 90 cm3) was added dropwise with stirring at room temper-ature. TLC analysis showed complete hydrolysis of the enaminone 9 to the enol of the aldehyde 10 within 45 min.Sodium cyanoborohydride (2.22 g, 35 mmol) was added in portions while maintaining the pH at 4.04.5 by automated addition of aq. hydrochloric acid (0.2 mol dm 3, using a pH- stat. The reaction mixture was stirred for 48 h at room temperature to afford a pale green solution. The solvent was removed in uucuo to afford an aqueous layer to which ethyl acetate (100 cm3) and saturated aq. sodium chloride (100 cm3) were added. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3 x 80 cm3). The organic layers were combined, washed with saturated aq. sodium chloride (50 cm3), 10 aq. citric acid (50 cm3) and water (50 cm3) and dried (Na,S04). The solvent was removed in uaeuo to afford a pale yellow foam (4.94 g, -86 crude recovery) which contained the trans and cis alcohols, 11 and 13 in a ratio of 5:2.The crude foam was purified by column chromatography on silica gel, using ethyl acetate-light petroleum (2 : 1, 40-60 "C) as eluent, to afford fractions of the diastereoisomeric alcohols in various ratios ( -46 yield). Column fractions containing 80-90 of the trans isomer and 80-90 of the cis isomer respectively were recrystallised from ethyl acetate-light petroleum (60-80 "C) to yield pure samples. The major product was tert-butyl (2S,4S)-N-tert-butoxy-carbonyl-4-hydroxymethylpyroglutamate11 (444 mg), mp 100- 102"C, aJi3 -19.2 (c 1.97 in CHCI,) (Found: C, 57.0; H, 8.1; N, 3.9. C,5H25N06 requires C, 57.1; H, 7.9; N, 4.4); m/z +ve FAB (NBA) 316 M + HI'; v,,,(KBr)/cm ' 3497 (OH), 1772 (imide) and 1740 (ester); 6,(360 MHz, C2HC1,-5 'H2O) 4.46 (1 H, dd, J,,,s 9.6, J2,3R 1.2, H-2), 3.94 (1 H, dd, J~A,~B 4.1, CHOH), 3.70 (I H, dd, J~R,~A11.3, J6~,4 11.3,J6,,45.6,CHOH),2.85(1 H,m,J4,3s 11.7,J4,3~9.0,J4,6~ 4.1,H-4),2.24(1H,ddd,J,s,,~13.2,J,S,29.6,J3S.411.7,H-3S), 2.10(1H,ddd,J3,,3sl3.2,J,R,2 1.5,J3~,49.0,H-3R),1,509H, s, OC(CH,), and 1.48 9 H, s, OC(CH,),; 6,(125.8 MHz, C2HC13) 174.8 and 170.2 (C=O), 149.2 (urethane), 83.6 OC(CH3)3, 82.4 OC(CH,)3, 61.5 (CHZOH), 58.1 (C-2), 43.9 (C-4), 28.0 C(CH,), and 24.8 (C-3).The minor component was tert-but~l(2S,4R)-N-tert-butoxycurbonyl-4-hydroxymethyl-pyroglutamate 13 (92 mg), mp 102-103 OC, a;2 -74 (c 0.73 in CHCI,) (Found: C, 57.3; H, 8.2; N, 4.2.C,,H2,N06 requires C, 57.1; H, 7.9; N, 4.4); m/z +ve FAB (NBA) 316 M + HI+; v,,,(KBr)/cm-' 3479 (OH), 1772 and 1705 (imide); 6,(360 MHz, C2HCl,-5 2H,0) 4.41 (1 H, dd, J2.3s9.3, J2.3, 6.3, H-2), 3.83 (1 H, dd, J~A,~B11.2, JbA.4 5.2, CHOH), 3.73 (1 H, dd, JbB,bA 11.2, J6B,46.1, CHOH), 2.76 (1 H, m, J4,3s7.5, J4.3~9.4, H-4), 2.47 (I H, dtd, J3S.3R13.4, J3s.4 7.5, JSS.2 9.3, H- 3S), 1.86 (1 H, dtd, J~R,~s13.4, J3,,4 9.4, J3~,26.3, H-3R), 1.48 9 H, s, OC(CH,), and 1.45 9 H, s, OC(CH,),; 6,(125.8 MHz, C2HC1,) 175.0 and 170.4 (C==O),149.3 (urethane), 83.7 OC(CH,),, 82.4 OC(CH,),, 62.3 (CH,O), 58.3 (C-2), 44.8 (C-4), 27.9 C( CH,), and 24.1 (C-3). tert-Butyl (2S)-N-tert- butoxycurbonylpyroglutamute8 was also obtained as a colour- less oil during column chromatography (870 mg, 17) and had a 'H NMR spectrum identical with that of an authentic sarnple.l2 (2S)-N-tevt-Butoxycarbonyl-4-methylideneproline14 Prepared using the method of Herdewijn et al.2o but proved to be a crystalline solid and not an oil as reported. The product was recrystallised from diethyl ether in 83 yield, mp 108-1 10 "C, Calk4 -55.3 (c 0.52 in CHCl,) (Found: C, 58.0; H, 7.4; N, 6.0. C,,H,,N04 requires C, 58.1; H, 7.5; N, 6.2);m/z +ve FAB, NBA 228 M + HI+; vmaX(KBr)/cm-' 1742 (urethane); 6,(500 MHz, C2HCl,, two rotational isomers) 9.00 (1 H, br s, COOH), 5.02 (2 H, s, olefinics), 4.52 and 4.40 (1 H, 2dd, J2,3s2.2, J2,3R 9.4, H-2), 4.08 (s, one rotational isomer) and 4.05 and 4.01 (2 x AB, JAB15.0, other rotational isomer) (total 2 H, H-5), 3.01 and 2.93 (1 H, 2 x dd, J3R.2 9.4, J,,,,, 16.3, H-3R), 2.78 and 2.69 (I H, 2 x br d, J,,,, 2.2, J,,,,, 16.3, H-3S) and 1.42 and 1.47 9 H, 2 x s, (CH,),C; 6,(125.8 MHz, C2HCl,, two rotational isomers) 178.01 and 176.02 (C=O), 155.37 and 153.83 (urethane), 142.80 and 142.04 (C-4), 108.15 (=CH,), 81.12 and 80.66 OC(CH,),, 58.83 and 58.62 (C-2), 50.89 and 50.50 (C-5),36.63 and 35.29 (C-3) and 28.34 and 28.22 C(CH,),.tert-Butyl(2S)-N-tert-butoxycarbonyl-4-methylideneprolinate Method A. tert-Butyl alcohol (0.30 cm3, 3 mmol), DMAP (0.013 g, 0.1 mmol) and dicyclohexylcarbodiimide (230 mg, 1.1 mmol) were added successively at 0 "C to (29-N-te1-t- butoxycarbonyl-4-methylideneproline14 (0.227 g, 1 mmol) dissolved in dry dichloromethane (10 cm3).The mixture was stirred overnight at room temperature and filtered. The filtrate was washed with 0.05 aq. citric acid (10 cm3) and water (10 cm3) and dried (Na2S04). The solvent was removed in uuc'uu and the residue was chromatographed on silica gel using dichloromethane-diethyl ether (13 :1) as eluent, to yield tert- butyl(2S)-N-tert-butoxycarbonyl-4-methylideneprolinute15 as a colourless oil (230 mg, 82), a;' -20.9 (c 1.01 in CHCl,); (Found: C, 63.7; H, 9.6; N, 5.0. C,sH2sN04 requires C, 63.6; H, 8.9; N, 4.9); m/z FAB 284 M + H +;v,,,(film)/cm~' I739 (ester) and 1692 (urethane); 6,(360 MHz, C2HC13; two rotational isomers) 5.01 and 4.98 (2 H, 2 x br s, olefinic), 4.37 and4.27(1 H,2 x dd, J2.3s2.5, J2,3,9.5,H-2),4.07and4.02(2 H, 2 X br S, H-5), 2.97 and 2.91 (1 H, 2 X m, J3~,29.5, J3,,3, tetrahydrofuran (2 rnol drn-,; 3 cm3, 6 mmol) and B2H, in tetrahydrofuran (1 rnol dm-,; 3 cm3, 3 mmol) were added dropwise over a period of 5 min at -5 "C to a 100 cm3 three- necked flask under a slight positive pressure of nitrogen.After stirring for 2 h at OOC, a solution of tert-butyl (29-N-tert- butoxycarbonyl-4-methylideneprolinate15 (283 mg, 1 mmol) in dry tetrahydrofuran (5 cm3) was added dropwise over a period of 5 min. The solution was stirred for 24 h at 20deg;C and the excess of disiamylborane was destroyed by addition of water (0.5 cm')). When hydrogen was no longer evolved at 0 "C, the reaction was oxidised by addition of aq.NaOH (3 rnol dm ,; 0.35 cm3, 1 mmol) followed by aq. H202 (30; 0.35 cm3, 5.25 mmol). After stirring for 30 min at 20 "C, water (20 cm3) was added and the solution was extracted with diethyl ether. The organic layer was washed with water and dried (Na2S04) and the solvent was removed in vacuo to give a colourless oil (555 mg). Purification by chromatography on silica gel using diethyl ether as eluent afforded tert-butyl(2S)-N-tert-butoxycarbony/-4-hydroxymethylprolinute 16 (1 57 mg, 52). This was used in the next step without further purification but the cis isomer, tert-butyl (2S,4S)-N-tert-butoxycarbonyl-4-hydroxymethylpro-linate 16 could be obtained by fractional crystallisation using light petroleum (40-60 "C) and was isolated as white crystals, mp 40.5-41.5 "C, a;' -59.2 (c 0.515 in CHC1,) (Found: C, 58.0; H, 9.1; N, 4.5.C,,H27N0s requires C, 59.8; H, 9.0; N, 4.65);m/z +ve FAB, NBS 302 M + H +;v,,,(KBr)/cm-' 1744 (ester) and 1672 (carbamate);6,(360 MHz, C2H,COC2H,, two rotational isomers) 4.08 (m, H-2 of one rotational isomer), 3.78 (t, J2.3 5.3, H-2 of other rotational isomer), 3.61 (I H, dd, J'R,~7.8, J,R,5s 10.4, H-5R), 3.52 (2 H, t, J6.4 6.0, CHZOH), 3.14and3.10(1 H,2 x t,J,s,,R;,s,49.4,H-5S),2.81and2.78(1 H, 2 x s, OH), 2.36 (2 H, m, H-3), 1.66 (1 H, m, H-4) and 1.42 and 1.39 18H,2 x s,C(CH,),. The temperature ofcoalescence of the two rotational isomers was found to be 333 K in DMSO by variable temperature 'H NMR spectroscopy. Method amp;using borane dimethylsulfide.Borane dimethyl- sulfide in tetrahydrofuran (2 rnol dm-,; 3 cm3, 6 mmol) was added dropwise to tert-butyl (2S)-N-tert-butoxycarbonyl-4-methylideneprolinate 15 (556 mg, 2 mmol) in dry tetrahydro- furan (10 cm3) over a period of 8 min at 0 "C under a positive pressure of nitrogen. The solution was stirred for 20 h at 20 "C. After cooling at 0 "C,ethanol (1 cm3) was added dropwise and the solution was stirred for 1 h until there was no further effervescence of hydrogen. Aq. sodium hydroxide (3 rnol drn-,; 0.7 cm3, 2 mmol) was added, followed by aq. H202(30; 0.7 cm3, 10.5 mmol) and the solution was stirred for 2 h at 20 "C. After addition of water (50 cm3), the mixture was extracted with ethyl acetate. The organic layer was washed with brine and 16.3,H-3R),2.56(1H,brd,J,s,22.5,J,s,,,16.3,H-3S)and1.47,dried (Na2S04).The solvent was removed in uacuo and the 1.45 and 1.44 18 H, 3 x s, C(CH,),; 6,(62.9 MHz, C2HC1,, two rotational isomers) 171.76 (C=O), 143.87 (urethane), 142.74 (C-4), 107.63 and 107.37 (=CH2), 81.22 OC(CH,,, 79.93 and 79.81 OC(CH,),, 59.66and 59.37 (C-2), 50.78 and 50.57 (C-5), 36.89 and 36.14 (C-3) and 28.38,28.30 and 27.92 C(CH,),. Method B. Di-tert-butyl dicarbonate (330 mg, 1.5 mmol), triethylamine (0.21 cm3, 1.5 mmol) and DMAP (13 mg, 0.1 mmol) were added successively at 0 "C to (29-N-tert-butyloxycarbonyl-4-methylideneproline14 (227 mg, 1 mmol) dissolved in acetonitrile (10 cm3, dried over CaH,) and the solution was stirred overnight at room temperature.The solvent was removed in uucuo and the residue was purified by chromatography on silica gel using light petroleum (60-80 "C)-ethyl acetate (4: 1) as eluent. The product tert-butyl(2S)-N-tert- butoxycarbonyl-4-methylideneprolinate15 was a colourless oil (238 mg, 84) with spectra identical to those of the sample prepared by method A. tert-Butyl(2S)-N-tert-butoxycarbonyl-4-hydroxymethyl-proiinate 16 Method A-using disiamylborane. 2-Methylbut-2-ene in residue was purified by chromatography on silica gel using light petroleum (60-80 "C)-ethyl acetate (1 :1) as eluent to yield tert- butyl (2S)-N-tert-buto.xycarbonyl-4-hydro.~ymethylprolinute 16 (341 mg, 57) with the same spectroscopic properties as the sample obtained by method A.tert-Butyl(2S)-N-tert-butoxycarbonyl-4-tert-butyldiphenyl-silyloxymethylprolinate 17 Dry dimethylformamide (1 cm3), imidazole (200 mg, 2.9 mmol) and tert-butyldiphenylsilyl chloride (0.4 cm3, 1.5 mmol) were added to the mixed diastereoisomers of tert-butyl (259-N-tert- butoxycarbonyl-4-hydroxymethylprolinate16 (345 mg, 1.14 mmol) at 0deg;C. The solution was stirred for 48 h at room temperature under nitrogen and, after addition of ethyl acetate (100 cm3), was washed with brine, 10 aq. citric acid and brine and dried (Na2S04). The solvent was removed in vacuo and the residue was purified by chromatography on silica gel using light petroleum (60-80 "C)Aichloromethane (3 :7) as eluent.The first fraction (Ph,Bu'SiOH) was isolated as white crystals and the second fraction afforded tert-butyl (2S)-N-tert- butoxycarbonyl-4-tert-butyldiphenylsilyloxymethylprolinate 17 J. Chem. Soc., Perkin Trans. I, I996 1135 as a colourless oil (486 mg, 79). Use of the pure (2S,4S)- alcohol 16 in the reaction gave tert-butyf (2S,4S)-N-tert- butoxycarbonyf-4-tert-butyldiphenyfsifyfoxymethyfprofinate17 as an oil, -35.8 (c 0.355 in CHCl,) (Found: C, 67.8; H, 8.2; N, 2.2. C,,H,,NO,Si requires C, 69.0; H, 8.4; N, 2.6); v,,,(film)/cm~' 1743 (ester) and 1704 (carbamate); 6,(500 MHz, C2HC13, two rotational isomers) 7.64 and 7.36 (10 H, 2 x m, aromatics), 4.17 and 4.12 (1 H, 2 x t, J2.3 7.9, H-2), 3.79 and 3.70 (1 H, 2 x dd, J5R,47.2, J5R,5, 10.5, H-5R), 3.64 (2 H, m, CH,OSi), 3.24 and 3.17 (1 H, 2 x dd, J,,,, 8.9, J,,,,, 10.5, H- 5S),2.41 (1 H,m, H-3S),2.34(1 H,m, H-3R), 1.76(1 H,m,H- 4), 1.47 and 1.45 and 1.45 and 1.44 18 H, 4 x s, (CH,),C, 1.06 and I .05 2 x s, 9 H, (CH,),CSi; 6,( 125.8 MHz, C2HCl,) 172.26 (M),153.90 (urethane), 135.52-127.69 (aromatics), 80.84 (CH,),CO, 79.79 and 79.50 (CH,),CO, 64.83 and 64.59 (CH20Si), 59.70 (C-2), 49.41 and 49.27 (C-5), 40.86 and 40.04 (C-4), 33.11 and 32.20 (C-3), 28.42,28.36,27.99 and 27.92 C(CH,),, 26.80 (CH,),CSi and 19.22 (CH,),CSi. Irradiation of 6 1.76 (H-4) gave a 21 NOE at 6 2.34 (H-3R), 0.7 at 6 3.24 (H-553and 1.5 at 6 3.64 (CH,O).Irradiation of 6 4.15 (H-2) gave a 1 NOE at 6 2.41 (H-3S) and 6 at 6 2.34 (H-3R).tert-Butyl(2S)-N-tert-butoxycarbonyl-4-tevt-butyldiphenyl-silyloxymethylpyroglutamate18 Method A, from the proline route. A solution of tert-butyl (2S)-N-tert-butoxycarbon yl-4-tert-butyldiphenylsilyloxymeth-ylprolinate 17 (602 mg, 1.12 mmol) in ethyl acetate (12 cm3) was added to a mixture of RuO2.H2O (60 mg, 0.45 mmol) and aq. NaIO, (10; 24 cm3). The solution was stirred vigorously overnight at room temperature. The layers were separated and the organic solution was treated with propan-2-01 (I cm3). Ethyl acetate (80 cm3) was added and the solution was washed with brine and dried (Na,SO,). The solvent was removed in vucuo and the residue was purified by column chromatography on silica gel using light petroleum (60-80 OC)-ethyl acetate (17: 3) as eluent.Starting material (48 mg) was recovered, followed by tert-butyl (2S)-N-tert-butoxycarhonyf-4-tert-butyf-diphenylsilyfoxymethyfpyroglutamate 18and 19 (22 1 mg, 39) as two separate diastereoisomers. The trans isomer, tert-butyf (2S,4S)-N-tert-butoxycarbonyl-4-tert-butyfdiphenyfsifyfoxy-methyfpyrogfutarnate19 was recrystallised from light petroleum (40-60deg;C) as white crystals (88 mg, 14), mp 97-99deg;C; -36.7 (c 0.4 in CHCl,) (Found: C, 67.1; H, 7.9; N, 2.4. C31H4,N06Si requires C, 67.2; H, 7.8; N, 2.5); m/z EI 496 M -C4H9+; v,,,(KBr)/cm-l 1754 and 1714 (imide) and 1732 (ester); SH(360 MHz, C2HCl,) 7.70-7.30 (10 H, m, aromatics), 4.50 (1 H, dd, J2,3s 9.7, J2.3, 2.9, H-2), 4.05 (1 H-4), 2.46 (1 H, dt, J3~,4;3~.29, J3,,3, 13.2, H-3R), 2.13 (I H, ddd, J,,., 7.3, J,,,, 8.4, J,,,,, 13.2, H-33, 1.51 and 1.46 El8 H, 2 x s, (CH,),C and 1.05 9 H, s, (CH,),C; 6,(125.8 MHz, C2HCl,) 173.03 and 170.63 (C=O), 149.74 (urethane), 135.9- 127.9 (aromatics), 83.62 (CH,),CO, 82.29 (CH,),CO, 62.78 (CH,OSi), 58.35 (C-2), 45.80 (C-4), 28.15 and 27.70 (CH,),C, 24.70 (C-3) and 19.53 (CH,),CSi.Two other products (77 mg) eluted from the column. The first showed 6,(360 MHz, C'HCl,) 7.61 and 7.31 (5 H, 2 x m, aromatics), 4.41 (I H, dd, J2,3A 2.2, J,,3~9.8, H-2), 4.25 (I H, dd, J6~,43.5, JAB10.8, CHOSi), 3.87 (1 H, dd, J6B,43.5, JAB 10.8, CHOSi), 3.3 (1 H, br, OH), 2.81 (1 H, m, H-4), 2.4 (1 H, m, H-3A), 2.07 (1 H, m, H-3B) and 1.48, 1.45 and 0.95 27 H, 3 x s, C(CH,),.Method B, from the pyroglutamate route. The diastereoiso- meric mixture of tert-butyl(2S,4RS)-N-tert-butoxycarbonyl-4-hydroxymethylpyroglutamate 11 and 13 (8.61 g, 27.3 mmol) was dissolved in dichloromethane (1 75 cm3) and DMAP (700 mg, 5.7 mmol) and triethylamine (9.7 cm3) were added at 0 "C with stirring. After 15 min, tert-butylchlorodiphenylsilane(8.52 cm3, 32.8 mmol) was added under nitrogen and the reaction mixture was stirred for 2 days. The mixture was concentrated in uacuo and dichloromethane (21 5 cm3) was added. The solution was washed with aq. hydrochloric acid (0.05 mol drn-,; 80 cm3). The aqueous layer was extracted with dichloromethane (3 x 200 cm3) and the organic layers were washed with water (250 cm3) and dried (Na,SO,).The solvent was removed in uucuo to yield a pale orange oil which was purified by chromatography on silica gel using light petroleum (40-60 "C)- ethyl acetate (17 :3) as eluent. The major component, tert-butyl (2S,4S)-N-tert-butoxycarbonyf-4-tert-butyfdiphenylsifyfoxy-methyfpyrogfutarnate 19was a solid, (5.15 g, 34) mp 88-89 "C, with spectra identical with those of the sample independently prepared by method A above. The minor component, tert-butyf (2S,4R)-N-tert-butoxycarbonyl-4-tert-butyfdiphenyfsifoxymeth-yfpyrogfutamate 18was a solid, mp 96-98 "C, (1.8 g, 12) with spectra identical to those of the sample prepared by method A above. 1-tevt-Butyl5-methyl(2S,4RS)-N-tert-butoxycarbonyl-4-tert-butyldiphenylsilyloxymethylglutamate21 and 22 tert-Butyl(2S,4S)-N-tert-butoxycarbonyl-4-tert-butyldiphenyl-silyloxymethylpyroglutamate 19 (150 mg, 0.271 mmol) was dissolved in methanol (10 cm3) and triethylamine (4.10 mg, 0.041 mmol) was added.The reaction was stirred for 6 days at room temperature and the solvents were removed in V~CUOto afford a pale yellow oil which was purified by column chromatography on silica gel, using diethyl ether- light petroleum (40-60 "C) as eluent, to yield three separate H, dd, J~A,~B compounds as colourless oils. The major component was a 4 : 110.2, J6~.4 4.7, CHOSi), 3.75 (1 H, dd, J~B,~A 10.2, J6~,43.4, CHOSi), 2.77 (1 H, m, J4,6~4.7, J4.6~3.4, J4.3~ 9.3, J4,3s 9.7, H-4), 2.42 (1 H, ddd, J,,,,, 13.2, J3,,2 9.7, J3s.4 9.7, H-39, 2.10 (1 H, ddd, J,,,,, 13.2, J3R,2 2.9, J3,,4 9.3, H-3R), 1.51 9 H, s, OC(CH3),, 1.48 9 H, s, OC(CH3), and 1.03 9 H, s, SiC(CH,),; amp;(I253 MHz, C2HCl,) 173.4 and 170.6 (C=O), 149.3 (urethane), 127.7-1 35.7 (aromatics), 83.2 and 82.2 OC(CH,),, 62.7 (CH20Si), 58.2 (C-2), 44.4 (C-4), 27.9 and 26.7 C(CH,),, 25.2 (C-3) and 19.2 SiC(CH,),.Irradiation at the resonance at 6 2.42 (H-39 resulted in a 10 NOE at 6 4.50 (H-2), whereas irradiation at 6 2.10 (H-3R) resulted in a 17 NOE at 6 2.77 (H-4). The cis isomer, tert-butyf (2S,4R)-N-tert-hutyfoxycarbonyf-4-tert-butyfdiphenyfsifyfoxy-rnethylpyrogfutamate 18 was obtained as white crystals, (1 33 mg, 2179, mp 100-102"C, -12.2 (c 0.43 in CHCI,); m/z EI 496.21 6 462, C,,H3,No6Si requires 496.21 5 542 M -Bur+; m/z +ve FAB, NBA 576 M + Na+ and 554 M + H +;v,,,(KBr)/cm-' 1789 (imide), 1743 (ester) and 1722 (urethane); 6,(360 MHz, C2HCl,) 7.65 and 7.40 (10 H, 2 x m, aromatics), 4.40 (1 H, dd, J2,3s 7.3, J2,3R 9, H-2), 3.93 (1 H, dd, J6B,46.5, JAB 10.3, CHOSi), 3.89 (1 H, dd, J6A,44.7, JAB 10.3, CHOSi), 2.77 (1 H, dddd, J6A,4 6.5, J4,,,8.4, J4,3R4.7 J6B.4 9, 1136 J.Chem. SOC.,Perkin Trans. 1,1996 mixture of the diastereoisomeric esters 21 and 22 (114 mg, 72) (Found: C, 65.4; H, 8.2; N, 2.1. C,,H,,NO,Si requires C, 65.6; H, 8.0; N, 2.4); m/z EI 528 M -C4H9+; vmaX(KBr)/cm-' 171 8 (ester); 6,(500 MHz, C'HCl,) 7.80-7.30 (10 H, m, aromatics), 5.05 and 5.00 I H, 2 x br m, (2S,4R)- and (2S,4S)-NH, 4.25 I H, m, (2S,4RS)-2-CH, 3.90-3.75 2 H, m, (2S,4RS)-CH20Si, 3.71 and 3.68 3 H, 2 x s, (2S,4S)-and (2S,4R)-OCH3, 2.71 l H, m, (2S,4RS)-H-4, 2.35 C0.8 H, dtd, (2S,4S)-H-3A, 2.15 and 2.00 C0.4 H, 2 x m, (2S,4R)-H-3A and H-3B, 1.75 C0.8 H, dtd, (2S,4S)-H-3B, 1.40-1.50 18 H, overlapping singlets, (2S,4RS)-0C(CH3), and I .08 and 1.05 9 H, (2S,4RS)-SiC(CH3),; selective irradiation of the multiplet at 6 2.71 (H-4) led to simplification of the C-3 protons of the major (2S,4S) diastereoisomer 21 (6 2.35, dtd and 6 1.75, dtd) to doublets of doublets, and of the multiplet at 6 3.80 (CH,O); 6,( 125.8 MHz, C2HCl,, major diastereoisomer only- minor diastereoisomer not observed above the background noise) 174.0 and 171.5 (C=O), 155.2 (urethane), 140.0-130.0 (aromatics), 82.0 and 79.6 OC(CH,),, 64.8 (CH,OSi), 52.6 (C-2), 51.8 (OCH,), 44.6 (C-4), 31.3 (C-3), 28.3 and 28.0 C(CH,),, 26.7 SiC(CH,), and 19.2 SiC(CH,),.The minor component, tert-butyl (2S,4S)-4-tert-butyldiphenyl-silyloxymethylpyroglutamate was obtained as a colourless oil (18 mg, 15); -3.25 (c 2.18 in CHCI,) (Found: C, 68.3; H, 7.9; N, 3.2. C26H3 J04Si requires C, 68.8; H, 7.7; N, 3.1); m/z +ve CI (NH,) 454 M + HI'; v,,,(film)/cm-' 3250 (NH, br d), 1738 (ester) and 1708 (lactamj; 6,(360 MHz, C2HCI3) 7.69-7.35 (10 H, m, aromatics), 4.13 (1 H, dd, J2,3, 9.1, J2.3~4.4, H-2), 3.99 (1 H, dd, J~A,~B10.1, J6~,44.8, CHOSi), 3.80 (1 H, dd, J~B,~A 3.3, CHOSi), 2.62 10.1, J6~,4 (1 H, m, J4.3~9.2, J4,6A 4.8, J4.6~3.3, H-4), 2.53 (1 H, ddd, J3s.2 9.1, J3,,3R 13.1,J3s.4 6.9, H-3S), 2.31 (1 H, ddd, J3R,2 4.4, J,R,,s 13.1,J3~,49.2, H-3R), I .48 9 H, OC(CH,), and 1.04 9 H, SiC(CH,),; 6,(90.6 MHz, C2HCI,) 177.3 and 171.4 (C=O), 135.7-1 27.7 (aromatics), 82.3 OC(CH,j,, 63.3 (CH20Si), 54.8 (C-2), 42.9 (C-4), 28.2 (C-3), 28.0 OC(CH3)3, 26.9 SiC(CH,), and 19.3 SiC(CH,),.1-tevt-Butyl5-methyl(2S,4RS)-N-tevt-butoxycarbonyl-4-tevt-butyldiphenylsilyloxymethyl4-ZHJglutamate21 and 22 C(CH3),;6,(360 MHz, C2HCI,) showed the presence of a-NH doublet 6 5.50 (1 H, JNH,~7.3) which exchanged upon addition of 2H20, and a C-2 proton at 6 4.21; 6,(125.8 MHz, C2H,02H) 177.1 and 173.5 (C=O), 158.0 (urethane), 82.7 and 80.5 OC(CH3),, 64.3 (CH,OH), 54.4 (C-2j, 46.2 (C-4), 31.5 ((2-3) and 28.7 and 28.3 OC(CH,),.tevt-Butyl(2S,4R)-N-tevt-butoxycarbonyl-sl-tevt-butyldiphenyl-silyloxymethylglutamic acid 24 ter t- Bu tyl(2S,4S)-N-tert-bu toxycar bon y l-4-ter t- but yldip henyl- silyloxymethylpyroglutamate 19 (600 mg, 1.08 mmol) was dissolved in tetrahydrofuran (stabilised with BHT, 16.2 cm3) and water (5.4 cm3). The solution was treated at 0deg;C with hydrogen peroxide (60 w/v; 0.123 cm3, 3.69 mmol) followed by lithium hydroxide (91.2 mg, 2.17 mmol) and stirred at 0 "C for 15 min to give a purple solution which was stirred for a further 3.5 h at room temperature. The reaction was quenched at 0 "C by addition of aq.sodium sulfite (1.5 mol drnp3; 1.6cm3j over a period of 20 min. The organic solvent was removed in tert-Butyl(2S,4S)-N-tert-butoxycarbonyl-4-tert-butyldiphenyl-vacua to afford an aqueous layer which was acidified to pH 3 by silyloxymethylpyroglutamate 19 (35 mg, 0.063 mmol) was dissolved in O-2Hmethanol (3 cm3) with triethylamine (9.60 mg, 0.095 mmol). The reaction was stirred for 5 days at room temperature and the solvents were removed in vacuo to afford a colourless oil which was dissolved in ethyl acetate (30 cm3j. The organic layer was washed with aq. sodium chloride (10; 20 cm3) and the aqueous layer was extracted with ethyl acetate (2 x 30 cm3). The organic layers were combined and dried (Na,SO,). The solvent was removed in V~CUOto yield the product as a colourless oil (36 mg) which was shown to contain a mixture of products by 'H NMR spectroscopic comparison of the crude product with that of authentic samples.These were unchanged tert-butyl (2S,4S)-N-tert-butoxycarbonyl-4-tert-butyldiphenylsilyloxymethylpyroglutamate19 ( -8), tert-butyl (2S,4S)-4-tert-butyldiphenylsilyloxymethylpyro-glutamate ( -16) and 1-tert-butyl 5-methyl (2S,4RS)-N-tert-butoxycarbonyl-4-tert-butyldiphenyl~ilyloxymethyl4-~K-glutamate 21 and 22 in a 2.3: I ratio of diastereoisomers and -76 yield. From the difference in the integration of the C-4 and C-3 protons of the crude product, it was evident that -20 deuterium had been incorporated at the C-4 position; 6,(38.4 MHz, CHCI,) 7.26 (C2HCI,) and 2.73 (2H-4); 6,(38.4 MHz, CHCI, + 5 CH,02H) 7.26 (C2HCI,), 2.78 (2H-4) and 1.48 (CH,02H). 1-tevt-Butyl(2S,4S)-N-tert-butoxycarbonyl-4-hydroxymethyl-glutamic acid 23 tert-Butyl (2S,4S)-N-tert-butoxycarbonyl-4-hydroxymethyl-pyroglutamate 11 (176 mg, 0.56 mmol) was dissolved in tetrahydrofuran (7 cm3) at 0 "C and aq. lithium hydroxide (1 mol drn-,; 0.73 cm3) was added dropwise with vigorous stirring over a period of 5 min.Stirring was continued for a further 20 min at 0 "C,ethyl acetate (25 cm3) and aq. sodium chloride (10; 10 cm3) were added to the reaction mixture, and the organic layer was separated. The aqueous layer was carefully acidified to pH 4-4.5 at 0deg;C with stirring by the careful dropwise addition of 10aq. citric acid.The aqueous layer was extracted with ethyl acetate (3 x 25 cm3) and the organic layers were combined, washed with 10 aq. sodium chloride and dried (Na2S04). The solvent was removed in vacuo to afford a white foam (98 mg, 53) which was crystallised from ethyl acetate-light petroleum (60-80 "C) to yield I-tert-butyl(2S,4S)- N-tert-butoxycarbonyl-4-hydroxymethylglutamic acid 23 as a white solid (62 mg, 3373, mp 128-1 30 "C; Cali2 -32.3(c 0.44 in MeOH); m/z +ve FAB, NBA 334 M + HI'; v,,,(KBr)/ cm-' 3200-3400 (NH, br d) and 1719 (acid); 6,(360 MHz, C2H,02H) 4.02 (1 H, dd, J2,3A 10.3, J2,3B 4.4, H-2), 3.52 (2 H, m, CHZOH), 2.60 (1 H, m, H-4), 2.12 (I H, dtd, J~A,~B13.9, J3A.2 10.3,J3,,4 3.8, H-3A), 1.68 (I H, dtd, J~B,~A13.9,J3~,24.3, J3B,410.0, H-3B), 1.44 9 H, s, C(CH,), and 1.41 9 H, s, dropwise addition of aq.hydrochloric acid (0.2 mol drn-,) and extracted with ethyl acetate (3 x 60 cm3). The organic layers were combined and dried (Na2S04).The solvent was removed in U~CUOto yield tert-butyl (2S,4R)-N-tert-butoxycarbonyl-4-tert-butyldiphenylsilyloxymethylglutamic acid 24as a colourless oil (403 mgj which was crystallised from light petroleum (60-80 "C) (355 mg, 57); mp 125-127 "C; Cali5 -2.52 (c 0.5 in CHCI,) (Found: C, 64.4; H, 7.9; N, 2.7. C,,H,,NO,Si requires C, 65.1; H, 7.9 N, 2.45); m/z +ve FAB, NBA 572 M + HI'; v,,, 1730 (ester) and 1712 (acid); 6,(360 MHz, C2HCI,) 7.63 and 7.40 (10 H, 2 x m, aromatics), 5.35 (I H, d, JNH,27.7, NH), 4.25 (1 H, m, H-2), 3.90 (1 H, dd, J6~,46.31, J6*,6B 9.55, CHOSi), 3.73 (I H, dd, J6~,4 9.55, CHOSi), 2.71 (I6.33, J~B,~A H, m, H-4), 2.18 (1 H, ddd, J3~,210.33,J3As3B 10.9, H-3A), 1.78 (I H, ddd, J,A,~B10.9, J3~,211.4, H-3Bj, 1.45 I8 H, 2 X S, C(CH,), and 1.04 9 H, s, SiC(CH,),; 6,(125.8 MHz, C2HC13) 176.07 and 17 1.03 (C=O), 156.43 (urethane), 135.53-127.73 (aromatics), 82.61 and 80.68 OC(CH,j,, 64.34 (CH,O), 52.5 (C-2), 44.8 (C-4), 32.72 (C-3), 28.2 C(CH,),, 26.75 C(CH3)3, 26.54 SiC(CH,), and 19.22 SiC(CH,),.tevt-Butyl(2S,4S)-tevt-butoxycarbonylamino-5-hydroxy-4-te~t-butyldiphenylsilyloxymethyl 5,5-2H2pentanoate 26 tert-Butyl (2S,4R)-N-tert-butoxycarbonyl-4-tert-butyldiphenyl-silyloxymethylglutamic acid 24 (100 mg, 0.175 mmol) was dissolved in tetrahydrofuran (0.81cm3) and the solution was cooled to -40 "C.Triethylamine (0.032 cm3, 0.23 mmol) was added, followed by dropwise addition of isobutyl chloroform- ate (0.027 cm3, 0.2 mmol) under nitrogen. A white sediment was formed in the reaction which was stirred for 1.5 h at -40 "C. The mixture was filtered under nitrogen and a mixture of sodium borodeuteride (NaB2H4; 22 mg, 0.58 mmolj in tetrahydrofuran (0.55 cm3) and 2H20 (0.064 cm3) was added dropwise to the filtrate at 0 "C with stirring. Effervescence was observed and a white sediment was obtained in the reaction mixture. Stirring was continued at room temperature for 1 h. The reaction was cooled to 0 "C and ethyl acetate (1 cm3) and aq. sodium chloride (10; 0.3 cm3) were added.The organic layer was washed with ice-cold aq. citric acid (10; 0.5 cm3) and aq. sodium chloride (10; 0.5 cm3) and dried (Na2S04). The solvents were removed in vacuoto afford a white foam (1 30 mg). On addition of ethyl acetate, a polymer was formed and the solution was centrifuged and chromatographed on silica gel, using light petroleum (40-60 "C)-ethyl acetate (1 : 1) as eluent to yield tert-butyl (2S,4S)-2-tert-butoxycarbonylamino-5-hydroxy-4-tert-butyldiphenylsilyloxymethylpentanoate26 as a colourless oil (66.3 mg, 68) (Found: C, 67.0; H, 7.9; N, 2.5. C,,H4,2H2N06Si requires c, 66.5 H, 8.1; N, 2.5); rn/z +ve FAB, NBA 560 M + HI+; 6,(360 MHz, C2HCI,) 7.66 and 7.42 (10 H, 2 x m, aromatics), 5.18 (I H, d, J.Chem. Soc.,Perkin Trans. 1,1996 1137 JNH,~ 7.46, NH), 4.26 (1 H, m, H-2), 3.73 (1 H, m, J~A,~B10.06, CHOSi), 3.65 (1 H, dd, J~,,,A10.06, J6,,, 5.79, CHOSi), 1.87 (2 H, m, H-4 and H-3A), 1.53 (1 H, m, H-3B), 1.44 lS H, 2s, C(CH,), and 1.07 9 H, s, SiC(CH,),; 6,(360 MHz, C6,H6) 7.73 and 7.24 (lo H, 2 x m, aromatics), 5.34 (1 H, d, JNH,27.29, NH), 4.55 (1 H, m, H-2), 3.63 (2 H, m, CHOSi), 1.93 (1 H, m, H-4), 1.87 (1 H, m, H-3A), 1.53 (I H, m, H-3B), 1.40 9 H, s, C(CH,),, 1.29 9 H, s, C(CH,), and 1.12 9 H, s, SiC(CH,),; 6,(125.8 MHz, C2HC1,) 172.03 (C=O), 155.72 (urethane), 135.55-128.74 (aromatics), 81.8 OC(CH,),, 79.76 OC(CH,), J, 66.24 (CH,O), 63.6 (m, C2H,0H), 52.62 (C-2), 39.19 (C-4), 32.18 (C-3), 28.28 C(CH,)J, 27.94 C(CH,),, 25.97 SiC(CH,), and 19.19 SiC(CH,),.tert-Butyl(2S,4R)-2-tevt-butoxycarbonylamino-5-hydroxy-4-tevt-butyldiphenylsilylox ymethylpentanoate Prepared as described for the labelled compound above, using sodium borohydride to reduce the mixed anhydride of the acid 24; m/z +ve FAB, NBA 558 M + HIrsquo;; 6,(360 MHz, C2HCI,) 7.67 and 7.4 (10 H, m, aromatics), 5.17 (1 H, d, JNH,, 7.7, NH), 4.26 (1 H, br, H-2), 3.76 (2 H, m, H-5), 3.73 (1 H, AB, JAB 10.15, CHOH), 3.65 (1 H, ABX, JAB 10.15, J4,5 5.68, CHOH), 1.87 (2 H, br, H-3A and H-4), 1.5 (1 H, br, H-3B), 1.44 18 H, s, C(CH,), and 1.07 9 H, s, SiC(CH,),; SH(360 MHz. C,2H,)7.74and7.24(10H,m,aromatics),5.30(1H,d,JN,,,8, NH), 4.55 (1 H, m, H-2), 3.65 (4 H, m, 2 x CH,O), 2.09 (1 H, m, H-4), 1.91 (2 H, m.H-3) and 1.39, 1.28 and 1.12 27 H, 3 x S, C(CH3),; SJ125.8 MHz, Crsquo;HCl,) 172.04 (C=O), 155.72 (urethane), 135.55-127.75 (aromatics), 81.81 and 79.75 OC(CH,),, 66.29 (CH,OSi), 64.37 (CH,OH), 52.58 (C-2), 39.46 (C-4), 32.21 (C-3), 28.27,26.83 and 25.96 C(CH,), and 19.18 SiC(CH,),. tevt-Butyl(2S,4R)-2-tevt-butoxycarbonylamino-5-hydroxy-4-tevt-butyldiphenylsi1yloxymethyl5,5-2H, pentanoate 31 The acid 30was prepared from the cis-silyl ether 18 as described above for the trans-acid 24. The product 30, m/z +ve FAB, NBA 572 M + HIrsquo;; 6,(360 MHz, C2HCI,) 7.6-7.3 (10 H, m, aromatics), 5.14 (1 H, d, JNH,,8, NH), 4.23 (I H, br, H-2), 3.88 (2 H, m, CH,O), 2.73 (1 H, m, H-4), 2.21 and 2.05 (2 H, 2 x m, H-3), 1.45 and 1.41 18 H, 2 x s, C(CH,), and 1.03 9 H, s, SiC(CH,),, was reduced using NaB2H, by the method used for the trans series to give the alcohol 31, m/z +ve FAB, NBA 560 M + HIrsquo;; 6,(360 MHz, C2HCI,) 7.7-7.4 (10 H, m, aromatics), 5.21 (1 H, br d, JNH,,7.5, NH), 4.18 (1 H, m, H-2), 3.74 (2 H, d, J3.97, CH,O), 1.9 (2 H, m, H-4 and H-3A), 1.61 (1 H, m, H-3B), 1.46 9 H, s, C(CH,),, 1.41 9 H, s, C(CH,), and 1.0 9 H, s, SiC(CH,),; 6,(125.8 MHz, C2HCI,) 172.07 (C=O), 155.52 (urethane), 135.55-1 19.64 (aromatics), 81.97 and 80.0 OC(CH,),, 65.06 (CH,O), 64.0 (m, C2H,0), 52.26 (C-2), 39.38 (C-4), 31.25 (C-3), 28.86, 28.28 and 27.98 3 x C(CH,), and 19.29 SiC(CH,),. tert-Butyl(2S,4S)-2-tevt-butoxycarbonylamino-5-hydroxy-4-hydroxymethyl 5,5-LH2pentanoate 27 1.83 (1 H, m, H-4), 1.77 (1 H, m, H-3A), 1.64 (I H, m, H-3B), 1.46 9 H, s, C(CH,), and 1.43 9 H, s, C(CH,),; Sc(125.8 MHz, C2HCI,) 171.96 (C=O), 155.78 (urethane), 82.2 OC(CH,,, 80.05 OC(CH,),, 64.7 (CH,OH), 64.19 (m, C2H20H), 52.28 (C-2), 39.05 (C-4), 31.86 (C-3) and 28.3 and 27.87 C(CH,),.tevt-Butyl(2S)-2-tevt-butoxycarbonylamino-5-hydroxy-4-hydroxyme t hylpent anoa te Prepared from the unlabelled compound using the method described above for the preparation of compound 27, a;rsquo;+ 6.08 (c 1 in CHCI,); m/z +ve FAB, NBA 320 M + HJ +; dH(360 MHz, C2HCI,) 4.22 (1 H, d, J2,3 5.65, H-2), 3.79 (1 H, dd, J5.4.4 4.1, J~A,~B10.85, H-5A), 3.76 (2 H, br, CH20H), 3.71 (1 H, dd, JsB,45.85, J5B,sA 10.85, H-5B), 3.03 (1 H, br d, OH) and 3.00 (1 H, br d, OH), 1.84 (1 H, m, H-4), 1.77 (1 H, m, H- 3A), 1.65 (1 H, m, H-3B) and 1.46 and 1.43 18 H, 2 x s, C(CH,),; amp;( 125.8, C2HCl,) 171.93 (C=O), 155.78 (urethane), 82.23 and 82.08 OC(CH,),J, 64.96 and 64.85 (2 x CH,OH), 52.28 (C-21, 39.25 (C-4), 31.96 (C-3) and 28.30 and 27.97 C(CH,,I.tevt-Butyl(2S,4R)-2-tert-butoxycarbonylamino-5-hydroxy-4-hydroxymethyl 5,5-,H, pentanoate 32 Prepared from the (2S,4R)-silyl ether 31 as described for the (2S,4S) compound 27 above; 6,(360 MHz, C2HCI,) 5.30 (1 H, d,JN,,, 5.49,NH),4.22(1 H,d, J4.64, H-2), 3.80(1 H,dd,J5Aq4 4.12, JSA,sB 10.84, H-5A), 3.71 (I H, dd, J5B,4 5.76, JSB,,, 10.84, H-5B), 2.41 (2 H, br d, 2 x CH,OH), 1.80 (1 H,m, H-3A), 1.64 (1 H, m, H-3B) and 1.47 and 1.44 18 H, 2 x s, C(CH,),; 6,( 128.5 MHz, C2HCI,) 171.91 (GO), 155.79 (urethane), 82.27 and 80.10 OC(CH,),, 65.03 (CH,O) and 63.88 (m, C2H,0H), 52.16 (C-2), 39.05 (C-4), 31.99 (C-3) and 28.29 and 27.96 lW333)31.Sodium (2S,4S)-5,5rsquo;-dihydroxy 5,5-,H, leucinate 4a tert-Butyl (2S,4S)-2-tert-butoxycarbonylamino-5-hydroxy-4-hydro~yrnethyl5,5-~H,pentanoate 27 (50 mg, 0.16 mmol) was dissolved in methanol (0.2 cm3) and trifluoroacetic acid (1 cm3) was added. The solution was stirred at room temperature overnight. The solvent was removed in vacuo to afford a colourless oil which was dried by azeotropic removal of residual trifluoroacetic acid with diethyl ether (8 x 10 cm3), (25 mg, 9573, 6,(360 MHz, 10 NaO2H-rsquo;H2O, referenced on H-3B) 3.21 (2 H, m, CH,OH), 2.92 (1 H, m, H-2), 1.38 (1 H, m, H-4), 1.23 (1 H, m, H-3A) and 1.08 (1 H, m, H-3B); S,( 125.8 MHz, 10 Na02H-2H20, normalised on C-3) 186.55 (C=O), 65.90 (CH,OH), 64.45 (m, C2H20H), 57.37 (C-2), 42.04 (C-4) and 37.25 (C-3).Sodium (2S)-5,5rsquo;-dihydroxyleucinate4 Prepared from the unlabelled alcohol tert-butyl (2S)-2-tert- butoxycarbonylamino-5-hydroxy-4-hydroxymethylpentanoate using the method described above for the labelled compound 4a, in/--FAB, H,O-glycerol 164 M + HIrsquo;; 6,(360 MHz, tert-Butyl (2S,4R)-2-tert-butoxycarbonylamino-5-hydroxy-4-10 Na02H-,H,0, tert-buty ldiphenylsilyloxymet hyl 5,5- H ,pen tanoate 26 (50 mg, 0.9 mmol) was dissolved in methanol (1.8 cm3) and ammonium fluoride (45.7 mg, 12.3 mmol) was added.The mixture was heated to reflux at 60 ldquo;C for 3.25 h.Water (2 cm3) was added and the solution was extracted with chloroform (3 x 10 cm3). The organic layers were dried (Na,SO,) and the solvent was removed in vacuo to yield an oil which was purified by silica gel column chromatography using ethyl acetate as eluent to yield a white solid (28.6 mg, 72). This solid was recrystallised from ethyl acetate-light petroleum mp 79-8 1 ldquo;C; a;rsquo; +8.71 (c 1 in CHCI,) (Found: C, 56.1; H, 9.15; N, 4.2. C,,H,,2H,N0, requires C, 56.1; H, 9.65; N, 4.4); m/z +ve FAB, NBA 322 M + HIrsquo;; 6,(360 MHz, C2HCl,) 5.33 (1 H, d, JNH,~7.88, NH), 4.20 (1 H, m, H-2), 3.74 (2 H, br, CHZOH), 1138 J. Chern.SOC.,Perkin Trans. I, 1996 unreferenced) 3.20 (4 H, m, 2 x CN,OH), 2.90 (1 H, m, H-2), 1.38 (1 H, m, H-4), 1.23 (1 H, m, H-3A) and 1.08 (1 H, m, H-3B); Sc(125.8 MHz, 10 Na02H-,H,0, normalised on C-3) 186.75 (C=O), 65.72 (CH,OH), 65.13 (CH,OH), 57.46 (C-2), 42.93 (C-4) and 37.25 (C-3) lit.,24 amp;(Na salt in rsquo;H,O, DSS), 65.0 and 64.42 (CH,OH), 56.88 (C-2), 42.33 (C-4) and 37.25 (C-3). Sodium (2S,4R)-5,5rsquo;-dihydroxy 5,5-*H2leucinate 4b Prepared from the diol32 using the method described above for the labelled compound 4a, 6,(360 MHz, 10 Na02H-,H,0, referenced on H-3B) 3.26 (2 H, m, H-6), 2.93 (1 H, m, H-2), 1.40 (1 H, m, H-4), 1.29 (1 H, m, H-3A) and 1.08 (1 H, m, H-3B); 6,(128.5 MHz, 10 Na02H-,H,0, normalised on C-3) 186.64 (C=O), 65.20 (CH,OH), 57.33 (C-2), 47.23 (C-4) and 7.25 (C-3). Acknowledgements We thank Dr R.A. August for preliminary experiments, the Royal Society for a post-doctoral fellowship (to P. H.), the SERC for a studentship (to J. A. K.) and the ERASMUS programme for a stipend (to X. D.). We thank Dr A. G. Avent for NMR spectrocopic analysis. References Part of this work has been reported as a preliminary communication: X. Durand, P. Hudhomme, J. A. Khan and D. W. Young, Tetruhedrron Lett., 1995, 36, 1351. 2 J. Stenflo, P. Fernlund, W. Egan and P. Roepstorff, Proc. Natl. Acad. Sci.,U.S.A., 1974, 71, 2730. 3 G. L. Nelsestuen, T. H. Zytkovicz and J. B. Howard, J. Biol. Chem., 1974,249,6347. 4 C. Vermeer, Biochem. J., 1990,266, 625 and references cited therein.5 S. Naganathan, R. Hershline, S. W. Ham and P. Dowd, J. Am. Chem. Soc., 1993, 115, 5839 and references cited therein. 6 J. Dubois, M. Gaudry, S. Bory, R. Azerad and A. Marquet, J. Bid. Chem.. 1983, 258, 7897. 7 P. Decottignies-Le-Markhal, C. Ducrocq, A. Marquet and R. Azerad, C. R. Acacl Sci. Pnris, 1984, 298, IT, 343. 8 P. Decottignies-Le-Marechal. C. Ducrocq, A. Marquct and R. Azerad, J. Bid. Chem., 1984, 259, 15 010. 9 C. Ducrocq, A. Righini-Tapie, R. Azerad, J. F. Green, P. A. Friedman, J. Beaucourt and B. Rousseau, J. Chern. Soc., Perkin Trans. I, 1986, 1323. 10 J. Dubois, C. Dugave, C. Foures, M. Kaminsky, J. Tabet, S. Bory, M. Gaudry and A. Marquet, Biochemistry, 1991, 30, 10 506. 11 T. H. Zytkovicz and G. L. Nelsestuen, J. Bid. Chom., 1975, 250, 2968. 12 R. A. August, J. A. Khan, C. M. Moody and D. W. Young, Tetrahedron Left., 1992, 33, 4617; J. Chem. Soc., Perkin Trans. I, 1996,507. 13 C. M. Moody and D. W. Young, Tetruhedron Lett., 1993,34,4661. 14 C. M. Moody, B. A. Slarkmann and D. W. Young, Tetraheclron Lett., 1994, 35, 5485. 15 C. M. Moody and D. W. Young, Tetrahedron Lett., 1994,35,7277. 16 A. N. Bowler, P. M. Doyle and D. W. Young, J. Chem. Soc., Chem Commun., 199 1, 3 14. 17 This process, first noted by us in 1987 (R. A. August, D.Phi1. Thesis, Sussex, 1987), has recently been independently observed for a reduced ester by T. Katoh, Y. Nagata, K. Arai, J. Minami and S. Terashima, Tetrahedron Lett., 1993,34, 5743. 18 M. Bethell and G. W. Kenner, J. Chem. SOC.,1965, 3850. 19 M. Bethell, D. R. Bigley and G. W. Kenner, Chem. Znd. (London), 1963,653. 20 P. Herdewijn, P. J. Claes and H. Vanderhaeghe, Ccin. J. Chem., 1982, 60, 2903. 21 P. Barraclough, P. Hudhomme, C. A. Spray and D. W. Young, Tetrahedron, 1995,51,4195. 22 J. Hondrelis, G. Lonergan, S. Voliotis and J. Matsoukas, Tetrahedron, 1990,46, 565. 23 S. Yoshifuji, K. Tanaka, T. Kawai and Y. Nitta, Chem. Pharm. Bull., 1985,33,5515. 24 J. Dubois, C. Foures, S. Bory, S. Falcou, M. Gaudry and A. Marquet, Tetrahedron, 199 1,47, 1001. Paper 5f07183F Received 3 I st October 1995 Accepted 24th January I996 J. Chem. SOC.,Perkin Trans. I, 1996 1139