J. CHEM. SOC. PERKIN TRANS. I 1994
`
`807
`
`Versatile Synthesis of Stereospecifically Labelled D-Amino Acids via Labelled
`Aziridines-Preparation of (2R,3S) - [3-2H and (2R,3R)- [2,3-2H,]-Serine;
`(2S,2'St3S,3'S) - [3,3' -2H2]- and (2S,2'S,3R,3'R) - [2,2',3,3' -2Hq]-Cystine; and
`(2S,3S) - [3-*H
`and (2S.3R)- [2,3-2H2]-p-Chloroalanine
`B. Svante Axelsson, Kevin J. O'Toole, Philip A. Spencer and Douglas W. Young *
`School of Chemistry and Molecular Sciences, University of Sussex, Falmer, Brighton BNl 9QJ, UK
`
`Stereospecifically j3-labelled protected 2-carboxyaziridines 2, with the stereochemistry of a D-
`amino acid at C-2, have been prepared by a chemicoenzymic synthesis. Preparation of the labelled
`malates 5, by hydration of fumaric acid using the enzyme fumarase or by amination with aspartase
`followed by nitrosation, was followed by conversion into the isoserines 3, by a process involving
`Curtius rearrangement with retention of stereochemistry at the chirally labelled primary centre.
`Protection and ring closure gave the aziridines 2, which, o n ring opening with the appropriate
`nucleophiles and deprotection, gave stereospecifically labelled samples of o-serine 16, o-cystine 20
`and j3-chloro- D-alanine 22.
`
`Naturally occurring amino acids overwhelmingly exist as the
`L-enantiomers, although D-amino acids do occur, with the L-
`enantiomers, in bacteria.2 Because D-amino acids, except in rare
` instance^,^ do not exist in mammals, enzymes which metabolize
`D-amino acids are seen as targets for antibacterial drugs. The
`mechanism of action of such enzymes is therefore of great
`interest for the design of inhibitors which may be of medicinal
`interest .
`Elucidation of the mechanism of action of enzymes which
`metabolise L-amino acids has been greatly advanced by studies
`of the stereochemical consequences of the enzymic reactions at
`the P-carbon atom of the substrate. Similar information on
`the corresponding reactions of D-amino acids 1 is, however,
`relatively rare.' We have, therefore, devised a versatile synthesis
`of D-amino acids which are stereospecifically labelled at the p-
`centre. This should allow the stereochemistry of metabolic
`reactions of D-amino acids to be elaborated.
`From the literature on the reactions of protected 2-
`carboxyaziridines 2 with heteronucleophiles, it appeared that, if
`we could prepare a suitably protected 2-carboxyaziridine 2 with
`D-amino acid stereochemistry at C-2 and stereospecific labelling
`at C-3, then a general synthesis of stereospecifically labelled D-
`amino acids 1 could be developed. Nucleophilic substitution of
`the aziridine 2 should occur with inversion of stereochemistry at
`the labelled primary centre C-3. Since the aziridines 2 might be
`accessed from labelled samples of (2S)-isoserine 3 by protection
`and cyclisation with inversion of stereochemistry at the centre
`C-2, synthesis of the labelled (2S)-isoserines 3 became our first
`synthetic goal.
`
`1
`
`2
`
`3
`
`It has long been known6 that the commercially available
`enzyme fumarase (EC 4.2.1.2) will catalyse the conversion of
`fumaric acid 4 into (2S)-malic acid 5 with anti addition of water.
`We therefore incubated unlabelled fumaric acid 4 with the
`enzyme in the minimum amount of 2 H 2 0 which would effect
`- 50% yield. Incubation of [2,3-2H2]fumaric acid, 4 HA = 2H,7
`reaction to obtain (2S,3R)-[3-2H,]malic acid, 5 HB = 2H, in
`
`in water gave (2S,3S)-[2,3-2H2]malic acid, 5 HA = 2H (Scheme
`1).
`Since yields were not high, and, occasionally led to mixtures
`
`containing unchanged fumaric acids which were difficult to
`remove, we found that an alternative method of preparation of
`the labelled samples of malic acid was preferable to using
`fumarase. Here samples of (2S,3R)-[3-2H, 1- and (2S,3S)-[2,3-
`2H2]-aspartic acid, 6 HB = 2H and 6 HA = 'H, respectively,
`in excellent yield using immobilised Escherichia
`were prepared
`coli by the method of Woodard.' These were treated with
`
`nitrous acid to yield the corresponding malic acids 5 in - 70%
`
`yield.
`Our first approach to labelled samples of isoserine 3 was to
`prepare the labelled monoamides, (2S)-P-malamic acids 9, by
`methods which had been successful for the preparation of the
`unlabelled material 'Oql' and to subject these, uia the 0-acetyl
`derivatives, to a Hofmann rearrangement which should, by
`precedent,I2 occur with retention of stereochemistry at the
`stereospecifically labelled primary carbon atom.
`We therefore prepared the diesters 7 from malic acid 5 by
`reaction with methanol and HCl. Care had to be taken during
`isolation of the product to prevent its thermal elimination to
`dimethyl fumarate. The diamides 8 were prepared from the
`diesters 7 in good yield and the 'H NMR spectra were indicative
`that the stereospecificity of labelling was intact.
`Initially, these spectra were run in 10% NaOZH/2H20 until
`it was realised that they were in fact identical with the
`spectra obtained for the p-malamic acids 9. It was seen that the
`spectra changed to those of the monoamides very quickly and
`so hydrolysis of the diamides 8 to the monoamides 9 was
`extremely rapid under these conditions. The 'H NMR spectra
`of the diamides were therefore routinely run in (CD,),SO
`([2H6]DMSO).
`The diamides 8 were now hydrolysed to the monoamides 9
`by using 1 mol aq. KOH. Since we found it to be extremely
`important in the subsequent Hofmann rearrangement step that
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`
`808
`
`J. CHEM. SOC. PERKIN TRANS. I 1994
`
`there be no anionic contaminant in the substrate it was essential
`to purify
`the monoamides 9 by careful
`ion-exchange
`chromatography. The monoamides were then converted into
`the corresponding 0-acetates and subjected in situ to Hofmann
`rearrangement by the method of Andruszkiewicz l 3 using
`freshly preparedl4 [bis(trifluoroacetoxy)iodo]benzene. The
`labelled isoserines 3 were obtained on hydrolysis (Scheme 2).
`Although the overall yield from the labelled malates was
`reasonable, the process involved several difficult and tedious
`steps. Thus, publication of a procedure involving the Curtius
`rearrangement and not requiring purification of intermediate
`products
`presented an attractive alternative, the Curtius
`rearrangement being expected to proceed with retention of
`stereochemistry at the migrating primary centre.16
`
`__t H o H - - H a -
`
`HA C02Me
`
`HA CONH;!
`
`H O R Z ; H
`
`HO2C
`
`HA
`
`Me02C
`
`HA
`
`H2NOC
`
`H A
`
`5
`
`7
`
`8
`
`The samples of labelled malic acid were therefore converted
`into the dioxolidinones 10 by reaction with paraformaldehyde
`and catalytic quantities of toluene-p-sulfonic acid (PTSA)
`(Scheme 3). These were converted without purification, via the
`acid chlorides 11 and azides 12, into labelled samples of
`isoserine 3 which had identical spectroscopic properties to the
`samples obtained by the Hofmann route. The Curtius and
`Hofmann rearrangements had therefore proceeded with the
`same stereochemical outcome at the migrating labelled primary
`centre. Overall yields were of the order of 30-39% for the
`four steps.
`
`H02C
`
`HA
`
`3
`Scheme 3
`
`0
`12
`
`Having achieved a reliable synthesis of stereospecifically
`labelled samples of our first target compound, we now investi-
`gated their conversion into the protected aziridines 2. The
`samples of isoserine 3 were therefore esterified to yield the
`corresponding methyl esters 13 in nearly quantitative yields by
`using thionyl dichloride and methanol and these were then
`converted into the N-trityl derivatives 14 by using 1 mole
`equivalent of trityl chloride with triethylamine in chloroform.
`These derivatives were treated with toluene-p-sulfonyl chloride
`in pyridine to yield the corresponding tosyl esters, which were
`cyclised without further purification to the N-tritylaziridines, 15
`HA = 2H and 15 HB = 2H, respectively (Scheme 4). The 'H
`
`I
`i Ii ----
`
`-.
`
`I/
`
`I
`
`(a) (iii)
`
`1
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`2.4 2.2 2.0 1.8 1.6 1.4 1.2
`6H/( 6,)
`Fig. 1 Part of (a) the 'H NMR spectrum in CDCl,, and (b) the 'H
`NMR spectrum in CHCl, of (i) methyl (2R)-N-tritylaziridine-2-
`carboxylate 15; (ii) methyl (2R,3R)-N-trityl-[3-'H&ziridine-2-car-
`boxylate 15 HB = 'H; and (iii) methyl (2R,3S)-N-trityl-[2,3-'Hz]azi-
`ridine-Zcarboxylate 15 HA = 'H
`
`and 2H NMR spectra of these compounds are shown in Fig. 1,
`indicating complete stereospecificity in every stage of the
`synthesis to this point.
`When the N-tritylaziridines 15 were heated to reflux with 20%
`aq. perchloric acid for 30 h, nearly quantitative yields of the
`corresponding samples of the labelled serine 16 were obtained.
`Since we had previously synthesized samples of (2S,3R)-[3-
`2H,]- and (2S,3S)-[2,3-2H,]-serine l 7 we were now in a position
`to confirm the stereochemistry assigned to the samples prepared
`via our aziridine synthesis. The 'H NMR spectra are shown in
`Fig. 2, and it can be seen that the spectrum of (2S,3R)-[3-
`2Hl]serine is identical with that of (2R,3S)-[3-2Hl]serine, the
`optical rotations being numerically equal but of opposite sign.
`Similarly the spectra of (2S,3S)-[2,3-2H2]- and (2R73R)-[2,3-
`2H,]-serine were identical and their rotations were those
`expected of enantiomers. This confirms the assumption that our
`synthesis involves retention of stereochemistry at the labelled
`primary centre in the Curtius rearrangement step, 12-3;
`inversion of stereochemistry at C-2 in the aziridine ring closure
`step, 14+15; and inversion of stereochemistry in the nucleo-
`philic ring-opening step, 15-+ 16.
`The N-tritylaziridines 15 were not reactive enough with
`other nucleophiles and so they were converted into the N-
`benzyloxycarbonyl derivatives 17 by deprotection using tri-
`fluoroacetic acid (TFA) in methanol-chlorofonn and then
`reaction with benzyl chloroformate under Schotten-Baumann
`conditions. The labelled aziridines 17 HA = 2H and 17 HB =
`2H were treated with benzyl mercaptan and boron trifluoride-
`diethyl ether to yield the adducts 18 HA = 2H and 18 H, = 2H,
`respectively in -40% yield. When these were deprotected by
`using refluxing 6 mol dm-3 HCI, labelled samples of S-
`benzylcysteine 19 HA = 2H and 19 HB = 2H were obtained.
`The S-benzyl protecting group was removed by using sodium in
`liquid ammonia to give reasonably clean labelled samples of
`cysteine before purification. Since this was partly oxidised
`
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`
`J. CHEM. SOC. PERKIN TRANS. 1 1994
`
`809
`
`13
`
`14
`
`15
`
`16
`
`Scheme 4
`
`17
`
`21
`
`22
`
`18
`
`H02C
`
`HB
`
`19
`
`20
`
`I
`
`sole product. The best conditions for production of the isomer
`using HCl were sonication in 6 mol dm--3 HCl but, when this
`
`. J L -
`
`(iii)
`
`Experimental
`M.p.s were determined on a Kofler hot-stage apparatus and are
`uncorrected. Optical rotations (given in units of lo-' deg cm2
`g-') were measured on a Perkin-Elmer PE241 polarimeter using
`a 1 dm pathlength micro cell. IR spectra were recorded on a
`Perkin-Elmer 1720 Fourier-transform
`instrument and UV
`spectra were recorded on a Philips PU8720 spectrophotometer.
`Mass spectra were recorded by Mr. A. Greenway using Kratos
`MS25 and Kratos MS80 instruments and on KS50 and VG7070
`instruments by Dr. S. Chotai at the Wellcome Research
`Laboratories, Beckenham. 3-NBA refers to 3-nitrobenzyl
`alcohol. All 'H NMR spectra were recorded on a Bruker
`WM360 instrument (360 MHz), I3C NMR spectra ('H-
`decoupled) were recorded by Dr. A. G. Avent on a Bruker AMX
`500 instrument (125.8 MHz), and 2H NMR spectra were
`recorded on a Bruker A-C 250SY instrument (38.4 MHz) by Mr.
`C. M. Dadswell. J-Values are given in Hz. 3-(Trimethylsily1)-
`propane-1-sulfonic acid (DSS), located at 6 0.0, was used as
`the internal standard for samples run in 20% DCl in D20. For
`all other NMR spectra, the residual solvent peak was used as
`reference. TLC was carried out on Merck Kieselgel60 F254 pre-
`coated silica gel plates of thickness 0.2 mm (ART 5554 and ART
`57 14). Column chromatography was performed using Merck
`Kieselgel 60 (230400 mesh-ART 9385). Ion-exchange resins
`were purchased in the chloride form from Aldrich (Dorset) and
`converted into the required form by passage of at least a five-
`fold excess of the relevant ion through a column of the resin,
`followed by washing with distilled water. Microanalyses were
`performed by Miss M. Patel, Sussex University, and by Mrs. P.
`Firmin, Wellcome Research Laboratories, Beckenham.
`
`4.6
`
`4.4
`
`4.2
`s,
`' H NMR spectra in 20% DCI/D20 of (i) (2R)-serine 16; (ii)
`Fig. 2
`(2R,3S)-[3-ZH,]serine 16 H, = 2H; (iii) (2R,3R)-[2,3-2H2]serine 16
`HA = *H; (iv) (2S)-serine; (v) (2S,3R)-[3-2H,]serine; and (vi) (2S,3S)-
`[2,3-2H,]serine
`
`4.0
`
`3.8
`
`during careful ion-exchange purification, however, we found it
`easier to oxidise the product by passing oxygen through the
`column fractions. Very clean samples of stereospecifically
`labelled D-cysteine 20 were obtained, in good yield.
`Our final target was to prepare stereospecifically labelled
`samples of the enzyme inhibitor P-chloroalanine 22. Previous
`work I 8 * l 9 indicated that ring opening of 3-unsubstituted 2-
`carboxyaziridine derivatives with HC1 was not entirely regio-
`specific, attack being at both a- and P-carbon atoms and, indeed,
`reaction of our N-tritylaziridine, 15, with HCl in ethanol and
`diethyl ether followed by deprotection gave a mixture of a- and
`P-chloroalanine. Change of N-protecting group and solvent
`altered the z/P ratio but failed to yield P-chloroalanine 22 as the
`
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`
`810
`
`(2S,3R)-[3-2Hl]Malic Acid 5 HB = ,H.-Method
`A.
`Fumaric acid 4 (1 5 g, 129 mmol) and dipotassium hydrogen
`phosphate (22 g, 126 mmol) were dissolved in water (1 dm3) and
`the pH of the solution was adjusted to 7.4 with 3 mol dm-3
`sodium hydroxide. The solution was lyophilised, and the
`residue was redissolved in ,H,O and relyophilised. The
`lyophilisation procedure was repeated twice to ensure maxi-
`mum exchange by deuterium. The residue was finally dissolved
`in ,H,O (100 cm3) to give a pD of 7.7 k- 0.2. Fumarase (Sigma,
`500 units) was added and the reaction mixture was incubated at
`28 "C for 5 days, the progress of the reaction being followed by
`observing the disappearance of 1290 nm in the UV spectrum.
`The enzyme was denatured by immersion in boiling water for 30
`min. The precipitated protein was removed by filtration, and
`the filtrate was titrated with 0.1 mol dm-, sodium hydroxide to
`the phenolphthalein end point and concentrated under reduced
`pressure to a volume of 10-20 cm3.
`The product was purified on a Dowex 1x2-200 (formate) ion-
`exchange column. Inorganic salts were eluted with water and
`(2S,3R)-[3-2H,]malic acid, 5 HB = ,H, was recovered by
`elution with 6% aq. formic acid. The product was a solid (9.3 g,
`(c 1.6,
`100 "C); [a]L3 -6.06
`54%), m.p. 97-98 "C (lit.,,'
`MeOH). The 'H NMR spectrum was identical with that
`described in Method B below, but in some incubations 'H NMR
`spectroscopic analysis showed the product to be contaminated
`with significant quantities of fumaric acid.
`Method B. Freshly prepared 30% aq. NaNO, (1 12.8 cm3) was
`added over a period of 20 min to a stirred solution of (2S,3R)-
`[3-2H,]aspartic acid 6 HB = ,H (10 g, 75 mmol) in 0.5 mol
`dmP3 H,SO,
`(376 cm3) at room temperature. The reaction
`mixture was stirred at room temperature for 2 h, the volume was
`
`reduced under reduced pressure to - 100 cm3, and Celite was
`
`added until a thick sludge was obtained. This was placed in an
`extraction thimble and extracted in a Soxhlet apparatus with
`diethyl ether during 48 h. The solvent was removed under
`reduced pressure to yield (2S,3R)-[3-2H,]malic acid 5 HB = ,H
`as a pale yellow solid (7.4 g, 7373, m.p. 97.5-99.O"C (lit.,,'
`100°C); [or];'.'
`-6.83
`(c 1.6, MeOH); m/z [+ve FAB
`(glycerol)] 136 ([M + HI+), v,,,(KBr)/cm-'
`3435br (OH),
`3000-2600br (COOH) and 1732 (COOH); 6,(10% NaOD in
`D,O) 1.93 (1 H, d, J3s,2 9.3, 3s-H) and 3.81 (1 H, d, J2,3s 9.3,
`2-H); dC(lO% NaOD in D,O) 183.53 and 182.25 (2 x CO,H),
`72.65 (C-2) and 44.86 (t, C-3).
`
`A. The
`(2S,3S)-[2,3-2H2]Mafic Acid 5 HA = 'H.-Method
`dideuteriated compound was prepared as above in 46% yield by
`using [2,3-2H,]fumaric acid 4 HA = ,H, in water. The spectra
`were identical with those reported below for method B.
`Method B. The product was prepared as above in 67% yield
`by using (2S,3S)-[2,3-2H,]aspartic acid 6 HA = ,H, and was a
`solid, m.p. 97-99.5 "C; [or]h2 -6.81 (c 1.6, MeOH); m/z [EI]
`137 ([M + HI+); v,,,(KBr)/cm-'
`3446br (OH), 3000-2600
`(COOH) and 1724 (COOH); 6,( 10% NaOD in D,O) 2.15 (1 H,
`s, 3R-H); dC(lO% NaOD in D,O) 183.52 and 182.28 (2
`x CO,H), 72.35 (t, C-2) and 44.78 (t, C-3).
`
`acid 5 (20 g, 149 mmol) was
`Dimethyl (2s)-Malate 7.-Malic
`dissolved in methanol (250 cm3). The solution was cooled to 0 "C
`in an ice-bath and was saturated with dry HCl gas. The reaction
`mixture was left at room temperature for 2 days, and the solvent
`was removed under reduced pressure to yield dimethyl (2s)-
`malate 7 as a pale green oil. If any solid starting material was
`present at this stage, the residue was redissolved in methanol,
`and the solution was resaturated with HCl gas and left for a
`further period until reaction was complete. Owing to a tendency
`for the product to dehydrate to fumaric acid, it was not purified
`further (21.8 g, 90%); [a]k2.5 -9.86
`( c 1.5, MeOH) (lit.,,'
`-6.85); m/z [EI] 163 ([M + HI+), 131 ([M - OCH,]+) and
`
`J. CHEM. soc. PERKIN TRANS. 1 1994
`3473br (OH) and
`103 ([M - C02CH3]+); v,a,(film)/cm-l
`1741 (ester); G,(CDCl,) 2.80 (1 H, dd, J3S.2 6.4, J3S,3R 16.4, 3s-
`H), 2.90 (1 H, dd, J 3 R . 2 4.5, J 3 R , 3 S 16.4, 3R-H), 3.72 (3 H, S,
`CO,Me), 3.8O(3H,s,CO2Me)and4.56(1 H,dd, J2,3s6.4, J 2 , 3 R
`4.5,2-H).
`Dimethyl (2S,3R)-[3-2H,]malate 7 HB = ,H was prepared
`as above in 85% yield by using (2S,3R)-[3-2H,]malic acid 5
`-9.63
`(c 1.5, MeOH); m/z [EI]
`HB = ,H, and had
`164 ([M + HI+), 132 ([M - OCH,]')
`and 104 ([M -
`C02CH3 I+); v,,,(film)/cm-l
`3465br (OH) and 1741 (ester);
`dH(CDC13) 2.80 (1 H, d, J3s.2 6.4, 3S-H), 3.72 (3 H, s, CO,Me),
`3.80 (3 H, s, C0,Me) and 4.56 (1 H, d, J2,3s 6.4,2-H).
`Dimethyl (2S,3S)-[2,3-2H,]malate 7 HA = ,H was prepared
`as above in 88% yield by using (2S,3S)-[2,3-2H,]malic acid 5
`- 10.5 (c 1.5, MeOH); m/z [EI]
`HA = ,H, and had
`165 ([M + HI+), 133 ([M - OCH,]+) and 105 (EM -
`3424br (OH) and 1737 (ester);
`CO,CH,] +); v,,,(film)/cm-'
`dH(CDC1,) 2.90 (1 H, s, 3S-H), 3.72 (3 H, s, C0,Me) and 3.81
`(3 H, s, C0,Me).
`
`(2s)-malate 7 (20 g, 123.45
`(2S)-Malarnide 8.-Dimethyl
`mmol) was dissolved in methanol (200 cm3) and the solution
`was cooled to 0 "C in an ice-bath. Liquid ammonia was added
`to the constantly stirred reaction mixture until the liquid
`volume had approximately doubled. The reaction mixture was
`then stirred for 2 h at room temperature to allow evaporation
`of the excess of ammonia, and was then left overnight in a
`refrigerator. The resulting crystals were filtered off, washed with
`cold water, and recrystallised from methanol-water (1 3 g, 80%),
`m.p. 156-157 "C (lit.,,' 157 "C); Calk3 -34.8
`(c 1.5, water)
`-37.9); m/z [+ve CI (NH,)] 133 ([M + HI+);
`(lit.,,'
`v,,,(KBr)/cm-'
`3413 (OH), 3373 and 3207 (NH) and 1657
`(amide); G,([2H,]DMSO) 2.20 (1 H, dd, J3s.2 9.5, J3S,3R 14.8,
`3S-H), 2.41 (1 H, dd, J3R.2 2.9, J,,,,, 14.8,3R-H), 4.14 (1 H, m,
`2-H), 5.56 (1 H, d, exch., JOH.2 5.8, OH) and 6.87,7.15,7.22 and
`7.31(4 x 1 H , 4 s,exch.,2 x CONH,).
`(2S,3R)-[3-2Hl]Malamide 8 H, = ,H was prepared as
`above in 76% yield from dimethyl (2S,3R)-[3-2H,]malate 7
`HB = ,H, and had m.p. 155.5-156.5 "C; Cali3 -38.3 (c 1.5,
`water); m/z [ + ve CI (NH,)] 134 ([M + H] +); v,,,(KBr)/cm-l
`3403 (OH), 3370 and 3200 (NH) and 1657 (amide);
`GH([~H~]DMSO) 2.18 (1 H, d, J3s.2 9.5, 3S-H), 4.14 (I H, dd,
`J2,3s 9.5, J2,0H 5.8, 2-H), 5.56 (1 H, d, JOH,, 5.8, OH) and 6.88,
`7.16, 7.22 and 7.32(4 x 1 H , 4 s,exch.,2 x CONH,).
`(2S,3S)-[2,3-2H,]Malamide 8 HA = ,H was prepared as
`above in 76% yield by using dimethyl (2S,3S)-[2,3-2H,]malate
`7 HA = ,H, and had m.p. 155-157 "C; [a]h3 -35.8 (c 1.2,
`water); m/z [+ve CI (NH,)] 135 ([M + HI'); v,,,(KBr)/
`cm-' 3404 (OH), 3390 and 3200 (NH) and 1656 (amide);
`~H(['H~]DMSO) 2.39 (1 H, S, 3R-H), 5.56 (1 H, br S, OH)
`and6.88,7.16,7.24and7.36(4 x 1 H,4s,exch.,2 x CONH,).
`
`(2S)-P-Malamic Acid 9.-(2S)-Malamide 8 (12 g, 90.91
`mmol) was dissolved in 1 mol dm-, potassium hydroxide (91
`cm3, 91 mmol) and the solution was heated to reflux for 3 h.
`Conc. hydrochloric acid (12 cm3) was added to the cooled
`reaction mixture, which was then left in a refrigerator overnight.
`Crystals precipitated. These were filtered off, and washed with
`ice-cold water. The yield at this stage was 8.7 g (72%). The
`product was pure except for the presence of chloride ions. The
`crystalline product was dissolved in water (10 cm3) and applied
`to a column of Dowex 1 X2-200 (OH -) ion-exchange resin. The
`column was eluted with water until no chloride ions could be
`detected in the eluent (silver nitrate-nitric acid). The product
`was eluted with 1% aq. acetic acid and was recrystallised from
`water (7.52 g, 62%); m.p. 148-149 "C (lit.," 149 "C);
`-9.33); m/z [+ve CI (NH,)] 134
`-9.4 (c 1.2, water) (lit.,"
`' 3404 (OH), 3250 and 3240 (NH),
`([M +HI+); v,,,(KBr)/cm
`
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`
`J. CHEM. SOC. PERKIN TRANS. 1 1994
`
`2500-3000 (COOH), 1720 (acid) and 1676 (amide); BH(lO%
`NaOD in D,O) 2.36 (1 H, dd, J3s,2 9.2, J3S,3R 15.3, 3S-H), 2.57
`(1 H, dd, J,,,, 3.6,J3R,3S 15.3,3R-H) and4.21 (1 H, m, J2.3s9.3,
`J 2 , 3 , 3.6, 2-H).
`(2S,3R)-[3-2Hl]-P-Malamic acid 9 HB = ,H was prepared as
`above, by using (2S,3R)-[3-2H,]malamide 8 HB = ,H, in 66%
`yield, m.p. 148-149 "C; [a]L3 -9.50 (c 1.4, water); m/z [ +ve
`CI (NH,)] 135 ([M + HI'); v,,,(KBr)/cm-l
`3398 (OH),
`3250 and 3240 (NH), 2500-3000 (COOH) and 1721 (acid);
`6,( 10% NaOD in D,O) 2.34 (1 H, d, J3s,2 9.1, 3S-H) and 4.21
`(1 H, d, J2,3,9.1,2-H).
`(2S,3S)-[2,3-2H,]-P-Malamic acid 9 HA = ,H was prepared
`as above, by using (2S,3S)-[2,3-2H,]malamide 8 HA = ,H, in
`66% yield, m.p. 148-149 "C; Calk3 -9.30 (c 1.0, water); m/z
`[+ve CJ (NH,)] 136 ([M + HI'); v,,,(KBr)/cm-'
`3402
`(OH), 3300 and 3200 (NH), 2500-3000 (COOH), 1736 (acid)
`and 1655 (amide); 6,(10% NaOD) 2.57 (1 H, s, 3R-H).
`
`(2S)-lsoserine 3.--Method A. (2S)-P-Malamic acid 9 (3 g,
`22.56 mmol) and pyridine (18 cm3, 225 mmol) were dissolved in
`constantly stirred acetonitrile (100 cm3) at 0 "C in an ice-bath.
`Acetic anhydride (2.3 cm3, 25 mmol) was added dropwise as
`the system was purged with nitrogen. The reaction mixture was
`stirred for I h at room temperature and was then diluted with
`water (1 00 cm3). Freshly prepared [bis(trifluoroacetoxy)iodo]-
`benzene (1 4.63 g, 33 mmol) was added and the reaction mixture
`was stirred for an additional 4 h at room temperature. The
`solvents were removed by heating (60 "C) under reduced
`pressure to yield an oil, which was dissolved in water (100 cm3)
`and extracted with diethyl ether (3 x 200 an3). The aqueous
`phase was diluted with acetone (1 00 cm3)xonc. hydrochloric
`acid (50 cm3) and was heated to reflux for 2 h. The solvents were
`removed under reduced pressure by heating (70 "C) to yield an
`oily residue, which was dissolved in water (10 cm3) and
`separated on a column of Dowex 1x2-200 (OH-). The pyridine-
`derived contaminants were eluted with water and then (2s)-
`isoserine was recovered by elution with 5% aq. acetic acid. The
`solvent was removed from the relevant fractions (as determined
`by TLC) under reduced pressure to yield a solid, which was
`recrystallised from methanol-water (1.56 g, 6673, m.p. 187-
`188 "C (lit.,', 188-189 "C); [a]h3 -31.4 (c 1.4, water) (lit.,13
`-32.2). Spectra were identical with those for the product
`prepared using Method B below.
`Method B. (2S)-Malic acid 5 (5.36 g, 40 mmol), para-
`formaldehyde (1.60 g, 53 mmol) and PTSA (41) mg; 0.23 mmol)
`were added to chloroform (40 cm3) and the reaction mixture
`was heated at reflux with azeotropic trapping of water for 4 h.
`The solvent was removed under reduced pressure. The
`remaining residue was heated to reflux with SOC1, (10 an3, 137
`mmol) for 1 h. After removal of the excess of SOCI, under
`reduced pressure, CCl, (20 cm3) was added and the solvent
`was removed under reduced pressure. The latter procedure was
`repeated several times to remove last traces of SOC1,.
`The residue was dissolved in acetone (40 cm3), cooled to
`- 15 "C using a salt-ice-bath, and added to a solution of sodium
`azide (3.5 g, 53.9 mmol) in water (12 cm3) at 0 "C. The reaction
`mixture was stirred at - 15 "C for 30 min. The acetone was
`removed under reduced pressure at 0 "C and the mixture was
`extracted with toluene (2 x 20 cm3). The organic phase was
`dried (MgSO,), and concentrated under reduced pressure to
`-20 cm3. The mixture was heated to 60"C, whereupon
`nitrogen was given off. After evolution of nitrogen had ceased,
`the mixture was heated at reflux for 15 min and the solvent was
`then removed under reduced pressure. 5 mol dm-, Hydrochloric
`acid (20 cm3) was added, and the reaction mixture was heated
`at reflux for 30 min. The solvent was removed under reduced
`pressure. Water (50 cm3) was added and the solvent was again
`removed under reduced pressure. The residue was dissolved
`
`81 1
`
`in water (10 cm3) and applied to a Dowex 1x2-200 (OH-)
`ion-exchange column. The column was eluted with water until
`the pH of the eluted water was neutral. The product was then
`recovered by elution with 5% aq. acetic acid. The solvent was
`removed from the relevant column fractions (as determined by
`TLC) under reduced pressure to yield (2S)-isoserine 3 as an
`orange solid (1.64 g, 3973, m.p. 188.5-190.5"C (lit.,13 188-
`189°C); [a];'.'
`-26.8 (c 1, water) (lit.,13 -32.2); m/z [+ve
`FAB (glycerol)] 106 ([M + HI'); v,,,(KBr)/cm-l
`3246 (OH),
`3200 (NH), 3000-2500 (COOH) and 1657 (acid); dH(D20)
`2.90 (1 H, dd, J3s.2 8.4, J3s.3, 13.1, 3S-H), 3.13 (I H, dd, J 3 R . 2
`~.~,J3R,3S13.1,3R-H)and4.01(1H,dd,J,,3,8.~,J,,3,~.~,2-H).
`(2S,3R)-[3-2Hl]Isoserine 3 HB = ,H was prepared by
`Method A above in 62% yield by using (2S,3R)-P-malamic acid
`9 HB = ,H, or by using Method B from (2S,3R)-[3-2Hl]malic
`acid 5 HB = 'H in 30% overall yield, m.p. 188-190 "C;
`- 26.4 (c 1, water); m/z [ + ve FAB (glycerol)] 107 ([M + H] +);
`vmax(KBr)/m-l 3400 (OH), 3070 (NH), 30W2500 (COOH)
`and 1650 (COOH); 6H(D20) 2.90 (1 H, d, J3s.2 8.4, 3S-H) and
`4.03 (1 H, d, J2,3s 8.4, 2-H).
`(2S,3S)-[2,3-2H,]Isoserine 3 HA = ,H was prepared by
`Method A above in 63% yield by using (2S,3S)-P-malamic acid
`9 HA = ,H, or by Method B by using (2S,3S)-[2,3-2H,]malic
`acid 5 HA = ,H, in 34% overall yield, m.p. 188-190 "C;
`-26.9
`(c 1, water); m/z [+ve FAB (glycerol)] 108
`([M + HI'); v,,,(KBr)/cm-'
`3246 (OH), 3050 (NH), 3000-
`2500 (COOH) and 1656 ( C m H ) ; dH(D20) 3.12 (1 H, s,
`3R-H).
`
`Methyl (2S)-Isoserinate Hydrochloride l3.--(2S)-Isoserine 3
`(1.32 g, 12.6 mmol) was added to a constantly stirred solution of
`methanol (20 cm3) and SOCI, (2.5 cm3, 34.3 mmol) at 0°C.
`When the starting material had dissolved, the reaction mixture
`was allowed to reach room temperature and was stirred for 22 h.
`The solvent was removed under reduced pressure, CCl, (10
`cm3) was added and the solvent was again removed under
`reduced pressure. The latter procedure was repeated several
`times to remove last traces of SOCI,. Methyl (2S)-isoserinate
`hydrochloride 13 was obtained as a pale brown solid (1.95 g,
`99%), m.p. 104-105 "C; [a];'." - 19.2 (c 1, water); m/z [+ve
`FAB (glycerol)] 120 ([M + HI');
`v,,,(KBr)/cm-l
`3537
`(OH), 3059 (NH) and 1746 (ester); c~,(~H,O) 3.10 (1 H, dd,
`J3s,2 8.4, J3s.3, 13.3, 3S-H), 3.31 (1 H, dd, J 3 R . 2 4.1, J 3 ~ , 3 s 13.3,
`3R-H), 3.67 (3 H, s, C0,Me) and 4.44 (1 H, dd, J2,3s 8.4, J 2 , 3 R
`4.1,2-H).
`Methyl (2S,3R)-[3-2Hl]isoserinate hydrochloride 13 HB =
`,If was prepared as above in 100% yield by using (2S,SR)-[3-
`2Hl]isoserine 3 HB = ,H. The product was a solid, m.p. 101.5-
`(c 1, water); m/z [+ve FAB
`103.5 "C;
`-18.2
`(glycerol)] 121 ([M + HI'); v,,(KBr)/cm-'
`3503 (OH),
`3050 (NH) and 1746 (ester); BH(D,O) 3.01 (1 H, d, J3s.2 8.4,
`3S-H), 3.60 (3 H, s, C0,Me) and 4.37 (1 H, d, J2,3s 8.4,2-H).
`Methyl (2S,3S)-[2,3-2H2]isoserinate hydrochloride 13 HA =
`,H was prepared as above in 97% yield by using (2S,3S)-[2,3-
`2H2]isoserine 3 HA = 'H. The product was a solid, m.p. 102-
`104 "C; [a];22.5 - 16.6 (c 1, water); m/z [ +ve FAB (glycerol)]
`122 ([M + HI'); v,,,(KBr)/cm-'
`3504 (OH), 3051 (NH) and
`1744 (ester); dH(,H20) 3.25 (1 H, s, 3R-H) and 3.62 (3 H, s,
`C0,Me).
`
`Methyl (2s)-N- Tritylisoserinate lrl.-Methy1(2S)-isoserinate
`hydrochloride 13 (1.95 g, 12.5 mmol) was dissolved in
`chloroform (10 cm3)-triethylamine (3.85 cm3, 27.6 mmol) at
`room temperature. The mixture was cooled to 0°C and a
`solution of trityl chloride (3.49 g, 12.5 mmol) in chloroform (10
`cm3) was added dropwise over a period of 30 min. The reaction
`mixture was stirred at 0°C under nitrogen for 24 h. It was
`washed successively with 10% aq. citric acid (2 x 15 cm3) and
`
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`

`
`812
`
`water (2 x 15 cm3), dried over Na2S04, and the solvent was
`removed under reduced pressure to yield theproduct as an off-
`+ 17.2 (c 1, CHCl,) (Found:
`white foam (4.08 g, 90%);
`C, 76.4; H, 6.5; N, 3.8. C2,H2,N03 requires C, 76.4; H, 6.4; N,
`3.9%); m/z [+ve CI (NH,)] 284 ([M - Ph]'); v,,,(film)/cm-'
`3485 (OH), 3317 and 3021 (NH) and 1709 (ester); G,(CDCl,)
`2.45 ( 1 H , dd, J ~ R ,
`2 3.7, J ~ R ,
`3s 1 2. I, 3R-H), 2.5 1 ( 1 H, dd, J3s.2 4.7,
`J3S,3R 12.1, 3S-H), 3.84 (3 H, s, C02Me), 4.26 (1 H, t, 2-H) and
`7.13-7.43 (15 H, m, CPh,).
`Methyl (2S,3R)-N-trityl-[3-2Hl]i~~~erinate 14 HB = 2H was
`prepared as above in 92% yield by using methyl (2S,3R)-[3-
`2Hl]isoserinate 13 HB = 2H. The product was a foam, [a];4.5
`+15.6 (c 1, CHCl,); m/z [+ve CI (NH,)] 285 ([M -Ph]+);
`v,,,(film)/cm-'
`3485 (OH), 33 17 and 3021 (NH) and 17 13 (ester);
`SH(CDC1,) 2.48 (1 H, d, J3S.2 4.8, 3S-H), 3.84 (3 H, s, C02Me),
`4.26 (1 H, d, J2,3s 4.8, 2-H) and 7.147.43 (15 H, m, CPh,).
`Methyl (2S,3S)-N-trityl-[2,3-2H2]isoserinate 14 HA = 2H
`was prepared in 97% yield by using methyl (2S,3S)-[2,3-
`2H2]isoserinate hydrochloride 13 HA = 2H. The product was a
`foam, [a];4,4.5 + 15.2 (c 1, CHCl,); m/z [+ve CI (NH,)] 286
`([M - Ph]');
`v,,,(film)/cm~'
`3486 (OH), 3317 and 3021
`(NH) and 1709 (ester); d,(CDCI,) 2.45 (1 H, s, 3R-H), 3.86
`(3 H, s, C0,Me) and 7.16-7.44 (15 H, m, CPh,).
`
`Methyl (2R)-N-Tritylaziridine-2-carboxylute 15.-Methyl
`(2S)-N-tritylisoserinate 14 (2.30 g, 6.37 mmol) was dissolved
`in pyridine (8 cm3, 99 mmol) and the solution was cooled to
`- 15 "C in a salt-ice-bath. Toluene-p-sulfonyl chloride (3.98 g,
`20.9 mmol) was added over a period of 30 min and the reaction
`mixture was stirred at 0 "C under nitrogen for 22 h. The solvent
`was removed under reduced pressure and the gummy residue
`was partitioned between ethyl acetate (20 cm3) and water (20
`cm3). The organic phase was washed successively with 10% aq.
`citric acid (20 cm3) and water (20 cm3), dried over Na2S0,, and
`the solvent was removed under reduced pressure to yield the
`intermediate tosyl compound as a gum.
`The gum was dissolved in a mixture of tetrahydrofuran (1 5
`cm3) and triethylamine (2.5 cm3, 17.9 mmol) and heated at
`reflux under nitrogen for 22 h. The solvent was removed under
`reduced pressure and the resulting product was dissolved in
`ethyl acetate (15 cm3) and washed successively with 10% aq.
`citric acid (2 x 10 cm3), 1 mol dm-, aq. sodium hydrogen
`carbonate (2 x 10 cm3) and water (2 x 10 cm3). The organic
`phase was dried over Na2S04 and the solvent was removed
`under reduced pressure to yield a dark red/brown solid.
`The crude product was dissolved in chloroform (10 cm3) and
`purified by silica gel column chromatography with (2:l)
`chloroform-light petroleum (60-80 "C) as eluent. The solvent
`was removed from the rele