Three approaches to the synthesis of L-leucine selectively labelled with carbon-13 or deuterium in either diastereotopic methyl group

Abstract

Three approaches to the synthesis of L-leucine selectively labelled with carbon-13 or deuterium in either diastereotopic methyl group as well as at C-3 and C-4 are described. In all three methods the stereogenic centre at C-2 was created with total stereocontrol via a one-pot, two-enzyme catalysed procedure involving hydrolysis and reductive amination of a 2-keto ester. However, the approaches vary in the synthesis of the isotopically labelled 2-keto esters and in the production of the stereogenic centre at C-4 which was achieved either via alkylation of a propionylated Evans’ auxiliary with labelled iodomethane or by the diastereoselective conjugate addition of a labelled organocopper reagent to crotonate tethered to a chiral sultam. The latter approach proved most efficient and using the (1R,2S,3R)-3-[N-phenylsulfonyl-N-(3,5-dimethyldiphenyl)aminobornan-2-ol ester 27, [5-¹³C]-L-leucine was prepared with >98% de at C-4 and in 49% overall yield from the first labelled intermediate 28.

---

Introduction

Isotopically labelled L-amino acids serve a vital role in a variety of studies in bioorganic chemistry.^1,2 For example, the selective labelling of either diastereotopic methyl group of L-leucine has enabled the elucidation of their fate during secondary metabolic reactions.^3–7 Leucine is also important in protein tertiary structure formation due to its participation in hydrophobic interactions. Assignment of the leucine pro-R and pro-S methyl group resonances in protein NMR spectra allows more precise definition of protein structure and the study of stereochemical aspects of associated molecular recognition phenomena.^8,9 Thus several groups have investigated approaches to the synthesis of leucine selectively labelled in either diastereotopic methyl group for example via biosynthesis,^10,11 using chemical and enzymatic resolutions,^5,6,7,11,12 and using starting materials from the chiral pool.¹³ Young and co-workers performed the first fully stereoselective synthesis of (2S,4R)-[5,5,5-²H₃]-leucine using (2S)-pyroglutamic acid as a chiral template ¹⁴ and more recently a similar approach has been reported by Nishiyama and co-workers.¹⁵ We have previously described a chemo-enzymatic route for the synthesis of L-leucine selectively labelled with deuterium and/or carbon-13 in either diastereotopic methyl group.¹⁶ Our strategy was versatile enabling the incorporation of isotopic labels at C-3 and C-4 as well as in either diastereotopic methyl group and gave total stereocontrol at C-2. However, there were technical difficulties in handling the volatile intermediates and only 86% de at C-4 was achieved. We now describe this approach in full as well as our more recent investigations which have led to the development of a new and efficient strategy for the synthesis of isotopically labelled L-leucine which not only overcomes the problems of handling volatile intermediates but also gives >98% de at C-4.

Results and discussion

Method 1. Synthesis of (2S,4R)-[5,5,5-²H₃]leucine 7

Our initial route ¹⁶ to the synthesis of L-leucine selectively labelled with deuterium and/or carbon-13 in either diastereotopic methyl group is shown in Scheme 1. First it was necessary to create the stereogenic centre which was to become C-4 in L-leucine and this was formed via alkylation of the known ^17,18 propionylated oxazolidinone 2 with CD₃I to give 3 (or ¹³CH₃I to give 8, Scheme 2). The optimum conditions for the reaction proved to be generation of the enolate with sodium hexamethyldisilazide (NaHMDS) and quenching with 10 equivalents of iodomethane giving the product in 90% yield. However, to minimise the quantity of expensive isotopically labelled iodomethane, further optimisation showed that 1.4 equivalents of ¹³CH₃I giving 76% yield of 8 was the best compromise. The alternative diastereomer 11 was prepared from (4S)-3-acetyl-4-isopropyloxazolidinone 9 by an initial alkylation with ¹³CH₃I followed by alkylation with iodomethane (Scheme 2). In the ¹H NMR spectra of 8 and 11 the signals assigned to 2′-CH₃ and 2′-¹³CH₃ were well resolved due to the large ¹³C–¹H coupling and integration of the signals at δ 1.22 (dd, J 128.3, 6.9 Hz in 8; dd, J 6.9, 5.0 Hz in 11) and at δ 1.15 (dd, J 6.7, 5.2 Hz in 8; dd, J 127.9, 6.7 Hz in 11) confirmed that selective alkylation of (4S)-3-propionyloxazolidinone had occurred giving a 13∶1 mixture of diastereomers.

Scheme 1 Reagents: i, BuLi, EtCOCl, 94%; ii, NaHMDS, CD₃I, 72%; iii, LiAlH₄, Et₂O; iv, Ph₃P, Br₂, PhNO₂; v, Mg, (CO₂Et)₂, Et₂O, THF, −78 °C, 14–60% from 3; vii, CRL, leucine dehydrogenase, FDH, HCO₂NH₄, 85%.

Scheme 2

With the required stereogenic centre established, the next stage was to cleave the auxiliary and to effect a two-carbon homologation to the 2-keto ester 6. Many methods are known for the synthesis of 2-keto esters and we favoured the reaction of a Grignard reagent with diethyl oxalate at low temperature.¹⁹ Reduction of 3 with lithium aluminium hydride cleaved the chiral auxiliary and liberated the labelled alcohol 4 which was converted to the bromide 5 using triphenylphosphine and bromine. Both alcohol 4 (bp ²⁰ca. 108 °C) and bromide 5 (bp ²⁰ca. 91 °C) are rather volatile necessitating the use of manifold trap techniques for their efficient manipulation. Nitrobenzene was used as the solvent in the conversion of 4 to 5 so that bromide 5 could be distilled from the reaction mixture and then used immediately to form the Grignard reagent, which was reacted in situ with diethyl oxalate to give the 2-keto ester 6 in variable yields (14–60% from 3).

The final stages of the synthesis involved hydrolysis of the ester and the reductive amination of the resultant 2-keto acid catalysed by a commercially available amino acid dehydrogenase. These enzymes require NADH; the cofactor may be recycled efficiently in situ according to the protocol of Shaked and Whitesides using a second commercially available enzyme, formate dehydrogenase (FDH), with the consumption of formate ions and the evolution of carbon dioxide.²¹ In our initial work ¹⁶ we converted α-keto ester 6 to the acid by saponification with sodium hydroxide. More recently we have adopted a milder procedure using a lipase isolated from Candida rugosa (CRL), as model reactions showed that it provides higher yields than saponification. Furthermore the reaction conditions of the lipase hydrolysis are compatible with those of the leucine dehydrogenase catalysed reductive amination.²² Thus a one-pot dual enzyme catalysed hydrolysis of 2-keto ester 6 and reductive amination of the resultant 2-keto acid gave (2S,4R)-[5,5,5-²H₃]-leucine 7 in 85% yield from 6. The methyl region of the ¹H NMR spectrum of 7 displayed only the doublet corresponding to the lower frequency methyl resonance of unlabelled L-leucine; this is consistent with previous assignments of the lower frequency resonance to the 4-pro-S and the higher frequency resonance to the 4-pro-R leucine methyl groups.^12,14 The ¹³C NMR spectrum of (2S,4R)-[5,5,5-²H₃]leucine 7 showed a singlet at δ_C 20.8 due to the methyl group and a quintet at δ_C 20.7, assigned to the trideuteromethyl group. On first inspection these are confusing observations as, in common with the ¹H NMR spectrum, the lower frequency ¹³C resonance has been assigned previously to the 4-pro-S and the higher frequency resonance to the 4-pro-R leucine methyl groups;^10,12,14 however, the apparent discrepancy is caused by an α-isotope shift ²³ of the trideuteromethyl group resonance, as noted by Young and co-workers.¹⁴

In principle this approach may be used for the synthesis of the complementary diastereomer, (2S,4S)-[5,5,5-²H₃]leucine by the route shown in part in Scheme 2 (using CD₃I). A more direct route would use the (+)-norephedrine derived auxiliary;¹⁷ however we found that in this case the key alkylation step proceeds in lower yield than with the L-valine derived auxiliary 1. Therefore for the synthesis of (2S,4S)-[5,5,5-²H₃]leucine we suggest the use of the D-valine derived auxiliary (i.e. the enantiomer of 1). This chemo-enzymatic approach to the synthesis of L-leucine has the further advantage that it enables the incorporation of carbon-13 at C-3 and/or C-4 using sodium [¹³C]acetate as the source of isotopic label (Scheme 2). For example, treatment of sodium [2-¹³C]acetate with pivaloyl chloride gives a mixed anhydride which on reaction with the lithium salt of 1 gives the acylated product 12 in 70% yield, the precursor to [4-¹³C]-L-leucine. In addition, the approach may also be modified for the inclusion of nitrogen-15.²⁴

Method 2. Synthesis of (2S,4R)-[5-¹³C]leucine 17

When designing routes to the synthesis of expensive isotopically labelled compounds it is essential that each stage is high yielding and reproducible. Although the above approach is versatile and most of the steps are indeed high yielding and simple to perform, the route is not ideal due to the problems inherent in the preparation and purification of the volatile bromide 5. Hence we required an alternative strategy for the two-carbon homologation of 2-methylpropanol to the key 2-keto ester. Our aim was to convert the alcohol to a good leaving group such as a sulfonate ester for coupling with the Umpolung 2-keto ester equivalent 2-ethoxycarbonyl-1,3-dithiane ²⁵ and finally hydrolysis of the resultant dithiane.

First the carbon-13 labelled derivative 8 was converted into alcohol 13 using a two-step lithium hydroperoxide cleavage ²⁶ – reduction sequence (Scheme 3), which gave a better yield than the direct reduction of 8 with lithium aluminium hydride and easy recovery of the Evans’ auxiliary 1 by acid–base extraction after the first step. Model reactions were carried out in order to determine the best mode of activation for the hydroxy group of alcohol 13 for reaction with the salt of 2-ethoxycarbonyl-1,3-dithiane. We found that the toluene-p-sulfonate, methanesulfonate and p-bromobenzenesulfonate all proved insufficiently reactive whereas the bromide and triflate were suitable leaving groups. Given the difficulties we experienced when using bromide 5 in Method 1, we chose triflate 14 as the alkylating agent. To our knowledge, the only previous example of the alkylation of a glyoxylic acid dithioacetal derivative with a triflate is found in Shiba and co-workers’ synthesis of 3-deoxy-D-manno-2-octulosonic acid (KDO).²⁷ Treatment of alcohol 13 with triflic anhydride, pyridine and carbon tetrachloride at 0 °C gave the required triflate 14 in 73% yield. The triflate 14 was used to alkylate 2-ethoxycarbonyl-1,3-dithiane (CBT) to provide 2-ethoxycarbonyl-2-{(2R)-[3-¹³C]-2-methylpropyl}-1,3-dithiane 15. It was not necessary to fully purify 15 and oxidative hydrolysis of crude 15 with N-bromosuccinimide (NBS) ^25,28 gave, after column chromatography, pure 2-keto ester 16 in 45% yield over the two steps.

Scheme 3 Reagents: i, a, LiOH, H₂O₂; b, LiAlH₄, Et₂O, 88%; ii, Tf₂O, pyridine, CCl₄, 73%; iii, BuLi, CDT, 56%; iv, NBS, Me₂CO, H₂O, 80%; v, CRL, leucine dehydrogenase, FDH, HCO₂NH₄, 79%.

2-Keto ester 16 was converted directly into (2S,4R)-[5-¹³C]leucine 17 in 79% yield, using our one-pot two-step hydrolysis–reductive amination sequence. As expected, the methyl region of the ¹H NMR spectrum of (2S,4R)-[5-¹³C]leucine 17 featured two signals with the downfield signal (δ 0.95) displaying the larger coupling (J 125 Hz) to carbon-13. The ¹³C NMR spectrum displayed one strongly enriched signal (δ 22.8) and, at higher field (δ 21.7), a weakly enriched signal {due to the minor diastereomer, (2S,4S)-[5-¹³C]leucine 25}, consistent with previous assignments of the leucine methyl groups.^10,12,14

Method 3. (2S,4S)-[5-¹³C]leucine 25

Although the second route to the synthesis of isotopically labelled L-leucine does overcome the problem of handling 1-bromo-2-methylpropane and each step of the synthesis is reliable and simple to perform, it is still not ideal. The diastereoselectivity at C-4 is only 86% and the overall yield for the synthesis of 2-keto ester 16 from the first isotopically labelled intermediate 8 is approximately 30%. Hence our third and final approach aimed to address these deficiencies and used different disconnections from the preceding two methods: the pivotal 2-keto ester came from a one-carbon homologation of labelled 3-methylbutanoic acid (isovaleric acid), itself ultimately derived from crotonic acid (Scheme 4).

Scheme 4

We proposed to synthesise the stereoselectively labelled 3-methylbutanoic acid by conjugate addition of a labelled organometallic reagent to a crotonic acid derivative, with a chiral auxiliary inducing the stereoselectivity. The application of Evans’ type auxiliaries in this context is uncommon,^18,29,30 although some success has been reported both by Hruby and co-workers ³¹ and Williams and co-workers.³² In contrast, Oppolzer and co-workers’ camphorsultam 18 is well documented as a suitable chiral auxiliary for conjugate additions.³³ Method 3 therefore began with the crotonylation of (1S)-1,10-camphorsultam 18 ^33b to provide the conjugate addition substrate 19 in 84% yield (Scheme 5).

Scheme 5 Reagents: i, NaH, crotonyl chloride, 84%, ii, (¹³CH₃)₂CuLi·PBu₃, 89%; iii, H₂O₂, LiOH, 93%; iv, Ph₃PCHCN 22, EDCI, DMAP 80%; v, O₃, MeOH, CH₂Cl₂, 76%; vi, CRL, leucine dehydrogenase, FDH, HCO₂NH₄, 81%.

Oppolzer and co-workers have reported the highly diastereoselective conjugate addition of Grignard reagents and organocuprates to substrates similar to 19.³³ However the addition of a methyl group is something of a special case: problems of 1,2 addition are suffered with Grignard reagents ^33c and low π-face discrimination with copper(I) catalysed Grignard reagents.^33e Oppolzer and co-workers minimised these difficulties by employing phosphine stabilised Gilman reagents (3 equivalents), which give good regio- and stereo-selectivity.^33d,e We needed to further optimise the conditions of our conjugate addition for we required not only maximum yield and maximum diastereoselectivity but also maximum isotope yield. Our investigations revealed that reduction of the quantity of Gilman reagent below 2.6 equivalents significantly lowered the reaction yield, as might be expected from inspection of the proposed mechanism.^33d Our optimised conditions were thus treatment of chiral crotonyl derivative 19 with 2.6 equivalents of labelled Gilman reagent {prepared from [¹³C]iodomethane, lithium metal and copper(I) iodide–tri-n-butylphosphine complex} at −78 °C, which gave the conjugate addition product 20 in 89% yield. The diastereoselectivity of this reaction was determined from integration of the signals assigned to 3′-CH₃ (δ 0.96, dd, J 6.6, 5.5 Hz) and 3′-¹³CH₃ (δ 0.97, dd, J 125.2, 6.6 Hz) in the ¹H NMR spectrum which indicated that a 10∶1 mixture of diastereomers (ca. 82% de) had been formed.

The auxiliary was cleaved with lithium hydroperoxide ^33g (and recovered in 85% yield) to liberate (S)-[4-¹³C]-3-methylbutanoic acid 21 in 93% yield. The one-carbon homologation of 21 was achieved via formation and ozonolysis of the β-ketocyanophosphorane 23 under the conditions described by Wasserman and Ho ³⁴ giving methyl (S)-[5-¹³C]-4-methyl-3-oxopentanoate 24. Finally the one-pot two-step enzymatic catalysed conversion of 2-keto ester 24 to (2S,4S)-[5-¹³C]leucine 25 proceeded smoothly giving an 81% yield of pure product after ion exchange chromatography. This approach may be readily adapted to the synthesis of the complementary diastereomer (2S,4R)-[5-¹³C]leucine 17 by starting from the commercially available (1R)-1,10-camphorsultam. This approach to the synthesis of either (2S,4S)- or (2S,4R)-[5-¹³C]leucine is high yielding and each step is simple to perform and reliable. The only downside of this route was the poor diastereoselectivity at C-4; thus we sought to improve this by using a different chiral auxiliary.

Helmchen and co-workers have described the use of (1R,2S,3R)-3-[N-phenylsulfonyl-N-(3,5-dimethylphenyl)amino]bornan-2-ol 26 as a (commercially available) chiral auxiliary for conjugate addition reactions.³⁵ Acylation of chiral auxiliary 26 with crotonyl chloride ^35b gave enoate 27 in 81% yield (Scheme 6). As in the case of the Oppolzer auxiliary, we wished to optimise the conjugate addition conditions to produce maximum de and minimum isotopic label wastage. Following Helmchen and Wegner we employed the Yamamoto reagent,^36,3713CH₃Cu·BF₃, in 7-fold excess (a 5-fold excess dramatically reduced the yield), derived from the organolithium with ether as solvent.^35a Our optimum conditions gave the conjugate addition product 28 in 79% yields; inclusion of tri-n-butylphosphine (to dissolve and stabilise the Yamamoto reagent) ^36,38 gave a similar result. NMR spectroscopy (in deuterochloroform, deuteroacetone or deuterobenzene) failed to detect the minor diastereomer (NMR spectroscopy of the conjugate addition product formed under sub-optimum reaction conditions revealed that the signals from each diastereomer’s 3′-CH₃ and 3′-¹³CH₃ are partially resolved) so we have achieved our objective and increased the de of the conjugate addition to above 98%.

Scheme 6 Reagents: i, Crotonyl chloride, molecular sieves, 81%; ii, ¹³CH₃Cu·BF₃, 79%.

The auxiliary was cleaved with aqueous potassium hydroxide in methanol ^35a (and recovered in 93% yield) to liberate (R)-[4-¹³C]-3-methylbutanoic acid in quantitative yield. Thus, using Helmchen’s auxiliary, we have improved Method 3 to produce a synthesis of (2S,4R)-[5-¹³C]leucine 17 with greater than 98% de and 39% overall yield from the first labelled intermediate 28. The complimentary diastereomer may be synthesised starting from the commercially available auxiliary (1R,2R,3S)-3-[N-phenylsulfonyl-N-(3,5-dimethylphenyl)amino]-2-bornanol.^35a

Conclusions

We have described three enantioselective syntheses of L-leucine that permit the selective labelling of either diastereotopic methyl group with carbon-13 or deuterium. These routes are extremely versatile and allow incorporation of isotopic labels at other sites and the appropriate method may be chosen for maximum de or maximum isotope yield. We consider the conjugate addition route (Method 3 using Helmchen’s auxiliary) the best of our three methods due to its comparative simplicity, high yields and excellent de.

Experimental

General experimental details have been described previously.³⁹ Cuprate reactions were conducted under argon. Copper(I) iodide was purified according to Taylor and Casy.⁴⁰ Copper(I) iodide–tri-n-butylphosphine complex was prepared according to the procedures of Taylor and Casy ⁴⁰ and Kauffman and Teter ⁴¹ from purified copper(I) iodide and freshly distilled tri-n-butylphosphine in 66% yield; mp 74–75 °C (lit.,⁴¹ 75 °C). All NMR spectra were run in CDCl₃ unless otherwise stated. J values are in Hz. [α]_D has units of 10⁻¹ deg cm² g⁻¹. The enzymes were purchased and stored as follows: lipase from Candida rugosa (CRL), Sigma, stored at 4 °C as a 10 000 eU ml⁻¹ solution in tris buffer (5 mM); formate dehydrogenase (FDH) from Candida boidinii, Boerhinger, stored at 4 °C; leucine dehydrogenase from Bacillus species, Sigma, stored at −20 °C; β-nicotinamide adenine dinucleotide hydride (NADH), Genzyme, stored at −20 °C.

General procedure for the one-pot hydrolysis and reductive amination of 2-keto esters

Potassium phosphate buffer (5 mM, 25 mL per mmol of substrate) was deoxygenated by bubbling through nitrogen for 0.5 h. CRL (10 000 eU per mmol of substrate) and the keto ester (1 eq.) in ethanol (1 mL per mmol of substrate) were added and the reaction mixture stirred at rt, maintaining the pH between 7.0 and 8.5 by the periodic addition of sodium hydroxide (1.0 or 0.1 M). Once the pH had stopped changing (ca. 5 h, ca. 1 eq. of sodium hydroxide having been added), ammonium formate (10 eq.), 1 M dithiothreitol (DTT) (1 μL per mmol of substrate), formate dehydrogenase (8 mg, 4 eU for less or 16 mg, 8 eU for more than 3 mmol of substrate), NADH (8 mg, 12 μmol for less or 16 mg, 24 μmol for more than 3 mmol of substrate) and leucine dehydrogenase (5 eU for less or 10 eU for more than 3 mmol of substrate) were added. The resultant solution was stirred at rt and maintained between pH 7.0 and 7.5 by the periodic addition of hydrochloric acid (1.0 or 0.1 M) until the pH remained static (ca. 7 days, ca. 0.8 eq. of hydrochloric acid having been added). The reaction mixture was concentrated in vacuo and the product isolated by ion exchange chromatography on Dowex ® 50WX8-100 (20 g per mmol of substrate), eluting first with water (3 × 40 mL per mmol of substrate) then conc. ammonia (2 × 100 mL per mmol of substrate). The conc. ammonia eluent was evaporated to dryness to yield the pure α-amino acid.

(4S)-4-Isopropyl-3-{(2R)-[3,3,3-²H₃]-2-methylpropanoyl}oxazolidin-2-one 3.. A solution of (4S)-4-isopropyl-3-propanoyloxazolidin-2-one 2 (558 mg, 3.0 mmol) in THF (6 mL) was added dropwise to a stirred solution of sodium hexamethyldisilazide (1.0 M in THF, 3.3 mL, 3.3 mmol) in THF (15 mL) at −78 °C. The reaction mixture was stirred at −78 °C for 1 h then [²H₃]-iodomethane (0.37 mL, 6.0 mmol) was added dropwise. The reaction mixture was stirred at −78 °C for 4 h then sat. ammonium chloride solution (15 mL) and water (6 mL) were added and the aqueous phase acidified to pH 2 with sulfuric acid. The product was extracted with ethyl acetate (3 × 60 mL). The combined extracts were washed successively with sat. sodium hydrogen carbonate solution (15 mL), sodium thiosulfate solution (15 mL) and brine (15 mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give an oil. The product was purified by column chromatography eluting with 2–10% ethyl acetate in light petroleum to afford a 13∶1 mixture of (4S)-4-isopropyl-3-{(2R)-[3,3,3-²H₃]-2-methylpropanoyl}oxazolidin-2-one 3 and (4S)-4-isopropyl-3-{(2S)-[3,3,3-²H₃]-2-methylpropanoyl}oxazolidin-2-one (437 mg, 72%) as a pale yellow oil; [α]_D +94.6 (c 4.1 in CH₂Cl₂) [lit.,⁴² unlabelled material +94.2 (c 1.1 in CH₂Cl₂)]; δ_H (270 MHz) 0.88 [3 H, d, J 7.0, (CH₃)₂CH], 0.91 [3 H, d, J 7.0, (CH₃)₂CH], 1.15, 1.22 [3 H, 2 × (d, J 6.8), major and minor diastereomer CH₃CHCO respectively], 2.34 [1 H, septet d, J 7.0 and 4.0, CH(CH₃)₂], 3.76 (1 H, br q, J 6.8, CHCO), 4.20 (1 H, dd, J 9.0 and 3.3, CH₂O), 4.28 (1 H, dd, J 9.0 and 8.2, CH₂O) and 4.45 (1H, ddd, J 8.2, 4.0 and 3.3, CHN); δ_C 14.2 [(CH₃)₂CH], 17.3 [(CH₃)₂CH], 18.3 (CH₃CHCO), 19.1 (quin, J 20, CD₃), 28.0 [CH(CH₃)₂], 31.8 (CHCO), 58.1 (CHN), 62.9 (CH₂O), 153.2 (NCO₂) and 176.9 (CON); m/z 202 (M⁺, 20%, 97% incorporation of three deuteriums), 159 (14), 130 (27), 86 (24), 74 (100) and 58 (60).

The above reaction was repeated using [¹³C]iodomethane as the electrophile to give a 13∶1 mixture of (4S)-4-isopropyl-3-{(2R)-[3-¹³C]-2-methylpropanoyl}oxazolidin-2-one 8 and (4S)-4-isopropyl-3-{(2S)-[3-¹³C]-2-methylpropanoyl}oxazolidin-2-one 11 as a pale yellow oil; [α]_D +89.9 (c 5.0 in CH₂Cl₂); δ_H (500 MHz) 0.88 [3 H, d, J 7.0, (CH₃)₂CH], 0.91 [3 H, d, J 7.0, (CH₃)₂CH], 1.15 (0.2 H, dd, J 127.9 and 6.9, 11¹³CH₃), 1.15 (2.8 H, dd, J 6.9 and 5.2, 8 CHCH₃¹³CH₃), 1.22 (2.8 H, dd, J 128.3 and 6.9, 8¹³CH₃), 1.22 (0.2 H, dd, J 6.9 and 5.0, 11 CHCH₃¹³CH₃), 2.34 [1 H, septet d, J 7.0 and 4.0, CH(CH₃)₂], 3.79 (1 H, septet d, J 6.9 and 5.0, CHCH₃¹³CH₃), 4.20 (1 H, dd, J 9.2 and 3.1, CH₂O), 4.27 (1 H, dd, J 9.2 and 8.4, CH₂O), and 4.45 (1 H, ddd, J 8.4, 4.0 and 3.1, CHN); δ_C 14.6 [CH(CH₃)₂], 17.8 [CH(CH₃)₂], 18.2 (CHCH₃¹³CH₃), 19.5 (CHCH₃¹³CH₃), 28.3 [CH(CH₃)₂], 32.5 (d, J 34, CHCH₃¹³CH₃), 58.3 (CHN), 63.2 (CH₂O), 153.6 (NCO₂) and 177.5 (d, J 2, CON); m/z 200 (M⁺, 24%, 93% incorporation of carbon-13), 185 (5), 157 (14), 130 (52), 86 (44), 78 (15), 72 (100) and 58 (66).

The above reaction was repeated using (4S)-4-isopropyl-3-{[3-¹³C]propanoyl}oxazolidin-2-one 10 and iodomethane giving a 13∶1 mixture of (4S)-4-isopropyl-3-{(2S)-[3-¹³C]-2-methylpropanoyl}oxazolidin-2-one 11 and (4S)-4-isopropyl-3-{(2R)-[3-¹³C]-2-methylpropanoyl}oxazolidin-2-one 8 as a pale yellow oil; [α]_D +84.0 (c 3.5 in CHCl₃); δ_H (500 MHz) 0.88 [3 H, d, J 7.0, (CH₃)₂CH], 0.91 [3 H, d, J 7.0, (CH₃)₂CH], 1.15 (2.8 H, dd, J 128.3 and 6.9, 11¹³CH₃), 1.15 [0.2 H, dd, J 6.9 and 5.0, 8 CH₃(¹³CH₃)CH], 1.22 (0.2 H, dd, J 127.9 and 6.9, 8¹³CH₃), 1.22 [2.8 H, dd, J 6.9 and 5.2, 11 CH₃(¹³CH₃)CH], 2.34 [1 H, septet d, J 7.0 and 4.0, CH(CH₃)₂], 3.79 (1 H, septet d, J 6.9 and 5.0, CHCH₃¹³CH₃), 4.20 (1 H, dd, J 9.2 and 3.1, CH HO), 4.27 (1 H, dd, J 9.2 and 8.4, CHHO), and 4.45 (1 H, ddd, J 8.4, 4.0 and 3.1, CHN); δ_C 14.6 [(CH₃)₂CH], 17.8 [(CH₃)₂CH], 18.2 (CHCH₃¹³CH₃), 19.6 (CHCH₃¹³CH₃), 28.4 [CH(CH₃)₂], 32.5 (d, J 34.9, CHCH₃¹³CH₃), 58.3 (CHN), 63.2 (CH₂O), 153.6 (NCO₂) and 177.5 (d, J 1.5, CON); m/z 200 (M⁺, 18%, 87% incorporation of carbon-13), 173 (3), 157 (15) 130 (52), 86 (42), 72 (100) and 58 (60).

Ethyl (4R)-[5,5,5-²H₃]-4-methyl-2-oxopentanoate 6.. A solution of (4S)-4-isopropyl-3-{(2R)-[3,3,3-²H₃]-2-methylpropanoyl}oxazolidin-2-one 3 (1.01 g, 5.0 mmol) in ether (5 mL) was added to a suspension of lithium aluminium hydride (380 mg, 10 mmol) in ether (50 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h then water (1 mL) was added carefully. The resultant mixture was filtered through a sinter funnel containing Celite, silica and magnesium sulfate, eluting with ether (100 mL). The filtrate was carefully concentrated in vacuo to ca. 10 mL. The product 4 and ether were distilled using a high vacuum manifold leaving behind the chiral auxiliary 1 (416 mg, 64%). The distillate was carefully concentrated in vacuo giving (2R)-[3,3,3-²H₃]-2-methylpropanol 4 as a colourless liquid. Bromine (0.26 mL, 5.0 mmol) was added dropwise to a solution of triphenylphosphine (1.31 g, 5.0 mmol) in nitrobenzene (25 mL). The bromine colour disappeared almost immediately, giving a yellow solution from which a white solid precipitated after ca. 5 min. A solution of the (2R)-[3,3,3-²H₃]-2-methylpropanol 4 in nitrobenzene (1 mL) was added and the flask was then connected to a trap at −78 °C and heated to 80 °C for 1 h, giving an orange solution. The reaction mixture was cooled to rt and quinoline (0.59 mL, 5.0 mmol) was added. The reaction mixture was stirred at rt for 15 min. The crude (2R)-[3,3,3-²H₃]-2-methylpropyl bromide 5 was distilled into a trap (−196 °C) under reduced pressure (0.04 mmHg). The distillate was redistilled under reduced pressure through a phosphorus pentoxide bulb and into a collection flask. THF (4 mL) was immediately added to the flask and the solution was warmed to rt. This solution of bromide 5 was added to vigorously stirred magnesium turnings (140 mg, 5.8 mmol). The Grignard reaction was heated under reflux for 10 minutes then added to a solution of diethyl oxalate (0.54 ml, 4.0 mmol) in ether (10 mL) and THF (10 mL) at −78 °C. The reaction mixture was stirred at −78 °C for 5 h then sat. ammonium chloride solution (4 mL) was added. The mixture was acidified to pH 2 with 1 M hydrochloric acid and the product extracted with ether (3 × 15 mL). The combined extracts were washed successively with sat. sodium hydrogen carbonate solution (4 mL) and brine (4 mL), dried over magnesium sulfate and concentrated in vacuo to give a yellow oil. The product was purified by column chromatography, eluting with 1% ethyl acetate in light petroleum, to give ethyl (4R)-[5,5,5-²H₃]-4-methyl-2-oxopentanoate 6 (175 mg, 22%) as a colourless oil; [α]_D +2.1 (c 1.0 in CHCl₃) (lit. data on unlabelled material ^43,44); δ_H (270 MHz) 0.89 (3 H, d, J 6.8, CH₃CH), 1.30 (3 H, t, J 7.1, OCH₂CH₃), 2.09 (1 H, m, 4-H), 2.64 (2 H, d, J 6.8, 3-H₂), and 4.24 (2 H, q, J 7.1, OCH₂CH₃); δ_C (68 MHz) 13.9 (OCH₂CH₃), 21.3 (quin, J 19, CD₃), 22.3 (CH₃CH), 23.9 (C-4), 47.7 (C-3), 62.3 (OCH₂), 161.3 (C-1), and 194.4 (C-2); m/z 161 (M⁺, 3%, 97% incorporation of 3 deuterium atoms), 116 (2), 97 (44), 88(44), 85 (50), 71 (72), 60 (39) and 57 (100).

(2S,4R)-[5,5,5-²H₃]Leucine 7.. The reaction was carried out as described in the general procedure using ethyl (R)-[5,5,5-²H₃]-2-oxo-4-methylpentanoate 6 (79 mg, 0.49 mmol) and ammonium formate (63 mg, 1.0 mmol). Purification by ion exchange chromatography gave (2S,4R)-[5,5,5-²H₃]leucine 7 (56 mg, 85%) as a white solid; [α]_D +12.5 (c 1.0 in 1 M HCl) [lit.,¹² (2R,4S)-diastereomer [α]_D −12.8 (c 0.47 in 6 M HCl)]; δ_H (500 MHz, D₂O) 0.87 (3 H, d, J 6.4, CH₃), 1.66 (2 H, m, 3-H₂), 1.76 (1 H, m, 4-H) and 3.97 (1 H, dd, J 8.1 and 6.4, CHNH₂); δ_C [68 MHz, D₂O + (CD₃)₂CO] 20.7 (quin, J 19, CD₃), 20.8 (CH₃), 23.6 (C-4), 38.7 (C-3), 51.3 (C-2), and 172.5 (C-1); m/z 134 (M⁺, 3%, 96% incorporation of 3 deuterium atoms), 117 (1), 89 (100) and 74 (39).

(4S)-4-Isopropyl-3-{[3-¹³C]propanoyl}oxazolidin-2-one 10.. A solution of sodium hexamethyldisilazide (1.0 M in THF, 1.1 ml, 1.1 mmol) was added to a stirred solution of (4S)-3-acetyl-4-isopropyloxazolidin-2-one 9 (345 mg, 2.0 mmol) in 1,2-dimethoxyethane (4 mL) at −78 °C. The reaction mixture was stirred at −78 °C for 20 min. [¹³C]Iodomethane (0.25 mL, 4.0 mmol) was added dropwise and the reaction mixture stirred at −78 °C for 3 h. Sat. ammonium chloride solution (5 mL) and water (2 mL) were added and the aqueous phase acidified to pH 2 with sulfuric acid (1 M). The product was extracted with ethyl acetate (3 × 20 mL). The combined organic extracts were washed with sat. sodium hydrogen carbonate solution (5 mL), sodium thiosulfate solution (5 mL) and brine (5 mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give a yellow oil. The crude product was purified by column chromatography to give (4S)-4-isopropyl-3-{[3-¹³C]propanoyl}oxazolidin-2-one 10 (203 mg, 54%) as a pale yellow oil; [α]_D +94.5 (c 4.0 in CH₂Cl₂) [lit.,¹⁷ unlabelled material +96.8 (c 8.7 in CH₂Cl₂)]; δ_H (500 MHz) 0.88 [3 H, d, J 6.8, (CH₃)₂CH], 0.92 [3 H, d, J 6.8, (CH₃)₂CH], 1.17 (3 H, dt, J 128.3 and 7.3, ¹³CH₃CH₂), 2.38 [1 H, septet d, J 7.3 and 3.9, CH(CH₃)₂], 2.91 (1 H, ddq, J 17.7, 7.3 and 4.4, ¹³CH₃CH₂) 2.98 (1 H, ddq, J 17.7, 7.3 and 4.4, ¹³CH₃CH₂), 4.22 (1 H, dd, J 9.1 and 3.0, CH₂O), 4.27 (1 H, dd, J 9.1 and 8.3, CH₂O) and 4.44 (1 H, ddd, J 8.3, 3.8 and 3.0, CHN); δ_C 8.4 (¹³CH₃), 14.6 (CH₃), 17.9 (CH₃), 28.3 [CH(CH₃)₂], 29.1 (d, J 35.2, ¹³CH₃CH₂), 58.3 (CHN), 63.3 (CH₂O), 154.1 (NCO₂) and 174.0 (d, J 3.1, CON); m/z 186 (M⁺, 7%, 88% incorporation of carbon-13), 143 (17), 113 (13), 85 (9), 71 (19) and 58 (100).

2-Ethoxycarbonyl-2-{(2R)-[3-¹³C]-2-methylpropyl}-1,3-dithiane 15.. Hydrogen peroxide solution (30%, 5.9 mL, 52 mmol) and lithium hydroxide monohydrate [875 mg, 21 mmol, in water (25 mL)] were added successively to a solution of (4S)-4-isopropyl-3-{(2R)-[3-¹³C]-2-methylpropanoyl}oxazolidin-2-one 8 (2.61 g, 13 mmol) in THF (50 mL) and water (25 mL) at 0 °C. After 2 h sodium sulfite [6.5 g in water (25 mL)] was added and the solution stirred at 0 °C for a further 15 min. The solution was adjusted to pH 9–10 with sat. sodium hydrogen carbonate solution, the THF evaporated and the residual aqueous solution extracted with dichloromethane (2 × 125 mL). The organic extracts were dried over sodium sulfate and evaporated to yield (4S)-4-isopropyloxazolidin-2-one 1 (1.50 g, 89%).

The aqueous solution was adjusted to pH 1–2 with 1 M sulfuric acid and extracted with ether (3 × 125 mL). The combined extracts were dried over sodium sulfate and evaporated to yield (2R)-[3-¹³C]-2-methylpropanoic acid ⁴⁵ (1.27 g, quantitative) as a colourless liquid; δ_H1.20 (3 H, dd, J 128.1 and 7.0, ¹³CH₃), 1.20 (3 H, dd, J 7.0 and 5.2, CH₃), 2.59 (1 H, septet d, J 7.0 and 4.6, CH) and 5.96 (br s, CO₂H); δ_C 18.7 (¹³CH₃), 33.8 (d, J 34, CH) and 183.4 (CO₂H).

A solution of (2R)-[3-¹³C]-2-methylpropanoic acid (1.16 g, 13.0 mmol) in ether (25 mL) was added dropwise to a stirred slurry of lithium aluminium hydride (0.99 g, 26 mmol) in ether (40 mL) at 0 °C and the resultant mixture stirred at 0 °C for 6 h. Water (6 mL) was added slowly to the mixture and the pH of the resultant grey slurry adjusted to 2–3 with 1 M sulfuric acid. The layers were separated and the aqueous layer extracted with ether (4 × 65 mL). The combined organic extracts were washed with 1 M sodium hydrogen carbonate solution (120 mL), dried over magnesium sulfate and filtered. This solution was distilled (50 °C, atmospheric pressure) to yield, as the residue, a mixture of (2R)-[3-¹³C]-2-methylpropan-1-ol 13 (861 mg, 88%, calculated from the ¹H NMR spectrum) and ether as a colourless liquid (lit. data on deuterated material ⁴⁶), δ_H (270 MHz) 0.91 (3 H, dd, J 124.8 and 6.7, ¹³CH₃), 0.91 (3 H, dd, J 6.8 and 5.3, CH₃), 1.21 (9 H, t, J 7.0, ether CH₃), 1.75 (1 H, nonet d, J 6.6 and 3.7, CH), 2.76 (1 H, br s, OH), 3.37 (2 H, dd, J 6.5 and 3.8, CH₂) and 3.48 (6 H, q, J 7.0, ether CH₂).

A solution of (2R)-[3-¹³C]-2-methylpropan-1-ol 13 [ca. 625 mg, ca. 8.3 mmol, carried through from the preceding step in ether–deuterochloroform (ca. 3 mL)] and pyridine (1.2 mL, 15 mmol) in carbon tetrachloride (17 mL) was added dropwise over ca. 40 min to a stirred solution of trifluoromethanesulfonic anhydride (2.4 mL, 14 mmol) in carbon tetrachloride (13 mL) at 0 °C. The resultant solution was stirred at 0 °C for a total of 55 min then water (30 mL) was added and the layers separated. The aqueous layer was extracted with dichloromethane (2 × 50 mL), the combined organic extracts dried over magnesium sulfate, filtered and evaporated until just dry, to yield crude (2R)-[3-¹³C]-2-methylpropyl trifluoromethanesulfonate 14 (1.26 g, ca. 73%) as a cloudy brown oil (lit. data on unlabelled material ⁴⁷), δ_H (270 MHz) 1.03 (3 H, dd, J 126.4 and 6.7, ¹³CH₃), 1.03 (3 H, dd, J 6.6 and 5.1, CH₃), 2.13 (1 H, nonet d, J 6.7 and 4.1, CH) and 4.31 (2 H, ddd, J 6.4, 3.8 and 0.5, CH₂).

A solution of 2-ethoxycarbonyl-1,3-dithiane (0.96 mL, 6.1 mmol) in THF (2.5 mL) was added dropwise to n-butyllithium (2.8 mL, 2.44 M solution in hexanes) in THF (5 mL) at −78 °C for 20 min. Crude (2R)-[3-¹³C]-2-methylpropyl trifluoromethanesulfonate 14 (1.26 g, ca. 6.1 mmol) was added dropwise and the mixture stirred at −78 °C for 15 min then at rt overnight. Sat. sodium hydrogen carbonate solution (50 mL) was added and the mixture extracted with ethyl acetate (5 × 60 mL). The combined organic extracts were washed successively with water (100 mL) and brine (100 mL), dried over magnesium sulfate, filtered and evaporated. Column chromatography of the resultant yellow oil with 1–5% ethyl acetate–light petroleum as eluent yielded pure 2-ethoxycarbonyl-2-{(2R)-[3-¹³C]-2-methylpropyl}-1,3-dithiane 15 (820 mg, 56%) as a colourless oil; δ_H (270 MHz) 0.94 (3 H, dd, J 125.1 and 6.4, ¹³CH₃), 0.94 (3 H, dd, J 6.5 and 5.4, CHCH₃), 1.33 (3 H, t, J 7.1, OCH₂CH₃), 1.86 (1 H, dtt, J 14.0, 12.5 and 3.3, SCH₂CHH^ax), 1.94–2.06 (3 H, m, 3-H₂ and 4-H), 2.14 (1 H, dtt, J 14.0, 4.5 and 2.5, SCH₂CHH^eq), 2.65 (2 H, ddd, J 14.5, 4.5 and 3.3, 2 × SCHH^eq), 3.26 (2 H, ddd, J 14.5, 12.5 and 2.5, 2 × SCHH^ax) and 4.24 (2 H, q, J 7.1, CO₂CH₂CH₃); δ_C 14.1 (CH₂CH₃), 23.7 (¹³CH₃ and CH₃), 24.8 (SCH₂CH₂), 25.2 (d, J 35, C-4), 27.9 (SCH₂), 47.0 (C-3), 53.1 (d, J 3, CS₂), 61.8 (OCH₂CH₃) and 171.4 (CO); followed by mixed fractions containing both 15 (319 mg, 21%) and 2-ethoxycarbonyl-1,3-dithiane, then pure 2-ethoxycarbonyl-1,3-dithiane.

Ethyl (4R)-[5-¹³C]-4-methyl-2-oxopentanoate 16.. A solution of 2-ethoxycarbonyl-2-{(2R)-[3-¹³C]-2-methylpropyl}-1,3-dithiane 15 (299 mg, 1.2 mmol) in acetone (4 mL) was added dropwise over ca. 5 min to a solution of N-bromosuccinimide (2.2 g, 12 mmol) in acetone (30 mL) and water (1.5 mL) at −5 °C and the solution stirred at −5 °C for a further 5 min. Sat. sodium sulfite solution (40 mL) was added and the solution extracted with light petroleum–dichloromethane (1∶1, 3 × 40 mL). The combined organics were washed successively with 1 M sodium hydrogen carbonate solution (40 mL), water (40 mL) and brine (40 mL), dried over magnesium sulfate, filtered and evaporated to yield ethyl (4R)-[5-¹³C]-4-methyl-2-oxopentanoate 16 (150 mg, 80%), as a colourless oil; δ_H 0.97 (3 H, dd, J 125.4 and 6.7, ¹³CH₃), 0.97 (3 H, dd, J 6.6 and 5.4, CHCH₃), 1.37 (3 H, t, J 7.2, OCH₂CH₃), 2.15–2.25 (1 H, m, 4-H), 2.72 (2 H, dd, J 6.9 and 4.1, 3-H₂) and 4.32 (2 H, q, J 7.2, OCH₂CH₃); δ_C 14.0 (OCH₂CH₃), 22.4 (¹³CH₃), 24.1 (d, J 29, C-4), 47.8 (C-3), 62.3 (OCH₂CH₃), 161.3 (C-1) and 194.4 (C-2).

(2S,4R)-[5-¹³C]Leucine 17.. The reaction was carried out as described in the general procedure using ethyl (4R)-[5-¹³C]-4-methyl-2-oxopentanoate 16 (340 mg, 2.1 mmol). Purification by ion-exchange chromatography gave (2S,4R)-[5-¹³C]leucine 17 (220 mg, 79%), as an off-white solid; mp 280–285 °C (decomp.) (lit.,²⁰ unlabelled leucine 293–295 °C); [α]_D +11.3 (c 1.56 in 6 M HCl) [lit.,⁴⁸ unlabelled leucine + 15.2 (c 1.58 in 6 M HCl)]; δ_H (400 MHz, D₂O) 0.94 (3 H, br t, J 5.5, CH₃), 0.95 (3 H, dd, J 125.1 and 5.7, ¹³CH₃), 1.63–1.77 (3 H, m, 3-H₂ and 4-H) and 3.71 (1 H, m, 2-H); δ_C (D₂O) 21.7 (slightly enriched CH₃), 22.8 (enriched CH₃), 25.0 (d, J 35, C-4), 40.6 (C3), 54.2 (d, J 3, C-2) and 176.3 (C-1); m/z (CI, aqueous methanolic solution) 133 [(M + H)⁺, 133.1057, C₅¹³CH₁₄NO₂ requires 133.1058, 28%] and 87 (100).

[¹³C]Methyllithium.. A solution of [¹³C]iodomethane (6.0 g, 42 mmol) in ether (26 mL) was added slowly to stirred lithium metal (wire, 1% sodium, freshly cut into small pieces and washed with light petroleum, 787 mg, 113 mmol) floating on ether (4 mL), at 0 °C, under an argon atmosphere. Effervescence occurred during the addition. The reaction mixture was stirred under an argon atmosphere at 0 °C for 30 min and then at rt for 4.5 h. The resultant [¹³C]methyllithium solution was left to settle (stored in a refrigerator) and its concentration (1.25 M) determined by quenching an aliquot (100 μL) with 0.1 M hydrochloric acid (5 mL) and back titration with 0.0926 M sodium hydroxide (phenolphthalein).

(1S)-N-{(3R)-[4-¹³C]-3-Methylbutanoyl}-1,10-camphorsultam 20.. A solution of [¹³C]methyllithium (0.46 M solution in Et₂O, 30.9 mL, 18.4 mmol) in ether (37 mL) was added dropwise to a stirred solution of copper(I) iodide–tri-n-butylphosphine complex (3.62 g, 9.2 mmol) in toluene (25 mL) at −5 °C. During the addition the reaction mixture became bright yellow then faded until colourless. The solution was stirred at −5 °C for 0.5 h, then cooled to −78 °C. A solution of (1S)-N-(E )-crotonyl-1,10-camphorsultam 19 ⁴⁹ (1.06 g, 3.5 mmol) in toluene (40 mL) was added slowly to the homocuprate solution (during the addition the reaction mixture turned yellow again) and the resultant mixture was stirred at −78 °C for 6 h. The reaction was quenched at −78 °C with a suspension of sat. ammonium chloride (40 mL) in THF (40 mL) and warmed to rt. The layers were separated and the aqueous layer extracted with ether (3 × 50 mL). The combined organic extracts were washed with sat. ammonium chloride solution (30 mL), dried over magnesium sulfate, filtered and evaporated. Column chromatography of the resultant cloudy white oil yielded a 10∶1 mixture of (1S)-N-{(3S)-[4-¹³C]-3-methylbutanoyl}-1,10-camphorsultam 20 and (1S)-N-{(3R)-[4-¹³C]-3-methylbutanoyl}-1,10-camphorsultam (1.0 g, 89%) as a white solid; mp 126–127 °C (lit.,⁵⁰ unlabelled material 129–130 °C); [α]_D −88.57 (c 1.18 in CHCl₃) [lit.,⁵⁰ unlabelled material −89.16 (c 1.14 in CHCl₃)]; δ_H (400 MHz) 0.95–0.99 (6 H, overlapping signals, 8-H₃ or 9-H₃ and CHCH₃), 0.96 (0.5 H,† dd, J 125.2 and 6.6, ¹³CH₃ minor diastereomer), 0.97 (3 H, s, CH₃), 0.97 (2.5 H,† dd, J 125.2 and 6.8, ¹³CH₃ major diastereomer), 1.16 (3 H, s, 8-H₃ or 9-H₃), 1.32–1.44 (2 H, m, 5-H and 6-H), 1.87–1.96 (3 H, m, 4-H, 5-H and 6-H), 2.08–2.10 (2 H, m, 3-H₂), 2.23 (1 H, nonet d, J 6.8 and 3.8, CHCH₃), 2.52 (1 H, ddd, J 15.6, 6.8 and 3.4, COCH H), 2.66 (1 H, ddd, J 15.7, 7.1 and 4.6, COCHH ), 3.43 (1 H, d, J 13.9, 10-H H), 3.49 (1 H, d, J 13.9, 10-HH ) and 3.88 (1 H, t, J 6.4, 2-H); δ_C (100 MHz) 19.9, 20.8 (C-8 and C-9), 22.3 (slightly enriched CH₃), 22.3 (enriched CH₃), 25.6 (d, J 35, CHCH₃), 26.5, 32.9 (C-5 and C-6), 38.6 (C-3), 44.2 (COCH₂), 44.7 (C-4), 47.7, 48.3 (C-1 and C-7), 53.1 (C-10), 65.2 (C-2) and 171.5 (CO); m/z 300 (M⁺, 300.1574, C₁₄¹³CH₂₅NO₃S requires 300.1589, 3%), 285 (1), 257 (22), 151 (12), 135 (25), 134 (25), 108 (17), 93 (16), 86 (100) and 58 (57).

(S)-[4-¹³C]-3-Methylbutanoic acid 21.. Hydrogen peroxide solution (30%, 3.3 mL, 29 mmol) and lithium hydroxide monohydrate (816 mg, 19 mmol) were added successively to a solution of (1S)-N-{(3S)-[4-¹³C]-3-methylbutanoyl}-1,10-camphorsultam 20 (3.65 g, 9.7 mmol) in THF (9 mL) and water (3 mL) at 0 °C. After stirring for 2 h at 0 °C and overnight at rt, the solution was cooled to 0 °C, sodium sulfite [3.7 g in water (20 mL)] was added and the solution stirred at 0 °C for a further 15 min. The solution was adjusted to pH 9–10 with sat. sodium hydrogen carbonate solution, the THF evaporated and the residual aqueous solution extracted with dichloromethane (2 × 100 mL). The organic extracts were dried over sodium sulfate and evaporated to yield crude (1S)-1,10-camphorsultam 18 (1.77 g, 85%). The aqueous solution was adjusted to pH 1–2 with 1 M sulfuric acid and extracted with ether (3 × 40 mL). The combined extracts were dried over sodium sulfate and evaporated to yield (S)-[4-¹³C]-3-methylbutanoic acid 21 ⁵¹ (930 mg, 93%) as a colourless liquid; δ_H (270 MHz) 0.99 (3 H, dd, J 6.6 and 5.3, CH₃), 0.99 (3 H, dd, J 125.2 and 6.6, ¹³CH₃), 2.05–2.17 (1 H, m, CH), 2.21–2.26 (2 H, m, CH₂) and 8.85 (1 H, br s, CO₂H); δ_C 22.4 (enriched CH₃), 25.5 (d, J 35, CH), 43.2 (CH₂) and 179.6 (d, J 3, CO₂H) .

Methyl (S)-[5-¹³C]-4-Methyl-3-oxopentanoate 24.. (Cyanomethylene)triphenylphosphorane hydrochloride (prepared as previously described⁵²) (9.45 g, 28 mmol) was dissolved in water (60 mL) and the solution washed with dichloromethane (60 mL). The aqueous layer was stirred with sodium hydroxide [1.68 g, 42 mmol in water (30 mL)] and dichloromethane (60 mL) for 10 min at rt. The layers were separated and the aqueous layer extracted with dichloromethane (60 mL). The combined organic extracts were dried over magnesium sulfate, filtered and evaporated until a precipitate just formed. Sufficient dichloromethane was added to redissolve the precipitate.

A mixture of (S)-[4-¹³C]-3-methylbutanoic acid 21 (0.96 g, 9.3 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (2.7 g, 14 mmol) and 4-dimethylaminopyridine (DMAP) (110 mg, 0.9 mmol) in dichloromethane (120 mL) was stirred at 0 °C for 10 min before the solution of (cyanomethylene)triphenylphosphorane 22 was added dropwise over ca. 20 min. The mixture was warmed to rt and stirred overnight. Water (200 mL) was added, the layers separated and the aqueous layer extracted with dichloromethane (2 × 200 mL). The combined organic extracts were washed successively with sat. sodium hydrogen carbonate solution (100 mL) and brine (100 mL), dried over magnesium sulfate, filtered and evaporated. Column chromatography of the resultant yellow oil with 5–10% ethyl acetate in dichloromethane as eluent yielded (S)-[6-¹³C]-5-methyl-3-oxo-2-triphenylphosphoranylidenehexanenitrile 23 (2.87 g, 80%) as an off-white solid; δ_H (270 MHz) 0.96 (3 H, dd, J 6.6 and 5.3, CH₃), 0.96 (3 H, dd, J 124.7 and 6.6, ¹³CH₃), 2.21 (1 H, nonet d, J 6.8 and 3.7, 5-H), 2.58 (2 H, dd, J 7.1 and 4.3, 4-H₂) and 7.46–7.66 (15 H, m, Ph); δ_C (67.9 MHz) 22.5 (enriched, CH₃), 25.8 (d, J 36, C-5), 48.1 (d, J 8, C-4), 49.3 (d, J 126, C-2), 122.6 (d, J 16, C-1), 123.3 (d, J 94, Ph ipso-C), 128.9 (d, J 13, aromatic), 132.8 (d, J 2, aromatic), 133.4 (d, J 10, aromatic) and 196.7 (C-2).

Ozone was passed through a stirred solution of (5S)-[6-¹³C]-5-methyl-3-oxo-2-triphenylphosphoranylidenehexanenitrile 23 (2.87 g, 7.4 mmol) in dichloromethane (60 mL) and methanol (30 mL) at −78 °C until the solution remained blue (ca. 2 h). The solution was then purged with oxygen, at −78 °C, until colourless and finally warmed to rt. The reaction mixture was evaporated to dryness and the resultant yellow solid purified by column chromatography with 25% light petroleum in chloroform as eluent to yield methyl (4S)-[5-¹³C]-4-methyl-3-oxopentanoate 24 (810 mg, 76%) as a colourless liquid (lit. data on unlabelled material ⁵³); δ_H (270 MHz) 0.97 (3 H, dd, J 6.6 and 5.3, CH₃), 0.97 (3 H, dd, J 125.4 and 6.7, ¹³CH₃), 2.20 (1 H, nonet d, J 6.7 and 3.8, 4-H), 2.73 (2 H, dd, J 6.8 and 4.2, 3-H₂) and 3.87 (3 H, s, CO₂CH₃); δ_C (67.9 MHz) 22.4 (enriched, CH₃), 24.2 (d, J 35, C-4), 47.9 (C-3), 52.8 (OCH₃), 161.7 (C-1) and 193.9 (C-2).

(2S,4S)-[5-¹³C]Leucine 25.. The reaction was carried out as described in the general procedure using methyl (4S)-[5-¹³C]-4-methyl-2-oxopentanoate 24 (810 mg, 5.6 mmol). Purification by ion-exchange chromatography gave (2S,4S)-[5-¹³C]leucine 25 (600 mg, 81%), as a pale yellow solid, mp 286 °C (decomp.); [α]_D −10.5 (c 2.0 in water) [lit.,⁵⁴ unlabelled leucine −10.7 (c 2.0 in water)]; δ_H (400 MHz, D₂O) 0.95 (3 H, dd, J 125.0 and 6.1, ¹³CH₃), 0.96 (3 H, br t, J 5.5, CH₃), 1.63–1.78 (3 H, m, 3-H₂ and 4-H) and 3.71 (1 H, m, 2-H); δ_C (67.9 MHz, D₂O) 20.8 (enriched CH₃), 22.0 (slightly enriched CH₃), 23.7 (d, J 35, C-4), 40.7 (C-3), 53.4 (C-2) and 175.4 (C-1).

(1R,2S,3R)-2-[(E )-Crotonyl]-3-[N-phenylsulfonyl-N-(3,5-dimethylphenyl)amino]bornan-2-ol 27.. Crotonyl chloride (2.0 mL, 21 mmol) was added to (1R,2S,3R)-3-[N-phenylsulfonyl-N-(3,5-dimethylphenyl)amino]bornan-2-ol 26 (1.19 g, 2.9 mmol) in carbon tetrachloride (100 mL) and the solution heated under reflux in the presence of 4 Å molecular sieves overnight. After the reaction mixture had been cooled to rt, the carbon tetrachloride was removed in vacuo. The residual oil was dissolved in ethyl acetate (20 mL) and washed with sat. sodium hydrogen carbonate solution (30 mL). The aqueous layer was extracted with ethyl acetate (30 mL). The combined organic extracts were dried over sodium sulfate, filtered and evaporated. The crude product was recrystallised from dichloromethane and light petroleum to give (1R,2S,3R)-2-[(E )-crotonyl]-3-[N-phenylsulfonyl-N-(3,5-dimethylphenyl)amino]bornan-2-ol 27 (1.12 g, 81%) as a white solid, mp 166–167 °C (data not previously reported ^33b); [α]_D +1.51 (c 1.96 in CHCl₃); ν_max (Nujol)/cm⁻¹ 1719 (CO), 1660 (C[double bond, length half m-dash]C), 1607 and 1593 (C[double bond, length half m-dash]C, Ar), δ_H (300MHz) 0.65, 0.78 and 0.99 (3 × 3 H, 3 × s, 8-H₃, 9-H₃ and 10-H₃), 1.08, 1.35, 1.53, 1.72 (each 1 H, m, 5-H₂ and 6-H₂), 1.93 (3 H, dd, J 7, 2, CHCH₃), 2.07 (3 H, br s, Ar-CH₃), 2.16 (1 H, m, 4-H), 2.26 (3 H, br s, Ar-CH₃), 3.82 (1 H, d, J 7, 3-H), 5.21 (1 H, d, J 7, 2-H), 5.91 (1 H, dq, J 15.5, 2, COCH), 6.03 (1 H, br s, Ar-H), 6.83 (2 H, br s, Ar-H), 7.03 (1 H, dq, J 15.5, 7, COCHCH ), 7.44 (5 H, m, Ar-H); δ_C 11.2, 20.8 and 21.2 (C-8, C-9 and C-10), 18.1 (CHCH₃), 21.1 (Ar-CH₃), 27.6 and 32.0 (C-5 and C-6), 47.4 and 50.2 (C-1 and C-7), 48.6 (C-4), 67.3 (C-3), 80.4 (C-2), 123.1 (COCH), 128.1, 128.3, 129.2, 132.4, 137.0 and 139.2 (C-Ar), 144.1 (COCHCH) and 165.4 (CO); m/z (CI) 482 [(M + H)⁺, 482.2372, C₂₈H₃₆NO₄S requires 482.2365, 12%], 396 (74), 341 (100), 254 (70), 226 (8).

(1R,2S,3R)-2-{(3R)-[4-¹³C]-3-Methylbutanoyl}-3-[N-phenylsulfonyl-N-(3,5-dimethylphenyl)-amino]bornan-2-ol 28.. Freshly prepared [¹³C]methyllithium (3.0 mmol) was added dropwise to copper(I) iodide (571 mg, 3.0 mmol) in ether (5.5 mL) at −10 °C. The mixture turned yellow and was stirred at −10 °C for 20 min, before cooling to −78 °C. Boron trifluoride–diethyl ether (0.37 mL, 3.0 mmol) was added dropwise and the yellow solution stirred at −78 °C for 15 min. A solution of (1R,2S,3R)-2-[(E )-crotonyl]-3-[N-phenylsulfonyl-N-(3,5-dimethylphenyl)amino]bornan-2-ol 26 (204 mg, 0.42 mmol) in ether (8 mL) was added dropwise and the reaction mixture stirred at −78 °C for 1 h, −40 °C for 3 h and finally −20 °C for 3 h. The reaction was quenched at −20 °C with sat. ammonium chloride solution (50 mL), before warming to rt. The layers were separated and the aqueous layer extracted with ether (3 × 50 mL). The combined organic extracts were washed with sat. ammonium chloride solution (50 mL), dried over sodium sulfate, filtered and evaporated to yield a yellow solid. Purification by column chromatography, eluting with 0–10% ethyl acetate in light petroleum gave (1R,2S,3R)-2-{(3R)-[4-¹³C]-3-methylbutanoyl}-3-[N-phenylsulfonyl-N-(3,5-dimethylphenyl)amino]bornan-2-ol 28 as a white solid (165 mg, 79%); mp 173–174 °C; [α]_D −3.8 (c 1.05 in CHCl₃); ν_max (CHCl₃)/cm⁻¹ 1733 (CO); δ_H [500 MHz; (CD₃)₂CO] 0.63, 0.79 and 0.94 (3 × 3 H, 3 × s, 8-H₃, 9-H₃ and 10-H₃), 0.99 (3 H, dd, J 125, 6.5, ¹³CH₃), 1.00 (3 H, dd, J 6.4, 5.3, CHCH₃), 1.22, 1.28, 1.54 and 1.70 (each 1 H, each m, 5-H₂ and 6-H₂), 2.04–2.34 (10 H, m, 2 × Ar-CH₃, 4-H, and COCH₂CH ), 4.00 (1 H, d, J 7.0, 3-H), 5.17 (1 H, d, J 7.0, 2-H), 6.05 (1 H, br s, Ar-H), 6.90 (1 H, br s, Ar-H), 6.95 (1H, br s, Ar-H), 7.43–7.64 (5 H, m, Ar-H); δ_C [75MHz, (CD₃)₂CO] 11.9, 21.1, 21.2, 21.8 and 25.5 (C-8, C-9, C-10 and 2 × Ar-CH₃), 22.9 (¹³CH₃), 23.0 (CHCH₃), 28.0 and 32.8 (C-5 and C-6), 44.2 (COCH₂), 48.0 and 50.7 (C-1 and C-7), 49.4 (C-4), 68.0 (C-3), 81.6 (C-2), 129.0, 129.2, 129.8, 133.5, 138.5 and 140.3 (C-Ar), 172.0 (CO); m/z (CI) 499 [(MH)⁺, 499.2714, C₂₈¹³CH₄₀NO₄S requires 499.2712, 13%], 396 (50), 358 (100), 254 (90), 85 (36).

Acknowledgements

We thank EPSRC for CASE studentships (RAH and NMK) and a postdoctoral fellowship (MDF ), University of Bristol for a Scholarship (AS) and Amersham International (NMK) and Zeneca Pharmaceuticals (RAH) for financial support. We are grateful to Drs R. G. Reid and P. Winton for valuable discussions.

References

1. N. M. Kelly, A. Sutherland and C. L. Willis, Nat. Prod. Rep., 1997, 14, 205.
2. F. J. Winkler, K. Kuhnl, R. Medina, R. Schwarz-Koske and H. L. Schmidt, Isot. Environ. Health Stud., 1995, 31, 161.
3. D. W. Young, Top. Stereochem., 1994, 21, 381.
4. N. Sitachitta, J. Rossi, M. A. Roberts, W. H. Gerwick, M. D. Fletcher and C. L. Willis, J. Am. Chem. Soc., 1998, 120, 7131.
5. R. Cardillo, C. Fuganti, D. Ghiringhelli, P. Grasselli and G. Gatti, J. Chem. Soc., Chem. Commun., 1977, 474.
6. C. Fuganti, P. Grasselli and G. Pedrocchi-Fantoni, Tetrahedron Lett., 1979, 20, 2453.
7. P. Anastasis, I. Freer, K. H. Overton, D. Picken, D. S. Rycroft and S. B. Singh, J. Chem. Soc., Perkin Trans. 1, 1987, 2427.
8. D. W. Young, Chem. Soc. Rev., 1994, 23, 119.
9. M. P. Crump, J. Crosby, C. E. Dempsey, J. A. Parkinson, M. Murray, D. A. Hopwood and T. J. Simpson, Biochemistry, 1997, 36, 6000.
10. S. R. Sylvester and C. M. Stevens, Biochemistry, 1979, 18, 4529; S. R. Sylvester, S. Y. Lan and C. M. Stevens, Biochemistry, 1981, 20, 5609.
11. D. J. Aberhart and B. H. Weiller, J. Labelled Compd. Radiopharm., 1983, 20, 663.
12. R. K. Hill, C. Abacherli and S. Hagishita, Can. J. Chem., 1994, 72, 110.
13. J. C. Shuttuck and J. Meinwald, Tetrahedron Lett., 1997, 38, 8461.
14. R. A. August, J. A. Khan, C. M. Moody and D. W. Young, Tetrahedron Lett., 1992, 33, 4617; R. A. August, J. A. Khan, C. M. Moody and D. W. Young, J. Chem. Soc., Perkin Trans. 1, 1996, 507.
15. M. Oba, T. Terauchi, A. Miyakawa, H. Kamo and K. Nishiyama, Tetrahedron Lett., 1998, 39, 1595.
16. N. M. Kelly, R. G. Reid, C. L. Willis and P. L. Winton, Tetrahedron Lett., 1995, 36, 8315.
17. D. A. Evans, J. Bartroli and T. L. Shih, J. Am. Chem. Soc., 1981, 103, 2127; D. A. Evans, M. D. Ennis and D. J. Mathre, J. Am. Chem. Soc., 1982, 104, 1737.
18. For reviews of chiral auxiliaries derived from amino acids, including Evans’ auxiliaries see: (a) D. J. Ager, I. Prakash and D. R. Schaad, Chem. Rev., 1996, 96, 835; (b) A. Studer, Synthesis, 1996, 793.
19. For reviews of α-keto esters and acids see: (a) A. J. L. Cooper, J. Z. Ginos and A. Meister, Chem. Rev., 1983, 83, 321; (b) L. Kovács, Recl. Trav. Chim. Pays-Bas, 1993, 112, 471.
20. J. Buckingham , S. M. Donaghy , J. I. G. Cadogan , R. A. Raphael and C. W. Rees , Dictionary of Organic Compounds, Chapman and Hall, New York, 1982..
21. Z. Shaked and G. M. Whitesides, J. Am. Chem. Soc., 1980, 102, 7104.
22. N. M. Kelly, R. G. Reid, C. L. Willis and P. L. Winton, Tetrahedron Lett., 1996, 37, 1517.
23. M. J. Garson and J. Staunton, Chem. Soc. Rev., 1979, 8, 539.
24. N. M. Kelly, B. C. O’Neill, J. Probert, G. Reid, R. Stephen, T. Wang, C. L. Willis and P. Winton, Tetrahedron Lett., 1994, 35, 6533.
25. E. L. Eliel and A. A. Hartmann, J. Org. Chem., 1972, 37, 505; G. S. Bates , in Encyclopedia of Reagents for Organic Synthesis, ed. L. A. Paquette, Wiley, Chichester, 1995, vol. 4, p. 2623..
26. D. A. Evans, T. C. Britton and J. A. Ellman, Tetrahedron Lett., 1987, 28, 6141.
27. M. Imoto, S. Kusumoto and T. Shiba, Tetrahedron Lett., 1987, 28, 6235.
28. E. J. Corey and B. W. Erickson, J. Org. Chem., 1971, 36, 3553.
29. For reviews of stereoselective conjugate additions see for example: (a) B. E. Rossiter and N. M. Swingle, Chem. Rev., 1992, 92, 771; (b) A. Alexakis , in Organocopper Reagents: A Practical Approach, ed. R. J. K. Taylor, Oxford University Press, Oxford, 1994, p. 159;; (c) P. Perlmutter , Conjugate Addition Reactions in Organic Synthesis, Pergamon, Oxford, 1992..
30. G. Pourcelot, J. Aubouet, A. Caspar and P. Cresson, J. Organomet. Chem., 1987, 328, C43.
31. (a) Y. Han and V. J. Hruby, Tetrahedron Lett., 1997, 38, 7317; (b) W. Yuan and V. J. Hruby, Tetrahedron Lett., 1997, 38, 3853; (c) X. Qian, K. C. Russell, L. W. Boteju and V. J. Hruby, Tetrahedron, 1995, 51, 1033and references therein..
32. (a) D. R. Williams, W. S. Kissel and J. J. Li, Tetrahedron Lett., 1998, 39, 8593; (b) D. R. Williams and J. J. Li, Tetrahedron Lett., 1994, 35, 5113.
33. (a) W. Oppolzer, R. J. Mills, W. Pachinger and T. Stevenson, Helv. Chim. Acta, 1986, 69, 1542; (b) M. Vandewalle, J. Van der Eycken, W. Oppolzer and C. Vullioud, Tetrahedron, 1986, 42, 4035; (c) W. Oppolzer, G. Poli, A. J. Kingma, C. Starkemann and G. Bernardinelli, Helv. Chim. Acta, 1987, 70, 2201; (d) W. Oppolzer, A. J. Kingma and G. Poli, Tetrahedron, 1989, 45, 479; (e) W. Oppolzer and A. J. Kingma, Helv. Chim. Acta, 1989, 72, 1337; (f) W. Oppolzer, Pure Appl. Chem., 1990, 62, 1241; (g) W. Oppolzer, Tetrahedron, 1987, 43, 1969.
34. H. H. Wasserman and W.-B. Ho, J. Org. Chem., 1994, 59, 4364.
35. (a) G. Helmchen and G. Wegner, Tetrahedron Lett., 1985, 26, 6051; (b) G. Helmchen and G. Wegner, Tetrahedron Lett., 1985, 26, 6047; (c) E. Urban, G. Riehs and G. Knühl, Tetrahedron Lett., 1995, 36, 4773; (d) E. Urban, G. Knühl and G. Helmchen, Tetrahedron, 1996, 52, 971.
36. Y. Yamamoto and K. Maruyama, J. Am. Chem. Soc., 1978, 100, 3240; Y. Yamamoto, S. Yamamoto, H. Yatagai, Y. Ishihara and K. Maruyama, J. Org. Chem., 1982, 47, 119.
37. T. Ibuka and Y. Yamamoto , in Organocopper Reagents: A Practical Approach, ed. R. J. K. Taylor, Oxford University Press, Oxford, 1994, p. 143..
38. W. Oppolzer, R. Moretti, T. Godel, A. Meunier and H. Löher, Tetrahedron Lett., 1983, 24, 4971.
39. J. A. MacRitchie, A. Silcock and C. L. Willis, Tetrahedron: Asymmetry, 1997, 8, 3895.
40. R. J. K. Taylor and G. Casy , in Organocopper Reagents: A Practical Approach, ed. R. J. K. Taylor, Oxford University Press, Oxford, 1994, p. 27..
41. G. B. Kauffman and L. A. Teter, Inorg. Synth., 1963, 7, 9.
42. A. S. Kende, K. Kawamura and M. J. Orwat, Tetrahedron Lett., 1989, 30, 5821.
43. J. Singh, T. P. Kissick and R. H. Mueller, Org. Prep. Proced. Int., 1989, 21, 501.
44. J. H. Babler, C. J. Marcuccilli and J. E. Oblong, Synth. Commun., 1990, 20, 1831.
45. K. A. Reynolds, D. O’Hagan, D. Gani and J. A. Robinson, J. Chem. Soc., Perkin Trans. 1, 1988, 3195.
46. J. K. MacDougall, M. C. Simpson and D. J. Cole-Hamilton, J. Chem. Soc., Dalton Trans., 1994, 3061.
47. M. F. Salomon, R. G. Salomon and R. D. Gleim, J. Org. Chem., 1976, 41, 3983.
48. Y. Shimojima, H. Hayashi, T. Ooka and M. Shibukawa, Tetrahedron, 1984, 40, 2519.
49. W. Oppolzer and J.-P. Barras, Helv. Chim. Acta, 1987, 70, 1666.
50. W. Oppolzer, O. Tamura and J. Deerberg, Helv. Chim. Acta, 1992, 75, 1965.
51. J. E. Baldwin, J. Lologer, W. Rastetter, N. Neuss, L. L. Huckstep and N. De La Higuera, J. Am. Chem. Soc., 1973, 95, 3796.
52. S. Trippett and D. M. Walker, J. Chem. Soc., 1959, 3874; G. P. Schiemenz and H. Engelhard, Chem. Ber., 1961, 94, 578.
53. H. Poisel, Chem. Ber., 1978, 111, 3136.
54. W. A. H. Huffman and A. W. Ingersoll, J. Am. Chem. Soc., 1951, 73, 3366.

---

Footnote

† Integral determined from decoupling experiment (irradiation at δ_H 2.23).

---