D-Glucose based syntheses of β-hydroxy derivatives of L-glutamic acid, L-glutamine, L-proline and a dihydroxy pyrrolidine alkaloid

Abstract

The β-hydroxy derivatives of L-glutamic acid, L-glutamine and L-proline, useful for peptide/protein studies, were synthesized starting from D-glucose. The C2 carbon in D-glucose provided the carboxylic acid functionality, while the amino and β-hydroxy groups of the amino acids were amenable from the C3 and C4 hydroxy groups of the sugar, respectively. The key intermediate with appropriate carbon framework of the target molecules was constructed by homologation of a suitable azido-D-glucofuranose derivative using the Arndt–Eistert reaction.

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Introduction

Amino acids and monosaccharides constitute the major building blocks of the complex molecular systems that are vital for life. There has been a constant effort to understand the structures and functions of such systems by synthesizing them either chemically or biologically.¹ Hence the importance of modified amino acids as ligating agents, in the synthesis of natural proteins has received considerable attention over the years.² These amino acids are also the key ingredients for the synthesis of modified proteins, that help in understanding the structure–activity relationship,³ and lantibiotic^4a,b study of the peptides of interest, besides providing peptidomimetic drugs.^4c Native chemical ligation (NCL) forms the basis of modern chemical synthesis of native, modified or cyclic peptides and proteins of moderate sizes,⁵ and is extensively used to synthesize complex protein targets.^2,6

Although the NCL approach has enriched peptide ligation chemistry, the required thiol/selenol-containing amino acids, which are essential, are accessible only through lengthy syntheses. The nonproteinogenic amino acids, possessing suitably placed (at the β/γ-position) hydroxy group(s) along the side chain are useful precursors of the corresponding thiol and selenol derivatives, required for NCL. A few commercially available hydroxy derivatives of natural amino acids, such as, β-hydroxy phenylalanine, β-hydroxy valine, and β-hydroxy leucine have been transformed to the corresponding thiol intermediates and used in NCL.⁷ Meanwhile, the β/γ-hydroxy derivatives of glutamic acid, glutamine, lysine, arginine, and aspartic acid have also been synthesized in different laboratories, and their mercapto derivatives are proven residues for the assembly of peptides using NCL.⁸ Moreover, many of these hydroxy amino acids are constituents of several natural products with intrinsic biological function.⁹ Overall, both as unnatural building blocks and target compounds, the β/γ-hydroxylated amino acids are attractive synthetic targets. Consequently, several target-specific¹⁰ as well as multi-target oriented¹¹ syntheses of the hydroxy amino acids have been reported.

Designing a common strategy, for various bioactive molecules has vital significance in organic synthesis. This can provide an economically accessible pathway to an array of discrete compounds from a single starting molecule.¹² The natural amino acids glutamic acid (Glu), glutamine (Gln) and proline (Pro) possess a similar five-carbon skeleton. It was hypothesized that the synthesis of suitable hydroxy derivatives of these may be realized using a common strategy. Hence, in view of our interest in modified amino acid synthesis, applicable for protein synthesis and study,^13a–c we formulated a general strategy for synthesizing the β-hydroxy derivatives of Glu (1a), Gln (1b) and Pro (1c) starting from inexpensive D-glucose. The corresponding β-hydroxy azido acids were also synthesized as the masked amino acids, because similar compounds are proven candidates for Staudinger ligation in peptides/proteins syntheses.¹⁴ In addition, several derivatives of 1a–1c, possessing different orthogonal ester protections (Me/allyl/benzyl) were synthesized so that they can be converted to free acids under different reaction conditions. Finally, in view of our interests on iminosugars,^13d,e we have transformed one of the intermediates into a biologically important pyrrolidine alkaloid 2. The chemical structures of the target compounds are shown in Fig. 1. Amongst the chosen targets, L-glutamate is an important nutrient in biochemical pathways like gluconeogenesis and ammonia detoxification,^15a and also plays a major role in learning, memory and neuronal development in mammalian central nervous system.^15b,c

Fig. 1 Chemical structures of the synthesized compounds.

Results and discussion

In the retrosynthetic analysis, we conceived that the C2 carbon in D-glucose would furnish the carboxylic acid functionality, while the C3 and C4 hydroxy groups would provide the required amino/azido and β-hydroxy groups, respectively, of the targeted β-hydroxy amino acid derivatives. The synthesis commenced with the known D-glucose-derived azido aldehyde 3,^13e,16 which was subjected to Pinnick oxidation (NaClO₂/NaH₂PO₄/30% H₂O₂)¹⁷ to furnish the azido acid 4 in 91% yield. The acid 4 was activated as a mixed anhydride using ethyl chloroformate, and subsequently reacted with CH₂N₂ in Et₂O to give the α-diazo ketone 5 in 80% yield. Wolff rearrangement¹⁸ of 5 in the presence of PhCO₂Ag and Et₃N in MeOH afforded the homologated methyl ester 6 (55%) that served as the common intermediate for all the target amino acid derivatives.

As the first application of 6, we attempted its conversion to the β-hydroxy glutamic acid derivatives. To this end, its 1,2-acetonide group was deprotected using aqueous trifluoroacetic acid (TFA), and the resultant hemiacetal was subjected to cleavage with NaIO₄ to yield the intermediate azido aldehyde. This on Pinnick oxidation afforded the glutamic acid derivative 7, containing a formylated C-3 hydroxy group (79%, over three steps). The formyl group in 7 could be selectively de-masked with aqueous saturated NaHCO₃ in THF to obtain the hydroxy acid 8 (89%). Compound 7 was also transformed to a fully masked Glu derivative 9 (84%) by reacting with allyl bromide in the presence of NaHCO₃ in anhydrous DMF. As above, the formyl group in 9 could be selectively removed with NaHCO₃ in THF at room temperature to obtain the β-hydroxy diester 10 in 87% yield.

Amino acid 10 is not suitable for Fmoc-SPPS as the side chain methyl ester is not easily cleavable under acidic/reduction condition. Hence it was thought of synthesizing a benzyl ester derivative 14 which would also serve as an ideal starting material for the various C3-substituted glutamic acid derivatives. For this, the ester function in 6 was hydrolyzed using LiOH in aqueous THF to afford the carboxylic acid 11 (86%), which on treatment with benzyl chloroformate (CbzCl) in the presence of Et₃N and 4-dimethylaminopyridine (DMAP) afforded the benzyl ester 12 in 63% yield. The ester 12 was directly transformed to the hydroxy azido acid 13 by a one-pot four-steps reaction sequence. Thus, acidic hydrolysis of the 1,2-acetonide function of 12, NaIO₄ cleavage of the resultant diol to the intermediate aldehyde, followed by Pinnick oxidation and alkaline hydrolysis furnished the desired C-3 hydroxy acid 13 in 71% yield. This was esterified with allyl bromide and NaHCO₃ to afford another glutamic acid precursor 14 in 87% yield. Compound 14 is a template on which all the functionalities except the β-hydroxy group is protected and thus is a suitable precursor for the synthesis of C-3 substituted glutamic acid derivatives, e.g. β-mercapto glutamic acid. Such a transformation has already been established from similar hydroxy derivatives of various amino acids.^7,8 Next, to confirm the stereochemistry at α and β-carbon in 14 it is necessary to convert it to a known derivative of β-hydroxy glutamic acid. For this azido acid 13 was opted as suitable substrate thus, a one pot reduction of azide functionality and debenzylation of ester using 10% Pd/C in MeOH–HCl afforded the fully unmasked β-hydroxy glutamic acid 1a in 95% yield (Scheme 1). The spectral and analytical data of 1a wherein agreement with that reported.^10q

Scheme 1 Synthesis of β-hydroxy glutamic acid derivatives.

For the synthesis of the β-hydroxy glutamine 1b, the benzyl amide of compound 11 was envisaged to serve as the masked amino equivalent of glutamine. Hence, compound 11 was coupled with benzylamine using HBTU and HOBt in the presence of diisopropylethylamine (DIEA) in DMF to afford the desired amide 15 (62%). This was transformed to the acid 16 (72%, over 4 steps), following the same sequence of reactions used to transform 12 to 13. The acid 16 was converted to the N-benzyl azido analogue of β-hydroxy glutamine ester 17 (79%) by a base-catalyzed reaction with allyl bromide. However, catalytic hydrogenation of 16 over 10% Pd–C in MeOH even under a pressurized (80 psi H₂) condition led to reduction of the azide functionality only, and furnished the hydrochloride of β-hydroxy glutamyl benzamide 18 instead of the fully unprotected β-hydroxy glutamine hydrochloride 1b. Our attempts to transform 16 to the desired product 1b with HCO₂NH₄/10% Pd–C/MeOH at room temperature as well as under reflux were also unsuccessful.

In an alternative method, the acid 11 was converted to the amide 19 (90%) with di-tert-butyl dicarbonate ((Boc)₂O), (NH₄)HCO₃·NH₂CO₂NH₄ and pyridine in MeCN. This was converted to the acid 20 (vide supra), which on catalytic hydrogenation afforded 1b in 90% yield. Azido acids, similar to 13, 16, and 20 are reported to be candidates for Staudinger ligation¹⁴ in peptide/protein synthesis (Scheme 2).

Scheme 2 Synthesis of β-hydroxy glutamine derivatives.

Next, we focused our attention to the synthesis of the β-hydroxy proline hydrochloride 1c and its derivative 23. It was also realized that the intermediates, generated in the process may be transformed to the pyrrolidine derivatives such as 2 that are of our own interest as bioactive iminosugars.^13d,e In this direction, compound 6 was subjected to a catalytic transfer hydrogenation (HCO₂NH₄/10% Pd–C/MeOH) to afford the bicyclic lactam 21 in 91% yield via a tandem azide reduction and cyclization. The lactam 21 was reduced with LiAlH₄ in THF under refluxing conditions, and the resultant amine functionality protected with CbzCl to furnish the N-Cbz protected bicyclic intermediate 22 in 57% yield (over two steps). The carbamate 22 was subsequently transformed to 1c as reported earlier.¹⁰ⁱ We also synthesized the allyl ester of β-hydroxy proline from 22 without any purification of the intermediates. For this, compound 22 was sequentially subjected to an acid-catalyzed ketal hydrolysis, NaIO₄ cleavage, Pinnick oxidation and alkaline hydrolysis to obtain the crude acid. After drying in vacuo, the acid was subjected to a base-catalyzed allylation to furnish the β-hydroxy proline allyl ester 23 in 65% yield (over five steps). It is worth noting that the β-hydroxy esters 14, and 23 could also serve as precursors for functional group transformations at the free hydroxy group, because the subsequent deallylation can be accomplished under neutral and non-reducing conditions using a Pd(II) catalyst (Scheme 3).

Scheme 3 Synthesis of β-hydroxy proline derivatives and a pyrrolidine alkaloid.

For the synthesis of the pyrrolidine iminosugar 2, the carbamate 22 was treated with aqueous TFA to unmask the acetonide group, and the resultant diol cleaved with NaIO₄ to yield an aldehyde, which on NaBH₄ reduction afforded the N-Cbz protected pyrrolidine 24. In the final step, the amino functionality in 24 was deprotected by catalytic hydrogenation over 10% Pd–C in MeOH to afford the desired dihydroxypyrrolidine 2 in 84% yield. Compound 2 is a versatile precursor for the 3,4-cis-substituted aza-sugars that show a wide range of biological activity. To our surprise unlike its enantiomer, only a few synthesis of 2 have been reported.¹⁹

Conclusions

In summary, we have devised an important strategy for the synthesis of β-hydroxy derivatives of glutamic acid, proline, glutamine, and a dihydroxy pyrrolidine alkaloid. Using this pathway different orthogonally protected hydroxy equivalent of glutamic acid, glutamine and proline are achievable. Noticeably, similar hydroxy amino acids with their functionalities protected as in 14, and 23 have been used for the synthesis of corresponding thiol derivatives and has been used for peptide ligation (NCL). Inexpensive reagents, cheap starting materials, and simple chemical transformations make this strategy a useful one for the synthesis of various protecting group variants of glutamine, glutamic acid and proline. Our efforts to transform the hydroxy derivatives to mercapto variants and their application in peptide synthesis are in progress and will be reported elsewhere.

Experimental section

(3aR,5S,6R,6aR)-6-Azido-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carboxylic acid 4

To a stirred solution of 3 (3.31 g, 15.52 mmol) in MeCN (50 mL) were added NaH₂PO₄ (0.484 g, 3.10 mmol) in H₂O (5 mL) and aqueous 30% H₂O₂ (2.3 mL, 17.1 mmol). The mixture was cooled to 0 °C, NaClO₂ (2.24 g, 24.84 mmol) in H₂O (6 mL) was dropwise added in 0.5 h and stirred at 20 °C till completion of the reaction (cf. 12 h, monitored by gas evolution). The reaction mixture was treated with sodium sulphate (1.00 g), and extracted with EtOAc (3 × 30 mL). Evaporation of solvent and column chromatography (silica gel, 10% MeOH/CHCl₃) of the residue gave 4 (3.25 g, 91%) as a thick liquid. R_f = 0.30 (30% MeOH/CHCl₃); [α]²⁵_D −31.3 (c 1.08, CHCl₃); ν_max/cm⁻¹: 3430, 2108, 1683 cm⁻¹; ¹H NMR: δ 8.19 (broad s, D₂O exchangeable, 1H), 6.01 (d, J = 3.4 Hz, 1H), 4.86 (d, J = 3.7 Hz, 1H), 4.67 (d, J = 3.4 Hz, 1H), 4.33 (d, J = 3.7 Hz, 1H), 1.48 (s, 3H), 1.32 (s, 3H); ¹³C NMR: δ 171.2, 113.1, 105.2, 82.9, 78.1, 66.5, 26.6, 26.2. Anal. calcd for C₈H₁₁N₃O₅: C, 41.92; H, 4.84; N, 18.33%. Found: C, 41.99; H, 4.90; N, 18.42%.

3-Azido-6-diazo-3,6-dideoxy-1,2-O-isopropylidine-5-keto-α-D-xylo-1,4-furanose 5

To a cooled (0 °C) and stirred solution of 4 (3.12 g, 13.62 mmol) in THF (45 mL) was sequentially added Et₃N (2.27 mL, 16.33 mmol) and ethyl chloroformate (1.43 mL, 14.97 mmol). After 15 min, the mixture was brought to room temperature and filtered through Celite-545. CH₂N₂ [prepared from N-nitrosomethyl urea (2.00 g, 19.40 mmol) and KOH (5 g)] in Et₂O (50 mL) was dropwise added to the filtrate at 0 °C in 0.5 h. After stirring at room temperature for 2 h, the mixture was concentrated in vacuo, and the residue purified by column chromatography (silica gel, 10% EtOAc/hexane) gave 5 (2.78 g, 80%) as a thick liquid. R_f = 0.35 (20% EtOAc/hexane); [α]²⁵_D −90.3 (c 1.14, CHCl₃); ν_max/cm⁻¹: 2105, 1720 cm⁻¹; ¹H NMR: δ 5.95 (d, J = 3.3 Hz, 1H), 5.81 (s, 1H), 4.71 (d, J = 3.1 Hz, 1H), 4.62 (d, J = 3.3 Hz, 1H), 4.35 (d, J = 3.1 Hz, 1H), 1.48 (s, 3H), 1.32 (s, 3H); ¹³C NMR: δ 191.1, 112.7, 105.1, 82.8, 82.5, 66.7, 54.7, 26.4, 26.0. Anal. calcd for C₉H₁₁N₅O₄: C, 42.69; H, 4.38; N, 27.66%. Found: C, 42.75; H, 4.44; N, 27.74%.

Methyl[(3aR,5R,6S,6aR)-6-azido-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yfl]acetate 6

To a stirred solution of 5 (1.00 g, 3.95 mmol) in anhydrous MeOH (15 mL) was dropwise added silver benzoate (0.290 g, 1.26 mmol) in Et₃N (3 mL). After stirring at 25 °C for 20 min, the mixture was concentrated in vacuo, and the residue purified by column chromatography (silica gel, 5% EtOAc/hexane) to obtain 6 (0.560 g, 55%) as a thick liquid. R_f = 0.52 (20% EtOAc/hexane); [α]²⁵_D −59.1 (c 1.17, CHCl₃); ν_max/cm⁻¹: 2105, 1737, 1208 cm⁻¹; ¹H NMR: δ 5.81 (d, J = 3.7 Hz, 1H), 4.63 (d, J = 3.7 Hz, 1H), 4.60–4.44 (m, 1H), 4.08 (d, J = 3.2 Hz, 1H), 3.67 (s, 3H), 2.87–2.59 (m, 2H), 1.47 (s, 3H), 1.28 (s, 3H); ¹³C NMR: δ 170.6, 112.1, 104.1, 83.5, 75.4, 66.7, 51.9, 33.5, 26.5, 26.1; ESI-MS: calcd for [C₁₀H₁₅N₃O₅ + Na]⁺: 280.09 Da. Found: 279.88 Da. Anal. calcd for C₁₀H₁₅N₃O₅: C, 46.69; H, 5.88; N, 16.33%. Found: C, 46.67; H, 5.93; N, 16.43%.

(2S,3R)-2-Azido-3-(formyloxy)-5-methoxy-5-oxopentanoic acid 7

A solution of 6 (0.702 g, 2.73 mmol) in TFA–H₂O (3.00 mL, 3 : 2) was stirred at 0 °C for 6 h. Azeotropic removal of TFA with toluene in vacuo afforded the intermediate hemiacetal (0.700 g, thick liquid), which was taken in acetone/water (10 mL, 9 : 1), cooled to 0 °C and NaIO₄ (0.640 g, 2.99 mmol) added. After stirring for 0.5 h, the reaction mixture was concentrated in vacuo, the residue extracted with CHCl₃ (3 × 10 mL), and the extract concentrated in vacuo to get the crude α-azido aldehyde (0.503 g, thick liquid). This was dissolved in MeCN (5 mL), treated successively with NaH₂PO₄ (0.08 g, 0.53 mmol) in H₂O (1 mL) and 30% H₂O₂ (0.40 mL, 2.95 mmol), cooled to 0 °C, and NaClO₂ (0.39 g, 4.36 mmol) in H₂O (1.5 mL) added into it in 20 min. After stirring at 20 °C till completion of the reaction (∼10 h, monitored by gas evolution), the reaction mixture was treated with sodium sulphate (0.20 g), and extracted with EtOAc (3 × 15 mL). Concentration of the extract in vacuo followed by column chromatography (silica gel, 10% MeOH/CHCl₃) of the residue gave 7 (0.500 g, 79% in three steps) as a thick liquid. R_f = 0.30 (30% MeOH/CHCl₃); [α]²⁵_D −6.00 (c 1.0, CHCl₃); ν_max/cm⁻¹: 2111, 1701 cm⁻¹; ¹H NMR: δ 9.31–8.71 (broad m, 1H, D₂O exchangeable), 8.00 (s, 1H), 5.84–5.65 (m, 1H), 4.31 (d, J = 2.2 Hz, 1H), 3.68 (s, 3H), 2.84 (dd, J = 6.8, 1.2 Hz, 2H); ¹³C NMR: δ 171.6, 170.2, 159.9, 69.5, 62.3, 52.3, 35.2. Anal. calcd for C₇H₉N₃O₆: C, 36.37; H, 3.92; N, 18.18%. Found: C, 36.42; H, 3.98; N, 18.28%.

(2S,3R)-2-Azido-3-hydroxy-5-methoxy-5-oxopentanoic acid 8

To a cooled (0 °C) and stirred solution of 7 (0.141 g, 0.61 mmol) in THF (3 mL) was added aqueous saturated NaHCO₃ (1 mL). After stirring for 0.5 h, the reaction mixture was concentrated in vacuo, the residue acidified to pH 1 with aqueous 1 N HCl, and extracted with EtOAc (6 × 10 mL). The combined organic extracts were dried, concentrated in vacuo to obtain a residue, which on column chromatography (silica gel, 20% MeOH/CHCl₃) gave 8 (0.110 g, 89%) as a thick liquid. R_f = 0.30 (30% MeOH/CHCl₃); [α]²⁵_D −26.0 (c 1.10, CHCl₃); ν_max/cm⁻¹: 3510, 2105, 1713, 1206 cm⁻¹; ¹H NMR: δ 6.64 (broad s, D₂O exchangeable, 2H), 4.72–4.55 (m, 1H), 4.00 (d, J = 2.3 Hz, 1H), 3.71 (s, 3H), 2.78 (dd, J = 16.6, 8.3 Hz, 1H), 2.63 (dd, J = 16.6, 4.8 Hz, 1H); ¹³C NMR: δ 172.4, 68.7, 64.7, 52.3, 37.8. Anal. calcd for C₆H₉N₃O₅: C, 35.47; H, 4.47; N, 20.68%. Found: C, 35.44; H, 4.52; N, 20.77.

5-Methyl 1-prop-2-en-1-yl(2S,3R)-2-azido-3-(formyloxy)pentanedioate 9

To a solution of 7 (0.500 g, 2.16 mmol) in DMF (3 mL) at 0 °C was added NaHCO₃ (0.45 g, 5.40 mmol) followed by allyl bromide (0.23 mL, 2.70 mmol). The reaction mixture was stirred to 25 °C for 12 h, DMF was removed in vacuo, the residue extracted with EtOAc (3 × 10 mL), the organic extract dried and concentrated in vacuo. The product was purified by column chromatography (silica gel, 10% EtOAc/hexane) to afford 9 (0.493 g, 84%) as a viscous liquid. R_f = 0.40 (20% EtOAc/hexane); [α]²⁵_D −11.0 (c 1.16, CHCl₃); ν_max/cm⁻¹: 3524, 2109, 1721, 1211 cm⁻¹; ¹H NMR: δ 7.96 (s, 1H), 6.00–5.70 (m, 2H), 5.43–5.21 (m, 2H), 4.69–4.63 (m, 2H), 4.23 (d, J = 2.9 Hz, 1H), 3.67 (s, 3H), 2.80 (d, J = 6.9 Hz, 2H); ¹³C NMR: δ 169.6, 167.2, 159.1, 130.8, 119.8, 69.3, 66.9, 62.3, 52.1, 35.1. Anal. calcd for C₁₀H₁₃N₃O₆: C, 44.28; H, 4.83; N, 15.49%. Found: C, 44.25; H, 4.80; N, 15.61%.

5-Methyl 1-prop-2-en-1-yl(2S,3R)-2-azido-3-hydroxypentanedioate 10

Following the procedure used for 8, deformylation of 9 (0.230 g, 0.84 mmol) with aqueous saturated NaHCO₃ (1.5 mL) in THF (5 mL) followed by usual work up and column chromatography (silica gel, 10% EtOAc/hexane) afforded 10 (0.180 g, 87%) as a thick liquid. R_f = 0.30 (20% EtOAc/hexane); [α]²⁵_D −17.0 (c 1.74, CHCl₃); ν_max/cm⁻¹: 3611, 2102, 1716, 1202 cm⁻¹; ¹H NMR: δ 6.08–5.82 (m, 1H), 5.50–5.21 (m, 2H), 4.82–4.69 (m, 2H), 4.65–4.49 (m, 1H), 3.88 (d, J = 2.6 Hz, 1H), 3.71 (s, 3H), 2.76 (dd, J = 16.3, 7.6 Hz, 1H), 2.75–2.25 (broad s, D₂O exchangeable, 1H), 2.58 (dd, J = 16.3, 4.5 Hz, 1H); ¹³C NMR: δ 172.1, 168.3, 131.0, 119.5, 68.9, 66.7, 64.9, 52.1, 37.8. Anal. calcd for C₉H₁₃N₃O₅: C, 44.44; H, 5.39; N, 17.28%. Found: C, 44.51; H, 5.46; N, 17.36%.

[(3aR,5R,6S,6aR)-6-Azido-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl]acetic acid 11

To a cooled (0 °C) solution of 6 (1.20 g, 4.66 mmol) in THF (40 mL) was dropwise added an aqueous 0.3 M solution of LiOH (0.58 g, 13.99 mmol) over 30 min. After completion of reaction (cf. TLC, 30 min), the pH of the mixture was adjusted to 5–6 with aqueous saturated citric acid (20 mL), and extracted with EtOAc (3 × 30 mL). The combined organic extracts were dried, concentrated, and the residue purified by column chromatography (silica gel, 10% MeOH/CHCl₃) to give 11 (0.980 g, 86%) as a thick liquid. R_f = 0.30 (30% MeOH/CHCl₃); [α]²⁵_D −31.4 (c 1.0, CHCl₃); ν_max/cm⁻¹: 3584, 2102, 1742 cm⁻¹; ¹H NMR: δ 9.00–7.81 (broad s, D₂O exchangeable, 1H), 5.86 (d, J = 3.7 Hz, 1H), 4.68 (d, J = 3.7 Hz, 1H), 4.62–4.50 (m, 1H), 4.10 (d, J = 3.2 Hz, 1H), 2.94–2.65 (m, 2H), 1.51 (s, 3H), 1.31 (s, 3H); ¹³C NMR: δ 175.8, 112.3, 104.2, 83.5, 75.1, 66.7, 33.6, 26.5, 26.2. Anal. calcd for C₉H₁₃N₃O₅: C, 44.44; H, 5.39; N, 17.28%. Found: C, 44.40; H, 5.41; N, 17.39%.

Benzyl[(3aR,5R,6S,6aR)-6-azido-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl]acetate 12

To a solution of 11 (0.210 g, 0.86 mmol) in CH₃CN (5 mL) was added Et₃N (0.27 mL, 1.93 mmol), CbzCl (0.245 mL, 1.72 mmol) and DMAP (0.05 g, 0.43 mmol). After stirring for 2 h at 25 °C, another portion of CbzCl (0.120 mL, 0.86 mmol) and DMAP (0.03 g, 0.22 mmol) were added and the mixture stirred overnight. It was concentrated in vacuo and extracted with EtOAc (3 × 15 mL). The combined organic extracts were sequentially washed with aqueous NaHCO₃ (5 mL) and water (5 mL), and dried. Concentration of extract and column chromatography (silica gel, 5% EtOAc/hexane) of the residue afforded 12 (0.180 g, 63%) as a viscous liquid. R_f = 0.51 (10% EtOAc/hexane); [α]²⁵_D −28.5 (c 1.0, CHCl₃); ν_max/cm⁻¹: 2110, 1734 cm⁻¹; ¹H NMR: δ 7.34 (s, 5H), 5.84 (d, J = 3.7 Hz, 1H), 5.14 (s, 2H), 4.65 (d, J = 3.7 Hz, 1H), 4.59 (ddd, J = 8.2, 6.3, 3.3 Hz, 1H), 4.09 (d, J = 3.3 Hz, 1H), 2.96–2.66 (m, 2H), 1.49 (s, 3H), 1.31 (s, 3H); ¹³C NMR: δ 170.0, 135.5, 128.6, 128.4, 128.2, 112.2, 104.2, 83.6, 75.5, 66.8, 66.7, 33.9, 26.5, 26.2; ESI-MS: calcd for [C₁₆H₁₉N₃O₅ + Na]⁺: 356.12 Da. Found: 355.89 Da. Anal. calcd for C₁₆H₁₉N₃O₅: C, 57.65; H, 5.75; N, 12.61%. Found: C, 57.63; H, 5.78; N, 12.70%.

(2S,3R)-2-Azido-5-(benzyloxy)-3-hydroxy-5-oxopentanoic acid 13

As described earlier, 12 (0.100 g, 0.30 mmol) was deacetalized with TFA–H₂O (3 mL, 3 : 2), the resultant diol cleaved with NaIO₄ (0.072 g, 0.33 mmol) in 10% aqueous acetone (5 mL) followed by oxidation with NaH₂PO₄ (0.01 g, 0.06 mmol), 30% H₂O₂ (50 μL, 0.30 mmol) and NaClO₂ (0.05 g, 0.49 mmol). The product was finally deformylated with aqueous saturated NaHCO₃ (1 mL). Usual workup and column chromatography (silica gel, 2% MeOH/CHCl₃) of the residue afforded 13 (0.060 g, 71% over four steps) as a thick liquid. R_f = 0.31 (10% MeOH/CHCl₃); [α]²⁵_D −20.0 (c 1.01, CHCl₃); ν_max/cm⁻¹: 2112, 1725 cm⁻¹; ¹H NMR: δ 7.33 (s, 5H), 6.91 (broad s, D₂O exchangeable, 2H), 5.13 (s, 2H), 4.71–4.60 (m, 1H), 3.96 (d, J = 2.1 Hz, 1H), 2.80 (dd, J = 16.6, 8.3 Hz, 1H), 2.64 (dd, J = 16.6, 4.7 Hz, 1H); ¹³C NMR: δ 172.4, 171.7, 135.1, 128.6, 128.5, 128.3, 68.7, 67.0, 64.8, 38.0. Anal. calcd for C₁₂H₁₃N₃O₅: C, 51.61; H, 4.69; N, 15.05%. Found: C, 51.58; H, 4.72; N, 15.13%.

5-Benzyl 1-prop-2-en-1-yl(2S,3R)-2-azido-3-hydroxypentanedioate 14

Following the procedure used for 9, the acid 13 (0.078 g, 0.27 mmol) was subjected to allylation using allyl bromide (29.0 μL) and NaHCO₃ (0.056 g) in DMF (1 mL). Usual workup and column chromatography (silica gel, 5% EtOAc/hexane) of the residue afforded 14 (0.075 g, 87%) as a thick liquid. R_f = 0.38 (20% EtOAc/hexane); [α]²⁵_D −24.3 (c 1.08, CHCl₃); ν_max/cm⁻¹: 2108, 1742 cm⁻¹; ¹H NMR: δ 7.34 (s, 5H), 6.05–5.80 (m, 1H), 5.42–5.29 (m, 2H), 5.15 (s, 2H), 4.72 (broad d, J = 5.8 Hz, 2H), 4.65–4.53 (m, 1H), 3.87 (d, J = 3.1 Hz, 1H), 3.14 (s, D₂O exchangeable, 1H), 2.79 (dd, J = 16.7, 8.4 Hz, 1H), 2.62 (dd, J = 16.7, 4.5 Hz, 1H); ¹³C NMR: δ 171.5, 168.2, 135.2, 131.0, 128.7, 128.5, 128.3, 119.4, 68.9, 66.9, 66.7, 64.9, 38.0; ESI-MS: calcd for [C₁₅H₁₇N₃O₅ + Na]⁺: 342.10 Da. Found: 341.85 Da. Anal. calcd for C₁₅H₁₇N₃O₅: C, 56.42; H, 5.37; N, 13.16%. Found: C, 56.48; H, 5.43; N, 13.23%.

(2S,3R)-3-Hydroxy-L-glutamic acid hydrochloride 1a

A mixture of 13 (0.085 g, 0.31 mmol) and 10% Pd–C (0.02 g) in methanolic HCl (10 mL) was stirred for 12 h under H₂ (80 psi). The catalyst was filtered through Celite-545 and washed with MeOH (3 × 10 mL), concentrated and the residue dried in vacuo to afford 1a (0.048 g, 95%) as a semisolid. [α]²⁵_D +14.7 (c 1.02, H₂O); ν_max/cm⁻¹: 3577, 1737 cm⁻¹; ¹H NMR: δ 4.66–4.58 (m, 1H), 4.10 (d, J = 3.3 Hz, 1H), 2.87 (dd, J = 16.4, 4.0 Hz, 1H), 2.72 (dd, J = 16.4, 8.7 Hz, 1H); ¹³C NMR: δ 174.1, 170.3, 65.5, 57.3, 38.4; ESI-MS: calcd for [C₅H₉NO₅ + Na]⁺: 186.04 Da. Found: 186.10 Da. Anal. calcd for C₅H₁₀ClNO₅: C, 30.09; H, 5.05; N, 7.02%. Found: C, 30.13; H, 5.10; N, 7.13%.

2-[(3aR,5R,6S,6aR)-6-Azido-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl]-N-benzylacetamide 15

To a solution of 11 (0.080 g, 0.33 mmol) in DMF at 25 °C was added HBTU (0.14 g, 0.37 mmol), HOBt monohydrate (0.06 g, 0.37 mmol) and DIEA (0.17 mL, 0.99 mmol). After stirring for 5 min, benzylamine (0.04 mL, 0.38 mmol) in DMF (0.40 mL) was added, and the mixture stirred for an additional 8 h. The mixture was concentrated in vacuo, the residue extracted with EtOAc (3 × 20 mL), the organic extract washed with water (3 × 5 mL) and brine (1 × 5 mL), and dried. Concentration of the extract in vacuo, and column chromatography (silica gel, 12% EtOAc/hexane) of the residue yielded 15 (0.068 g, 62%) as a thick liquid. R_f = 0.55 (50% EtOAc/hexane); [α]²⁵_D −24.0 (c 1.05, CHCl₃); ν_max/cm⁻¹: 2103, 1689 cm⁻¹; ¹H NMR: δ 7.31–7.23 (m, 2H), 7.22–7.17 (m, 3H), 6.29 (broad s, D₂O exchangeable, 1H), 5.79 (d, J = 3.7 Hz, 1H), 4.59 (d, J = 3.7 Hz, 1H), 4.56–4.49 (m, 1H), 4.41 (dd, J = 14.8, 5.8 Hz, 1H), 4.34 (dd, J = 14.8, 5.6 Hz, 1H), 3.98 (d, J = 3.0 Hz, 1H), 2.62 (dd, J = 15.2, 7.7 Hz, 1H), 2.53 (dd, J = 15.2, 5.8 Hz, 1H), 1.43 (s, 3H), 1.25 (s, 3H); ¹³C NMR: δ 170.0, 137.7, 128.7, 127.6, 127.5, 112.4, 104.3, 83.4, 76.0, 67.1, 43.7, 36.2, 26.5, 26.2; ESI-MS: calcd for [C₁₆H₂₀N₄O₄ + Na]⁺: 355.13 Da. Found: 354.93 Da. Anal. calcd for C₁₆H₂₀N₄O₄: C, 57.82; H, 6.07; N, 16.86%. Found: C, 57.80; H, 6.05; N, 16.93%.

(2S,3R)-2-Azido-5-(benzylamino)-3-hydroxy-5-oxopentanoic acid 16

Following the procedure used for the synthesis of 13, compound 15 (0.110 g, 0.33 mmol) was deacetalized with TFA–H₂O (3 mL, 3 : 2), the resultant diol cleaved with NaIO₄ (0.08 g, 0.37 mmol) in 10% aqueous acetone (5 mL) followed by oxidation with NaH₂PO₄ (0.01 g), 30% H₂O₂ (35 μL) and NaClO₂ (0.05 g). The product was finally deformylated with aqueous saturated NaHCO₃ (1 mL). Usual workup and column chromatography (silica gel, 10% MeOH/CHCl₃) of the residue afforded 16 (0.067 g, 72% in four steps) as a thick liquid. R_f = 0.35 (30% MeOH/CHCl₃); [α]²⁵_D −19.0 (c 1.04, CHCl₃); ν_max/cm⁻¹: 3570, 2107, 1737, 1685 cm⁻¹; ¹H NMR: δ 7.34–7.23 (m, 5H), 7.18 (broad s, D₂O exchangeable, 1H), 4.43 (s, 1H), 4.34 (s, 2H), 3.74 (s, 1H), 2.49 (s, 2H); ¹³C NMR: δ 173.9, 172.0, 138.4, 128.1, 127.1, 126.7, 69.4, 67.5, 42.7, 39.9. Anal. calcd for C₁₂H₁₄N₄O₄: C, 51.80; H, 5.07; N, 20.13%. Found: C, 51.87; H, 5.13; N, 20.25%.

Prop-2-en-1-yl(2S,3R)-2-azido-5-(benzylamino)-3-hydroxy-5-oxopentanoate 17

Following the procedure described earlier, 16 (0.06 g, 0.22 mmol) was reacted with allyl bromide (20 μL) in the presence of NaHCO₃ (0.04 g) in DMF (2 mL). Usual workup and column chromatography (silica gel, 8% EtOAc/hexane) of the residue gave 17 (0.055 g, 79%) as a thick liquid. R_f = 0.41 (30% EtOAc/hexane); [α]²⁵_D −16.4 (c 0.55, CHCl₃); ν_max/cm⁻¹: 2105, 1744 cm⁻¹; ¹H NMR: δ 7.38–7.17 (m, 5H), 6.13–5.69 (m, partially D₂O exchangeable, 2H), 5.44–5.15 (m, 2H), 4.67 (broad d, J = 5.8 Hz, 2H), 4.62–4.46 (m, 1H), 4.38 (d, J = 5.7 Hz, 2H), 3.75 (d, J = 3.2 Hz, 1H), 2.58 (dd, J = 15.4, 8.9 Hz, 1H), 2.37 (dd, J = 15.4, 3.8 Hz, 1H), 1.58 (broad s, D₂O exchangeable, 1H); ¹³C NMR: δ 170.8, 168.4, 137.6, 131.1, 128.8, 127.8, 127.7, 119.4, 69.6, 66.7, 65.1, 43.7, 39.1; ESI-MS: calcd for [C₁₅H₁₈N₄O₄ + Na]⁺: 341.12 Da. Found: 340.92 Da. Anal. calcd for C₁₅H₁₈N₄O₄: C, 56.60; H, 5.70; N, 17.60%. Found: C, 56.55; H, 5.68; N, 17.69%.

(1S,2R)-4-(Benzylamino)-1-carboxy-2-hydroxy-4-oxobutan-1-aminium chloride 18

Catalytic hydrogenation of 16 (0.048 g, 0.17 mmol) over 10% Pd–C (0.015 g) in MeOH (5 mL) using H₂ (80 psi) gave 18 (0.044 g, 89%) as a semi-solid. [α]²⁵_D +22.0 (c 1.00, CHCl₃); ν_max/cm⁻¹: 1713, 1406 cm⁻¹; ¹H NMR: δ 7.44 (d, J = 3.9 Hz, 2H), 7.38 (s, 3H), 4.72–4.64 (m, 1H), 4.44 (s, 2H), 4.19 (d, J = 3.7 Hz, 1H), 2.82–2.76 (m, 1H), 2.70 (dd, J = 14.8, 8.8 Hz, 1H); ¹³C NMR: δ 173.6, 171.7, 139.5, 130.5, 129.2, 129.0, 67.8, 59.0, 44.8, 41.8; ESI-MS: calcd for [C₁₂H₁₆N₂O₄ + H]⁺: 253.11 Da. Obsd: 253.90 Da. Anal. calcd for C₁₂H₁₇ClN₂O₄: C, 49.92; H, 5.93; N, 9.70%. Found: C, 49.88; H, 5.96; N, 9.78%.

2-[(3aR,5R,6S,6aR)-6-Azido-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl]acetamide 19

To a solution of 11 (0.210 g, 0.87 mmol) in CH₃CN (10 mL) was added (Boc)₂O (0.246 g, 1.13 mmol), and NH₄HCO₃ (0.161 g, 2.04 mmol) to give a cloudy mixture. After adding pyridine (0.05 mL, 0.60 mmol), the mixture was stirred at room temperature till completion of the reaction (∼for 5 h, cf. TLC). The mixture was concentrated in vacuo, the residue extracted with EtOAc (3 × 15 mL), the organic extract washed with water (5 mL) and dried. Concentration of the extract in vacuo, and column chromatography (silica gel, 30% EtOAc/hexane) of the residue afforded 19 (0.189 g, 90%) as colorless crystals. mp: 122–125 °C; R_f = 0.45 (80% EtOAc/hexane); [α]²⁵_D −23.1 (c 1.18, MeOH); ν_max/cm⁻¹: 2111, 1665 cm⁻¹; ¹H NMR: δ 5.81 (d, J = 3.7 Hz, 1H), 4.72 (d, J = 3.7 Hz, 1H), 4.56 (td, J = 6.9, 3.1 Hz, 1H), 4.04 (d, J = 3.1 Hz, 1H), 2.63 (dd, J = 15.2, 7.3 Hz, 1H), 2.55 (dd, J = 15.2, 6.6 Hz, 1H), 1.45 (s, 3H), 1.29 (s, 3H); ¹³C NMR: δ 172.1, 110.2, 102.7, 81.9, 74.6, 65.6, 33.0, 23.8, 23.4; ESI-MS: calcd for [C₉H₁₄N₄O₄ + Na]⁺: 265.09 Da. Found: 265.90 Da. Anal. calcd for C₉H₁₄N₄O₄: C, 44.63; H, 5.83; N, 23.13%. Found: C, 44.59; H, 5.87; N, 23.25%.

(2S,3R)-5-Amino-2-azido-3-hydroxy-5-oxopentanoic acid 20

Following the procedure used for the synthesis of 16, compound 19 (0.08, 0.33 mmol) was deacetalized with TFA–H₂O (3 mL, 3 : 2), the resultant diol subjected to oxidative cleavage with NaIO₄ (0.08 g, 0.37 mmol) followed by oxidation with NaH₂PO₄ (0.01 g), 30% H₂O₂ (35 μL) and NaClO₂ (0.05 g). The product was deformylated using NaHCO₃ (1 mL) in THF (5 mL). Usual workup and purification by column chromatography (silica gel, 10% MeOH/CHCl₃) of the residue gave 20 (0.04 g, 66% over four steps) as a thick liquid. R_f = 0.45 (30% MeOH/CHCl₃); [α]²⁵_D −14.4 (c 1.00, MeOH); ν_max/cm⁻¹: 2110, 1690 cm⁻¹; ¹H NMR: δ 4.50 (broad s, 1H), 3.85 (d, J = 1.5 Hz, 1H), 2.49 (dd, J = 14.7, 8.2 Hz, 1H), 2.42 (dd, J = 14.7, 5.0 Hz, 1H); ¹³C NMR: δ 174.5, 170.6, 69.0, 65.3, 39.4. Anal. calcd for C₅H₈N₄O₄: C, 31.92; H, 4.29; N, 29.78%. Found: C, 31.95; H, 4.26; N, 29.87%.

(2S,3R)-3-Hydroxy-L-glutamine hydrochloride 1b

The procedure for transforming 13 to 1a was followed for the catalytic hydrogenation of 20 (0.02, 0.11 mmol) using 10% Pd/C (0.015 g) and H₂ (80 psi) to give 1b as a semisolid (0.019 g, 90%). [α]²⁵_D +9.1 (c 1.08, H₂O); ν_max/cm⁻¹: 3440, 1708 cm⁻¹; ¹H NMR: δ 4.71–4.60 (m, 1H), 4.20 (d, J = 3.8 Hz, 1H), 2.77 (dd, J = 15.1, 4.1 Hz, 1H), 2.67 (dd, J = 15.1, 8.8 Hz, 1H); ¹³C NMR: δ 174.8, 169.9, 65.8, 57.1, 39.3; ESI-MS: calcd for [C₅H₁₀N₂O₄ + Na]⁺: 185.05 Da. Found: 185.00 Da. Anal. calcd for C₅H₁₁ClN₂O₄: C, 30.24; H, 5.58; N, 14.11%. Found: C, 30.29; H, 5.64; N, 14.23%.

(3aR,4aR,7aS,7bR)-2,2-Dimethylhexahydro-6H-[1,3]dioxolo[4,5]furo[3,2-b]pyrrol-6-one 21

A mixture of 6 (0.300 g, 1.16 mmol), 10% Pd/C (0.03 g) and HCO₂NH₄ (0.220 g, 3.50 mmol) in MeOH (15 mL) was refluxed for 2 h till completion of the reaction (cf. TLC). The mixture was filtered through Celite-545, the residue washed with MeOH (10 mL) and concentrated in vacuo. Column chromatography (silica gel, EtOAc) of the residue yielded 21 (0.210 g, 91%) as a white solid. mp: 174–176 °C; R_f = 0.30 (EtOAc); [α]²⁵_D +24.8 (c 1.00, CHCl₃); ν_max/cm⁻¹: 1658, 1413 cm⁻¹; ¹H NMR: δ 7.45 (broad s, D₂O exchangeable, 1H), 5.88 (d, J = 3.8 Hz, 1H), 4.90 (t, J = 3.8 Hz, 1H), 4.58 (d, J = 3.8 Hz, 1H), 4.08 (d, J = 4.2 Hz, 1H), 2.60–2.32 (m, 2H), 1.46 (s, 3H), 1.27 (s, 3H); ¹³C NMR: δ 177.0, 112.1, 106.0, 83.2, 77.9, 63.9, 38.0, 26.9, 26.4. Anal. calcd for C₉H₁₃NO₄: C, 54.26; H, 6.58; N, 7.03%. Found: C, 54.29; H, 6.63; N, 7.10%.

(3aR,4aR,7aS,7bR)-2,2-Dimethylhexahydro-3aH-[1,3]dioxolo[4,5]furo[3,2-b]-N-carboxybenzylpyrrole 22

To a stirred and ice-cold suspension of LiAlH₄ (0.071 g, 1.88 mmol) in dry THF (5 mL) was added 21 (0.150 g, 0.75 mmol) in dry THF (10 mL) in 5 min. The mixture was stirred further for 15 min at 0 °C, allowed to attain room temperature (25 °C), and then refluxed. After 3 h, the mixture was cooled to 25 °C, EtOAc (7 mL) added into it slowly followed by aqueous saturated NH₄Cl (1 mL) and stirred for 1 h. It was filtered through Celite-545 by washing with 20% MeOH–EtOAc, and the filtrate evaporated in vacuo to give the corresponding amine. The amine was dissolved in MeOH–water (3 : 1, 10 mL), cooled to 0 °C, NaHCO₃ (0.176 g, 2.10 mmol) and CbzCl (0.25 mL, 1.75 mmol) were successively added into it. After 3 h, the mixture was concentrated in vacuo, and the residue extracted with CH₂Cl₂ (3 × 10 mL). The organic extract was dried, concentrated in vacuo, and the residue purified by column chromatography (silica gel, 7% EtOAc/hexane) to give 22 (0.136 g, 57% over two steps) as a colorless thick liquid. R_f = 0.50 (20% EtOAc/hexane); [α]²⁵_D −55.6 (c 1.06, CHCl₃); ν_max/cm⁻¹: 1671, 1420 cm⁻¹; ¹H NMR: δ 7.49–7.25 (m, 5H), 5.80 (s, 1H), 5.20–5.01 (m, 2H), 4.92–4.60 (m, 2H), 4.20 (s, 1H), 3.77–3.60 (m, 1H), 3.40–3.23 (m, 1H), 2.06 (dd, J = 13.6, 6.0 Hz, 1H), 1.91–1.69 (m, 1H), 1.49 (s, 3H), 1.28 (s, 3H); ¹³C NMR: δ 154.4, 136.6, 128.5, 128.0, 127.9, 111.8, 106.0, 84.5, 82.2, 67.6, 67.0, 45.5, 30.0, 27.1, 26.5; ESI-MS: calcd for [C₁₇H₂₁NO₅ + Na]⁺: 342.13 Da. Found: 341.93 Da. Anal. calcd for C₁₇H₂₁NO₅: C, 63.94; H, 6.63; N, 4.39%. Found: C, 63.89; H, 6.67; N, 4.47%.

(2S,3R)-3-Hydroxyproline hydrochloride 1c

Following the procedure used for the synthesis of 16, compound 22 (0.160 g, 0.50 mmol) was deacetalized with TFA–H₂O (3 mL, 3 : 2), the resultant diol subjected to oxidative cleavage with NaIO₄ (0.110 g, 0.50 mmol), followed by oxidation with NaH₂PO₄ (0.01 g), 30% H₂O₂ (35 μL), NaClO₂ (0.05 g) and subsequent deformylation with aqueous saturated NaHCO₃ (1 mL) in THF (5 mL). The resultant acid was hydrogenated with H₂ (80 psi) over 10% Pd–C (0.015 g) to afford 1c (0.045 g, 54% over five steps) as a pale-yellow semi-solid. [α]²⁵_D −12.3 (c 1.04, H₂O); ν_max/cm⁻¹: 3576, 1715 cm⁻¹; ¹H NMR: δ 4.86 (t, J = 4.0 Hz, 1H), 4.44 (d, J = 4.0 Hz, 1H), 3.50–3.71 (m, 2H), 2.39–2.29 (m, 1H), 2.26–2.18 (m, 1H); ¹³C NMR: δ 169.2, 70.8, 66.3, 44.0, 32.9. Anal. calcd for C₅H₁₀ClNO₃: C, 35.83; H, 6.01; N, 8.36%. Found: C, 35.80; H, 6.07; N, 8.44%.

Prop-2-en-1-yl(2S,3R)-N-carboxybenzyl-3-hydroxypyrrolidine-2-carboxylate 23

As described before, 22 (0.064 g, 0.20 mmol) was deacetalized using TFA–H₂O (3 mL, 3 : 2), the resultant diol oxidatively cleaved with NaIO₄ (0.047 g, 0.22 mmol) followed by oxidation with NaH₂PO₄ (0.006 g), 30% H₂O₂ (21 μL), NaClO₂ (0.03 g), and the formyl group unmasked with aqueous NaHCO₃ (1 mL) in THF (5 mL). The resultant crude acid was dried in vacuo and allylated with allyl bromide (21.6 μL) and NaHCO₃ (0.04 g) in DMF (3 mL). The mixture was concentrated in vacuo, the residue extracted with EtOAc (3 × 10 mL), the organic extract washed with water (2 × 5 mL) and dried. Concentration of the extract in vacuo, and column chromatography of the residue (silica gel, 15% EtOAc/hexane) gave 23 (0.040 g, 65% over five steps) as a thick liquid. R_f = 0.36 (50% EtOAc/n-hexane); [α]²⁵_D +28.00 (c 1.00, CHCl₃); ν_max/cm⁻¹: 1715, 1215 cm⁻¹; ¹H NMR: δ 7.45–7.22 (m, 5H), 6.08–5.60 (m, 1H), 5.48–4.91 (m, 4H), 4.86–4.34 (m, 4H), 3.81–3.40 (m, 2H), 2.84 (broad s, D₂O exchangeable, 1H), 2.24–1.82 (m, 2H); ¹³C NMR: δ 169.8, 154.4, 136.2, 131.8, 128.4, 128.0, 127.8, 118.5, 72.2, 67.2, 65.9, 63.8, 44.4, 32.0; ESI-MS: calcd for [C₁₆H₁₉NO₅ + Na]⁺: 328.11 Da. Found: 327.96 Da. Anal. calcd for C₁₆H₁₉NO₅: C, 62.94; H, 6.27; N, 4.59%. Found: C, 63.01; H, 6.25; N, 4.66%.

(2R,3R)-2-(Hydroxymethyl)-N-carboxybenzylpyrrolidin-3-ol 24

A solution of 22 (0.100 g, 0.31 mmol) in TFA–H₂O (3.00 mL, 3 : 2) was stirred at to 0 to 10 °C for 1.5 h. TFA was removed azeotropically with toluene in vacuo to afford the hemiacetal as a thick liquid. To a cooled (0 °C) solution of the crude hemiacetal in acetone–water (9 : 1, 5 mL) was added NaIO₄ (0.073 g, 0.34 mmol). After stirring for 30 min, the reaction mixture was concentrated in vacuo, and the residue extracted with CHCl₃ (3 × 10 mL) to get the crude aldehyde (0.09 g) as a thick liquid. This was dissolved in THF–H₂O (4 : 1, 5 mL), cooled to 5 °C and NaBH₄ (0.015 g, 0.40 mmol) in H₂O (0.5 mL) was added to it. After stirring for 30 min, the mixture was concentrated in vacuo, the residue extracted with EtOAc (2 × 10 mL), the organic extract washed with water (2 × 5 mL) and dried. Concentration of the extract in vacuo, and column chromatography of the residue (silica gel, 20% EtOAc/hexane) yielded 24 (0.05 g, 63% over three steps) as a thick liquid. R_f = 0.30 (60% EtOAc/hexane); [α]²⁵_D −11.5 (c 1.20, CHCl₃); ν_max/cm⁻¹: 1673, 1421 cm⁻¹; ¹H NMR: δ 7.41–7.28 (m, 5H), 5.12 (q, J = 12.4 Hz, 2H), 4.51 (d, J = 3.7 Hz, 1H), 4.09–3.80 (m, 3H), 3.54 (t, J = 6.3 Hz, 2H), 2.85–2.34 (m, D₂O exchangeable, 2H), 2.11–1.85 (m, 2H); ¹³C NMR: δ 154.5, 136.4, 128.5, 128.1, 128.0, 72.8, 67.2, 61.9, 44.6, 32.9; ESI-MS: calcd for [C₁₃H₁₇NO₄ + Na]⁺: 274.10 Da. Found: 274.11 Da. Anal. calcd for C₁₃H₁₇NO₄: C, 62.14; H, 6.82; N, 5.57%. Found: C, 62.18; H, 6.87; N, 5.66%.

(2R,3R)-2-(Hydroxymethyl)pyrrolidin-3-ol 2

A mixture of 24 (0.080 g, 0.31 mmol) and 10% Pd/C (0.02 g) in MeOH (10 mL) was stirred under H₂ (80 psi) for 6 h. The catalyst was filtered through Celite-545 by washing with MeOH (20 mL), and the filtrate concentrated in vacuo to afford 2 (0.030 g, 84%) as a thick liquid. R_f = 0.15 (40% MeOH/CHCl₃); [α]²⁵_D +11.5 (c 1.14, H₂O); ν_max/cm⁻¹: 3480 cm⁻¹; ¹H NMR: δ 4.57 (s, 1H), 3.99 (dd, J = 12.0, 5.1 Hz, 1H), 3.87 (dd, J = 12.0, 8.2 Hz, 1H), 3.58–3.51 (m, 1H), 3.50–3.40 (m, 1H), 3.38–3.32 (m, 1H), 2.30–2.20 (m, 1H), 2.11–2.03 (m, 1H); ¹³C NMR: δ 70.5, 64.5, 58.6, 43.1, 33.1; ESI-MS: calcd for [C₅H₁₁NO₂ + H]: 118.08 Da. Found: 118.19 Da. Anal. calcd for C₅H₁₁NO₂: C, 51.26; H, 9.46; N, 11.96%. Found: C, 51.33; H, 9.38; N, 12.05%.

References

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra01340b

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