α-Chymotrypsin and L-acylase aided synthesis of 5-hydroxypipecolic acid via Jacobsen's hydrolytic kinetic resolution of epoxy amino acids

Abstract

Diethyl malonate derivatives were used to synthesize racemic 2-amino-5-hexenoic acid. These racemic 2-amino-5-hexenoic acid (homoallylyglycine) derivatives were efficiently resolved aided by α-chymotrypsin or L-acylase, giving rise to L- and D-enantiomers. These isolated enantiomerically pure amino acids with tert-butoxycarbonyl (Boc) protection were oxidised with 3-chloroperbenzoic acid. The oxidation gave rise to inseparable diastereomeric epoxides due to a newly generated chiral center at the C⁵ carbon. The isolation of one of the diastereomeric epoxides was possible by selectively converting the remaining diastereomer into a dihydroxyl compound catalysed by Jacobsen's hydrolytic kinetic resolution (HKR). The isolated epoxide was regioselectively attacked by LiBr to give vicinal halohydrin, with bromide attacking the terminal C⁶ carbon. Boc deprotection of the halohydrin led to intramolecular cyclization by attack of free amine at the C⁶ carbon, generating a single isomer of 5-hydroxypipecolic acid which was effortlessly recovered in good yield after re-protection of the amine with the Boc group. Similarly the dihydroxyl compound isolated earlier was converted to a halohydrin with iodine at the C⁶ carbon. This was feasible by efficient regioselective mono-tosylation with catalytic Bu₂SnO followed by iodine substitution. This was utilized to synthesize the 5-hydroxypipecolic acid derivative in the same described sequence consisting of Boc removal by acid treatment, cyclization, reprotection and purification. Finally the same sequence was repeated with the D-isomer and two diastereomers were isolated.

---

1. Introduction

5-Hydroxypipecolic acid (5-hydroxypiperidine-2-carboxylic acid) (1, Fig. 1), a vital non-proteinogenic cyclic δ-hydroxy-α-amino acid found in plants^1,2 and animals,³ occurs as a metabolic by-product. Derivatives of 5-hydroxypipecolic acid are constituents of a tumor necrosis-converting enzyme inhibitor, TNF-α (3, Fig. 1).⁴ Recently cis-5-hydroxypipecolic acid (2, Fig. 1) was utilized as a precursor for the synthesis of β-lactamases inhibitor MK-7655 ⁵ (4, Fig. 1), which combined with Merck's Primaxin® is in phase II clinical trials for the treatment of gram-negative bacterial infections.⁶ It is also employed in the synthesis of 5-guanidino pipecolates, as a constrained arginine mimetic and exhibited weak to moderate inhibition of nitric oxide synthase (NOS).⁷ cis-5-hydroxypipecolic acid demonstrates inhibition of Aspergillus spp. responsible for spoilage of stored grains.⁸ It is also reported to display potent inhibitory effects on human platelet aggregation induced by serotonin.⁹

Fig. 1 5-Hydroxypipecolic acid and its derivatives.

Due to its above mentioned biological relevance and ability to induce rigidity owing to its cyclic structure, several groups have attempted the synthesis of 5-hydroxypipecolic acid.^7,10–14 In this context glycine amino acid residues functionalized with epoxide side chains are synthetically valuable precursors for generation of cyclic amino acids. We recently synthesized 4-hydroxyproline isomers from epoxide derived from 2-amino-4-pentenoic acid (allyl glycine), by selective intramolecular reaction of amine on C⁵ carbon.¹⁵ Similarly the synthesis of 5-hydroxypipecolic acid can be achieved by selective attack of amine group on C⁶ carbon of 2-amino-5-hexenoic acid epoxide (homoallyl glycine). Several reports exists on the synthesis of 5-hydroxypipecolic acid derivatives via intramolecular reactions of epoxy amino acid.^1,16 However these synthetic procedures yield an unresolved diastereomeric mixture of desired cis- and trans-5-hydroxypipecolic acids,^1,16,17 along with the undesirable 5-hydroxymethylprolines.^1,17 A straightforward method generating 5-hydroxypipecolic acid derivatives bypassing the laborious need to isolate the cis- and trans-diastereomers in the final stages combined with elimination of the formation of regioisomeric proline is desired. In this paper we would like to present the synthetic scheme circumventing the drawbacks imposed by the established synthetic procedures involving generation of 5-hydroxypipecolic acid by intramolecular reaction of epoxy amino acid. To the best of our knowledge this is the first work reporting the synthesis of 5-hydroxy pipecolic acid wherein the 2S (L-) and 2R (D-) isomers were efficiently separated by enzymatic kinetic resolution. Whereas the epoxide precursors that give rise to cis- and trans-isomers of 5-hydroxypipecolic acid were separated via Jacobsen's Co catalyzed HKR.

2. Results and discussion

L-acylase and α-chymotrypsin catalyzed enzymatic resolution of 2-amino-5-hexenoic acid derivatives

Enantiomerically pure 2-amino-5-hexenoic acid derivatives were synthesized by enzymatic resolution of acetyl amino acid and tert-Boc amino acid ester, with L-aminoacylase and α-chymotrypsin respectively (Scheme 1).

Scheme 1 Enzymatic resolution of 2-amino-5-hexenoic and derivatives.

Scheme 2 m-CPBA oxidation of the alkene side chain of (S)-9.

Scheme 3 Jacobsen's hydrolytic kinetic resolution of (2S,5S)-11 and (2S,5R)-11.

Scheme 4 Formation of (2S,5R)-hydroxypipecolic acid by intramolecular cyclization.

Diethyl acetamidomalonate (5) was reacted with excess of 4-bromo-1-butene in the presence of sodium ethoxide in absolute ethanol to obtain the alkylated diester. The diester was half saponified and decarboxylated simultaneously, by treating with solid NaOH in EtOH : H₂O mixture (1 : 1, v/v) and refluxing for 24 h. This gave the desired racemic acetyl amino acid (RS)-6, (47% overall yield based on 5) suitable for the L-acylase catalyzed enzymatic resolution.

An aqueous solution of (RS)-6, maintained at 38 °C, with pH 7.5 adjusted with LiOH (aq.) was treated with L-aminoacylase from Aspergillus genus.¹⁸ After 24 h, the solution was concentrated and acidified with 1 M HCl to pH 3 and extracted with EtOAc to remove the unreacted (R)-6. The remaining acidic solution was passed through ion exchange resin (DOWEX® 50WX8) to separate (S)-7, which was selectively eluted with NH₄OH (1 M). Due to high water solubility of the acetyl amino acid the yields were less, but pure D- and L-2-amino-5-hexenoic acid were obtained with good chemical and enantiomeric purity ascertained by ¹H NMR and chiral HPLC.

Subsequently diethyl(2-Boc-amino)malonate (8), was treated with excess 4-bromo-1-butene in presence of sodium ethoxide to obtain alkylated diester. The later was selectively saponified with solid LiOH to obtain the half acid half ester, which was successfully decarboxylated in refluxing toluene to obtain (RS)-9 (59% overall yield with respect to 8).

Enzymatic resolution was carried by suspending the racemic (RS)-9 in phosphate buffer (pH 8, 0.1 mmol) at 38 °C and treating with α-chymotrypsin. To avoid slight deviance of pH caused by formation of (S)-10, minimal amount of NH₄OH (1 M) was added and maintained at pH 8. After 24 h, the mixture was cooled to room temperature and alkalinity was enhanced by addition of (4% NaHCO₃) followed by extraction with EtOAc to recover unreacted (R)-9 (51%). The remaining alkaline aqueous solution was subsequently acidified with solid citric acid to pH 3, extracted with EtOAc to obtain (S)-10 (46%).

m-CPBA oxidation (Scheme 2)

Kinetically resolved Boc-amino acid (S-10) was esterified with ethyl bromide in the presence of Et₃N to obtain (S)-9 in 84% yield. This fully protected Boc-amino acid ester with unsaturated side chain was epoxidized with excess 3-chloroperbenzoic acid (m-CPBA) by treating the solution of (S)-9 in DCM with excess m-CPBA at 0 °C, followed by 24 h stirring at room temperature. Subsequently excess m-CPBA was cautiously reduced with 10% Na₂SO₃ at 0 °C. The DCM layer was separated and washed (4% NaHCO₃ and brine), concentrated and chromatographed (SiO₂) to obtain 11 (90%, yield).

The resulting epoxide was a diastereomeric mixture (2S,5R)-11 and (2S,5S)-11 due to the newly generated chiral centre at C⁵ carbon. However the diastereomers moved as a single spot with same R_f value on TLC and could not be separated.¹H NMR analysis in (CDCl₃) did not show any set of distinguishable peaks for the individual diastereomers. In this context m-CPBA oxidation of similar epoxides is believed to produce 1 : 1 mixture of both diastereomers, nonetheless it was reported that these diastereomers were inseparable.^16,19

Hydrolytic kinetic resolution (HKR) of epoxy amino acids^20,21 (Scheme 3)

To aid the separation of the diastereomeric epoxide, 11 was treated with (0.55 eq.) of H₂O in the presence of (0.5 mol%) of (RR)-Co-Salen and AcOH, at room temperature in THF. This produced the chiral diol (2S,5S)-12 and chiral epoxide (2S,5R)-11 which had a considerable R_f difference. In fact the chiral epoxide (2S,5R)-11 and chiral diol (2S,5S)-12, were easily separated by silica gel column chromatography in good yield, 47% and 46%, respectively.

tert-butoxycarbonyl (Boc) removal and intramolecular cyclization (Scheme 4)

Next, the chiral epoxide (2S,5R)-11 was treated with LiBr in the presence of AcOH in THF, which attacked the C⁶ carbon selectively giving rise to bromohydrin 13.²² This bromo amino acid was isolated in good yield (92%) after (SiO₂) purification, which was suitable for synthesis of 5-hydroxypipecolic acid. To aid the intramolecular cyclization, Boc group of 13 was removed by treating it with TFA. After 1 h TFA was removed to obtain, 14. TFA in quantitative yield which was not purified owing to its hydrophilic character, instead it was used for next step directly.

14. TFA, was dissolved in THF at room temperature and treated with DIEA (2 eq.) and stirred. DIEA treatment leads to the neutralization of TFA and simultaneously gives rise to highly nucleophilic and free amine. This amine will attack intramolecularly the C⁶ carbon, thereby producing the 5-hydroxypipecolic acid. Due to the formation of HBr after intramolecular cyclization, another equivalent of DIEA was required to neutralize this and complete the cyclization. TLC analysis at the end of 4 h, (butanol/pyridine/acetic acid/H₂O, 4 : 1 : 1 : 2, v/v/v/v) by staining with ninhydrin showed the absence of the reactant and appearance of a yellow spot, which is characteristic of the cyclic amino acids. This confirmed that the nitrogen intramolecularly attacked the side chain to produce the piperidine ring, i.e., (2S,5R)-15. To aid the purification process the nitrogen of the piperidine ring was again reprotected with Boc group by treatment with Boc₂O in presence of DIEA to generate the Boc-5-hydroxypipecolic acid derivatives. 5-hydroxypipecolic acid, (2S,5R)-16 was separated as an oil (80%) by silica gel column chromatography (50% EtOAc in hexane).

Subsequently the oil (2S,5R)-16 was converted to 1.HCl. The spectral data, melting point and specific rotation of 1.HCl, were in good agreement with the reported values.²³

Regioselective halogenation, N-Boc removal and intramolecular cyclization

In order to transform the chiral diol 12 into 5-hydroxypipecolic acid, it was necessary to transform 12 into amino acid with a good leaving group at C⁶ carbon similar to 13. This was achieved by selective tosylation of diol by treating with TsCl in the presence of catalytic Bu₂SnO (30 mol%), DMAP (cat.) and triethylamine at −10 °C, yielding the mono tosylated product in almost quantitative yield.^24–26 The temperature was maintained between −10 °C to 0 °C in order to retain the acid labile Boc group. The tosyl group in 17 was replaced with iodo group by refluxing it in the presence of NaI (6 eq.) in acetone. After 8 h, acetone was replaced with EtOAc washed with (sat. Na₂S₂O₃), brine and chromatographed on (SiO₂) resulting (2S,5S)-18 (95%) used for synthesis of 5-hydroxypipecolic acid (Scheme 5).

Scheme 5 Formation of (2S,5S)-hydroxypipecolic acid by intramolecular cyclization (i) TFA; (ii) DIEA, THF; (iii) Boc₂O, DIEA, THF.

To aid the intramolecular cyclization Boc group of 18 was removed by treating with TFA at room temperature. After 1 h the solution was concentrated to obtain Boc-deprotected amino acid salt of TFA in quantitative yield, which was used for subsequent step directly.

After dissolving in THF at room temperature and treating it with DIEA (2 eq.) free amine was generated. The free amine attacked the C⁶ carbon intramolecularly generating 5-hydroxypipecolic acid. After 3 h, to aid the purification process the nitrogen of the amino acid was again reprotected with Boc group by treating with Boc₂O in presence of DIEA to generate the Boc-5-hydroxypipecolic acid derivatives. The fully protected 5-hydroxypipecolic acid (2S,5S)-19 was easily separated as an oil by silica gel column chromatography (50% EtOAc in hexane) in 76% yield. Subsequently the oil (2S,5S)-19 was converted to 2.HCl. The spectral data, melting point and specific rotation of 2.HCl, were in good agreement with the reported values.^27,28

Synthesis of (2R,5R)-20 and (2R,5S)-21

Finally the synthesis of the (2R,5R) and (2R,5S)- isomers of the 5-hydroxypiecolic acid was easily achieved starting from (2R)-9 (Scheme 6), [ESI-S2†].

Scheme 6 Formation of (2R,5R) and (2R,5S) isomers by intramolecular cyclization.

3. Experimental section

General

Reagents, dry solvents, and enzymes were from commercial sources. DOWEX 50WX8 (50–100) was used as an ion exchange resin, after washing with 1 M HCl. (R,R)-(−)-N,N′-Bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminocobalt(II) was purchased from Sigma-Aldrich®. TLC was visualized by one of the following, UV (F254), I₂, H₃(PMo₁₂O₄) or ninhydrin. Chiral HPLC analysis were performed on Column I: CHIRALPAK IA column (4.6 × 250 mm, Diacel Chemical Industries, Ltd.), n-hexane : 2-propanol : TFA (97 : 3 : 0.1, v/v/v) with a flow rate of 1 mL min⁻¹, detection at 220 nm; Column II: CHIRALCEL OD column (4.6 × 250 mm, Diacel Chemical Industries, Ltd.), n-hexane : 2-propanol : TFA (90 : 10 : 0.1) with a flow rate of 1 mL min⁻¹, detection at 220 nm. ¹H were measured in CDCl₃, CD₃OD or D₂O at 500 or 400 MHz and ¹³C NMR were measured in CDCl₃, CD₃OD or D₂O at 125 MHz. Normal- and high resolution-FAB mass were measured routinely. Optical rotations were measured using Jasco Polarimeter (P-1010). Melting point (m.p.) were recorded by As One melting point instrument (Model Number ATM-01).

Kinetic resolutions by L-acylase from Aspergillus genus

(RS)-2-(acetamido)-5-hexenoic acid (RS)-6. Sodium metal (5.10 g, 222.0 mmol) was added to a flask containing 195 mL of absolute ethanol maintained at 0 °C. After complete dissolution of sodium, 5 (43.5 g, 200 mmol) was added in one portion and the resulting solution was refluxed. After 3 h to this refluxing solution 4-bromo-1-butene (25.0 g, 185 mmol) was added and continued to reflux. After 24 h the mixture was concentrated and the residue dissolved in EtOAc (600 mL) and washed with 10% citric acid, 4% NaHCO₃, brine, dried (MgSO₄), filtered and evaporated to get the alkylated diester.

This diester was dissolved in 1 : 1 (v/v), mixture of ethanol and water (230 mL) at 0 °C, and to this solution solid NaOH (7.0 g, 175 mmol) was added, this solution was then refluxed. After 24 h the ethanol was removed and the resulting solution was dissolved in 4% NaHCO₃ and extracted with diethyl ether to remove the unreacted starting material. Then the remaining alkaline aqueous solution was acidified to pH 3 with 1 M HCl and extracted with EtOAc (20 mL × 20). The combined organic layer was washed with brine, dried (MgSO₄), filtered and evaporated to give (RS)-6 as a white powder (15 g, 47% w.r.t. 5). M.p. 119–121 °C [lit.²⁹ 106–108 °C]; ¹H NMR (400 MHz, CD₃OD) 5.89–5.79 (m, 1H), 5.10–5.00 (m, 2H), 4.37 (m, 1H), 2.14 (m, 2H), 1.99 (s, 3H), 1.96–1.71 (m, 2H); ¹³C NMR (125 MHz, CD₃OD) 175.6, 173.5, 138.5, 116.2, 53.3, 32.1, 31.2, 22.5; LRMS-FAB (m/z) 343 (67), 194 (58), 172 (100), 126 (68), 83 (68); HRMS-FAB (m/z) calcd for C₈H₁₄N₁O₃ ([M + H]⁺)172.0974 found 172.0909.

(S)-2-Amino-5-hexenoic acid (S)-7 and (R)-2-(acetamido)-5-hexenoic acid (R)-6. (RS)-6 (3.00 g, 17.5 mmol) and CoCl₂·6H₂O (14 mg, 50 μmol) was dissolved in 60 mL of H₂O at 38 °C, while maintaining pH at 7.5 with aqueous LiOH. L-aminoacylase from Aspergillus genus (0.8 g) was then added and stirred. The pH was monitored at 7.5 with small amount of NH₄OH (1 M). After 24 h, the reaction mixture was concentrated, acidified to pH 2–3 with 1 M HCl. The unreacted (R)-6 was extracted with EtOAc, washed with brine, dried (MgSO₄), filtered and evaporated to give (R)-6 (1.23 g, 41%). (Column II. ee 88%).

The remaining acidic aqueous phase was applied to a column of ion exchange resin (DOWEX® 50WX8). After sample charging and elution with 1 M HCl, followed by H₂O, finally the compound (S)-7 was eluted with 1 M NH₄OH. Evaporation of this eluant afforded (S)-7 as white solid (0.72 g, 32%) (after N acetylation, Column II. ee > 99%). ¹H NMR (D₂O, 500 MHz) 5.84–5.76 (m, 1H), 5.12–5.03 (m, 2H), 3.72–3.69 (m, 1H), 2.15–2.11 (m, 2H), 1.99–1.85 (m, 2H); ¹³C NMR (D₂O, 125 MHz) 174.7, 137.0, 115.9, 54.3, 29.7, 28.6; LRMS-FAB (m/z) 130 (100).

Kinetic resolutions by α-chymotrypsin

(RS)-ethyl 2 N-(tert-butoxycarbonyl)-2-amino-5-hexenoate (RS)-9. Metallic sodium (0.92 g, 39.9 mmol) was added to EtOH (17.0 mL) at 0 °C and stirred until complete dissolution of sodium. 8 (10.0 g, 36.3 mmol) was then added at the same temperature and stirred for 30 min. Finally 4-bromo-1-butene (5.6 mL, 54.8 mmol) was added in one portion and the resulting yellow solution was refluxed for 24 h. After 24 h, the solution was partitioned between H₂O and EtOAc, extracting the desired product into EtOAc. The EtOAc was then washed with 10% citric acid, brine, dried (MgSO₄), filtered and evaporated to obtain an oil (9.51 g), which was suspended in EtOH : H₂O (18 : 8, v/v) and cooled to 0 °C. This solution was treated with solid LiOH·H₂O (1.33 g, 31.8 mmol) and stirred overnight at 0 °C. The solution was then mixed with 4% NaHCO₃ to make the solution alkaline and extracted with diethyl ether to remove any unreacted starting material. The remaining alkaline solution was then acidified to pH 3 with solid citric acid and extracted with EtOAc. The EtOAc was then washed with brine, dried (MgSO₄), filtered and evaporated to give the mono ester mono acid (8.30 g). This was dissolved in toluene (25 mL) and refluxed. After 18 h the solvent was evaporated at 40–50 °C to give a crude mixture which directly purified by silica gel column chromatography (hexane : EtOAc, 80 : 20, v/v) to give (RS)-9 (5.5 g, 59%, after 3 steps). ¹H NMR (500 MHz, CDCl₃) 5.84–5.76 (m, 1H), 5.07–5.00 (m, 2H), 4.31–4.30 (m, 1H), 4.21 (m, 2H), 2.15–2.09 (m, 2H), 1.95–1.88 (m, 1H), 1,75–1.68 (m,1H), 1.45 (s, 9H) 1.28 (m, 3H); ¹³C NMR (125 MHz, CDCl₃) 172.8, 155.3, 137.1, 115.6, 79.8, 61.3, 53.1, 32.1, 29.5, 28.3, 14.2; LRMS-FAB (m/z) 258 (50), 230 (21), 202 (100), 158 (93) and 128(18); HRMS-FAB (m/z) calcd for C₁₃H₂₄ N₁O₄ ([M + H]⁺) 258.1705, found 258.1707.

(R)-Ethyl 2 N-(tert-butoxycarbonyl)-2-amino-5-hexenoate (R)-9 and (S) 2 N-(tert-butoxycarbonyl)-2-amino-5-hexenoic acid (S)-10. (RS)-9 (4.80 g, 18.6 mmol) was suspended in phosphate buffer (360 mL, 0.1 M; pH 8.00), warmed to 38 °C and α-chymotrypsin (28 mg, 40 units per mg) was added in one portion. The resulting mixture was stirred at 38 °C for 24 h while monitoring the pH 8 with little amount of (1 M NH₄OH). After 24 h the above solution was made more alkaline by addition of 4% NaHCO₃ and was extracted with EtOAc (20 mL × 10). The combined EtOAc layer (200 mL) was washed with brine, dried (MgSO₄), filtered and evaporated to give (R)-9 (2.42 g, 51%). (Column I. ee > 64%); [α]²⁵_D = −30° (c = 2.44, CH₂Cl₂). The remaining alkaline solution was then acidified to pH 3, with solid citric acid and extracted with EtOAc (30 mL × 10). The combined EtOAc layer (300 mL) was washed with brine, dried (MgSO₄), filtered and evaporated to give (S)-10 (1.95 g, 46%). (Column I. ee > 99%); [α]²⁰_D = −1.18° (c = 1.30, CH₃OH). {lit.³⁰[α]²⁰_D = −1.1° (c = 1.30, CH₃OH)}; ¹H NMR (500 MHz, CD₃OD) 5.81–5.73 (m, 1H), 5.01–4.98 (m, 3H), 4.04–4.02 (m, 1H), 2.18–2.08 (m, 2H), 1.91–1.85 (m, 1H), 1.75–1.68 (m, 1H), 1.44 (s, 9H); ¹³C NMR (125 MHz, CD₃OD) 175.0, 156.7, 137.1, 114.6, 79.1, 52.9, 30.8, 29.7, 27.3; LRMS-FAB (m/z) 275 (35), 252 (16), 230 (17), 174 (100) 130 (65); HRMS-FAB (m/z) calcd for C₁₁H₂₀N₁O₄ ([M + H]⁺) 230.1392, found 230.1336.

m-CPBA oxidation

(S)-Ethyl 2 N-(tert-butoxycarbonyl)-2-amino-5-hexenoate (S)-9. (S)-10 (1.31 g, 5.72 mmol) was dissolved in DMF (15 mL) and cooled with an ice-water bath. This solution was treated with triethylamine (1.20 mL, 8.58 mmol) and finally ethyl bromide (2.6 mL, 35 mmol). The resulting solution was then allowed to attain room temperature and continued to stir. After 24 h the solution was concentrated to get a crude mixture as an oil. This crude mixture was purified by silica gel column chromatography (hexane : EtOAc, 80 : 20, v/v) to give (S)-9 (1.24 g, 84%). [α]_D²⁵ = +8.53° (c = 2.46, CH₂Cl₂). [¹H NMR same as (RS)-9].

Ethyl 2-[(tert-butoxycarbonyl)amino]-4-(2-oxiranyl)butanoate, (2S,5R)-11 and (2S,5S)-11. (S)-9 (1.2 g, 4.67 mmol) was dissolved in DCM 20.0 mL and cooled to 0 °C with ice-water bath. To this solution m-CPBA (1.57 g, 7.0 mmol) was added in one portion and stirred. The ice-water bath was removed once the m-CPBA completely dissolved and allowed to stir at room temperature. After 24 h the crude reaction mixture was again cooled with in an ice-water bath, treated with 10% aqueous Na₂SO₃ (30 mL), then the solution was stirred vigorously to reduce the excess m-CPBA. After 2 h the organic phase was separated and washed with 4% NaHCO₃, brine, dried (MgSO₄), filtered and evaporated to get a clear oil. This was purified by silica gel column chromatography (hexane : EtOAc, 70 : 30, v/v) to get pure 11 as a clear oil (1.15 g, 90%). ¹H NMR (500 MHz, CDCl₃) 5.10–5.05 (m, 1H), 4.32 (m, 1H) 4.20 (q, 2H), 2.93 (m, 1H), 2.76 (m, 1H), 2.49 (m, 1H) 1.99 (m, 1H) 1.8 (m, 1H), 1.68 (m, 1H) 1.51 (m, 1H), 1.45 (s, 9H), 1.29 (t, 3H); ¹³C NMR (125 MHz, CDCl₃) 172.5 (172.4), 155.4, 79.9, 61.5 (61.4), 53.2 (53.0), 51.6 (51.5), 47.1 (47.0), 29.2, 28.4, 28.3, 14.2(14.1); LRMS-FAB (m/z) 274 (42), 218 (100), 174 (49); HRMS-FAB (m/z) calcd for C₁₃H₂₄N₁O₅ ([M + H]⁺) 274.1654, found 274.2659.

Jacobsen's hydrolytic kinetic resolution (HKR)

Ethyl 2-[(tert-butoxycarbonyl) amino]-4-(2-oxiranyl)butanoate (2S,5R)-11 and ethyl 2-[(tert-butoxycarbonyl) amino]-5,6-dihydroxyhexanoate, (2S,5S)-12. 11 (1 g, 3.65 mmol) was taken in a vial equipped with a stir bar, to this was added (RR)-Co-Salen (0.5 mol%, 12 mg), followed by acetic acid (30 μL) and THF (0.3 mL). Finally H₂O (2 mmol, 36 μL) was added in one portion at room temperature and stirred. After 48 h, the solution was directly purified by silica gel column chromatography, eluting the column by (EtOAc : hexane, 30 : 70, v/v) gave (2S,5R)-11 (0.47 g, 47%) as a clear oil. Further elution with (EtOAc : hexane : methanol, 60 : 40 : 2, v/v/v) gave (2S,5S)-12 (0.49 g, 46%). ¹H (500 MHz, CDCl₃) 5.25–5.14 (m, 0.66H), 4.36 (m, 0.73H), 4.21 (q, 2H), 3.75 (br s, 0.91H), 3.64 (br s, 0.96H), 3.46 (m, 0.98H), 2.03–1.93 (m, 1.90H), 1.84–1.68 (m, 0.82H), 1.60–1.48 (m, 5H), 1.45, 1.44 (both s, total 9H) 1.29 (t, 3H); ¹³C NMR (125 MHz, CDCl₃) 173.0 (172.8), 155.8, 80.0 (79.9), 71.7 (71.5), 66.6, (66.5), 61.4, 53.4, 29.0, 28.7, 28.3, 14.1; LRMS-FAB (m/z) 356 (33), 354 (35), 300 (53), 298 (55), 256 (98), 254 (100); HRMS-FAB (m/z) calcd for C₁₃H₂₆N₁O₆ ([M + H]⁺) 292.1760, found 292.1767.

Synthesis of (2S,5R)-5-hydroxypipecolic acid derivatives (Scheme 4)

Ethyl 2-[(tert-butoxycarbonyl) amino]-5-hydroxy-6-bromohexanoate (2S,5R)-13. 11 (0.46 g, 1.71 mmol) was dissolved in THF (15 mL) and cooled to 0 °C. To this solution AcOH (0.30 mL, 5.25 mmol) was added followed by LiBr (0.24 g, 2.76 mmol). The solution was then warmed to room temperature and stirred. After 16 h, when the TLC analysis showed the complete disappearance of the epoxide, the solution was concentrated in vacuum and the residue taken in EtOAc. Washed with 4% NaHCO₃, brine, dried (MgSO₄), filtered and evaporated to get crude product which was purified by silica gel column chromatography eluting with (EtOAc : hexane, 40 : 60 v/v) to get 13 (0.55 g, 92%). ¹H NMR (500 MHz, CDCl₃) 5.21 (bd, 1H), 4.37 (m, 1H), 4.21 (m, 2H), 3.82 (br s, 1H), 3.50–3.38 (m, 2H), 2.87 (bd, 1H), 2.05–1.91 (m, 1H), 1.86–1.54 (m, 3H), 1.45 (s, 9H), 1.29 (t, 3H); ¹³C NMR (125 MHz, CDCl₃), 172.5, 155.7, 80.2, 70.8, 61.5, 53.1, 40.1, 30.6, 29.7, 28.3, 14.2; LRMS-FAB (m/z) 356 (33), 354 (35), 300 (53), 298 (55), 256 (98), 254 (100); HRMS-FAB (m/z) calcd for C₁₃H₂₅N₁O₅Br₁ ([M + H]⁺) 354.0916, found 354.0881.

1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate (2S,5R)-16. 13 (0.26 g, 0.73 mmol) was dissolved in TFA (5 mL) and allowed to stand at room temperature. After 1 h when the reaction was complete (TLC) the excess TFA was evaporated. The crude product was redissolved in diethyl ether and evaporated to remove excess TFA, to get 14. TFA as a white sticky oil.

14. TFA was dissolved in THF (5 mL) and to this DIEA (0.25 mL, 1.46 mmol) was added, and the resulting solution was stirred at room temperature. After 4 h the solution was treated with additional DIEA (0.13 mL, 0.73 mmol) and Boc₂O (0.32 g, 1.46 mmol). The resulting solution stirred at room temperature. After 12 h the solution was concentrated and the residue was dissolved in ethyl acetate and washed with 4% NaHCO₃, 10% citric acid, brine, dried (MgSO₄), filtered and evaporated to get an oil purified by silica gel column chromatography (EtOAc : hexane, 50 : 50, v/v) to get 16 as an oil (0.16 g, 80%). ¹H (500 MHz, CDCl₃) 4.91, 4.74 (both br s, 1H), 4.20 (m, 2H), 4.08–3.95 (m, 2H), 3.24–3.10 (m, 1H), 2.17–2.00 (m, 2H), 1.89–1.76 (m, 2H), 1.47, 1.45 (both s, total 9H); ¹³C (125 MHz, CDCl₃) 171.7 (171.5), 156.5 (156.2), 80.4, 61.1, 54.7 (53.5), 48.1 (47.1), 28.2, 27.2 (26.9), 20.3, 14.3; LRMS-FAB (m/z) 274 (57), 218 (100), 174 (100), 144 (50); HRMS-FAB (m/z) calcd for C₁₃H₂₄ N₁O₅ ([M + H]⁺) 274.1654, found 274.1656.

Synthesis of (2S,5S) 5-hydroxypipecolic acid derivatives (Scheme 5)

Ethyl 2-[(tert-butoxycarbonyl)amino]-5-hydroxy-6-tosylhexanoate (2S,5S)-17. 12 (0.45 g, 1.53 mmol) was dissolved in DCM (25.0 mL) and cooled to −10 °C, to this stirred solution triethylamine (0.25 mL, 1.8 mmol) followed by Bu₂SnO (0.11 g, 0.46 mmol, 30 mol%) and DMAP (10 mg, cat.) were added. The solution was then stirred for 15 min at −10 °C finally TsCl (0.32 g, 1.68 mmol) was added portion wise over a period of 1 h. The resulting solution was stirred for an additional 1 h. At the end of 2 h, the reaction was quenched with 4% NaHCO₃ and DCM was replaced with EtOAc. The EtOAc layer was washed with brine, dried (MgSO₄), filtered and evaporated to give a crude mixture purified by silica gel column chromatography (EtOAc : hexane, 30 : 70 to 50 : 50, v/v) to give 17 (0.67 g, 97%). ¹H (500 MHz, CDCl₃) 7.80 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 5.10 (br d, 1H), 4.27 (m, 1H), 4.19 (m, 2H), 4.12 (m, 1H), 4.00 (m, 1H), 3.88 (m, 2H), 2.46 (s, 3H), 1.91–1.77 (m, 2H), 1.55–1.46 (m, 2H), 1.43 (s, 9H); ¹³C (125 MHz, CDCl₃) 172.5, 155.5, 145.1, 132.6, 130.0, 128.0, 80.0, 73.7, 68.8, 61.5, 53.0, 28.7, 28.3, 21.7, 14.2; LRMS-FAB (m/z) 446 (15), 390 (42), 347 (37), 346 (100); HRMS-FAB (m/z) calcd for C₂₀H₃₂ N₁O₈S₁ ([M + H]⁺) 446.1849, found 446.1864.

Ethyl 2-[(tert-butoxycarbonyl)amino]-5-hydroxy-6-iodohexanoate (2S,5S)-18. 17 (0.55 g, 1.23 mmol) was dissolved in acetone (30 mL) and to this solution NaI (1 g, 6.67 mmol) was added and the resulting solution was refluxed for 8 h. After 8 h, the acetone was replaced with EtOAc and washed with sat. Na₂S₂O₃, brine, dried (MgSO₄), filtered and evaporated to give an oil purified by silica gel chromatography (EtOAc : hexane, 50 : 50, v/v) to give 18 (0.47 g, 95%). ¹H (500 MHz, CDCl₃) 5.11 (bd, 1H), 4.32 (m, 1H), 4.21 (m, 2H), 3.58 (m, 1H), 3.36 (m, 1H), 3.22 (m, 1H), 2.16 (bd, 1H), 1.93–1.92 (m, 1H), 1.82–1.80 (m, 1H), 1.70–1.66 (m,1H), 1.60–1.53 (m, 1H), 1.45 (s, 9H), 1.28 (m, 3H); ¹³C NMR (125 MHz, CDCl₃) 172.5, 155.5, 80.1, 70.3, 61.5, 53.1, 32.1, 29,1, 28.3, 15.8, 14.2; LRMS-FAB (m/z) 402 (35), 346 (69), 302 (100), 284 (37); HRMS-FAB (m/z) calcd for C₁₃H₂₅N₁O₅I₁ ([M + H]⁺) 402.0777, found 402.0794.

1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxylate (2S,5S)-19. 18 (0.262 g, 0.65 mmol) was dissolved in TFA (4 mL) and allowed to stand at room temperature. After 1 h when the reaction was complete (TLC) the excess TFA was evaporated, the crude product was redissolved in diethyl ether and evaporated to remove excess TFA, to obtain Boc deprotected product as a salt of TFA, which was dissolved in THF (5 mL) and to this DIEA (0.23 mL, 1.30 mmol) was added, and the resulting solution was stirred at room temperature. After 4 h when the reaction was complete which was confirmed by TLC, the solution was treated with additional DIEA (0.12 mL, 0.65 mmol) and Boc₂O (0.28 g, 1.30 mmol) and the resulting solution stirred at room temperature. After 12 h the solution was concentrated and the residue was dissolved in ethyl acetate and washed with 4% NaHCO₃, 10% citric acid, brine, dried (MgSO₄), filtered and evaporated to get an oil purified by silica gel column chromatography (EtOAc : hexane, 50 : 50, v/v) to get 19 as an oil (0.13 g, 76%). ¹H NMR (500 MHz, CDCl₃) 4.87–4.58 (m, 1H), 4.28–4.04 (m, 3H), 3.71–3.58 (m, 1H), 2.82–2.64 (m, 1H), 2.35–2.19 (m, 1H) 2.04–1.94 (m, 1H), 1.78–1.67 (m, 1H), 1.62–1.53 (m, 1H), 1.47, 1.44 (both s, total 9H); ¹³C NMR (125 MHz, CDCl₃) 171.5 (171.4), 155.5 (155.2), 80.5, 66.8 (66.7), 61.3, 54.0 (52.8), 48.5 (47.6), 30.5 (30.0), 28.3, 25.0 (24.8), 14.2; LRMS-FAB (m/z) 274 (70), 218 (60), 174 (100), 144 (45); HRMS-FAB (m/z) calcd for C₁₃H₂₄ N₁O₅ ([M + H]⁺) 274.1654, found 274.1658.

4. Conclusion

Starting from a common intermediate all the four isomers of 5-hydroxypipecolic acid (2S,5R), (2S,5S), (2R,5S) and (2R,5R) were synthesized from commercially available malonate derivatives. Enantiomerically pure 2-amino-5-hexenoic acids were obtained by enzymatic resolution of the racemic amino acid ester (α-chymotrypsin) or acetyl aminoacid (L-acylase). Oxidation of the unsaturated side chain of the enantiomerically pure L-2-amino-5-hexenoic acid with m-CPBA, generated the diastereomeric, inseparable epoxides. Co catalysed hydrolytic kinetic resolution converted the diastereomeric epoxide into separable components, each of them were correspondingly transformed into C⁶ halo amino acids. Followed by, intramolecular nucleophilic attack of the amino –NH₂ group at C⁶ carbon generating the respective 5-hydroxypipecolic acid diastereomers. Similarly starting from D-2-amino-5-hexenoic acid, two diastereomers of 5-hydroxypipecolic acid were synthesized. Ultimately synthesis of a single diastereomer was successfully achieved surpassing the difficulty of isolation of the cis- and trans-diastereomers, in a facile fashion. Application of these compound to synthesize biologically important cyclic tetra peptides are being investigated which will be reported in future.

Acknowledgements

We are grateful to the Center for Instrumental Analysis, Kyushu Institute of Technology (KITCIA) for ¹H NMR and ¹³C NMR spectra. We also like to thank Dr. Tamaki Kato for helping us in chiral HPLC analysis.

References

1. B. Witkop and C. M. Foltz, J. Am. Chem. Soc., 1957, 79, 192–197.
2. J. Despontin, M. Marlier and G. Dardenne, Phytochemistry, 1977, 16, 387–388.
3. C. E. Polan, W. G. Smith, C. Y. NG, R. H. Hammerstedt and L. M. Henderson, J. Nutr., 1967, 91, 143–150.
4. M. A. Letavic, M. Z. Axt, J. T. Barberia, T. J. Carty, D. E. Danley, K. F. Geoghegan, N. S. Halim, L. R. Hoth, A. V. Kamath, E. R. Laird, L. L. Lopresti-Morrow, K. F. McClure, P. G. Mitchell, V. Natarajan, M. C. Noe, J. Pandit, L. Reeves, G. K. Schulte, S. L. Snow, F. J. Sweeney, D. H. Tan and C. H. Yu, Bioorg. Med. Chem. Lett., 2002, 12, 1387–1390.
5. S. P. Miller, Y.-L. Zhong, Z. Liu, M. Simeone, N. Yasuda, J. Limanto, Z. Chen, J. Lynch and V. Capodanno, Org. Lett., 2014, 16, 174–177.
6. T. A. Blizzard, H. Chen, S. Kim, J. Wu, R. Bodner, C. Gude, J. Imbriglio, K. Young, Y.-W. Park, A. Ogawa, S. Raghoobar, N. Hairston, R. E. Painter, D. Wisniewski, G. Scapin, P. Fitzgerald, N. Sharma, J. Lu, S. Ha, J. Hermes and M. L. Hammonda, Bioorg. Med. Chem. Lett., 2014, 24, 780–785.
7. L. L. Corre, J. C. Kizirian, C. Levraud, J. L. Boucher, V. Bonnet and H. Dhimane, Org. Biomol. Chem., 2008, 6, 3388–3398.
8. S. A. Brenner and J. T. Romeo, Appl. Environ. Microbiol., 1986, 51, 690–693.
9. L. Mester, L. Szabados, M. Mester and N. Yadav, Planta Med., 1979, 35, 339–341.
10. C.-S. Sun, Y.-S. Lin and D.-R. Hou, J. Org. Chem., 2008, 73, 6877–6880.
11. D. Scarpi, L. Bartali, A. Casini and E. G. Occhiato, Eur. J. Org. Chem., 2013, 2013, 1306–1317.
12. R. Hara, N. Uchiumi, N. Okamoto and K. Kino, Biosci., Biotechnol., Biochem., 2014, 78, 1384–1388.
13. P. N. M. Botman, F. J. Dommerholt, R. de Gelder, Q. B. Broxterman, H. E. Schoemaker, F. P. J. T. Rutjes and R. H. Blaauw, Org. Lett., 2004, 6, 4941–4944.
14. P. D. Bailey and J. S. Bryans, Tetrahedron Lett., 1988, 18, 2231–2234.
15. S. Krishnamurthy, T. Arai, K. Nakanishi and N. Nishino, RSC Adv., 2014, 4, 2482–2490.
16. S. Hoarau, J. L. Fauchère, L. Pappalardo, M. L. Roumestant and P. Viallefont, Tetrahedron: Asymmetry, 1996, 7, 2585–2593.
17. D. S. Kemp and T. P. Curran, J. Org. Chem., 1986, 51, 2377–2378.
18. J. E. Baldwin, C. Hulme and C. J. Schofield, J. Chem. Res., Miniprint, 1992, 1517–1526.
19. P. Allevi, M. Galligani and M. Anastasia, Tetrahedron: Asymmetry, 2002, 13, 1901–1910.
20. S. E. Schaus, B. D. Brandes, J. F. Larrow, M. Tokunaga, K. B. Hansen, A. E. Gould, M. E. Furrow and E. N. Jacobsen, J. Am. Chem. Soc., 2002, 124, 1307–1315.
21. M. Tokunaga, J. F. Larrow, F. Kakiuchi and E. N. Jacobsen, Science, 1997, 277, 936–938.
22. J. S. Bajwa and R. C. Anderson, Tetrahedron Lett., 1991, 32, 3021–3024.
23. 1.HCl m.p. 215–217, [α]²³_D = −10.8 (c = 0.6, H₂O), {lit.¹³ 212–215 °C; lit.¹¹ [α]²³_D =−9.5 (c = 0.59, H₂O)}.
24. R. B. Kamble, S. H. Gadre and G. M. Suryavanshi, Tetrahedron Lett., 2015, 56, 1263–1265.
25. M. J. Martinelli, N. K. Nayyar, E. D. Moher, U. P. Dhokte, J. M. Pawlak and R. Vaidyanathan, Org. Lett., 1999, 1, 447–450.
26. For selective mono mesylation see: J. Marin, C. Didierjean, A. Aubry, J.-P. Briand and G. Guichard, J. Org. Chem., 2002, 67, 8440–8449.
27. M.p. 188–192 °C, [α]²⁰_D = −23.1 (c = 1, H₂O), {lit.²⁸ m.p. 195–196 °C; [α]²⁰_D = −21.9 (c = 1, H₂O)}.
28. C. Herdeis and E. Heller, Tetrahedron Lett., 1993, 4, 2085–2094.
29. Y. A. Lin, J. M. Chalker, N. Floyd, G. J. L. Bernardes and B. G. Davis, J. Am. Chem. Soc., 2008, 130, 9642–9643.
30. K. Ramesh, S. Surprenant and W. D. Lubell, J. Org. Chem., 2005, 70, 3838–3844.

---

Footnote

† Electronic supplementary information (ESI) available: Synthesis of (2R) isomers, ¹H NMR and ¹³C NMR spectra. See DOI: 10.1039/c5ra09207h

---