Asymmetric Synthesis of the C15–C32 Fragment of Alotamide and Determination of the Relative Stereochemistry

Alotamide is a cyclic depsipetide isolated from a marine cyanobacterium and possesses a unique activation of calcium influx in murine cerebrocortical neurons (EC50 4.18 µM). Due to its limited source, the three stereocenters (C19, C28, and C30) in its polyketide fragment remain undetermined. In this study, the first asymmetric synthesis of its polyketide fragment was achieved. Four relative possible diastereomers were constructed with a boron-mediated enantioselective aldol reaction and Julia–Kocienski olefination as the key steps. Comparison of 13C NMR spectra revealed the relative structure of fragment C15–C32 of alotamide.


Introduction
Recently, several active secondary metabolites have been isolated from marine cyanobacterium and some of these metabolites demonstrate excellent bioactivities such as cytotoxic, antimicrobial, and antiprotozoal properties [1,2]. For example, apratoxins display potent cytotoxicity against several cancer cells at the nanoscale level and have become the new lead compounds in anticancer drug discovery [3][4][5][6].
Alotamide was also isolated from the marine cyanobacterium Lyngbya bouillonii in 2009 [7]. It is a cyclic depsipeptide and structurally has two parts. The northern part is a tripetide that consists of N-Me-Val, Cys-derived thiazolene ring, and Pro and the southern part is a special unsaturated polyketide with three undetermined stereocenters (C19, C28, and C30). Functionally, alotamide is a unique calcium influx activator in murine cerebrocortical neurons (EC 50 4.18 µM). Given that calcium overload is involved in physiological processes and may lead to several nervous diseases such as AD and epilepsy, this compound has gained increasing attention as a new neurotoxin from the marine resource [8]. In view of the limited natural source, a concise synthetic strategy of alotamide should be developed. In this study, we described the first asymmetric synthesis of its polyketide fragment C15-C32 and obtained four possible diastereomers. The relative stereochemistry was assigned after the NMR comparison.

Results
The chemical structure of alotamide is shown in Figure 1. The southern polyketide with three undetermined stereocenters established eight possible isomers and only four needed to be evaluated for relative stereochemical determination. In this regard, we set C19 as R and listed four diastereomers, which are shown in Figure 2 (1a-1d). According to the retrosynthetic analysis (Scheme 1), the dihydroxy unit would arise from an asymmetric aldol reaction and the Julia-Kocienski olefination would be applied to form the diene part. The polyketide fragment would be separated into two subunits, 2 and 3, which both could be prepared from commercial compounds. Scheme 1. Retrosynthetic analysis.

Results
The chemical structure of alotamide is shown in Figure 1. The southern polyketide with three undetermined stereocenters established eight possible isomers and only four needed to be evaluated for relative stereochemical determination. In this regard, we set C19 as R and listed four diastereomers, which are shown in Figure 2 (1a-1d).

Results
The chemical structure of alotamide is shown in Figure 1. The southern polyketide with three undetermined stereocenters established eight possible isomers and only four needed to be evaluated for relative stereochemical determination. In this regard, we set C19 as R and listed four diastereomers, which are shown in Figure 2 (1a-1d). According to the retrosynthetic analysis (Scheme 1), the dihydroxy unit would arise from an asymmetric aldol reaction and the Julia-Kocienski olefination would be applied to form the diene part. The polyketide fragment would be separated into two subunits, 2 and 3, which both could be prepared from commercial compounds.  According to the retrosynthetic analysis (Scheme 1), the dihydroxy unit would arise from an asymmetric aldol reaction and the Julia-Kocienski olefination would be applied to form the diene part. The polyketide fragment would be separated into two subunits, 2 and 3, which both could be prepared from commercial compounds.

Results
The chemical structure of alotamide is shown in Figure 1. The southern polyketide with three undetermined stereocenters established eight possible isomers and only four needed to be evaluated for relative stereochemical determination. In this regard, we set C19 as R and listed four diastereomers, which are shown in Figure 2 (1a-1d). According to the retrosynthetic analysis (Scheme 1), the dihydroxy unit would arise from an asymmetric aldol reaction and the Julia-Kocienski olefination would be applied to form the diene part. The polyketide fragment would be separated into two subunits, 2 and 3, which both could be prepared from commercial compounds.
Compound 2 was prepared from commercial lactone 4 and treated with LiOH to open the lactone ring (Scheme 2) [9]. The resulting compound was subjected to TBS protection and condensation with Evan's protocol 6 [10] in sequence to obtain 7 in 71% yield over three steps. Compound 7 was treated with NaHMDS/MeI at −78 • C to obtain the desired R-methyl 8 in 89% yield (dr = 12:1). After the reduction of 8 by LiBH 4 [11], alcohol 9 was oxidized into the corresponding aldehyde, which was immediately refluxed with the Wittig reagent 10 to generate olefin 11. In this reaction, the E/Z selectivity reached 30:1 and the little Z isomer was separated in the following DIBAL-H reduction. The sequential oxidation with IBX and Pinnick reaction generated a free acid and the allyl protection was conducted to acquire 12 in 64% yield for the above four steps. After the deprotection of the TBS group, the Mitsunobu reaction was applied to convert the compound 13 into tetrazole 15 in 94% yield. Then the SPT part was oxidized by H 2 O 2 (20 eq) in the presence of (NH 4 ) 6 Mo 7 O 24 (0.2 eq) to obtain the desired 2 without the Z isomer [12]. Compound 2 was prepared from commercial lactone 4 and treated with LiOH to open the lactone ring (Scheme 2) [9]. The resulting compound was subjected to TBS protection and condensation with Evan's protocol 6 [10] in sequence to obtain 7 in 71% yield over three steps. Compound 7 was treated with NaHMDS/MeI at −78 °C to obtain the desired R-methyl 8 in 89% yield (dr = 12:1). After the reduction of 8 by LiBH4 [11], alcohol 9 was oxidized into the corresponding aldehyde, which was immediately refluxed with the Wittig reagent 10 to generate olefin 11. In this reaction, the E/Z selectivity reached 30:1 and the little Z isomer was separated in the following DIBAL-H reduction. The sequential oxidation with IBX and Pinnick reaction generated a free acid and the allyl protection was conducted to acquire 12 in 64% yield for the above four steps. After the deprotection of the TBS group, the Mitsunobu reaction was applied to convert the compound 13 into tetrazole 15 in 94% yield. Then the SPT part was oxidized by H2O2 (20 eq) in the presence of (NH4)6Mo7O24 (0.2 eq) to obtain the desired 2 without the Z isomer [12]. The different bases were tested through olefination (Table 1). When 1 eq KHMDS (entry 1) was applied, the yield was 50% and numerous reactants 2 and 3 remained. The product diene was an E/Z mixture with E/Z selectivity of up to 15:1. When the amount of KHMDS was increased to 1.5 eq, the yield improved and the two reactants remained in small quantities. When 2 eq KHMDS was used, the two reactants were completely consumed and the yield reached 86% while the high selectivity (15:1) was maintained. Other bases such as LiHMDS were also tried, but the resulting E/Z selectivity was low. We also exchanged the aldehyde and SO2PT functional groups and subjected them to olefination (entry 5). The yield of the Z isomer greatly increased and the selectivity was 1.5:1 [15,16]. Having completed the construction of diene 21a, we started to prepare the dihydroxy unit (Scheme 4). To our delight, under the condition of the HF/Py complex, we fairly achieved the alcohol 22 and oxidized it to corresponding aldehyde 23 by DMP prior to the aldol reaction (deprotection of PMB group in this step led to a complex mixture). The different bases were tested through olefination (Table 1). When 1 eq KHMDS (entry 1) was applied, the yield was 50% and numerous reactants 2 and 3 remained. The product diene was an E/Z mixture with E/Z selectivity of up to 15:1. When the amount of KHMDS was increased to 1.5 eq, the yield improved and the two reactants remained in small quantities. When 2 eq KHMDS was used, the two reactants were completely consumed and the yield reached 86% while the high selectivity (15:1) was maintained. Other bases such as LiHMDS were also tried, but the resulting E/Z selectivity was low. We also exchanged the aldehyde and SO 2 PT functional groups and subjected them to olefination (entry 5). The yield of the Z isomer greatly increased and the selectivity was 1.5:1 [15,16]. The different bases were tested through olefination (Table 1). When 1 eq KHMDS (entry 1) was applied, the yield was 50% and numerous reactants 2 and 3 remained. The product diene was an E/Z mixture with E/Z selectivity of up to 15:1. When the amount of KHMDS was increased to 1.5 eq, the yield improved and the two reactants remained in small quantities. When 2 eq KHMDS was used, the two reactants were completely consumed and the yield reached 86% while the high selectivity (15:1) was maintained. Other bases such as LiHMDS were also tried, but the resulting E/Z selectivity was low. We also exchanged the aldehyde and SO2PT functional groups and subjected them to olefination (entry 5). The yield of the Z isomer greatly increased and the selectivity was 1.5:1 [15,16]. Having completed the construction of diene 21a, we started to prepare the dihydroxy unit (Scheme 4). To our delight, under the condition of the HF/Py complex, we fairly achieved the alcohol 22 and oxidized it to corresponding aldehyde 23 by DMP prior to the aldol reaction (deprotection of PMB group in this step led to a complex mixture). Having completed the construction of diene 21a, we started to prepare the dihydroxy unit (Scheme 4). To our delight, under the condition of the HF/Py complex, we fairly achieved the alcohol 22 and oxidized it to corresponding aldehyde 23 by DMP prior to the aldol reaction (deprotection of PMB group in this step led to a complex mixture). IPCBCl-controlled aldol reaction [17,18] was selected to install the C28 stereo-center ( Table 2). The application of (-)-IPCBCl at −20 °C successfully generated 24b with the C28 (S) configuration in 59% yield (dr = 99:1). The chiral reactant was changed into (+)-IPCBCl, which afforded the C28 (R) product as expected and maintained the high diastereoselectivity (dr = 98:2). LiHMDS also proceeded and a 1:1 mixture was obtained in this aldol reaction. After TBS protection followed by the reduction of the combination of BH3-DMS and CBS catalysts, we obtained 26a (dr = 4:1, determined by 1 H NMR) and 26b (dr = 17:1, determined by 1 H NMR), respectively. Compounds 26c (dr = 3.6:1, determined by 1 H NMR) and 26d (dr = 14:1, determined by 1 H NMR) were achieved from 25b with the similar dr value (Scheme 5). IPCBCl-controlled aldol reaction [17,18] was selected to install the C28 stereo-center ( Table 2). The application of (−)-IPCBCl at −20 • C successfully generated 24b with the C28 (S) configuration in 59% yield (dr = 99:1). The chiral reactant was changed into (+)-IPCBCl, which afforded the C28 (R) product as expected and maintained the high diastereoselectivity (dr = 98:2). LiHMDS also proceeded and a 1:1 mixture was obtained in this aldol reaction. IPCBCl-controlled aldol reaction [17,18] was selected to install the C28 stereo-center ( Table 2). The application of (-)-IPCBCl at −20 °C successfully generated 24b with the C28 (S) configuration in 59% yield (dr = 99:1). The chiral reactant was changed into (+)-IPCBCl, which afforded the C28 (R) product as expected and maintained the high diastereoselectivity (dr = 98:2). LiHMDS also proceeded and a 1:1 mixture was obtained in this aldol reaction. After TBS protection followed by the reduction of the combination of BH3-DMS and CBS catalysts, we obtained 26a (dr = 4:1, determined by 1 H NMR) and 26b (dr = 17:1, determined by 1 H NMR), respectively. Compounds 26c (dr = 3.6:1, determined by 1 H NMR) and 26d (dr = 14:1, determined by 1 H NMR) were achieved from 25b with the similar dr value (Scheme 5). After TBS protection followed by the reduction of the combination of BH 3 -DMS and CBS catalysts, we obtained 26a (dr = 4:1, determined by 1 H NMR) and 26b (dr = 17:1, determined by 1 H NMR), respectively. Compounds 26c (dr = 3.6:1, determined by 1 H NMR) and 26d (dr = 14:1, determined by 1 H NMR) were achieved from 25b with the similar dr value (Scheme 5). IPCBCl-controlled aldol reaction [17,18] was selected to install the C28 stereo-center ( Table 2). The application of (-)-IPCBCl at −20 °C successfully generated 24b with the C28 (S) configuration in 59% yield (dr = 99:1). The chiral reactant was changed into (+)-IPCBCl, which afforded the C28 (R) product as expected and maintained the high diastereoselectivity (dr = 98:2). LiHMDS also proceeded and a 1:1 mixture was obtained in this aldol reaction. After TBS protection followed by the reduction of the combination of BH3-DMS and CBS catalysts, we obtained 26a (dr = 4:1, determined by 1 H NMR) and 26b (dr = 17:1, determined by 1 H NMR), respectively. Compounds 26c (dr = 3.6:1, determined by 1 H NMR) and 26d (dr = 14:1, determined by 1 H NMR) were achieved from 25b with the similar dr value (Scheme 5). The stereochemistries of the 1,3-diol part in 26a-26d were confirmed by their 13 C NMR chemical shifts of the corresponding acetonides 28a-28d (Scheme 6). Syn-diol acetonide preferred a chair conformation and two ketal methyl groups were significantly different (e.g., 19.94 and 30.46 ppm for 28a). Meanwhile anti-diol acetonide preferred a twist-boat conformation and two similar methyl groups existed (e.g., 25.24 and 25.13 ppm for 28b) [19][20][21]. After the sequential methylation and removal of the TBS group from 26a-26d, we successfully furnished the four desired analogues 1a-1d (Scheme 7). The total yield was 2.5% for 1a from the lactone and 2.7% for 1b, 2.5% for 1c, and 2.6% for 1d.
After the sequential methylation and removal of the TBS group from 26a-26d, we successfully furnished the four desired analogues 1a-1d (Scheme 7). The total yield was 2.5% for 1a from the lactone and 2.7% for 1b, 2.5% for 1c, and 2.6% for 1d. Scheme 7. Synthesis of 1a-1d.

Discussion
A careful comparison between four isomers and alotamide was conducted. The differences in 13 C NMR chemical shifts are shown in Figure 3a. From C15 to C28, several significant variations (Δδ > 1 ppm) existed between all four isomers and alotamide possibly because of the difference between the "straight-chain" mode in our analogues and the "ring" mode in the original structure. From C28 to C32, the dihydroxy unit is a linear chain both in our analogues and natural compound. Thus, comparing the data in this portion is suitable for relative stereochemistry determination.
At the same time, several obvious variations in 13 C spectra were observed between 1,3-syn isomers (1a and 1c) and 1,3-anti isomers (1b and 1d) from C29 to C32 (Figure 3a). Therefore, we chose 1a and 1b to represent the syn-analogues and anti-analogues to distinguish syn-configurations and anti-configurations (Figure 3b and 3c).
A significant variation at C30 position of 1b (Δδ > 4 ppm) and a closer correlation of 1a at C29, C31 and C32 in the 13 C spectra revealed the syn configuration in the C28-C30 unit. In addition, smaller variations with alotamide in 1 H spectra were noticed for analogue 1a at protons H29A and H29B (differences between alotamide and 1a and 1b at proton H30 were not plotted in Figure 3c because the differences were all greater than 0.4 ppm and insignificant). This finding confirmed the 1,3-syn structure in the dihydroxy unit.

Discussion
A careful comparison between four isomers and alotamide was conducted. The differences in 13 C NMR chemical shifts are shown in Figure 3a. From C15 to C28, several significant variations (∆δ > 1 ppm) existed between all four isomers and alotamide possibly because of the difference between the "straight-chain" mode in our analogues and the "ring" mode in the original structure. From C28 to C32, the dihydroxy unit is a linear chain both in our analogues and natural compound. Thus, comparing the data in this portion is suitable for relative stereochemistry determination.
At the same time, several obvious variations in 13 C spectra were observed between 1,3-syn isomers (1a and 1c) and 1,3-anti isomers (1b and 1d) from C29 to C32 (Figure 3a). Therefore, we chose 1a and 1b to represent the syn-analogues and anti-analogues to distinguish syn-configurations and anti-configurations (Figure 3b,c). Two 1,3-syn isomers 1a and 1c were also compared. Their 1 H spectra were the same and the differences in 13 C NMR chemical shifts are listed in Table 3. A closer correlation of 1c was observed especially at the sites C20, C29, and C31. Thus, it appeared that 1c (19R, 28S, 30R) most closely fit the original natural alotamide. The total synthesis of alotamide with fragment 1c and another (19S, 28R, 30S) enantiomer is in progress. A significant variation at C30 position of 1b (∆δ > 4 ppm) and a closer correlation of 1a at C29, C31 and C32 in the 13 C spectra revealed the syn configuration in the C28-C30 unit. In addition, smaller variations with alotamide in 1 H spectra were noticed for analogue 1a at protons H29A and H29B (differences between alotamide and 1a and 1b at proton H30 were not plotted in Figure 3c because the differences were all greater than 0.4 ppm and insignificant). This finding confirmed the 1,3-syn structure in the dihydroxy unit. Two 1,3-syn isomers 1a and 1c were also compared. Their 1 H spectra were the same and the differences in 13 C NMR chemical shifts are listed in Table 3. A closer correlation of 1c was observed especially at the sites C20, C29, and C31. Thus, it appeared that 1c (19R, 28S, 30R) most closely fit the original natural alotamide. The total synthesis of alotamide with fragment 1c and another (19S, 28R, 30S) enantiomer is in progress.

Materials and Methods
All anaerobic and moisture-sensitive manipulations were carried out with standard Schlenk techniques under argon. Solvents were dried and distilled by standard procedures. 1 H-NMR and 13 C-NMR spectra were recorded in CDCl 3 on a Bruker Ascend-400 400 MHz or Bruker Ascend-500 500 MHz at room temperature. Chemical shifts (δ) are reported in ppm and are referenced to chloroform (δ 7.26 ppm for 1H, δ 77.16 ppm for 13 C). Data for NMR spectra are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, quint. = quintet, sext. = sextet, m = multiplet, br = broad signal, J = coupling constant in Hz. HRMS were recorded on an Agilent 6530-Q-TOF mass spectrometer equipped with an Agilent 1260-HPLC. Optical rotations were measured on a PerkinElmer 241 MC polarimeter.

(R)-3-(4-((Tert-Butyldimethylsilyl)Oxy)Butanoyl)-4-Phenyloxazolidin-2-One (7)
To a solution of the lactone (5 g, 58 mmol) in MeOH (50 mL) at room temperature was added LiOH (2.44 g, 58 mmol) and stirred overnight. The solvent was removed from the reaction and the residue was dissolved in dimethylformamide (50 mL) at 0 • C. Imidazole (8 g, 116 mmol) was added to this solution, which was followed by TBSCl (8.7 g, 58 mmol) in two portions over 15 min. The reaction mixture was warmed to room temperature overnight with stirring and then diluted with 1 M HCl and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine and dried over Na 2 SO 4 . The organic layer was removed under vacuum and the crude acid was used without further purification.
To a stirred solution of the acid in dry THF (200 mL) at 0 • C under argon, Et 3 N (20 mL, 145 mmol) and PivCl (7.1 mL, 58 mmol) were added sequentially. After 1 h stirring at 0 • C, LiCl (0.62 g, 14.5 mmol), followed by oxazolidinone 6 (9.4 g, 58 mmol), were added. The reaction was continued for 1 h at 0 • C and another 2 h at room temperature prior to quenching with a saturated NH 4 Cl solution (50 mL) and extracted with DCM (2 × 200 mL). The combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated in vacuum. Purification by column chromatography (PE/EA = 9:1) afforded compound 7 (

(R)-4-((Tert-Butyldimethylsilyl)Oxy)-2-Methylbutan-1-Ol (9)
To an ice-cold solution of compound 8 (13.8 g, 36.6 mmol) in THF (100 mL) moist with a catalytic amount of water, LiBH 4 (1.2 g, 54.9 mmol) was added portion wise under argon. After 12 h of stirring at room temperature, the reaction was quenched cautiously with a saturated NH 4 Cl solution (50 mL) and then distilled under a reduced pressure followed by extraction with DCM. The combined organic solution was dried over Na 2 SO 4 and concentrated in vacuum. Purification by column chromatography (PE/EA = 9:1) provided pure compound 9 (6.86 g, 86%) as a colorless oil [11]. 1   To a stirred solution of 9 (6.86 g, 31.5 mmol) in DMSO (50 mL) IBX (10.6 g, 37.8 mmol) was added. After 1 h stirring at 40 • C, the reaction was quenched with water (50 mL) and extracted with ether (2 × 100 mL). The combined organic layers were washed with brine, dried over Na 2 SO 4 , and removed under vacuum. The residue was refluxed with 10 (22 g, 63 mmol) in toluene (100 mL) at 80 • C for 3 h and the solvent was removed under vacuum. Purification by column chromatography (PE/EA = 40:1) provided pure compound 11 (7 g, 75%) as a colorless oil [22]. 1   To a stirred solution of compound 11 (7 g, 23.6 mmol) in dry DCM (60 mL) at −78 • C under argon, DIBAL-H (1.5 M solution in toluene, 18.0 mL, 27.0 mmol) was added dropwise. After 1 h, the reaction mixture was quenched with aqueous sodium-potassium tartrate solution (20 mL) and warmed up to room temperature before being extracted with DCM (2 × 100 mL). The combined organic extracts were washed with water and brine, dried over Na 2 SO 4 , and concentrated in vacuum. Purification by column chromatography (PE/EA = 8:1) gave alcohol (4.8 g, 79%) as a colorless oil.
To a stirred solution of above alcohol in DMSO (50 mL), IBX (6.2 g, 22 mmol) was added. After 1 h stirring at 40 • C, the reaction was quenched with water (50 mL) and extracted with ether (2 × 100 mL). The combined organic layers were washed with brine, dried over Na 2 SO 4 , and removed under vacuum. The crude anhydride was used without further purification.
To a stirred solution of above acid in dry DMF (50 mL) allylBr (3.2 g, 37.2 mmol) and K 2 CO 3 (5.1 g, 37.2 mmol) were added separately. After being stirred for 12 h, the mixture was quenched with a saturated aqueous solution of NH 4 Cl and extracted with EA (100 mL × 3). The combined organic layers were washed with brine, dried over Na 2 SO 4 , filtrated, and concentrated. The residue was purified by column chromatography on silica gel (PE/EA = 40:1) to give 12 (4.7 g, 81%) for three steps as a colorless oil.
To a stirred solution of crude 3 and compound 2 (1.  To a stirred solution of 21a (1.0 g, 2.6 mmol) in dry THF (5 mL), HF/Py complex (1 mL) was added at 0 • C. After being stirred for 1 h, the mixture was quenched with a saturated aqueous solution of NaHCO 3 and extracted with DCM (20 mL × 3). The combined organic layers were washed with 1 M HCl, brine, dried over Na 2 SO 4 , filtrated, and concentrated. The residue was purified by column chromatography on silica gel (PE/EA = 6:1) to give 22 (550 mg, 76%) as a colorless oil.   and NaHCO 3 (240 mg, 2.9 mmol) was added sequentially. After being stirred for 30 min, the mixture was carefully quenched with a solution of saturated aqueous NaHCO 3 and Na 2 S 2 O 3 . The resulting mixture was extracted with DCM (20 mL × 3) and the combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated. The crude aldehyde 23 was used without future purification.
To a stirred solution of (+)-IPCBCl (1.8 M in heptane, 1.08 mmol, 0.6 mL) in dry ether (3 mL) at −0 • C under argon, Et 3 N (0.2 mL, 1.44 mmol) and acetone (80 uL, 1.08 mmol) were added sequentially. After 1 h stirring at −0 • C, aldehyde 23 in ether (2 mL) was added at −78 • C. The reaction was continued further for 1 h at −78 • C and another 12 h at −20 • C prior to quenching with a mixture of PH 7 buffer (1 mL), methanol (1 mL), and H 2 O 2 (1 mL). The mixture was warmed up to room temperature before being extracted with ether (2 × 10 mL). The combined organic extracts were washed with water and brine, dried over Na 2 SO 4 , and concentrated in vacuum. Purification by column chromatography (PE/EA = 4:1) created 24a (132 mg, 55%) as a colorless oil.  To a stirred solution of (−)-IPCBCl (1.7 M in heptane, 1.08 mmol, 0.64 mL) in dry ether (3 mL) at 0 • C under argon, Et 3 N (0.2 mL, 1.44 mmol) and acetone (80 uL, 1.08 mmol) were added sequentially. After 1 h stirring at 0 • C, aldehyde 23 in ether (2 mL) was added at −78 • C. The reaction was continued further for 1 h at −78 • C and another 12 h at −20 • C prior to quenching with a mixture of PH 7 buffer (1 mL), methanol (1 mL), and H 2 O 2 (1 mL). The mixture was warmed up to room temperature before being extracted with ether (2 × 10 mL). The combined organic extracts were washed with water and brine, dried over Na 2 SO 4   To a stirred solution of compound 24a (132 mg, 0.4 mmol) in dry DCM (3 mL) at −78 • C under argon, 2,6-lutidine (0.23 mL, 2 mmol) and TBSOTf (0.35 mL, 1.5 mmol) were added sequentially. After 1 h, the reaction mixture was quenched with aqueous NaHCO 3 (5 mL) and warmed up to room temperature before being extracted with DCM (2 × 10 mL). The combined organic extracts were washed with 1 M HCl and brine, dried over Na 2 SO 4      To a solution of (R)-Me-CBS (1 M in toluene, 0.05 mL, 0.05 mmol) in dry THF (5 mL) was slowly added BH 3 ·DMS (12 uL, 0.13 mmol) at −40 • C. After being stirred for 30 min at the same temperature, a solution of 25a (57 mg, 0.13 mmol) in THF (2 mL) was slowly added. After being stirred for 2 h at −40 • C, the mixture was diluted with MeOH. The resulting mixture was concentrated and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine, dried, filtrated, and concentrated. The residue was purified by column chromatography on silica gel (PE/EA = 15:1) to give a mixture of 26a and 26b (4:1, 48 mg, 83%) as a colorless oil (pure major isomer 26a can be obtained by repeating the purification on silica gel To a solution of (S)-Me-CBS (1 M in toluene, 0.1 mL, 0.1 mmol) in dry THF (5 mL), BH 3 ·DMS (24 uL, 0.25 mmol) was slowly added at −40 • C. After being stirred for 30 min at the same temperature, a solution of 25a (110 mg, 0.24 mmol) in THF (2 mL) was slowly added. After being stirred for 2 h at −40 • C, the mixture was diluted with MeOH. The resulting mixture was concentrated and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine, dried, filtrated, and concentrated. The residue was purified by column chromatography on silica gel (PE/EA = 15:1) to create 26b (92 mg, 85%) as a colorless oil. 1  To a solution of (S)-Me-CBS (1 M in toluene, 0.05 mL, 0.05 mmol) in dry THF (5 mL), BH 3 ·DMS (12 uL, 0.13 mmol) was slowly added at −40 • C. After being stirred for 30 min at the same temperature, a solution of 25b (55 mg, 0.12 mmol) in THF (2 mL) was slowly added. After being stirred for 2 h at −40 • C, the mixture was diluted with MeOH. The resulting mixture was concentrated and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine, dried, filtrated, and concentrated. The residue was purified by column chromatography on silica gel (PE/EA = 15:1) to give a mixture of 26c and 26d (3.6:1, 44 mg, 80%) as a colorless oil (pure major isomer 26c can be obtained by repeating the purification on silica gel  To a solution of (R)-Me-CBS (1 M in toluene, 0.1 mL, 0.1 mmol) in dry THF (5 mL), BH 3 ·DMS (24 uL, 0.25 mmol) was slowly added at −40 • C. After being stirred for 30 min at the same temperature, a solution of 25b (113 mg, 0.25 mmol) in THF (2 mL) was slowly added. After being stirred for 2 h at −40 • C, the mixture was diluted with MeOH. The resulting mixture was concentrated and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine, dried, filtrated, and concentrated. The residue was purified by column chromatography on silica gel (PE/EA = 15:1) to provide 26d (100 mg, 88%) as a colorless oil. 1

Synthetic Procedure of 27a-27d
To a stirred solution of 26a (35 mg) in dry THF (2 mL), the HF/Py complex (0.7 mL) was added at 0 • C. After being stirred for 1 h, the mixture was quenched with a saturated aqueous solution of NaHCO 3 and extracted with DCM (10 mL × 3). The combined organic layers were washed with 1 M HCl, brine, dried over Na 2 SO 4 , filtrated, and concentrated. The residue was purified by a column chromatography on silica gel (PE/EA = 2:1) to create 27a (22 mg, 84%) as a colorless oil. 1  Compound 27b (20.9 mg, 80%) was obtained as a colorless oil. 1 13