Total Synthesis and Stereochemical Assignment of Nostosin B

Nostosins A and B were isolated from a hydrophilic extract of Nostoc sp. strain from Iran, which exhibits excellent trypsin inhibitory activity. Nostosin A was the most potent natural tripeptide aldehyde as trypsin inhibitor up to now. Both r- and s-2-hydroxy-4-(4-hydroxy-phenyl)butanoic acid (Hhpba) were prepared and incorporated into the total synthesis of nostosin B, respectively. Careful comparison of the NMR spectra and optical rotation data of synthetic nostosin B (1a and 1b) with the natural product led to the unambiguous identification of the r-configuration of the Hhpba fragment, which was further confirmed by co-injection with the authentic sample on HPLC using both reversed phase column and the chiral AD-RH column.


Introduction
Nostosin A and B were isolated from a hydrophilic extract of Nostoc sp. strain obtained from a paddy field in Iran ( Figure 1) [1]. Both nostosin A and B showed potent inhibitory activity toward porcine trypsin, with IC 50 values of 0.35 and 55 µM respectively. Structurally and functionally, nostosin A and B could be classified as natural small linear peptides that act as trypsin inhibitors [2][3][4][5]. These small peptides structurally feature an arginine motif at the C-terminal, which has been proven to be crucial for their interactions with the protease targets [6][7][8][9][10][11]. Surprisingly, nostosin A was more potent than the commercially available trypsin inhibitor leupeptin (IC 50 = 0.5 µM), rendering it as the most potent tripeptide natural trypsin inhibitor up to now.
Both nostosin A and B contain three subunits, 2-hydroxy-4-(4-hydroxyphenyl)butanoic acid (Hhpba), L-Ile and L-Arg, the C-terminal of nostosin B exists as the reduced form of nostosin A, which dramatically diminished its biological activity (158-fold compared to nostosin A), similar to aeruginosin 298 A [12][13][14]. The absolute stereochemistry of Hhpba was not elucidated [1]. Although the authors of isolation paper prefer that Hhpba possesses R-configuration according to the statistical data of the stereochemistry of similar residue of 39 trypsin inhibitors isolated from natural microorganisms, the docking results of both nostosins A and B with trypsin made no obvious difference whatever the Ror S-Hhpba was adopted [1].
Small peptide aldehydes had long been used as proteasome inhibitors, further development of these compounds as antibacterial, anticancer, or other therapeutic reagents have also been reported,

Results
Nostosins A and B are distinctive trypsin inhibitors isolated from nature, however the C-2 stereochemistry of Hhpba has not been defined, which hampers the further development of nostosins as biological probes or lead candidates. As part of our research interests [18][19][20][21][22][23][24][25][26][27][28][29], we decided to verify the absolute stereochemistry of nostosins via the total synthesis of both stereoisomers of nostosin B 1. Since the Hhpba fragment was located at the N-terminal of nostosin B, we decided to prepare this tripeptide via a 1 + 2 strategy, the late-stage introduction of Hhpba made the synthesis more convenient for the preparation of two stereoisomers ( Figure 2). The arginine motif in nostosin B could be easily obtained from Pbf-L-Arg 4.

Synthesis of the Right-Hand Dipeptide
The synthesis was commenced from the reduction of the carboxylic group of N ω -Pbf-L-arginine 4, the resulted amino alcohol was coupled with Fmoc-L-isoleucine 3. The reaction was facilitated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxy-7-azabenzotriazole in the presence of N,N-diisopropylethylamine in dichloromethane to produce dipeptide 5 in 80% yield over two steps. The primary alcohol of 5 was protected by tert-butyldimethylsilyl chloride to give silyl ether 6 in 81% yield. Removal of the fluorenyl-9-methoxycarbonyl group via treatment of 6 with diethylamine in acetonitrile provided amine 7, which was ready for the next step of transformation (Scheme 1).

Results
Nostosins A and B are distinctive trypsin inhibitors isolated from nature, however the C-2 stereochemistry of Hhpba has not been defined, which hampers the further development of nostosins as biological probes or lead candidates. As part of our research interests [18][19][20][21][22][23][24][25][26][27][28][29], we decided to verify the absolute stereochemistry of nostosins via the total synthesis of both stereoisomers of nostosin B 1. Since the Hhpba fragment was located at the N-terminal of nostosin B, we decided to prepare this tripeptide via a 1 + 2 strategy, the late-stage introduction of Hhpba made the synthesis more convenient for the preparation of two stereoisomers ( Figure 2). The arginine motif in nostosin B could be easily obtained from Pbf-L-Arg 4.
Mar. Drugs 2017, 15, 58 2 of 13 production, which could have potential for the treatment of neuronal injury and neurodegenerative diseases [15][16][17]. Herein, we report our total synthesis and stereochemical assignment of nostosin B.

Results
Nostosins A and B are distinctive trypsin inhibitors isolated from nature, however the C-2 stereochemistry of Hhpba has not been defined, which hampers the further development of nostosins as biological probes or lead candidates. As part of our research interests [18][19][20][21][22][23][24][25][26][27][28][29], we decided to verify the absolute stereochemistry of nostosins via the total synthesis of both stereoisomers of nostosin B 1. Since the Hhpba fragment was located at the N-terminal of nostosin B, we decided to prepare this tripeptide via a 1 + 2 strategy, the late-stage introduction of Hhpba made the synthesis more convenient for the preparation of two stereoisomers ( Figure 2). The arginine motif in nostosin B could be easily obtained from Pbf-L-Arg 4.

Synthesis of the Right-Hand Dipeptide
The synthesis was commenced from the reduction of the carboxylic group of N ω -Pbf-L-arginine 4, the resulted amino alcohol was coupled with Fmoc-L-isoleucine 3. The reaction was facilitated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxy-7-azabenzotriazole in the presence of N,N-diisopropylethylamine in dichloromethane to produce dipeptide 5 in 80% yield over two steps. The primary alcohol of 5 was protected by tert-butyldimethylsilyl chloride to give silyl ether 6 in 81% yield. Removal of the fluorenyl-9-methoxycarbonyl group via treatment of 6 with diethylamine in acetonitrile provided amine 7, which was ready for the next step of transformation (Scheme 1).

Synthesis of the Right-Hand Dipeptide
The synthesis was commenced from the reduction of the carboxylic group of N ω -Pbf-L-arginine 4, the resulted amino alcohol was coupled with Fmoc-L-isoleucine 3. The reaction was facilitated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxy-7-azabenzotriazole in the presence of N,N-diisopropylethylamine in dichloromethane to produce dipeptide 5 in 80% yield over two steps. The primary alcohol of 5 was protected by tert-butyldimethylsilyl chloride to give silyl ether 6 in 81% yield. Removal of the fluorenyl-9-methoxycarbonyl group via treatment of 6 with diethylamine in acetonitrile provided amine 7, which was ready for the next step of transformation (Scheme 1).
To prepare the Hhpba fragment, we initially took advantage of malic acid via the Friedel-Crafts reaction [33], anticipating that both R-and S-Hhpba could be originated from D-and L-malic acid, respectively. According to literature precedent, both acetoxysuccinic anhydride and 2-acetoxy-butanoyl chloride had been used to react with benzene or methoxy-substituted benzenes, providing 2-hydroxy-4-aryl-4-oxobutanoates in moderate yields [34,35]. To eliminate the interference of the acetyl group on acetoxysuccinic anhydride in the process of Friedel-Crafts reaction, we decided to protect the hydroxyl group of 9 with benzyl group (Scheme 2).

Scheme 2. Attempt synthesis of Hhpba by Friedel-Crafts reaction.
Esterification of L-malic acid 8 was performed using standard conditions. Hydroxyl group directed regio-selective reduction of the alpha-methyl ester, followed by acid catalyzed cyclization produced lactone 9 at a 77% yield [36,37]. O-benzylation of the sensitive hydroxyl group of 9 was achieved using benzyl trichloroacetimidate (BTCA) in the presence of a catalytic amount of triflic acid [38], affording compound 10 at a 75% yield. The Friedel-Crafts reaction between 10 and benzyl phenyl ether was then performed under different reaction conditions (Table 1).
Using aluminum trichloride as Lewis acid, toluene as solvent, the Friedel-Crafts reaction was carried out either under reflux or at room temperature (entries 1 and 2), both conditions provided no desired product with only decomposition of starting material. When the reaction solvent was changed to dichloroethane (entries 3 and 4), or Lewis acid was changed to trifluoroborane diethyletherate (entry 5), there was still no desired product and the starting material was not recoverable. While molecular sieves (4 Å) did not destroy the substrate, nor did they promote the Friedel-Drafts reaction (entry 6).
To prepare the Hhpba fragment, we initially took advantage of malic acid via the Friedel-Crafts reaction [33], anticipating that both R-and S-Hhpba could be originated from D-and L-malic acid, respectively. According to literature precedent, both acetoxysuccinic anhydride and 2-acetoxy-butanoyl chloride had been used to react with benzene or methoxy-substituted benzenes, providing 2-hydroxy-4-aryl-4-oxobutanoates in moderate yields [34,35]. To eliminate the interference of the acetyl group on acetoxysuccinic anhydride in the process of Friedel-Crafts reaction, we decided to protect the hydroxyl group of 9 with benzyl group (Scheme 2).
To prepare the Hhpba fragment, we initially took advantage of malic acid via the Friedel-Crafts reaction [33], anticipating that both R-and S-Hhpba could be originated from D-and L-malic acid, respectively. According to literature precedent, both acetoxysuccinic anhydride and 2-acetoxy-butanoyl chloride had been used to react with benzene or methoxy-substituted benzenes, providing 2-hydroxy-4-aryl-4-oxobutanoates in moderate yields [34,35]. To eliminate the interference of the acetyl group on acetoxysuccinic anhydride in the process of Friedel-Crafts reaction, we decided to protect the hydroxyl group of 9 with benzyl group (Scheme 2).

Scheme 2. Attempt synthesis of Hhpba by Friedel-Crafts reaction.
Esterification of L-malic acid 8 was performed using standard conditions. Hydroxyl group directed regio-selective reduction of the alpha-methyl ester, followed by acid catalyzed cyclization produced lactone 9 at a 77% yield [36,37]. O-benzylation of the sensitive hydroxyl group of 9 was achieved using benzyl trichloroacetimidate (BTCA) in the presence of a catalytic amount of triflic acid [38], affording compound 10 at a 75% yield. The Friedel-Crafts reaction between 10 and benzyl phenyl ether was then performed under different reaction conditions (Table 1).
Using aluminum trichloride as Lewis acid, toluene as solvent, the Friedel-Crafts reaction was carried out either under reflux or at room temperature (entries 1 and 2), both conditions provided no desired product with only decomposition of starting material. When the reaction solvent was changed to dichloroethane (entries 3 and 4), or Lewis acid was changed to trifluoroborane diethyletherate (entry 5), there was still no desired product and the starting material was not recoverable. While molecular sieves (4 Å) did not destroy the substrate, nor did they promote the Friedel-Drafts reaction (entry 6).

Scheme 2. Attempt synthesis of Hhpba by Friedel-Crafts reaction.
Esterification of L-malic acid 8 was performed using standard conditions. Hydroxyl group directed regio-selective reduction of the alpha-methyl ester, followed by acid catalyzed cyclization produced lactone 9 at a 77% yield [36,37]. O-benzylation of the sensitive hydroxyl group of 9 was achieved using benzyl trichloroacetimidate (BTCA) in the presence of a catalytic amount of triflic acid [38], affording compound 10 at a 75% yield. The Friedel-Crafts reaction between 10 and benzyl phenyl ether was then performed under different reaction conditions (Table 1).
Using aluminum trichloride as Lewis acid, toluene as solvent, the Friedel-Crafts reaction was carried out either under reflux or at room temperature (entries 1 and 2), both conditions provided no desired product with only decomposition of starting material. When the reaction solvent was changed to dichloroethane (entries 3 and 4), or Lewis acid was changed to trifluoroborane diethyletherate (entry 5), there was still no desired product and the starting material was not recoverable. While molecular sieves (4 Å) did not destroy the substrate, nor did they promote the Friedel-Drafts reaction (entry 6). We then turned to develop an asymmetric strategy for Hhpba fragments using commercial available aldehyde 14 and 12 as starting material (Scheme 3). Thus, protection of vanillin 12 with benzylbromide in the presence of potassium carbonate and tetrabutylammonium iodide in acetonitrile, followed by reduction of the aldehyde with sodium borohydride in methanol and bromination of the corresponding benzyl alcohol with hydrogen bromide in dichloromethane, afforded compound 13 at a 94% yield over three steps. The ylide, generated by deprotonation with n-butyllithium of the intermediate by refluxing of 13 with triphenylphosphine, was reacted with aldehyde 14 to forge alkene 15 at a 65% yield [39]. Hydrogenation of 15 with palladium on charcoal in ethyl acetate in the presence of sodium bicarbonate smoothly saturated the double bond with the benzyl ether untouched, giving compound 16 at a 97% yield. The acetonide was subsequently removed using p-toluenesulfonic acid in methanol, the primary alcohol was masked using trityl chloride in dichloromethane in the presence of trimethylamine to afford the secondary alcohol 17 at a 92% yield over two steps. Protection of the free alcohol with benzyl bromide, followed by releasing the primary alcohol, afforded compound 18 at a 67% yield. The synthesis of Hhpba was accomplished via mild oxidation using 2,2,6,6-tetramethylpiperidoxyl, iodobenzene diacetate in water, and dichloromethane [40], producing S-2 at an 84% yield.  We then turned to develop an asymmetric strategy for Hhpba fragments using commercial available aldehyde 14 and 12 as starting material (Scheme 3). Thus, protection of vanillin 12 with benzylbromide in the presence of potassium carbonate and tetrabutylammonium iodide in acetonitrile, followed by reduction of the aldehyde with sodium borohydride in methanol and bromination of the corresponding benzyl alcohol with hydrogen bromide in dichloromethane, afforded compound 13 at a 94% yield over three steps. The ylide, generated by deprotonation with n-butyllithium of the intermediate by refluxing of 13 with triphenylphosphine, was reacted with aldehyde 14 to forge alkene 15 at a 65% yield [39]. Hydrogenation of 15 with palladium on charcoal in ethyl acetate in the presence of sodium bicarbonate smoothly saturated the double bond with the benzyl ether untouched, giving compound 16 at a 97% yield. The acetonide was subsequently removed using p-toluenesulfonic acid in methanol, the primary alcohol was masked using trityl chloride in dichloromethane in the presence of trimethylamine to afford the secondary alcohol 17 at a 92% yield over two steps. Protection of the free alcohol with benzyl bromide, followed by releasing the primary alcohol, afforded compound 18 at a 67% yield. The synthesis of Hhpba was accomplished via mild oxidation using 2,2,6,6-tetramethylpiperidoxyl, iodobenzene diacetate in water, and dichloromethane [40], producing S-2 at an 84% yield.

Scheme 3. Stereocontrolled synthesis of S-Hhpba.
With S-2 in hand, we set forth to prepare its R-enantiomer (Scheme 4). Treatment of acid S-2 with iodomethane and potassium carbonate in N,N-dimethylformamide, followed by hydrogenation to remove the benzyl ethers and subsequent re-protection of the phenol as benzyl ether, produced methyl ester 19 at an 85% yield over three steps. Mitsunobu reaction of the secondary alcohol 19 with 4-nitrobenzoic acid (pNBA) in the presence of triphenylphosphine and diethyl azodicarboxylate in tetrahydrofuran gave ester 20 at a 78% yield. A two-step removal of the protection groups was performed at this stage, first treatment of diester 20 with potassium carbonate in methanol produced ent-19, which demonstrated the integrity of stereochemistry by comparison of the analytical data with compound 19. Further hydrolysis of the methyl ester of ent-19 gave compound 21 at an 85% overall yield. With S-2 in hand, we set forth to prepare its R-enantiomer (Scheme 4). Treatment of acid S-2 with iodomethane and potassium carbonate in N,N-dimethylformamide, followed by hydrogenation to remove the benzyl ethers and subsequent re-protection of the phenol as benzyl ether, produced methyl ester 19 at an 85% yield over three steps. Mitsunobu reaction of the secondary alcohol 19 with 4-nitrobenzoic acid (pNBA) in the presence of triphenylphosphine and diethyl azodicarboxylate in tetrahydrofuran gave ester 20 at a 78% yield. A two-step removal of the protection groups was performed at this stage, first treatment of diester 20 with potassium carbonate in methanol produced ent-19, which demonstrated the integrity of stereochemistry by comparison of the analytical data with compound 19. Further hydrolysis of the methyl ester of ent-19 gave compound 21 at an 85% overall yield.

Total Synthesis of Nostosin B
With both S-and R-Hhpba (S-2 and 21) in hand, we were ready to complete the total synthesis of nostosin B and confirm its absolute stereochemistry.

Scheme 5.
Completion of the total synthesis of nostosin B (both 1a and 1b).

Discussion
The NMR spectra data for nostosin B 1a and 1b were very similar, but both were slightly different from the reported spectra of the natural product  1.0, MeOH)). From these analytical data, we believe that nostosin 1b was the real structure of the natural product, the slight differences on NMR spectra should be arisen from the different experimental conditions for data acquisition, i.e., the concentration of sample and pH of the solution.
To further identify the stereochemistry of nostosin B, the synthetic samples (1a and 1b) were co-injected with the authentic sample (natural product sample) using high-performance liquid

Total Synthesis of Nostosin B
With both S-and R-Hhpba (S-2 and 21) in hand, we were ready to complete the total synthesis of nostosin B and confirm its absolute stereochemistry.

Total Synthesis of Nostosin B
With both S-and R-Hhpba (S-2 and 21) in hand, we were ready to complete the total synthesis of nostosin B and confirm its absolute stereochemistry.

Scheme 5.
Completion of the total synthesis of nostosin B (both 1a and 1b).

Discussion
The NMR spectra data for nostosin B 1a and 1b were very similar, but both were slightly different from the reported spectra of the natural product . From these analytical data, we believe that nostosin 1b was the real structure of the natural product, the slight differences on NMR spectra should be arisen from the different experimental conditions for data acquisition, i.e., the concentration of sample and pH of the solution.
To further identify the stereochemistry of nostosin B, the synthetic samples (1a and 1b) were co-injected with the authentic sample (natural product sample) using high-performance liquid Scheme 5. Completion of the total synthesis of nostosin B (both 1a and 1b).

Discussion
The NMR spectra data for nostosin B 1a and 1b were very similar, but both were slightly different from the reported spectra of the natural product . From these analytical data, we believe that nostosin 1b was the real structure of the natural product, the slight differences on NMR spectra should be arisen from the different experimental conditions for data acquisition, i.e., the concentration of sample and pH of the solution.
To further identify the stereochemistry of nostosin B, the synthetic samples (1a and 1b) were co-injected with the authentic sample (natural product sample) using high-performance liquid chromatography (Figure 3). Both reverse phase column and chiral column gave the same results, indicating that synthetic nostosin B 1b has identical retention time with natural nostosin B, unambiguously defining the stereochemistry of Hhpba as R-configuration. chromatography ( Figure 3). Both reverse phase column and chiral column gave the same results, indicating that synthetic nostosin B 1b has identical retention time with natural nostosin B, unambiguously defining the stereochemistry of Hhpba as R-configuration.

General Experiment
Non-aqueous reactions were performed under a nitrogen or argon atmosphere and all reaction vessels were oven-dried. Solvents were distilled prior to use: dichloromethane (DCM), triethylamine and diisopropylethylamine (DIPEA) from CaH2, tetrahydrofuran (THF) from Na/benzophenone. TLCs were carried out using pre-coated sheets (Qingdao silica gel 60-F250, 0.2 mm, Qingdao, China) and visualized at 254 nm, and stained in ninhydrin or phosphomolybdic acid solution followed by heating. Flash column chromatography was performed on E. Qingdao silica gel 60 (230-400 mesh ASTM) using the indicated solvents. Optical rotations were measured on AUTOPOL I automatic polarimeter (Rudolph Research Analytical, Hackettstown NJ 07840, USA). NMR spectra were recorded on Bruker spectrometers (Bruker BioSpin AG, Industriestrasse 26, 8117 Fällanden, Switzerland). Chemical shifts were reported in parts per million (ppm), relative to the signals of solvent residue. Data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad), integration and coupling constant. ESI mass spectra were obtained using a AB QSTAR Elite mass spectrometer (International Equipment Trading Ltd. Mundelein, IL 60060, USA).

Synthesis of the Right-Hand Dipeptide Fragment
NaBH4 (2.13 g, 56.27 mmol) was suspended in THF (100 mL) at 0 °C, L-Pbf-Arg 4 (10.00 g, 23.45 mmol) was added in one portion. A solution of I2 (5.95 g, 23.45 mmol) in THF (20 mL) was dropwise added to the above amino acid solution within 0.5 h. After gas evolution ceased, the

General Experiment
Non-aqueous reactions were performed under a nitrogen or argon atmosphere and all reaction vessels were oven-dried. Solvents were distilled prior to use: dichloromethane (DCM), triethylamine and diisopropylethylamine (DIPEA) from CaH 2 , tetrahydrofuran (THF) from Na/benzophenone. TLCs were carried out using pre-coated sheets (Qingdao silica gel 60-F 250 , 0.2 mm, Qingdao, China) and visualized at 254 nm, and stained in ninhydrin or phosphomolybdic acid solution followed by heating. Flash column chromatography was performed on E. Qingdao silica gel 60 (230-400 mesh ASTM) using the indicated solvents. Optical rotations were measured on AUTOPOL I automatic polarimeter (Rudolph Research Analytical, Hackettstown NJ 07840, USA). NMR spectra were recorded on Bruker spectrometers (Bruker BioSpin AG, Industriestrasse 26, 8117 Fällanden, Switzerland). Chemical shifts were reported in parts per million (ppm), relative to the signals of solvent residue. Data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad), integration and coupling constant. ESI mass spectra were obtained using a AB QSTAR Elite mass spectrometer (International Equipment Trading Ltd. Mundelein, IL 60060, USA).

Synthesis of the Right-Hand Dipeptide Fragment
NaBH 4 (2.13 g, 56.27 mmol) was suspended in THF (100 mL) at 0 • C, L-Pbf-Arg 4 (10.00 g, 23.45 mmol) was added in one portion. A solution of I 2 (5.95 g, 23.45 mmol) in THF (20 mL) was dropwise added to the above amino acid solution within 0.5 h. After gas evolution ceased, the reaction mixture was heated to reflux for 18 h. The reaction temperature was cooled to room temperature, and methanol was carefully added to quench the reaction until the reaction solution became clear. Stir was continued for 0.5 h, the solution was concentrated in vacuo. The residue was dissolved by aqueous solution of KOH (50 mL, 20% in water) and stirred for 4 h, then the solution was extracted by CH 2 Cl 2 (100 mL × 3). The combined organic layers were washed with water (50 mL), dried over anhydrous sodium sulfate and concentrated in vacuo to afford the amino alcohol (6.28 g, 65%) as crude oil.
To a solution of amino alcohol (0.58 g, 1.42 mmol) in CH 2 Cl 2 (20 mL), Fmoc-Ile-OH 3 (0.50 g, 1.42 mmol), HOAt (0.29 g, 2.12 mmol), and EDCI (0.27 g, 1.42 mmol) were sequentially added at 0 • C, after DIPEA (0.47 mL, 2.83 mmol) was added via a syringe, the reaction mixture was brought to room temperature and stirred for 16 h. Volatiles were removed in vacuo. The residue was dissolved in ethyl acetate (150 mL) and washed with saturated aqueous solution of NH 4 Cl (30 mL), NaHCO 3 (30 mL), and brine (30 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography to give dipeptide 5 (0.84 g, 80%) as yellow oil.
Compound 5 (0.53 g, 0.70 mmol) was dissolved in DCM (5 mL) at 0 • C, after imidazole (0.24 g, 3.5 mmol) and TBSCl (0.21 g, 1.40 mmol) were added, the reaction mixture was brought to room temperature and stirred for 16 h. The reaction mixture was diluted by DCM (50 mL), the organic solution was washed with saturated aqueous solution of NH 4 Cl (30 mL), brine (30 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography to afford compound 6 (0.49g, 81%).  Compound 6 (55 mg, 0.064 mmol) was dissolved in CH 3 CN (2 mL) and cooled to 0 • C, after diethylamine (0.07 mL, 0.64 mmol) was added, the reaction mixture was brought to room temperature and monitored by TLC. Upon the consumption of all starting materials, the reaction mixture was concentrated in vacuo. The residue was dissolved in DCM (2 mL) and concentrated in vacuo, these procedures were repeated twice. The residue was dried under high vacuum for 1 h to give the crude amine 7, which was used directly without further purification.

Synthesis of S-Hhpba (S-2)
Compound 15 [39] (3.55 g, 11.42 mmol) was dissovled in ethyl acetate (50 mL), Pd-C (355 mg, 10% on charcoal) and sodium bicarbonate (10.00 g, 114.24 mmol) were added. The reaction vessel was sealed and the atmosphere was changed to hydrogen. The reaction mixture was stirred at room temperature under hydrogen (balloon) atmosphere and monitored by thin layer chromatography. Upon completion of the starting material, the reaction mixture was filtered through a pad of silica gel and eluted with ethyl acetate (20 mL × 2). The combined organic filtrate was washed with brine (30 mL), dried over anhydrous sodium sulfate and concentrated in vacuo to afford compound 16  41 (s, 3H). 13  Compound 16 (0.50 g, 1.60 mmol) was dissolved in methanol (10 mL) and cooled to 0 • C, after PTSA (30 mg, 0.16 mmol) was added, the reaction mixture was stirred at room temperature for 16 h. The reaction solution was concentrated in vacuo, the residue was dissolved in ethyl acetate (50 mL) and washed with saturated aqueous solution of sodium bicarbonate (50 mL) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography to afford the desired diol compound S1 (0.43 g, 99%) as clear oil.  Diol compound S1 (136 mg, 0.50 mmol) was dissolved in DCM (5 mL) and cooled to 0 • C, after triethyl amine (0.35 mL, 2.5 mmol) and triphenylmethyl chloride (0.15 g, 0.53 mmol) were added, the reaction mixture was stirred at room temperature for 4 h. The reaction was diluted with DCM (50 mL) and extracted with brine (50 mL). Layers were separated, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by flash chromatography to afford compound 17  Compound 18 (0.21 g, 0.59 mmol) was dissolved in DCM-H 2 O (3 mL, 2:1) at 0 • C. DAIB (16 mg, 0.08 mmol) and TEMPO (5 mg, 0.29 mmol) were sequentially added, the reaction mixture was stirred in dark for 16 h at room temperature. The reaction was diluted by DCM (50 mL) and washed with brine (20 mL). The organic layer was separated, dried over sodium sulfate, and concentrated in vacuo. The residue was purified by flash chromatography to provide acid S-2 (0.19 g, 84%) as clear oil.
[α] 20 D = −15.  (21) Compound S-2 (40 mg, 0.11 mmol) was dissolved in DMF (2 mL) at 0 • C, Cs 2 CO 3 (30 mg, 0.21 mmol) and CH 3 I (33 µL, 0.53 mmol) were added at 0 • C. The reaction was brought to room temperature and stirred for 16 h. The reactant was diluted by ethyl acetate (50 mL). The organic solution was washed with brine (30 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by flash chromatography to give the corresponding methyl ester S3 ( Methyl ester S3 (60 mg, 0.154 mmol) was dissolved in ethyl acetate (2 mL), after Pd/C (6 mg, 10% Pd on charcoal) was added under a protective flow of nitrogen, the reaction vessel was sealed and purged by hydrogen. The reaction mixture was stirred under hydrogen atmosphere (balloon) for 1 h. The reaction was monitored by TLC. Upon the complete consumption of starting material, the reaction solution was filtrated through a pad of celite and rinsed by ethyl acetate (5 mL Intermediate S4, obtained from last step of reaction without further purification, was dissolved in acetonitrile (2 mL) at 0 • C. K 2 CO 3 (30 mg, 0.21 mmol) was added to the solution, 10 min later, benzyl bromide (85 µL, 0.72 mmol) and TBAI (5 mg, 0.014 mmol) were sequentially added. The reaction mixture was stirred at room temperature for 16 h, and quenched by addition of saturated aqueous solution of NH 4 Cl (10 mL). Volatiles were removed in vacuo. The residue was extracted by ethyl acetate (30 mL × 2). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by flash chromatography to afford compound 19 ( Triphenylphosphine (40 mg, 0.133 mmol) was added to a solution of compound 19 (10 mg, 0.033 mmol) and 4-nitrobenzoic acid (11 mg, 0.066 mmol) dissolved in THF (2 mL) at 0 • C, after DEAD (10 µL, 0.066 mmol) was added, the reaction was stirred at room temperature for 16 h and then quenched by addition of saturated aqueous solution of NH 4 Cl (10 mL). Volatiles were removed in vacuo. The residue was extracted by ethyl acetate (30 mL × 2). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by flash chromatography to produce ester 20 ( (2 mL) at 0 • C, the reaction mixture was then brought to room temperature and stirred for 10 h. Volatiles were removed in vacuo, the residue was dissolved in ethyl acetate (50 mL). The organic solution was washed with brine (30 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography to afford the secondary alcohol compound ent-19 (20 mg, 85%).
[α] 20 D = −10.2 (c 1.0, CHCl 3 ). LiOH (7 mg, 0.17 mmol) was added to a solution of the above secondary alcohol (10 mg, 33 µmol) dissolved in THF-MeOH-H 2 O (3 mL, v/v/v = 1/1/1) at 0 • C. The reaction mixture was stirred at room temperature for 3 h and concentrated in vacuo. The residue was diluted with water (5 mL) and extracted with Et 2 O (20 mL), the organic phase was discarded, the aqueous phase was adjusted to pH 3 with dilute HCl and extracted by ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give acid 21 (8 mg, 91%), which was ready for next step of transformation.

Synthesis of Nostosin B 1a and 1b
S-2 (20 mg, 0.053 mmol) and 7 (51 mg, 0.08 mmol) were dissolved in DCM (2 mL) at 0 • C, HOAT (14 mg, 0.11 mmol) and HATU (40 mg, 0.11 mmol) were added, followed by addition of DIPEA (44 µL, 0.26 mmol). The reaction mixture was warmed to room temperature and stirred for 16 h. Volatiles were removed in vacuo. The residue was dissolved in ethyl acetate (100 mL), washed with saturated aqueous solution of NH 4 Cl (30 mL), NaHCO 3 (30 mL) and brine (30 mL), dried over Na 2 SO 4 , and concentrated in vacuo.  Tripeptide 22 (72 mg, 0.072 mmol) was dissolved in ethyl acetate (2 mL), Pd/C (8 mg, 10%) was added. Hydrogen was bubbled for 1 h at room temperature. The conversion of reaction was monitored by TLC. The mixture was concentrated and passed through a silica column. The filtrate was concentrated in vacuo, the residue was dried under high vacuum for 2 h and then it (56 mg, 0.068 mmol) was dissolved in DCM (2 mL), TFA (0.5 mL) was added at 0 • C. The reaction was stirred at room temperature for 2 h, volatiles were removed in vacuo. The residue was purified by HPLC and lyophilized to give nostosin B 1a (17 mg, 74%) as a powder.  13  Author Contributions: Z.X. and T.Y. conceived and designed the experiments; J.F. performed the experiments; Y.M. and Z.Z. helped on data collection and analyses; X.W. contributed to scientific discussion; Z.X. and T.Y. wrote the paper.