Total synthesis of odoamide , a novel cyclic depsipeptide , from an Okinawan marine cyanobacterium †

Odoamide is a novel cyclic depsipeptide with highly potent cytotoxic activity isolated from the Okinawan marine cyanobacterium Okeania sp. It contains a 26-membered macrocycle composed of a fatty acid moiety, a peptide segment and isoleucic acid. Four possible stereoisomers of the odoamide polyketide substructure were synthesised using a chiral pool approach. The first total synthesis of odoamide was also successfully achieved. The structure of synthetic odoamide was verified by comparing its NMR spectra with those of the natural product.


Introduction
Many peptide secondary metabolites derived from natural resources show attractive biological activities. 1 Because of their favourable drug-like properties including good membrane permeability and biostability, 2 a number of synthetic and medicinal chemistry studies of macrocyclic peptides and highly N-methylated peptides have been carried out. 3 Among them, aurilide-class cyclic depsipeptides exhibit highly potent antiproliferative activity against cancer cell lines (Fig. 1). The first 26-membered cyclic depsipeptide, aurilide (1a), was isolated from the sea hare Dolabella auricularia. 4 The related depsipeptides, aurilide B (1b) and C (1c), from Lyngbya majuscula also show potent cytotoxicity. 5 Kulokekahilide-2 (2) is a similar cytotoxic depsipeptide from a marine mollusk, Philinopsis speciosa, which has two conformers of the 26membered macrocycle in dichloromethane. 6 Lagunamide A (3a) and B (3b) from Lyngbya majuscula exhibit antimalarial activity against Plasmodium falciparum at submicromolar concentrations. 7 Lagunamide C (3c) 8 and palau'amide (4) 9 exhibit cytotoxicity at nanomolar concentrations comparable to other aurilide-class depsipeptides, although these peptides have unique 27-membered and 24-membered macrocycles, respectively. The configurations of the component amino acids of the depsipeptides were investigated by chiral HPLC, chiral GC-MS, and Marfey's analyses, 10 while the stereoselective synthesis and the NMR analysis facilitated the determination of the absolute stereochemistries of the fatty acid substructure. In some cases, the structure was verified or revised through synthetic studies of natural products and their stereoisomers. 11 Odoamide (5) is a novel cyclic depsipeptide from the Okinawan marine cyanobacterium Okeania sp. (Fig. 2A), which shows highly potent cytotoxic activity against HeLa S 3 cell lines. 12 The overall structure of the 26-membered macrocycle is similar to those of aurilide-class depsipeptides, and comprises three substructures: a fatty acid moiety, a peptide segment (Ala-D-MePhe-Sar-Ile-MeAla) and isoleucic acid. At the initial stage of this study, the absolute configurations of the constituent amino acids and isoleucic acid in 5 were determined by chiral HPLC analysis and Marfey's analysis. The absolute configuration of the 5-hydroxy group of the polyketide part was determined by Mosher's method, 13 while the remaining configurations of the polyketide were ambiguous. In this study, we carried out a synthetic study of odoamide to verify its structure and complete stereochemistry.
The synthetic strategy is illustrated in Scheme 1. During the cyclisation of the linear peptide, epimerisation and dimer formation are often problematic. 3e,f, 14 To avoid the less reactive process of N-methylated amide (CO-NMe) or ester bond formation compared with standard peptide bond (CO-NH) formation, we chose macrocyclisation of the Ala and D-alloisoleucic acid residues of the linear precursor 6 for odoamide (5). 11d Peptide 6 could be prepared by coupling of alcohol 7, MeAla 8 and tetrapeptide 9, which could be obtained by standard solid-phase peptide synthesis. Alcohol 7 could be synthesised by coupling of D-allo-isoleucic acid ester 11 15 with carboxylic acid 10.

Results and discussion
Synthesis of the polyketide substructure of odoamide The stereochemistries of the polyketide part were unknown when we started this study. Therefore, it was necessary to synthesise all the possible polyketide substructures of odoamide 5. The polyketide substructure in lagunamide A (3a), a closely related structural analogue of 5, has 5S,7R-dihydroxy and 6S,8S-dimethyl groups. Additionally, aurilide-class depsipeptides 1a-c, 2, and 3a,b possess the syn-1,3-diol moiety with a 5S-hydroxy configuration. On the basis of the structures of these related molecules, we expected that the plausible stereochemical configuration of the natural odoamide 5 was 5S,6S,7R,8S. Among these four stereocentres, the configuration at the C8-methyl group was ambiguous because attempts to determine it based on derivatisation and NMR analysis of odoamide (5) were unsuccessful. It was also desirable to confirm the stereochemistry of the C6-methyl group. Therefore, we designed four possible methyl esters 12a-d as polyketide substructure substrates (Fig. 2B).
Methyl esters 12a,b were synthesised from (S)-Roche ester 13 according to a similar process described in previous reports by us and others (see the ESI †). 11a,12 Preparation of (5S,6R,7R,8S)-ester 12c and (5S,6R,7R,8R)-ester 12d started from the commercially available (R)-Roche ester ent-13 in a similar manner (Scheme 2). (R)-Roche ester ent-13 was converted to alcohol ent-15 via benzyl protection 16 and LiAlH 4mediated reduction. After Swern oxidation, an n-Bu 2 BOTfmediated Evans aldol reaction 17 of the resulting aldehyde provided the syn-aldol products 16c and 17d. The requisite stereochemistries at the C8 chiral centre in 12c and 12d were generated at this step by using propionyl-and pentanoyl-oxazolidinones, respectively. TBS protection of the secondary alcohol in 16c and 17d followed by removal of the chiral auxiliary with LiBH 4 gave alcohols 20c 18 and 21d. Swern oxidation of 20c and the subsequent Wittig reaction of the resulting aldehyde with ethylidene-triphenylphosphorane provided olefin 22c as an E/Z isomeric mixture. Hydrogenation of 22c in the presence of Pd/C afforded the key alcohol 23c with a threo/ threo-configuration. Separately, tosylation of 21d followed by LiAlH 4 -mediated reduction afforded benzyl ether 24d, which was converted to the corresponding alcohol 23d (with a threo/ erythro-configuration) by hydrogenation. Swern oxidation of 23c followed by a Mukaiyama aldol reaction 19 with 1-methoxy-2-methyl-1-trimethylsiloxy-1,3-butadiene (25) 20 produced methyl ester 12c with a (5S)-hydroxy group (dr >99 : 1). 21 Ester 12d was obtained from 23d by using the identical protocol.
The stereochemistry of the 5-hydroxy group in alcohol 12a was confirmed by the NMR analysis of the corresponding acetonide (Scheme 3). TBS deprotection of 26a 22 and 12a provided 1,3-diols 27a and 28a, which were treated with 2,2dimethoxypropane in the presence of PPTS to give acetonides 29a and 30a, respectively. It is known that 13 C NMR chemical shifts of the ketal methyl groups in syn-and anti-1,3-diol acetonides are different. 23 A syn-acetonide shows different chemical shifts for the two ketal methyl groups (e.g., 19.5 and 30.0 ppm for 30a) because of its predominant chair conformation. In contrast, an anti-acetonide shows close chemical shifts (e.g., 23.5 and 25.2 ppm for 29a), because the antiisomer exists in a twist-boat conformation to avoid the 1,3diaxial interaction that would be present in the chair conformation. Accordingly, it was demonstrated that 1,3-diol 28a, the precursor of acetonide 30a has the desired 1,3-syn configuration. Of note, esters 12a-d were employed as the key substrates for the stereochemical assignment of the polyketide substructure in 5 in our previous research. 12 Manipulations of esters 12a-d including DIBAL-mediated reductive transformation provided the corresponding triol derivatives. The comparative NMR analysis between the natural product-derived triol and synthetic triols demonstrated that the polyketide substructure had the 5S,6S,7R,8S configuration (see the ESI †). 12

Conclusions
In this study, the total synthesis of odoamide was completed via the synthesis of four possible polyketide substructures 12a-d. The NMR spectra of the synthetic peptide 5a were identical to those of the natural odoamide 5. Accordingly, the full structural assignment and first total synthesis of odoamide were achieved.

Organic & Biomolecular Chemistry Paper
This

Experimental section
Synthetic general method NMR spectra were recorded using a JEOL ECA-500 spectrometer. Chemical shifts are reported in δ ( ppm) relative to Me 4 Si (in CDCl 3 ) as an internal standard. 13 C NMR spectra were referenced to the residual solvent signal. Melting points were measured by a hot stage melting point apparatus (uncorrected). Exact mass (HRMS) spectra were recorded on a Shimadzu LC-ESI-IT-TOF-MS instrument. IR spectra were recorded on a JASCO FT/IR-4100 spectrometer. Optical rotations were measured with a JASCO P-1020 polarimeter. For flash chromatography, Wakogel C-300E (Wako) was employed.
For analytical HPLC, a Cosmosil 5C18-ARII column (4.6 × 250 mm, Nacalai Tesque, Inc.) was employed with a linear gradient of CH 3 CN (with 0.1% (v/v) TFA, except for the analysis of final products 5a,b using solvents without TFA) in H 2 O at a flow rate of 1 cm 3 min −1 , and eluting products were detected by UV at 220 nm. Preparative HPLC was performed using a Cosmosil 5C18-ARII preparative column (20 × 250 mm, Nacalai Tesque, Inc.) at a flow rate of 8 cm 3 min −1 . The purity of peptides 5a,b was determined by HPLC analysis (>95%). The synthetic procedures for esters 12a,b were described in our previous report. 12 Methyl (R)-3-benzyloxy-2-methylpropanoate (ent-14). To a stirred solution of ent-13 (9.9 g, 83.8 mmol) in CH 2 Cl 2 (210 cm 3 ) under argon were added benzyl 2,2,2-trichloroacetimidate (17.1 cm 3 , 92.2 mmol) in cyclohexane (420 cm 3 ) and triflic acid (3.0 cm 3 , 33.5 mmol) at 0°C. After 10 min, the reaction mixture was warmed to room temperature and stirred for 18 h. The precipitated trichloroacetamide was filtered off. The filtrate was washed with saturated aqueous NaHCO 3 and brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (50 : 1 to 10 : 1) to give compound ent-14 (14.8 g, 85%) as a colorless oil. The spectral data were in good agreement with those previously reported. 28 (S)-3-Benzyloxy-2-methylpropan-1-ol (ent-15). To a stirred suspension of LiAlH 4 (4.0 g, 105.9 mmol) in THF (175 cm 3 ) under argon was added dropwise a solution of ent-14 (14.7 g, 70.6 mmol) in THF (175 cm 3 ) at 0°C. After stirring for 1 h, the reaction mixture was poured into a saturated aqueous solution of sodium potassium tartrate at 0°C and stirred overnight at room temperature. The whole mixture was extracted with Et 2 O and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (9 : 1 to 3 : 1) to give compound ent-15 (10.3 g, 81%) as a colorless oil. The spectral data were in good agreement with those previously reported. 28 (R)-4-Benzyl-3-[(2R,3S,4R)-5-benzyloxy-3-hydroxy-2,4-dimethylpentanoyl]oxazolidin-2-one (16c). To a stirred solution of oxalyl chloride (0.32 cm 3 , 3.72 mmol) in CH 2 Cl 2 (7.4 cm 3 ) under argon was added DMSO (0.53 cm 3 , 7.44 mmol) in CH 2 Cl 2 (1.2 cm 3 ) at −78°C. After stirring for 30 min, a solution of ent-15 (334.7 mg, 1.86 mmol) in CH 2 Cl 2 (6.4 cm 3 ) was added dropwise and stirred at −78°C for 1 h. i-Pr 2 NEt (1.62 cm 3 , 9.3 mmol) was added and the reaction mixture was stirred at 0°C for 30 min. The mixture was quenched with saturated aqueous NH 4 Cl. The whole mixture was extracted with CH 2 Cl 2 and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure to give the corresponding aldehyde, which was used without further purification.
After stirring for 1 h, the reaction mixture was warmed to 0°C and stirred for 30 min. To this solution was added the above aldehyde in CH 2 Cl 2 (3.9 cm 3 ) at −78°C. After stirring for 1 h, the mixture was warmed to −10°C and stirred for 1 h. The mixture was quenched with pH 7.0 phosphate buffer solution (1.8 cm 3 ) and 30% H 2 O 2 in MeOH (1 : 2, 4.2 cm 3 ) and stirred overnight at room temperature. The whole mixture was concentrated under reduced pressure and extracted with CH 2 Cl 2 . The extract was washed with saturated aqueous NaHCO 3 , and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (5 : 1 to 3 : 1) to give compound 16c (610.7 mg, 80%, dr >15 : 1) as a colorless oil. The minor isomer was removed by column chromatography: (R)-4-Benzyl-3-[(2R,3S,4R)-5-benzyloxy-3-(tert-butyldimethylsilyloxy)-2,4-dimethylpentanoyl]oxazolidin-2-one (18c). To a stirred solution of 16c (14.1 g, 34.3 mmol) in CH 2 Cl 2 (137 cm 3 ) under argon were added TBSOTf (9.5 cm 3 , 41.2 mmol) and 2,6lutidine (7.9 cm 3 , 68.6 mmol) at 0°C. The reaction mixture was warmed to room temperature and stirred for 2.5 h. The reaction was quenched with 1 N HCl. The whole mixture was extracted with CH 2 Cl 2 and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (9 : 1) to give compound 18c  (2S,3R,4R)-5-Benzyloxy-3-(tert-butyldimethylsilyloxy)-2,4dimethylpentan-1-ol (20c). To a stirred solution of 18c (22.4 g, 42.5 mmol) in THF (213 cm 3 ) and MeOH (5.2 cm 3 , 127.6 mmol) under argon was added LiBH 4 (2.78 g, 127.6 mmol) at 0°C. After stirring for 10 min, the reaction mixture was warmed to room temperature. After 4 h, the mixture was cooled to 0°C and quenched with saturated aqueous NH 4 Cl. The whole mixture was extracted with EtOAc and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (9 : 1) to give compound 20c {[(2R,3R,4S)-1-(Benzyloxy)-2,4-dimethylhept-5-en-3-yl]oxy}(tertbutyl)dimethylsilane (22c). To a stirred solution of oxalyl chloride (0.11 cm 3 , 1.30 mmol) in CH 2 Cl 2 (6.5 cm 3 ) under argon was added DMSO (0.18 cm 3 , 2.60 mmol) in CH 2 Cl 2 (0.43 cm 3 ) at −78°C. After stirring for 30 min, a solution of 20c (228.9 mg, 0.65 mmol) in CH 2 Cl 2 (2.2 cm 3 ) was added dropwise and stirred at −78°C for 1.5 h. i-Pr 2 NEt (0.57 cm 3 , 3.25 mmol) was added and the reaction mixture was stirred at 0°C for 30 min. The mixture was quenched with saturated aqueous NH 4 Cl. The whole mixture was extracted with CH 2 Cl 2 and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure to give the corresponding aldehyde, which was used without further purification. To a stirred suspension of ethyltriphenylphosphonium bromide (508.6 mg, 1.37 mmol) in THF (5.5 cm 3 ) under argon was added n-BuLi (1.6 mol dm −3 in hexane; 0.81 cm 3 , 1.30 mmol) at room temperature. After stirring for 30 min, a solution of the above aldehyde in THF (1.3 cm 3 ) was added and the reaction mixture was stirred for 1.5 h. The mixture was quenched with saturated aqueous NaHCO 3 . The whole mixture was extracted with EtOAc and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was filtered through a short pad of silica gel with hexane-EtOAc (9 : 1).  (2R,3R,4S)-3-(tert-Butyldimethylsilyloxy)-2,4-dimethylheptan-1-ol (23c). To a stirred solution of 22c (1.3 g, 3.7 mmol) in EtOH (37.0 cm 3 ) was added 10% Pd/C (787.5 mg, 0.7 mmol) at room temperature and the mixture was treated with H 2 gas (1 atm). After stirring for 1 h, the reaction mixture was filtered through Celite. The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (10 : 1) to give compound 23c The stirring was continued for 1 h at this temperature and for additional 1.5 h at room temperature. Then, pentane and cold saturated NaHCO 3 were added to the reaction mixture. The whole mixture was extracted with pentane and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure to give compound 25, which was used without further purification. 20 To a stirred solution of oxalyl chloride (0.060 cm 3 , 0.70 mmol) in CH 2 Cl 2 (3.5 cm 3 ) under argon was added DMSO (0.099 cm 3 , 1.40 mmol) in CH 2 Cl 2 (0.23 cm 3 ) at −78°C. After stirring for 30 min, a solution of 23c (96.4 mg, 0.35 mmol) in CH 2 Cl 2 (1.2 cm 3 ) was added dropwise and stirred at −78°C for 1.5 h. i-Pr 2 NEt (0.49 cm 3 , 2.8 mmol) was added and the reaction mixture was stirred at 0°C for 30 min. The mixture was quenched with saturated aqueous NH 4 Cl. The whole mixture was extracted with CH 2 Cl 2 and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure to give the corresponding aldehyde, which was used without further purification. To a stirred solution of the above aldehyde in CH 2 Cl 2 (2. mixture was warmed to room temperature and stirred for 15 min. Then, saturated aqueous NaHCO 3 was added to the mixture at 0°C. The whole mixture was extracted with CH 2 Cl 2 and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (20 : 1 to 10 : 1) to give compound 12c 1 mmol) at room temperature. After stirring for 1 h, the reaction was quenched with saturated aqueous NH 4 Cl. The whole mixture was extracted with CH 2 Cl 2 and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the precipitated white solid was filtered off. The filtrate was concentrated under reduced pressure to give the corresponding tosylate, which was used without further purification. To a stirred suspension of LiAlH 4 (2.2 g, 57.3 mmol) in THF (100 cm 3 ) under argon was added dropwise a solution of the above tosylate in THF (91 cm 3 ) at 0°C. After stirring for 10 min, the reaction mixture was warmed to room temperature. After 5 h, the reaction mixture was poured into a saturated solution of sodium potassium tartrate at 0°C and stirred at room temperature for 1 h. The whole mixture was extracted with Et 2 O and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the resulting residue was purified by flash chromatography over silica gel with hexane-EtOAc (100 : 0 to 70 : 1) to give compound 24d (4.9 g, 70%) as a colorless oil: To a stirred solution of 31 (1.14 g, 2.6 mmol) in MeOH (17 cm 3 ) and THF (17 cm 3 ) was added 1 N LiOH (17 cm 3 ) at 0°C. The reaction mixture was warmed to 30°C and stirred overnight. The mixture was concentrated under reduced pressure and EtOAc and 1 N HCl were added to the residue. The whole mixture was extracted with EtOAc and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was filtered through a short pad of silica gel with hexane-EtOAc (3 : 1) to give 10, which was used without further purification. To a stirred solution of acid 10 in CH 2 Cl 2 (12.8 cm 3 ) were added MNBA (1.32 g, 3.8 mmol), DMAP (935.0 mg, 7.7 mmol) and 11 (959.0 mg, 3.8 mmol) at room temperature. After stirring overnight, the mixture was quenched with 1 N HCl. The whole mixture was extracted with CH 2 Cl 2 and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (10 : 1) to give compound 32 0.12 mmol) in THF (0.80 cm 3 ) and pyridine (0.20 cm 3 ) was added HF·pyridine (0.50 cm 3 ) at 0°C. The reaction mixture was warmed to room temperature and stirred overnight. The reaction mixture was poured into saturated aqueous NaHCO 3 at 0°C. The whole mixture was extracted with EtOAc, and the extract was washed with brine and 1 N HCl, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (2R,3S)-3-Methyl-1-oxo-1-(2-oxo-2-phenylethoxy)pentan-2-yl (5S,6S,7R,8S,E)-2,6,8-trimethyl-7-[(N-methyl-L-alanyl)oxy]-5-(methylthiomethoxy)undec-2-enoate (33). Fmoc-MeAla-Cl was synthesised by using the identical procedure reported previously. 26 To a stirred solution of Fmoc-MeAla-OH (227.7 mg, 0.70 mmol) in CH 2 Cl 2 (3.9 cm 3 ) were added DMF (0.0054 cm 3 , 0.070 mmol) and SOCl 2 (0.508 cm 3 , 7.0 mmol) at room temperature. After stirring for 1 h, the mixture was concentrated under reduced pressure to give Fmoc-MeAla-Cl, which was used without further purification. To a stirred solution of 7 (152.6 mg, 0.28 mmol) and the above Fmoc-MeAla-Cl in 1,2dichloroethane (2.8 cm 3 ) was added i-Pr 2 NEt (0.244 cm 3 , 1.40 mmol) at room temperature. The reaction mixture was warmed to 40°C and stirred for 14 h. The mixture was cooled to room temperature and quenched with saturated aqueous NH 4 Cl. The whole mixture was extracted with CH 2 Cl 2 and the extract was washed with brine, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was filtered through a short pad of silica gel with hexane-EtOAc (9 : 1 to 3 : 1) to give crude Fmoc-protected amine, which was used without further purification. To a stirred solution of the above protected amine in MeCN (7.0 cm 3 ) was added Et 2 NH (2.3 cm 3 ) at 0°C. The reaction mixture was warmed to room temperature and stirred for 1.5 h. The mixture was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (3 : 1 to 1 : 2) to give compound 33 (94.9 mg, 54%) as a yellow oil: [ Linear peptides (6a,b). To a stirred solution of 33 (59.5 mg, 0.094 mmol), peptide 9 (185.2 mg) and HOAt (38.4 mg, 0.28 mmol) in CH 2 Cl 2 (3.1 cm 3 ) was added EDCI·HCl (54.1 mg, 0.28 mmol) at 0°C. The reaction mixture was warmed to room temperature and stirred for 24 h. The mixture was quenched with saturated aqueous NaHCO 3 . The whole mixture was extracted with CH 2 Cl 2 and the extract was washed with saturated aqueous NH 4 Cl, and dried over MgSO 4 . The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography over silica gel with hexane-EtOAc (1 : 1 to 2 : 3) to give peptide 34 as a 1.4 : 1 diastereomixture, which was used without further purification. To a stirred solution of 34 in AcOH/EtOAc/H 2 O (60 : 35 : 5, 4.3 cm 3 ) was added Zn (92.2 mg, 1.4 mmol) at room temperature. After stirring for 8 h, the reaction mixture was filtered through Celite, and 1 N HCl was added to the filtrate. The whole mixture was extracted with EtOAc and the extract was washed with brine, and dried over MgSO 4 . After the filtrate was concentrated under reduced pressure, AcOH was removed by azeotropic distillation with toluene to give the corresponding carboxylic acid, which was used without further purification. To a stirred solution of the above acid in MeCN (2.4 cm 3 ) was added Et 2 NH (0.80 cm 3 ) at 0°C. The reaction mixture was warmed to room temperature and stirred for 1.5 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by reverse-phase preparative HPLC (59% CH 3 CN in 0.1% TFA solution) to give linear peptides 6a (29.6 mg, 30% from 33) and 6b (24.0 mg, 24% from 33) as a colorless powder.