First Total Syntheses of Beauvericin A and allo-Beauvericin A

Abstract

The first total syntheses of beauvericin A and allo-beauvericin A were achieved. N-Methyl-l-phenylalanine, (2R)-hydroxylvaleric acid, and (2R,3S)- or (2R,3R)-2-hydroxy-2-methylpentanoic acid were linked and cyclized to form the target natural products. The structure of synthetic beauvericin A was confirmed by X-ray crystallographic analysis. NMR data of the synthetic beauvericins were identical with those of the reported natural products. These results secure the structures of natural products, as originally proposed in the isolation studies.

Introduction

Beauvericins (BEAs) are a class of cyclohexadepsipeptide natural products and have attracted much attention due to their unique structural features and biological activities including antimicrobial, insecticidal, anthelmintic, herbicidal, antihaptotactic, anticholesterol, and anticancer activities (Figure 1A).¹ BEA (1) isolated from Beauveria bassiana in 1967 is the first example of BEAs.² The structure of 1 is characterized by the cyclic depsipeptide with C3 symmetry in which l-N-methyl phenylalanine (MePhe, 3) and (2R)-hydroxylvaleric acid (Hiv, 4) are repetitively linked. The absolute structures of 3 and 4 were proposed by the chemical degradation of 1 and analysis of the degradation products. Hydrolysis of 1 gave 3 and 4. The optical rotatory dispersion spectra of the resulting 3 and 4 were compared with those of the authentic samples, suggesting the absolute stereochemistry of MePhe (3) and Hiv (4) to be 2S and 2R, respectively. The cyclic structure of 1 was also confirmed by total synthesis.³

Figure 1.

(A) Structures of beauvericin (BEA, 1), beauvericin A (BEAA, 2a), and allo-beauvericin A (allo-BEAA, 2b). (B) Structures of the amino acid unit MePhe (3), 2-hydroxyacid units (2R)-Hiv (4), (2R,3S)-Hmp (5a), and (2R,3R)-Hmp (5b). (2R)-Hiv (4) is biosynthetically provided from l-valine (6) with the formal inversion of the (S)-amino acid stereochemistry of 6.⁶ Proposed biosynthesis of (2R)-5ab from l-amino acids 7ab is done according to the biosynthesis of 4 from l-valine (6).

Several natural and artificial beauvericin congeners have been reported.¹ Beauvericin A (BEAA, 2a) and allo-beauvericin A (allo-BEAA, 2b) are representative examples. BEAA (2a) is a natural congener of 1 isolated from the culture broth of B. bassiana in 1995 (Figure 1).⁴allo-BEAA (2b) is an artificial congener produced by the precursor-directed biosynthesis method feeding of l-allo-isoleucine (7b) to a fermentation broth of Paecilomyces tenuipes BCC 1614 (Figure 1B).⁵ BEAAs 2ab consisted of MePhe (3), Hiv (4), and 2-hydroxy-3-methylpropionic acids (Hmps, 5ab). The structures of 5ab were proposed to be (2R,3S)-Hmp (5a) for 2a and (2R,3R)-Hmp (5b) for 2b based on the hypothetical biosynthetic pathway.^4,5 Previous biosynthetic studies of (2R)-Hiv (4) showed that 4 was generated from l-valine (6) via 2-keto-3-methylbutylic acid (Figure 1B).⁶ Similarly, it was proposed that (2R,3S)-Hmp (5a) and (2R,3R)-Hmp (5b) were presumably biosynthesized from naturally occurring l-isoleucine (7a) and l-allo-isoleucine (7b), respectively.

In this study, we report the first syntheses of 2ab. NMR data of synthetic 2ab were identical with those of the reported data. These results secure the structures of 2ab as originally proposed in the isolation studies. Only a small structural difference appears in the Hmp moieties of 2ab. To determine the stereochemistry of 2ab, total synthesis using stereochemically defined synthetic methods would be one of the useful and powerful approaches. In addition, the structure determination of 2ab provides benefits in consideration of the structure–activity relationship of beauvericins. Beauvericins are mycotoxin and often result in food poisoning.¹ Analysis and monitoring of beauvericins in food have become an important subject in terms of the engagement of risk assessment to human and animal health. In this context, synthetic samples of 2ab would be useful tools for analytical studies.

Results and Discussion

Retrosynthetic analyses of BEAA (2a) and allo-BEAA (2b) are depicted in Scheme 1. These target molecules would deliver from acyclic precursors 8ab, which are disconnected to three fragments, 9, 10a, and 10b. These fragments would be prepared by esterification of commercially available N-Fmoc-MePhe-OH (11) with Hiv-OBn (12),^3a (2R,3S)-Hmp-OBn (13a),⁷ or (2R,3R)-Hmp-OBn (13b).⁸

Scheme 1. Retrosynthesis of BEAA (2a) and allo-BEAA (2b).

N-Fmoc-MePhe-OH (11) was condensed with Hiv-OBn (12)^3a using 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI) in the presence of 4-dimethylaminopyridine (DMAP) to give 9 in 78% yield (Scheme 2). In a similar manner, (2R,3S)-Hmp-OBn (13a)⁷ and (2R,3R)-Hmp-OBn (13b)⁸ were coupled with 11 to give 10a and 10b, respectively.

Scheme 2. Synthesis of 9 and 10ab.

Total synthesis of BEAA (2a) was achieved by coupling of 9 and 10a (Scheme 3). Benzyl ester 9 was converted to acid 14 by hydrogenation in the presence of Pd/C under a hydrogen atmosphere. Treatment of 14 with 1-chloro-N,N,2-trimethyl-1-propenylamine (Ghosez’s reagent)⁹ provided acid chloride 15, which was reacted with amine 16 prepared from 9 in the presence of diisopropylethylamine (DIPEA) to give 17 in 76% yield. In a similar manner, acyclic hexadepsipeptide 21a was prepared by coupling of acid chloride 20a prepared from (2R,3S)-Hmp-OBn (10a) and amine 18 prepared from 17 in 78% yield. Sajiki et al.,¹⁰ reported the mild removal of that N-Fmoc under the hydrogenation reaction condition in the presence of acetonitrile.⁹ Under the condition, N-Fmoc and Bn protecting groups of 21a were simultaneously removed to provide a free amino acid, which was treated with Ghosez’s reagent to give acid chloride 8a. The resulting compound 8a was treated with DIPEA to furnish BEAA (2a). The structure of 2a was unambiguously determined by X-ray crystallographic analysis (Figure 2).¹¹ In a similar manner, allo-BEAA (2b) was synthesized by coupling of Fmoc-MePhe-OH (11), Hiv-OBn (12), and (2R,3R)-Hmp-OBn (13b), followed by cyclization. ¹H and ¹³C NMR data of the synthetic 2ab are identical with those of the naturally occurring 2a(3) and artificial biosynthetic product 2b(4) synthesized by the precursor-directed biosynthesis, respectively (Tables 1 and 2). These results secure the originally proposed structures of BEAA (2a) and allo-BEAA (2b), which possess (2R,3S)-Hmp (5a) and (2R,3R)-Hmp (5b), respectively.

Scheme 3. Total Synthesis of BEAA (2a) and allo-BEAA (2b).

Figure 2.

ORTEP structure of synthetic BEAA (2a). Supplementary crystallographic data for the structure have been deposited at the Cambridge Crystallographic Data Centre (No. CCDC 2311751).¹¹

Table 1. ¹H NMR Data for Natural and Synthetic 2ab (CDCl₃, 400 MHz).

	BEAA (2a)		allo-BEAA (2b)
position	natural4	synthetic	biosynthetic5	synthetic
Hiv	2 units	2 units	2 units	2 units
2	4.93 (1H, d, 6.2)	4.89 (1H, d, 7.2)	4.86 (2H, d, 8.5)	4.86 (2H, d, 8.5)
	4.96 (1H, d, 6.5)	4.91 (1H, d, 7.2)
3	2.04 (2H, m)	2.00 (2H, m)	1.98 (2H, m)	1.98 (2H, m)
4	0.43 (3H, d, 6.4)	0.40 (3H, d, 6.5)	0.41 (6H, d, 6.7)	0.41 (6H, d, 6.8)
	0.45 (3H, d, 6.5)	0.42 (3H, d, 6.5)
4′	0.76 (3H, d, 6.6)	0.77 (3H, d, 6.7)	0.81 (6H, d, 6.6)	0.81 (6H, d, 6.6)
	0.77 (3H, d, 6.8)	0.76 (3H, d, 6.7)
Hmp	1 unit (2R,3S)	1 unit (2R,3S)	1 unit (2R,3R)	1 unit (2R,3R)
2	5.04 (1H, d, 7.7)	5.00 (1H, d, 7.8)	4.96 (1H, d, 9.1)	4.97 (1H, d, 9.0)
3	1.78 (1H, m)	1.73 (1H, m)	1.79 (1H, m)	1.79 (1H, m)
4	0.70 (2H, m)	0.68–0.72 (2H, m)	1.37 (1H, m)	1.37 (1H, m)
			0.94 (1H, m)	0.93 (1H, m)
5	0.68 (3H, m)	0.62–0.70 (3H, m)	0.75 (3H, t, 7.4)	0.75 (3H, t, 7.4)
3-Me	0.81 (3H, d, 6.8)	0.80 (3H, d, 6.6)	0.32 (3H, d, 6.8)	0.30 (3H, d, 6.8)
NMePhe	3 units	3 units	3 units	3 units
2	5.43 (3H, m)	5.51 (3H, m)	5.67 (1H, dd, 12, 4.8)	5.65 (1H, dd, 12, 5.0)
			5.51 (2H, m)	5.48 (2H, m)
3	3.35 (3H, m)	3.36 (3H, m)	3.38 (3H, dd, 14.6, 4.8)	3.37 (3H, dd, 14.6, 4.9)
	2.99 (3H, m)	2.89–2.99 (3H, m)	2.95 (3H, m)	2.90–3.00 (3H, m)
5–9	7.22 (15H, m)	7.11–7.30 (15H, m)	7.16–7.28 (15H, m)	7.12–7.31 (15H, m)
N–CH3	2.95 (3H, s)	2.96 (3H, s)	3.00 (3H, s)	2.99 (3H, s)
	2.99 (6H, s)	3.00 (6H, s)	3.03 (3H, s)	3.00 (3H, s)
			3.05 (3H, s)	3.04 (3H, s)

Table 2. ¹³C NMR Data for 2ab (CDCl₃, 101 MHz).

	BEAA (2a)		allo-BEAA (2b)
position	natural4	synthetic	biosynthetic5	synthetic
Hiv	2 units	2 units	2 units	2 units
1 C=O	169.94 or 169.24	169.96 or 169.94 or 169.91 or 169.48 or 169.44	169.78, 169.64	169.64
2	75.4	75.52, 75.48	75.64	75.63
3	29.7	29.71, 29.69	29.74, 29.70	29.75, 29.72
4	17.54	17.46, 17.43	17.43	17.48, 17.43
4′	18.29	18.28, 18.26	18.32	18.35, 18.34
Hmp	1 unit (2R,3S)	1 unit (2R,3S)	1 unit (2R,3R)	1 unit (2R,3R)
1 C=O	169.94 or 169.24	169.96 or 169.94 or 169.91 or 169.48 or 169.44	169.61	169.92
2	74.22	74.30	74.27	74.25
3	35.81	35.84	35.62	35.62
4	24.47	24.40	24.53	24.55
5	11.31	11.30	10.63	10.63
3-Me	14.34	14.35	13.27	13.27
NMePhe	3 units	3 units	3 units	3 units
1 C=O	169.94 or 169.24	169.96 or 169.94 or 169.91 or 169.48 or 169.44	169.99, 169.94, 169.92	169.97, 169.92, 169.89
2	57.53, 57.52	57.30, 57.16	57.36, 56.64	57.35, 56.64
3	34.83, 34.68	34.79, 34.68, 34.62	34.84, 34.73, 34.62	34.87, 34.75, 34.64
4	136.64	136.60, 136.59, 136.56	136.64, 136.58, 136.48	136.66, 136.60, 136.49
5–9	128.92, 128.53, 126.76	128.86, 128.83, 128.82, 128.51, 128.45, 126.75	128.81, 126.77, 128.53	128.84, 128.82, 128.80, 128.53, 126.78
N–CH3	32.43, 32.37	32.22, 32.19	32.33, 31.86	32.35, 31.87

In summary, we have achieved the first total syntheses of BEAA (2a) and allo-BEAA (2b) starting from stereochemically defined staring materials 11, 12, and 13ab. Comparison of NMR data of synthetic 2ab was identical with those of the reported data described in the isolation studies. These results confirmed stereochemistry of the Hmp moieties of 2ab and provided additional supports for the biosynthetic pathway of Hmps 5ab where l-isoleucine (7a) and l-allo-isoleucine (7b) were biosynthetically transformed to 5ab via the formal inversion of the stereochemistry. It was reported that 2a and 2b displayed antibacterial activities against Mycobacterium tuberculosis (MIC, 1.6 and 0.8 μg/mL).⁵ We preliminarily tested the potency of antibacterial activity of 2a against Mycobacterium smegmatis. However, 2a did not show potent activities at >10 μg/mL. Further biological studies are ongoing in our laboratory.

Experimental Section

General Experimental Procedures

FTIR spectra were measured on a JASCO FT/IR-6200 infrared spectrophotometer. ¹H NMR spectra were recorded on a Bruker AVANCE III HD 400 (400 MHz) or JEOL JNM-ECZ 400S (400 MHz) spectrometer. Chemical shifts for the ¹H NMR spectra were reported relative to CHCl₃ (δ 7.26) in CDCl₃. ¹³C NMR spectra were recorded on a Bruker AVANCE III HD 400 (101 MHz) or JNM-ECZ 400S spectrometer. Chemical shifts for ¹³C NMR spectra were reported relative to CHCl₃ (δ 77.0) in CDCl₃. High-resolution mass spectra (HRMS) were obtained on a JEOL JMS-T100LP instrument for electrospray ionization (ESI). X-Ray crystallographic analysis was performed on a Rigaku AFC11/Saturn 724+ CCD diffractometer with monochromated Mo-Kα radiation (λ = 0.710747 Å). Optical rotations were taken on a JASCO P-1030 polarimeter with a sodium lamp (D line) using CHCl₃ or MeOH of spectrochemical analysis grade. Melting points were determined with Yanaco MP-21 melting point apparatus. All reactions were monitored by thin-layer chromatography (TLC), which was performed with precoated plates (silica gel 60 F-254, 0.25 mm thickness, manufactured by Merck). TLC visualization was accompanied using a UV lamp (254 nm) or a charring solution (ethanolic phosphomolybdic acid, ethanolic p-anisaldehyde, and butanoic ninhydrin). A Daiso IR-60 1002W (40/63 μm) was used for flash column chromatography on silica gel. All reagents and solvents were purchased from either Aldrich Chemical Co., Inc., Kanto Kagaku Co., Inc., Merck & Co., Inc., Nacalai Tesque Company, Ltd., Peptide Institute, Tokyo Chemical Industry Co., Ltd., or FUJIFILM Wako Pure Chemical Corporation, Ltd. and used without further purification unless otherwise indicated. Dichloromethane (CH₂Cl₂) was distilled from phosphorus pentoxide (P₂O₅). Tetrahydrofuran (THF) of anhydrous grade was used. Antibacterial activity test against M. smegmatis was performed according to the reported procedure.¹²

N-Fmoc-MePhe-(2R)-Hiv-OBn (9)

To a stirred solution of 12 (0.93 g, 4.45 mmol) in CH₂Cl₂ (25 mL) were added 11 (2.14 g, 5.34 mmol), DMAP (0.65 g, 5.34 mmol), and EDCI·HCl (1.02 g, 5.34 mmol) at 0 °C under Ar. The mixture was stirred for 14 h at rt and quenched with 1 M aqueous HCl (12 mL) at 0 °C. After separating the organic layers, the water layer was extracted with CH₂Cl₂ (x3). The combined organic layers were washed with saturated aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (n-hexane:EtOAc = 5:1) to give 9 (2.06 g, 78%) as a colorless viscous oil. [α]²⁴_D −44 (c 0.60, CHCl₃). FTIR (neat) ν_max (cm^–1) 2966, 1742, 1703, 1452, 1194, 1129, 1022, 741, 699. ¹H NMR (400 MHz, CDCl₃, mixture of rotamers) δ 7.76 (2H, d, J = 7.5 Hz), 7.55–7.32 (9H, m), 7.31–7.17 (6H, m), 7.06 (1H, d, J = 6.7 Hz), 5.31–5.21 (1.6H, m), 5.15 (1H, dd, J = 12.1, 9.4 Hz), 5.00–4.90 (1H, m), 4.86 (0.4H, d, J = 4.0 Hz), 4.39–4.10 (3H, m), 3.43 (0.6H, dd, J = 14.7, 5.4 Hz), 3.27 (0.4H, dd, J = 14.6, 5.0 Hz), 3.04 (0.6H, dd, J = 14.7, 10.9 Hz), 2.90–2.80 (0.4H, m), 2.88 (1.8H, s), 2.86 (1.2H, s), 2.26 (1H, m,), 0.96 (1.8H, d, J = 6.9 Hz), 0.90 (4.2H, d, J = 6.9 Hz). ¹³C NMR (101 MHz, CDCl₃, mixture of rotamers) δ 170.8, 170.6, 169.1, 168.9, 156.7, 156.1, 144.0, 143.8, 141.24, 141.20, 136.9, 135.2, 135.1, 128.76, 128.72, 128.56, 128.52, 128.47, 128.41, 127.62, 127.57, 127.03, 127.00, 126.95, 126.8, 126.7, 125.1, 125.0, 124.9, 124.8, 120.0, 119.94, 119.91, 119.87, 77.5, 77.4, 67.8, 67.6, 67.1, 67.0, 59.8, 59.6, 47.2, 47.0, 34.9, 34.7, 31.2, 31.0, 30.1, 30.0, 18.7, 17.04, 16.98. HRMS (ESI) m/z 614.25189 [M + Na]⁺ (calcd for C₃₇H₃₇NO₆Na, 614.25186).

N-Fmoc-MePhe-(2R,3S)-Hmp-OBn (10a)

To a stirred solution of 13a (0.50 g, 2.25 mmol) in CH₂Cl₂ (15 mL) ware added 11 (1.1 g, 2.70 mmol), DMAP (0.30 g, 2.48 mmol), and EDCI·HCl (0.42 g, 2.70 mmol) at 0 °C under Ar. The mixture was stirred for 14 h at rt, quenched with 1 M aqueous HCl (12 mL) at 0 °C. After separating the organic layers, the water layer was extracted with CH₂Cl₂ (x3). The combined organic layers were washed with the saturated aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (n-hexane:EtOAc = 5:1) to give 10a (0.99 g, 75%) as a colorless viscous oil. [α]²⁴_D −37 (c 1.2, CHCl₃). FTIR (neat) ν_max (cm^–1) 2964, 1742, 1704, 1452, 1196, 1132, 741, 699. ¹H NMR (400 MHz, CDCl₃) δ 7.75 (2H, d, J = 7.4 Hz), 7.53–7.16 (15H, m), 7.07 (1H, d, J = 6.6 Hz), 5.30–5.10 (3.2H, m), 5.04 (0.4H, d, J = 3.2 Hz), 4.97 (0.4H, dd, J = 10.8, 5.0 Hz), 4.37–4.15 (2.6H, m), 4.11 (0.4H, t, J = 6.5 Hz), 3.41 (0.6H, dd, J = 14.7, 5.4 Hz), 3.27 (0.4H, dd, J = 14.6, 5.0 Hz), 3.03 (0.6H, dd, J = 14.7, 11.0 Hz), 2.93–2.82 (0.4H, m), 2.87 (3H, s), 2.01 (1H, m), 1.43–1.14 (2H, m), 0.90–0.80 (6H, m). ¹³C NMR (101 MHz, CDCl₃, mixture of rotamers) δ 170.8, 170.6, 169.4, 169.2, 156.7, 156.1, 144.01, 143.97, 143.9, 143.8, 141.24, 141.20, 136.9, 135.2, 135.1, 128.8, 128.7, 128.61, 128.58, 128.53, 128.49, 128.45, 128.4, 127.63, 127.58, 127.04, 127.00, 126.96, 126.8, 126.7, 125.2, 125.0, 124.9, 124.8, 119.94, 119.91, 119.88, 75.6, 75.5, 67.8, 67.7, 67.14, 67.07, 59.9, 59.6, 47.2, 47.0, 36.5, 36.4, 34.9, 34.7, 31.1, 31.0, 26.0, 14.3, 14.2, 11.6. HRMS (ESI) m/z 628.26755 [M + Na]⁺ (calcd for C₃₈H₃₉NO₆Na, 628.26751).

N-Fmoc-MePhe-(2R,3R)-Hmp-OBn (10b)

To a stirred solution of 13b (0.22 g, 0.967 mmol) in CH₂Cl₂ (6.0 mL) were added 11 (0.54 g, 1.35 mmol), DMAP (0.15 g, 1.26 mmol), and EDCI·HCl (0.26 g, 1.35 mmol) at 0 °C under Ar. The mixture was stirred for 14 h at rt, quenched with 1 M aqueous HCl (3.0 mL) at 0 °C. After separating the organic layers, the water layer was extracted with CH₂Cl₂ (x3). The combined organic layers were washed with the saturated aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (n-hexane:EtOAc = 5:1) to give 10b (0.41 g, 70%) as a colorless viscous oil. [α]²⁴_D −40 (c 0.80, CHCl₃). FTIR (neat) ν_max (cm^–1) 2964, 1743, 1703, 1452, 1309, 1193, 741, 699. ¹H NMR (400 MHz, CDCl₃) δ 7.76 (2H, d, J = 7.4 Hz), 7.53–7.18 (15H, m), 7.05 (1H, d, J = 6.6 Hz), 5.30–4.88 (4H, m), 4.37–4.04 (3H, m), 3.41 (0.6H, dd, J = 14.7, 5.4 Hz), 3.25 (0.4H, dd, J = 14.6, 5.1 Hz), 3.03 (0.6H, dd, J = 14.7, 10.9 Hz), 2.92–2.77 (0.4H, m), 2.87 (1.8H, s), 2.84 (1.2H, s), 2.00 (1H, m), 1.38 (1H, m), 1.20 (1H, m), 0.95–0.75 (6H, m). ¹³C NMR (101 MHz, CDCl₃, mixture of rotamers) δ 170.8, 170.6, 169.2, 168.9, 156.7, 156.1, 144.0, 143.9, 143.8, 141.25, 141.21, 136.9, 135.2, 135.1, 128.8, 128.7, 128.60, 128.57, 128.53, 128.48, 128.4, 127.63, 127.59, 127.04, 127.00, 126.96, 126.8, 126.7, 125.1, 125.0, 124.9, 124.8, 120.0, 119.92, 119.88, 67.8, 67.6, 67.1, 67.0, 59.8, 59.6, 47.2, 47.1, 36.5, 34.9, 34.7, 31.2, 31.0, 24.5, 24.4, 15.4, 11.5, 11.4. HRMS (ESI) m/z 628.26751 [M + Na]⁺ (calcd for C₃₈H₃₉NO₆Na, 628.26751).

N-Fmoc-MePhe-(2R)-Hiv-MePhe-(2R)-Hiv-OBn (17)

To a stirred solution of 9 (250 mg, 0.423 mmol) in THF (0.5 mL) was added Et₂NH (1.0 mL) at rt. The mixture was stirred for 1.5 h and concentrated under reduced pressure. The crude amine 16 was used for the next condensation step without further purification.

To a mixture of Pd/C (50.0 mg, 10% on charcoal) in THF (1.5 mL) was added a solution of 9 (250 mg, 0.423 mmol) in THF (3.0 mL). The suspension was stirred for 1.5 h at rt under H₂ (balloon) and filtered through a thin Celite pad. The filtrate was concentrated under reduced pressure. The crude acid 14 was used for the next step without further purification. To a stirred solution of the crude acid 14 in CH₂Cl₂ (1.0 mL) was added Ghosez’s reagent (152 μL, 1.16 mmol) at 0 °C. The mixture was stirred for 20 min at the same temperature to form acid chloride 15in situ. This acid chloride 15 was used for the next condensation step without further purification. A stirred solution of crude amine 16 in CH₂Cl₂ (1.0 mL) was added to a solution of the acid chloride 15 in CH₂Cl₂ (1.0 mL) at 0 °C. The mixture was stirred for 20 min at the same temperature, and then N,N-diisopropylethylamine (258 μL, 1.52 mmol) was added to the mixture at 0 °C. The mixture was stirred for 17 h at rt, quenched with H₂O, and extracted with CH₂Cl₂ (x3). The combined organic layers were washed with brine, dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (n-hexane:EtOAc = 4:1) to give 17 (275 mg, 76%) as a viscous oil. [α]²⁴_D −47 (c 1.9, CHCl₃). FTIR (neat) ν_max (cm^–1) 2966, 1739, 1704, 1666, 1453, 1195, 1025, 741, 698. ¹H NMR (400 MHz, CDCl₃) δ 7.77–7.70 (2H, m), 7.55–7.05 (21H, m), 5.69 (1H, dd, J = 12.1, 4.7 Hz), 5.26 (1H, d, J = 12.1 Hz), 5.19 (0.6H, J = 11.1, 5.1 Hz), 5.14 (1H, d, J = 12.1 Hz), 5.04–4.88 (2.4H, m), 4.33–4.05 (3H, m), 3.47 (1H, dd, J = 14.9, 4.7 Hz), 3.39 (0.6H, dd, J = 14.6, 5.2 Hz), 3.27 (0.4H, dd, J = 14.6, 4.7 Hz), 3.08–2.84 (2H, m), 2.94–2.90 (6H, m), 2.30 (1H, m), 1.56 (1H, m), 1.07–0.95 (6H, m), 0.72–0.60 (3H, m), 0.55–0.45 (3H, m). ¹³C NMR (100 MHz, CDCl₃, mixture of rotamers) δ 170.6, 170.4, 170.3, 169.9, 169.63, 169.60, 169.30, 169.27, 156.7, 156.2, 144.1, 144.0, 143.9, 143.8, 141.2, 137.1, 136.61, 136.58, 135.2, 128.7, 128.63, 128.59, 128.53, 128.50, 128.47, 127.6, 127.5, 127.04, 127.00, 126.96, 126.85, 126.82, 126.7, 126.6, 125.2, 125.1, 125.0, 124.9, 119.93, 119.88, 119.85, 77.8, 75.5, 75.3, 67.7, 67.6, 67.1, 59.9, 59.7, 57.3, 47.2, 47.0, 34.9, 34.7, 34.6, 31.7, 31.3, 31.1, 30.1, 29.4, 29.3, 19.04, 18.95, 18.61, 18.55, 17.0, 16.9, 16.5, 16.3. HRMS (ESI) m/z 875.38846 [M + Na]⁺ (calcd for C₅₂H₅₆N₂O₉Na, 875.38835).

N-Fmoc-MePhe-(2R,3S)-Hmp-NMePhe-(2R)-Hiv-MePhe-(2R)-Hiv-OBn (21a)

To a stirred solution of 17 (41.0 mg, 47.8 μmol) in THF (0.5 mL) was added Et₂NH (1.0 mL) at rt. The mixture was stirred for 1.5 h and concentrated under reduced pressure. The crude amine 18 was used for the next condensation step without further purification. To a mixture of Pd/C (6.0 mg, 10% on charcoal) in THF (0.2 mL) was added a solution of 10a (32.0 mg, 0.0528 mmol) in THF (0.5 mL). The suspension was stirred for 1.5 h at rt under H₂ (balloon) and filtered through a thin Celite pad. The filtrate was concentrated under reduced pressure. The crude acid 19a was used for the next step without further purification. To a stirred solution of the crude acid 19a in CH₂Cl₂ (0.5 mL) was added Ghosez’s reagent (17 μL, 0.131 mmol) at 0 °C. The mixture was stirred for 20 min at the same temperature to form acid chloride 20ain situ. This acid chloride 20a was used for the next condensation step without further purification.

A stirred solution of crude amine 18 in CH₂Cl₂ (0.5 mL) was added to a solution of the acid chloride 20a in CH₂Cl₂ (0.5 mL) at 0 °C. The mixture was stirred for 20 min at the same temperature, and then N,N-diisopropylethylamine (29 μL, 0.172 mmol) was added to the mixture at 0 °C. The mixture was stirred for 17 h at rt, quenched with H₂O, and extracted with CH₂Cl₂ (x3). The combined organic layers were washed with brine, dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated under reduced pressure. The crude residue was roughly purified by silica gel flash column chromatography (n-hexane:EtOAc = 3:1) to give 21a (42.0 mg, 48%). [α]²⁴_D −44 (c 2.0, CHCl₃). FTIR (neat) ν_max (cm^–1) 3727, 3706, 3627, 3599, 2965, 1739, 1704, 1666, 1455, 1198, 1021, 740, 698. ¹H NMR (400 MHz, CDCl₃) d 7.80–7.70 (2H, m), 7.55–7.10 (26H, m), 5.80–5.55 (2H, m), 5.30–5.10 (3H, m), 5.09–4.90 (2H, m), 4.45–4.00 (3H, m), 3.50–3.20 (3H, m), 3.10–2.80 (11H, m), 2.04 (1H, m), 1.50 (2H, m), 1.40–1.20 (2H, m), 0.98 (3H, d, J = 6.8 Hz), 0.95–0.80 (6H, m), 0.76 (2H, d, J = 6.9 Hz), 0.72 (1H, d, J = 6.9 Hz), 0.66 (2H, d, J = 6.9 Hz), 0.63 (1H, d, J = 6.9 Hz), 0.54 (3H, d, J = 6.8 Hz), 0.51–0.43 (3H, m). ¹³C NMR (101 MHz, CDCl₃, mixture of rotamers) δ 170.6, 170.3, 170.2, 170.1, 170.0, 169.8, 169.6, 156.7, 144.0, 143.92, 143.86, 143.8, 141.19, 141.17, 139.6, 137.2, 136.9, 136.7, 136.64, 136.58, 135.2, 129.3, 128.8, 128.73, 128.66, 128.63, 128.61, 128.55, 128.51, 128.44, 128.39, 127.9, 127.81, 127.76, 127.6, 127.5, 127.0, 126.9, 126.8, 126.75, 126.6, 125.2, 125.1, 125.0, 124.5, 124.32, 120.28, 120.04, 120.01, 119.91, 119.87, 119.8, 75.9, 75.8, 75.6, 75.3, 71.1, 67.7, 67.6, 67.28, 67.26, 67.14, 67.1, 59.9, 59.63, 59.59, 59.55, 57.32, 57.25, 48.6, 47.1, 47.0, 36.5, 36.4, 34.9, 34.8, 34.7, 34.6, 32.0, 31.9, 31.6, 31.4, 31.2, 31.1, 29.4, 29.3, 29.2, 25.92, 25.89, 25.7, 19.1, 19.0, 18.9, 16.4, 16.2, 15.99, 15.95, 15.9, 14.23, 14.2, 14.1, 13.9, 13.3, 11.7, 11.6. HRMS (ESI) m/z 1150.54046 [M + Na]⁺ (calcd for C₆₈H₇₇N₃O₁₂Na, 1150.54049).

N-Fmoc-MePhe-(2R,3R)-Hmp-MePhe-(2R)-Hiv-MePhe-(2R)-Hiv-OBn (21b)

To a stirred solution of 17 (53.0 mg, 62.1 μmol) in THF (0.5 mL) was added Et₂NH (1.0 mL) at rt. The mixture was stirred for 1.5 h at the same temperature and concentrated under reduced pressure. The crude amine 18 was used for the next condensation step without further purification.

To a mixture of Pd/C (10.0 mg, 10% on charcoal) in THF (1.0 mL) was added a solution of 10b (54.0 mg, 89.1 μmol) in THF (1.0 mL). The suspension was stirred for 1.5 h at rt under H₂ (balloon) and filtered through a thin Celite pad. The filtrate was concentrated under reduced pressure. The crude acid 19b was used for the next step without further purification. To a stirred solution of the crude acid 19b in CH₂Cl₂ (0.5 mL) was added Ghosez’s reagent (22 μL, 167 μmol) at 0 °C. The mixture was stirred for 20 min at the same temperature to form acid chloride 20bin situ. This acid chloride 20b was used for the next condensation step without further purification.

A stirred solution of crude amine 18 in CH₂Cl₂ (0.5 mL) was added to a solution of the acid chloride 20b in CH₂Cl₂ (0.5 mL) at 0 °C. The mixture was stirred for 20 min at the same temperature, and then N,N-diisopropylethylamine (38 μL, 224 μmol) was added to the mixture at 0 °C. The mixture was stirred for 17 h at rt, quenched with H₂O, and extracted with CH₂Cl₂ (x2). The combined organic layers were washed with brine, dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated under a reduced pressure. The crude residue was purified by silica gel flash column chromatography (n-hexane:EtOAc = 3:1) to give 21b (53.0 mg, 75%) as a viscous oil. [α]²⁴_D −48 (c 1.2, CHCl₃). FTIR (neat): ν_max (cm^–1) 2965, 1738, 1704, 1665, 1453, 1021, 741, 699. ¹H NMR (400 MHz, CDCl₃) δ 7.74 (2H, t, J = 7.7 Hz), 7.55–7.05 (26H, m), 5.64 (2H, td, J = 12.9, 4.5 Hz), 5.26 (1H, d, J = 12.1 Hz), 5.19 (1H, m), 5.14 (1H, d, J = 12.1 Hz), 4.35–4.00 (3H, m), 3.55–3.20 (3H, m), 3.10–2.90 (3H, m), 3.03 (3H, s), 2.91 (6H, s), 2.06 (1H, m), 1.54 (2H, m), 1.42 (2H, m), 0.98 (3H, d, J = 6.9 Hz), 0.89 (3H, t, J = 7.4 Hz), 0.76 (1.8H, d, J = 6.9 Hz), 0.72 (1.2H, d, J = 6.9 Hz), 0.66 (1.8H, d, J = 6.9 Hz), 0.63 (1.2H, d, J = 6.9 Hz), 0.55 (3H, d, J = 6.7 Hz), 0.47 (3H, dd, J = 9.0, 6.7 Hz). ¹³C NMR (101 MHz, CDCl₃, mixture of rotamers) δ 170.6, 170.3, 170.2, 170.13, 170.11, 170.05, 169.8, 169.6, 169.3, 156.7, 156.2, 144.1, 143.94, 143.85, 141.2, 137.2, 136.9, 136.8, 136.6, 135.2, 128.8, 128.74, 128.68, 128.62, 128.57, 128.54, 128.49, 128.45, 127.5, 127.11, 127.06, 127.0, 126.96, 126.9, 126.8, 126.7, 126.6, 125.2, 125.1, 125.0, 124.9, 119.92, 119.88, 119.8, 75.8, 75.5, 75.3, 67.73, 67.66, 67.1, 59.9, 59.6, 57.4, 57.3, 47.2, 47.1, 36.6, 34.9, 34.8, 34.7, 34.6, 32.02, 31.97, 31.7, 31.2, 31.1, 29.4, 29.3, 29.2, 24.1, 19.1, 19.0, 18.9, 16.4, 16.3, 16.1, 16.0, 15.3, 11.7. HRMS (ESI) m/z 1150.54061 [M + Na]⁺ (calcd for C₆₈H₇₇N₃O₁₂Na, 1150.54049).

Beauvericin A (BEAA, 2a)

To a suspension of Pd/C (26 mg, 10% on charcoal) in MeOH/THF (0.8 mL, 1:1 v/v) were added a solution of 21a (88.0 mg, 0.0783 mmol) in MeOH/THF (0.8 mL, 1:1) and MeCN (20 μL, 0.392 mmol) under H₂. The mixture was stirred for 18 h at rt under H₂ (balloon) and filtered through a thin Celite pad. The Celite pad was washed with EtOAc. The filtrate was concentrated under reduced pressure. The acid chloride 8a was employed for the cyclization reaction without purification.

To a solution of the acid chloride 8a in CH₂Cl₂ (12 mL) was added Ghosez’s reagent (28 μL, 0.215 mmol) at 0 °C. The mixture was stirred for 20 min at 0 °C, and then N,N-diisopropylethylamine (36 μL, 0.282 mmol) was added at room temperature. The mixture was stirred for 17 h at rt, diluted with water, and extracted with CH₂Cl₂ (x2). The combined organic layers were washed with brine, dried in MgSO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by high-performance C18 reversed phase silica gel column chromatography (5C18-AR-2, Nacalai tesque, Inc., H₂O/MeCN = 3:7) to give 2a as a white powder (17.2 mg, 28%). [α]²⁵_D +62 (c 1.13, CHCl₃, [α]²⁵_D +49 (c 0.50, MeOH) [lit. Five [α]²⁵_D +57 (c 0.18, MeOH)]. mp 156–157 °C. FTIR (neat): ν_max (cm^–1) 2964, 1743, 1662, 1180, 747, 699. HRMS (ESI) m/z 820.41545 [M + Na]⁺ (calcd for C₄₆H₅₉N₃O₉Na, 820.41490).

allo-Beauvericin A (allo-BEAA, 2b)

To a suspension of Pd/C (26 mg, 10% on charcoal) in MeOH/THF (0.8 mL, 1:1) were added a solution of 21b (40 mg, 0.0354 mmol) in MeOH/THF (0.8 mL, 1:1) and MeCN (9.4 μL, 0.180 mmol) under H₂. The mixture was stirred for 18 h at rt under H₂ (balloon) and filtered through a thin Celite pad. The Celite pad was washed with EtOAc. The filtrate was concentrated under reduced pressure. The acid chloride 8b was employed for the cyclization reaction without purification.

To a solution of the acid chloride 8b in CH₂Cl₂ (6.0 mL) was added Ghosez’s reagent (13 μL, 0.0974 mmol) at 0 °C. The mixture was stirred for 20 min at 0 °C, and then N,N-diisopropylethylamine (20 μL, 0.127 mmol) was added at room temperature. The mixture was stirred for 17 h at rt, diluted with water, and extracted with CH₂Cl₂ (x2). The combined organic layers were washed with brine, dried over MgSO₄, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by high-performance C18 reversed phase silica gel column chromatography (5C18-AR-2, Nacalai Tesque, Inc., H₂O/MeCN = 3:7) to give 2b as a white powder (5.9 mg, 21%). [α]²⁵_D +60 (c 0.64, CHCl₃), [α]²⁵_D +41 (c 0.50, MeOH) [lit. Five [α]²⁵_D +60 (c 0.20, MeOH)]. mp 159–160 °C. FTIR (neat): ν_max (cm^–1) 2964, 1747, 1663, 1182, 1017, 744, 699. HRMS (ESI) m/z 820.41467 [M + Na]⁺ (calcd for C₄₆H₅₉N₃O₉Na, 820.41490).

X-Ray Crystallographic Analysis of BEAA (2a)

A colorless block crystal of 2a from hexanes at room temperature was mounted on the CryoLoop with Palaton oil and placed in the N₂ stream at 110 K. Unit cell dimensions and data reduction were done by using the CrysAlisPro software package (Rigaku Oxford Diffraction, 2020). Absorption corrections were applied using the Multi Scan method. The structures were solved using direct methods (SHELXS-97, Sheldrick GM. University of Gottingen; Germany:1997) and refined by full-matrix least-squares on F² using SHELXL-2018/3 (Sheldrick GM. University of Gottingen; Germany: 2018). The X-ray data have been deposited at the Cambridge Crystallographic Data Center (CCDC 2311751).¹¹

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c00278.

• Experimental details and copies of ¹H and ¹³C NMR spectra (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This work was financially supported from JSPS KAKENHI (JP20H02869 to T.S. and JP23K06051 to A.N.) and research grants from MAYEKAWA HOUONKAI Foundation, FY2023 Fellowship of The Kyoto Technoscience Center Foundation, and Asahi Group Foundation (to A.N.). Fellowship support was provided by Ono Scholarship Foundation (to S.E). This research was also partially supported by Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP23ama121054 (to M.A.).

The authors declare no competing financial interest.