Lower Homologues of Ahpatinin, Aspartic Protease Inhibitors, from a

Jun 24, 2014 - ABSTRACT: Two linear peptides, ahpatinin Ac (1) and ahpatinin Pr. (2), were isolated together with the known ahpatinin iBu, pepstatin A...
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Lower Homologues of Ahpatinin, Aspartic Protease Inhibitors, from a Marine Streptomyces sp. Yi Sun,† Kentaro Takada,† Yuichi Nogi,‡ Shigeru Okada,† and Shigeki Matsunaga*,† †

Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan ‡ Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Natsushima, Yokosuka, Kanagawa 237-0061, Japan S Supporting Information *

ABSTRACT: Two linear peptides, ahpatinin Ac (1) and ahpatinin Pr (2), were isolated together with the known ahpatinin iBu, pepstatin Ac, pepstatin Pr, and pepsinostreptin from a Streptomyces sp. derived from a deep-sea sediment. The structure of ahpatinin Pr (2) was assigned by interpretation of NMR data and HPLC analysis of the hydrolysate after converting to the DNP-L-Val derivative. During the LCMS analysis of the acid hydrolysate, products arising from the retro-aldol cleavage of the statine and Ahppa units in 2 were observed and could facilitate the determination of the absolute configuration of the statine class of nonproteinogenic amino acids. Both ahpatinin Ac (1) and ahpatinin Pr (2) potently inhibited pepsin and moderately inhibited cathepsin B.

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s a part of our program to discover anticancer lead compounds from marine organisms, we examined the culture extract of Streptomyces sp. ACT232, which exhibited significant inhibitory activity against cathepsin B, a thiol protease considered a promising target of anticancer agents.1,2 Bioassay-guided fractionation of the extract afforded six moderate inhibitors. The structural study showed that the active compounds were all related to pepstatin3−5 and ahpatinin.6 As expected from the structural similarity to pepstatin, these compounds exhibited significant inhibitory activity against pepsin, an aspartic protease. In this contribution we describe the isolation and structure elucidation of two new compounds of the ahpatinin class. The bacterial culture medium and the mycelia were extracted separately with EtOAc and acetone, respectively, and the extracts were concentrated in vacuo. Each extract was fractionated by ODS column chromatography followed by several rounds of reversed-phase HPLC to yield ahpatinin Ac (1) and ahpatinin Pr (2), together with four known compounds, ahpatinin iBu,6,7 pepstatin Ac,5 pepstatin Pr,5 and pepsinostreptin.8,9 The molecular formula of ahpatinin Pr (2) was determined to be C35H57N5O9 by HRFABMS. The 1H NMR spectrum in CD3OH (Table 1) exhibited five amide protons. Further analysis of the 1H NMR spectral data in conjunction with the HSQC data revealed the presence of a monosubstituted benzene ring, five methines attached to nitrogen atoms, two oxygenated methines, five aliphatic methylenes, three aliphatic methines, seven doublet methyls, and a triplet methyl (Table 1). Spin systems assignable to Ala and two Val residues were inferred from the COSY data. There was a spin system attributable to a propionyl group. The remaining spin systems © XXXX American Chemical Society and American Society of Pharmacognosy

resembled Leu and Phe residues attached to a coupled oxygenated methine and a deshielded methylene pair. These partial structures suggested the presence of one each of statine (Sta)3 and 4-amino-3-hydroxy-5-phenylpentanoic acid (Ahppa) residues.6 Even though couplings between the β- and γ-protons could not be observed in the COSY spectrum due to their close chemical shifts, the presence of Sta and Ahppa residues was confirmed by the cross-peak between H2-α and Cγ in the Received: April 15, 2014

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dx.doi.org/10.1021/np500337m | J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. NMR Spectroscopic Data (600 MHz) for 1 and 2 ahpatinin Ac (1)a δC, type Val-1d

Val-2d

Sta

Ala

CO α β γ γ′ NH

171.1, 57.9, 30.2, 18.3, 19.3,

C CH CH CH3 CH3

CO α β γ γ′ NH

170.8, 58.2, 30.2, 18.2, 19.2,

C CH CH CH3 CH3

CO α β γ δ

170.8, 39.5, 69.1, 51.0, 38.8,

C CH2 CH CH CH2

ε ζ ζ′ NH

24.4, CH 21.8, CH3 23.5, CH3

CO α β

170.9, C 48.5, CH 18.5, CH3

NH Ahppa

CO α β γ δ

δH (J in Hz) 4.16, m 1.93, m 0.80, dc 0.81, dc 8.00, d (8.5) 4.11, m 1.93, m 0.82, dc 0.82, dc 7.95, d (8.5) 2.12, m 3.83, m 3.79, m 1.24, m 1.36, m 1.50, m 0.78, dc 0.81, dc 7.57, d (8.5) 4.21, m 1.10, d (6.0) 7.88, d (7.2)

e

no 39.9, CH2

67.8, CH 54.3, CH 36.8, CH2

1.87, m 1.94, 3.63, 3.76, 2.63,

m m m m

ahpatinin Pr (2)b δC, type 174.0, 60.5, 31.5, 18.8, 19.9,

C CH CH CH3 CH3

173.6, 60.9, 31.5, 18.8, 19.9,

C CH CH CH3 CH3

173.7, 41.2, 71.2, 53.2, 41.0,

C CH2 CH CH CH2

25.7, CH 22.2, CH3 23.6, CH3

174.8, C 50.7, CH 18.2, CH3

acyl

139.5, 128.1, 129.2, 125.9,

C CH CH CH

CO α β

169.4, C 22.6, CH3

7.23, m 7.20, m 7.12, m 7.55, d (8.1) 1.84, s

4.17, 2.07, 0.96, 0.99, 8.00,

t (7.7) m d (6.7) d (6.6) d (7.6)

4.14, 2.07, 0.97, 0.95, 8.02,

t (7.7) m d (6.6) d (6.7) d (7.5)

2.28, 3.98, 3.88, 1.33, 1.56, 1.60, 0.88, 0.90, 7.76,

m m m m m m d (6.4) d (6.4) d (9.3)

Figure 1. Sequential HMBC and NOESY correlations in 2.

tion reagent was chosen because it was reported to give the best result among several variants of Marfey’s reagents.10 The (3S,4S)- and (3S,4R)-isomers of both statine and Ahppa were prepared following the protocol reported by Molinski et al.11,12 The LCMS analysis showed the presence of L-Val, L-Ala, (3S,4S)-statine, and (3S,4S)-Ahppa. Unexpectedly we observed the presence of L-Leu and L-Phe in the hydrolysate, which were likely to be derived from statine and the Ahppa residue, respectively, through a retro-aldol reaction.12 Ahpatinin Ac (1) had the molecular formula C34H54N5O9, which was smaller than that of 2 by a CH2 unit. The 1H NMR spectrum of 1 was almost identical with that of 2 except for the presence of a singlet methyl group (δH 1.85, H3-34) instead of the terminal ethyl group. 2D NMR data including HMBC indicated that the N-terminus was acetylated. Otherwise the structure of 1 was identical with that of 2 (Table 1). All of the compounds isolated in this study exhibited moderate inhibitory activity against cathepsin B, with IC50 values between 10 and 29 μM (Table 2). The structural similarity of 1 and 2 with pepstatin,14 a potent aspartic protease inhibitor, prompted us to examine the inhibitory activity against pepsin, an aspartic protease. As expected, 1 and 2, as well as pepstatin Ac and pepstatin Pr, inhibited pepsin with IC50 values between 11 and 50 nM. Our strain of marine-derived Streptomyces produced only homologues of pepstatin and ahpatinin with short acyl groups. The isolation of these congeners raised a problem of identification of compounds that had been isolated when high-field NMR spectroscopy was not available. Because of the lack of NMR spectroscopic data, it was not possible to directly confirm the identification of the four previously reported compounds. However, due to the reported enzyme inhibitory activities and their isolation from other Streptomyces strains, it was highly likely that our compounds are identical to the those in the literature.9 In our structural study we observed the retroaldol fragmentation and oxidation of statine and Ahppa residues during acid hydrolysis, which permitted the assignment of the C-4 absolute configuration of these residues by Marfey’s analysis. If we could combine this information with the NMR method to assign the relative configuration of these residues,12,13 it will be possible to assign the total absolute configuration of statine and its relatives without using synthetic standards.

4.32, q (7.6) 1.28, d (7.1) 8.08, d (7.1)

180.0, C 41.5, CH2

69.9, CH 56.1, CH 38.4, CH2

2.80, m 1 2, 6 3, 5 4 NH

δH (J in Hz)

140.0, 130.4, 129.1, 127.1,

C CH CH CH

177.3, C 29.8, CH2 10.4, CH3

2.18, dd (15.1, 3.4) 2.28, m 3.94, m 4.00, m 2.81, dd (13.5, 8.2) 2.94, dd (13.5, 6.8) 7.24, 7.43, 7.14, 7.67,

m m m d (8.5)

2.27, m 1.12, t (7.6)

a

In DMSO-d6. bIn CD3OH. cJ-value was not determined due to overlapped signals. dMethyl signals of the two valine residues were not assigned to each residue. eNot observed.

HMBC spectrum (Figure 1). The sequence of the abovementioned residues was established by interpretation of the NOESY and HMBC data; all of the sequential NOESY crosspeaks (NH to αH) were observed (Figure 1). The absolute configuration of 2 was studied by the LCMS analysis of the acid hydrolysate after converting to the dinitrophenyl (DNP)-L-Val derivatives. This chiral derivatiza-

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EXPERIMENTAL SECTION

General Experimental Procedures. General procedures are described elsewhere.15 ESI mass spectra were measured on a JEOL B

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Table 2. Inhibitory Activities (IC50 Values) against Pepsin and Cathepsin B pepsin (μM) cathepsin B (μM) a

ahpatinin Ac (1)

ahpatinin Pr (2)

ahpatinin iBu

pepstatin Ac

pepstatin Pr

pepsinostreptin

0.011 15

0.050 25

NTa 26

0.011 17

0.018 10

NT 29

NT: not tested. mixtures of n-hexane and EtOAc (9:1, 30 mL × 2; 8:2, 30 mL × 2; 1:1, 30 mL × 2). The N-Boc-5-isobutylpyrrolidine-2,4-dione eluted in fractions 4 and 5 (yield 109 mg). A 55 mg portion of this material was dissolved in a mixture of CH2Cl2 and TFA (3:2, 2.5 mL) and left at rt for 10 min. To the reaction product dissolved in CH2Cl2−AcOH (9:1, 2 mL) was added NaBH4 (15 mg) and left at rt for 10 min. The reaction mixture was dried, applied to a column of SiO2 (1.5 × 2 cm), and eluted with 10 mL each of CH2Cl2, CH2Cl2−MeOH (98:2), CH2Cl2−MeOH (95:5), CH2Cl2−MeOH (9:1), and CH2Cl2−MeOH (8:2). Fractions 3 and 4 were combined to afford a mixture of (4S,5S)and (4R,5S)-4-hydroxy-5-isobutylpyrrolidin-2-one (22 mg): 1H NMR (600 MHz, DMSO-d6) (4S,5S)-isomer (major product) δ 7.57 brs, 4.90 (d, J = 4.9 Hz; OH), 4.16 m, 3.49 (brq, J = 6.3 Hz), 2.39 (dd, J = 6.1, 16.5 Hz), 1.94 (dd, J = 2.7, 16.5 Hz), 1.68 m, 1.37 m, 1.28 m, 0.88 (3H, d, J = 6.5 Hz), 0.85 (3H, d, J = 6.5 Hz); (4R,5S)-isomer (minor product) δ 7.73 brs, 5.13 (d, J = 4.6 Hz; OH), 3.85 m, 3.24 (brt, J = 6.3 Hz), 2.44 (dd, J = 6.7, 16.8 Hz), 1.89 (dd, J = 3.7, 16.8 Hz), 1.68 m, 1.24 m, 1.19 m, 0.88 (3H, d, J = 6.5 Hz), 0.85 (3H, d, J = 6.5 Hz). An 8 mg portion of this mixture was dissolved in 6 N HCl (1 mL), kept at 110 °C for 1.5 h, and evaporated to dryness to afford a mixture of statine isomers (12 mg): 1H NMR (600 MHz, D2O) (3S,4S)isomer (major product) δ 4.00 m, 3.22 m, 2.66 m, 2.47 m, 1.59 m, 1.41 (2H, m), 0.83 (3H, m), 0.81 (3H, m); (3R,4S)-isomer (minor product) δ 4.24 m, 3.35 m, 2.55 m, 2.44 m, 1.59 m, 1.41 (2H, m), 0.83 (3H, m), 0.81 (3H, m). Ahppa (refs 11 and 12). The (3S,4S)- and (3R,4S)-isomers of Ahppa were prepared from Boc-L-Phe following a published procedure to prepare the mixture of statine isomers: 1H NMR (600 MHz, D2O) (3S,4S)-isomer (major product) δ 7.2−7.3 (5H, m), 4.04 m, 3.48 m, 3.04 m, 2.78 m, 2.69 m, 2.53 m; (3R,4S)-isomer (minor product) δ 7.2−7.3 (5H, m), 4.29 m, 3.60 m, 3.04 m, 2.69 m, 2.64 m, 2.51 m. LC-MS Analysis of the DNP-Val Derivatives of Amino Acids (ref 10). Compound 2 (0.1 mg) was dissolved in 6 N HCl (0.1 mL) and heated at 110 °C overnight. The reaction mixture was evaporated to dryness. To the product were added 200 μL of 0.6 M NaHCO3 and 150 μL of a 3% FDNP-Val solution in EtOH. The solution was left at rt overnight, neutralized with 2 N HCl, and analyzed by LCMS with the following HPLC conditions: column, Cosmosil π nap, ⦶ 2.0 × 100 mm; 10% MeCN for 2 min, and the MeCN concentration was raised to 50% in 20 min. Retention times and the molecular ion [M + H]+ for the DNP-L-Val derivatives of the standard amino acids (min, m/z): LAla (24.2, 371), D-Ala (26.8, 371), L-Val (27.9, 399), D-Val (30.8, 399), L-Leu (29.9, 413), D-Leu (33.0, 413), L-Phe (31.0, 447), D-Phe (33.4, 447), (3S,4S)-Sta (26.8, 457), (3S,4R)-Sta (27.0, 457), (3S,4S)-Ahppa (27.8, 491), (3S,4R)-Ahppa (28.1, 491), (3R,4S)-Ahppa (31.1, 491), (3R,4S)-Sta (31.1, 457), (3R,4R)-Sta (31.3, 457), (3R,4R)-Ahppa (31.3, 491). Retention times for the acid hydrolysate of 2 (min): 24.2 (m/z 371), 26.8 (m/z 457), 27.7 (m/z 491), 27.9 (m/z 399), 29.9 (m/ z 413), 31.0 (m/z 447). Cathepsin B Inhibition Assay. The cathepsin B inhibitory assay was performed according to a modification of the method in the literature.17 The enzyme (cathepsin B from bovine spleen, Sigma C6286) was stocked at 1 unit/mL in 50 mM MES pH 6.0 and 0.1% Brij-35. The enzyme solution was diluted 100 times with the buffer before use. The mixture of 4 μL test sample solution, 100 μL of the enzyme solution, and 50 μL of 25 μM fluorescent substrate (Z-ArgArg-AMC, Peptide Institute, Inc.) in DMSO was incubated at 37 °C for 30 min. The fluorescence of the liberated AMC was measured with an excitation at 345 nm and emission at 440 nm. Pepsin Inhibition Assay. The pepsin assay was performed by a slight modification of a method described in the literature.18 Pepsin from porcine gastric mucosa (Sigma P7012) and MOCAc-Ala-Pro-Ala-

JMS-T 100LC. FAB mass spectra were measured on a JEOL JMS700T MStation. All HRFABMS data were measured using NBA with LiCl as a matrix. LC-MS data were obtained using a Bruker amaZon SL-TA equipped with a Shimadzu LC system. HPLC was carried out on a Shimadzu LC 20AT with an SCL-10Avp controller and an SPD10Avp detector. Fluorescence for the pepsin assay was measured with a Molecular Devices Spectra MAX GEMINI apparatus. Collection and Identification. Deep-sea sediments were collected by the manned submersible “SHINKAI 2000” system off Hatsu-shima, Sagami-Bay, Japan, at a depth of 1174 m, in December 2001. The sediment sample was stored in a sterilized sampler, frozen with liquid nitrogen, and transported to the laboratory, where it was kept frozen until processed. The Streptomyces sp. ACT232 was isolated from this sample. The taxonomy of the strain was determined by 16S rRNA phylogenetic analysis using 27F and 1492R primers, and the sequence was deposited in the DNA Data Bank of Japan (DDBJ, accession no. AB968434). Fermentation, Extraction, and Isolation. Streptomyces sp. ACT232 was cultured in 60 × 500 mL Erlenmeyer flasks each containing 150 mL of ISP medium 2 (yeast extract 0.6 g, malt extract 1.5 g, glucose 0.6 g) at 27 °C on rotary shakers at 150 rpm. After 4 days of culture, the fermentation broth was harvested and centrifuged to separate the mycelia from the aqueous layer. The mycelia and the aqueous layer were extracted with acetone and EtOAc, respectively. Each extract was concentrated in vacuo and separately subjected to ODS flash column chromatography (5 × 30 cm) eluting with 2 L each of 20%, 40%, 60%, 80%, and 100% (v/v) MeOH in H2O. The fractions that eluted with 60% MeOH were combined and fractionated by chromatography on a Sephadex LH-20 column (CHCl3−MeOH, 1:1) followed by ODS-HPLC with gradient elution from 45% to 60% aqueous MeOH with 0.2% AcOH to yield six fractions (A−F). Fractions D and E were further purified by ODS-HPLC using 27% and 25% MeCN in H2O, respectively, to afford ahpatinin Pr (2, 2.0 mg), ahpatinin iBu (1.6 mg), pepsinostreptin (1.2 mg), and pepstatin Ac (1.8 mg). Fraction F was purified by ODS-HPLC using 55% MeOH in H2O to yield ahpatinin Ac (1, 1.2 mg) and pepstatin Pr (1.1 mg). Ahpatinin Ac (1): colorless solid; [α]25D −27 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 252 (2.6), 264 (2.7) nm; 1H NMR (DMSO-d6) and 13C NMR (DMSO-d6), see Table 1; HRFABMS m/z 690.4231 [M + 2Li − H]+ (calcd for C34H54N5O9Li2, 690.4242). Ahpatinin Pr (2): colorless solid; [α]25D −21 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 252 (2.6), 261 (2.7) nm; 1H NMR (CD3OH) and 13C NMR (CD3OH), see Table 1; HRFABMS m/z 698.4299 [M + Li]+ (calcd for C35H57N5O9Li, 698.4317). FDNP-Val (ref 10). To a solution of L-Val (88 mg, 0.75 mmol) in 0.6 M NaHCO3 (4.0 mL) was added 1,5-difluoro-2,4-dinitrobenzene (305 mg) in a mixture of acetone and H2O (10 and 7.5 mL, respectively) and stirred at room temperature (rt) overnight. The reaction mixture was dried and applied to a column of SiO2 (1.5 × 4.0 cm) and eluted with CHCl3−MeOH (9:1, 30 mL), CHCl3−MeOH (8:2, 30 mL), and CHCl3−MeOH−H2O (7:3:0.5, 30 mL × 2). The fraction eluted with CHCl3−MeOH (8:2) and the first fraction eluted with CHCl3−MeOH−H2O (7:3:0.5) were combined to afford FDNPL-Val. FDNP-D-Val was prepared in the same way starting with D-Val. Statine (refs 11 and 12). To a solution of Boc-L-Leu (229 mg, 0.92 mmol) in CH2Cl2 (5 mL) were added Meldrum’s acid (146 mg, 1.01 mmol), DMAP (157 mg, 1.3 mmol), and EDC-HCl (211 mg, 1.11 mmol) and stirred at rt for 1 h. The reaction mixture was washed with 5% KHSO4 (three times) and brine, dried over MgSO4, and evaporated to dryness. The product was dissolved in MeCN (10 mL), and the solution was refluxed for 1 h. After evaporating the solvent, the product was applied to a column of SiO2 (1.5 × 3 cm) and eluted with C

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(13) Preciado, A.; Williams, P. G. J. Org. Chem. 2008, 73, 9228− 9234. (14) Tumminello, F. M.; Bernacki, R. J.; Gebbia, N.; Leto, G. Med. Res. Rev. 1993, 13, 199−208. (15) Akiyama, T.; Takada, K.; Oikawa, T.; Matsuura, N.; Ise, Y.; Okada, S.; Matsunaga, S. Tetrahedron 2013, 69, 6560−6564. (16) Jeong, Y.-G.; Moloney, M. G. Synlett 2009, 2487−2491. (17) Hiwasa, T.; Fujita-Yoshigaki, J.; Shirouzu, M.; Koide, H.; Sawada, T.; Sakiyama, S.; Yokokawa, S. Cancer Lett. 1993, 69, 161− 165. (18) Kondo, H.; Shibano, Y.; Amachi, T.; Cronin, N.; Oda, K.; Dunn, B. M. J. Biochem. 1998, 124, 141−147.

Lys-Phe-Phe-Arg-Leu-Lys (Dnp)-NH2 (Peptide Institute Inc. 3216-v) were used as enzyme and fluorescent substrate, respectively. A 1 μL portion of a test solution dissolved in MeOH was diluted with 50 μL of 0.1 M ammonium formate buffer (pH 3.0), followed by the addition of 100 μL of the pepsin solution (1 μg/mL pepsin in the buffer). Fluorescent substrate solution (50 μL; 10 μM) was added into the reaction mixtures and incubated at 30 °C for 20 min. Pepsin activity was measured by the fluorescence of the released MOCAc-Ala-ProAla-Lys-Phe peptide at 393 nm upon excitation at 328 nm.

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ASSOCIATED CONTENT

* Supporting Information S

NMR data for compounds 1, 2, ahpatinin iBu, pepstatin Ac, pepstatin Pr, and pepsinostreptin. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 81-3-5841-5297. Fax: 81-3-5841-8166. E-mail: assmats@ mail.ecc.u-tokyo.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemical Biology of Natural Products” and JSPS KAKENHI Grant Nos. 25252037, 25712024, and 25660163 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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REFERENCES

(1) Gondi, C. S.; Rao, J. S. Expert Opin. Ther. Targets 2013, 17, 281− 291. (2) Mason, S. D.; Joyce, J. A. Trends Cell Biol. 2011, 21, 228−237. (3) Morishima, H.; Takita, T.; Aoyagi, T.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1970, 23, 263−265. (4) Kunimoto, S.; Aoyagi, T.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1972, 25, 251−255. (5) Aoyagi, T.; Yagisawa, Y.; Kumagai, M.; Hamada, M.; Morishima, H.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1973, 26, 539−541. (6) Omura, S.; Imamura, N.; Kawakita, K.; Mori, Y.; Yamazaki, Y.; Masuma, R.; Takahashi, Y.; Tanaka, H.; Huang, L.-Y.; Woodruff, H. B. J. Antibiot. 1986, 39, 1079−1085. (7) Ahpatinin iBu corresponds to either ahpatinin A or B. The structures of ahpatinins A and B were reported based on the fragment ions by EIMS, which cannot assign the structure of the acyl chains.6 (8) Kanematsu, T.; Sugino, H.; Asano, T.; Kakinuma, A. J. Takeda Res. Lab. 1976, 35, 128−135. (9) The structures of these four compounds had been assigned by analysis of fragment ions in mass spectrometry.5,6,8 Because their NMR spectroscopic data have not been reported, we report their NMR data in the Supporting Information. (10) Bhushan, R.; Kumar, R. Anal. Bioanal. Chem. 2009, 394, 1697− 1705. (11) Molinski, T. F.; Reynolds, K. A.; Morinaka, B. I. J. Nat. Prod. 2012, 75, 425−431. (12) With this observation we reasoned that the absolute configuration of statine and its variants could be determined without preparation of standard nonproteinogenic amino acids. A combination of Marfey’s analysis of the acid hydrolysate to analyze the amino acid fragment liberated from a statine-related amino acid and the empirical 1 H NMR method proposed by Preciado and Williams permits the assignment of the relative configuration of this class of nonproteinogenic amino acids.13 Unfortunately the methyl ester of 2 as well as pepstatin methyl ester was not sufficiently soluble in CDCl3, the solvent of choice for this 1H NMR analysis. D

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